Anti-CD25 antibody and Anti-CD25 antibody-drug conjugate

Abstract

The present invention addresses the problem of providing: an antibody that binds to CD25 and has an internalization activity; an antibody-drug conjugate which contains said antibody and has an anti-tumor activity; a medicine which includes said antibody-drug conjugate and has a therapeutic effect on tumors; and a method for treating a tumor using said antibody, said antibody-drug conjugate, or said medicine. Provided is a CD25 antibody or an antigen-binding fragment of the antibody, characterized by (1) not having an IL-2 blocking ability, and (2) having an internalization activity into CD25-expressing cells by binding to CD25. The antibody or antigen-binding fragment of the antibody can be conjugated with a cytotoxically active compound to exhibit ability to remove regulatory T cells and / or ability to promote proliferation of granzyme-positive CD8-positive cells.

Description

Anti-CD25 antibodies and anti-CD25 antibody-drug conjugates

[0001] The present invention relates to an anti-CD25 antibody that has the ability to bind to CD25, a method for producing the anti-CD25 antibody, an antibody-drug conjugate containing the antibody, an antitumor agent containing the antibody-drug conjugate, and the like.

[0002] Regulatory T cells (Tregs) are a subset of CD4+ T cells that play a role in suppressing immune responses and establishing and maintaining self-immune tolerance (Non-Patent Document 1). They account for approximately 10% of peripheral CD4+ T cells, and Forkhead box P3 (FoxP3) has been identified as a master transcription factor (Non-Patent Document 2). Scurfy mice, which have a FoxP3 mismutation, have been shown to exhibit excessive proliferation of CD4+ cells and elevated cytokines (Non-Patent Documents 3 and 4). Furthermore, mutations in the FoxP3 gene in humans are known to cause Treg dysfunction, resulting in an autoimmune disorder known as IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome (Non-Patent Document 5). These findings suggest that Tregs play an important role in immune tolerance.

[0003] Cancer is a disease that can be one of the leading causes of death (Non-Patent Document 6), and can become fatal due to damage to normal tissues caused by abnormal proliferation of cancer cells. Characteristics of cancer cells include unregulated cell proliferation, resistance to apoptosis, and migration to other tissues (Non-Patent Document 7). Another characteristic of cancer cells is their ability to evade immune responses against them.

[0004] Cancer cells accumulate genetic mutations, including not only driver mutations but also passenger mutations, due to genetic instability. Abnormal proteins transcribed and translated from these mutations are presented as neoantigens. Because these neoantigens are distinct from autoantigens, immune cells would normally recognize them as non-normal autologous cells and eliminate them (Non-Patent Document 8). However, the tumor microenvironment creates an immune-suppressive environment in various ways, including the high expression of immunosuppressive cytokines such as TGF-β and IL-10, immunosuppressive molecules such as PD-1 and PD-L1, and the infiltration of immunosuppressive cells, which is thought to make it difficult for immune cells to eliminate cancer cells (Non-Patent Document 9). In recent years, immune checkpoint inhibitors such as anti-PD-1 antibodies have shown promising results in clinical trials and have been approved for the treatment of various advanced cancers, including melanoma and non-small cell lung cancer (NSCLC) (Non-Patent Documents 10 and 11). However, immune checkpoint inhibitors only benefit a limited number of patients, and one of the reasons for this is that the aforementioned immunosuppressive cells (especially Tregs) are thought to be the main cells suppressing tumor immunity. The presence of Tregs within tumors suppresses the activity of effector T cells (Teff) and cytotoxic T cells (CTLs), preventing the elimination of cancer cells as foreign bodies and contributing to the worsening of the disease. It has also been reported that the balance between Tregs and CTLs within tumors correlates with prognosis (Non-Patent Document 12).

[0005] CD25 (also known as the IL-2 receptor α chain, Tac antigen) is a type I transmembrane protein with two extracellular Sushi domains (Non-Patent Documents 13, 14), and is known as the IL-2 receptor α chain. Upon binding with IL-2, a factor known to play various roles, including T cell differentiation and proliferation, CD25 transduces IL-2 signals into the cell (Non-Patent Document 15). It has been reported that CD25 is expressed at low levels on limited immune cells, such as activated T cells and B cells (Non-Patent Documents 16, 17), but is highly expressed on Tregs (Non-Patent Document 18). Furthermore, it has been shown that the immunosuppressive activity of Tregs correlates with the intensity of CD25 expression (Non-Patent Document 18). It has been reported that the role of CD25 in Tregs is that IL-2 signaling is essential for their survival (Non-Patent Document 19), and that by binding to and consuming IL-2, it reduces the opportunity for other immune cells to bind IL-2 (Non-Patent Document 19). Furthermore, CD25 has also been reported to be expressed in blood cancer cells such as non-Hodgkin's lymphoma (Non-Patent Document 20).

[0006] Several anti-CD25 antibodies have been created to date. Among these, there are two types: antibodies that can bind to CD25 while maintaining its IL-2 signaling, and antibodies that bind by inhibiting IL-2 signaling. For example, Anti-Tac (anti-human CD25 antibody) is an antibody that inhibits IL-2 signaling, while 7G7B6 (anti-human CD25 antibody) is an antibody that does not inhibit IL-2 signaling. It has been reported that Anti-Tac inhibits IL-2-dependent T cell proliferation, while 7G7B6 does not (Non-Patent Document 21). Basiliximab, an antibody that inhibits IL-2 signaling, is believed to suppress T cell-mediated immune responses and is used to prevent rejection in organ transplants. Therefore, anti-CD25 antibodies that inhibit IL-2 signaling suppress immune activation. On the other hand, anti-CD25 antibodies that do not inhibit IL-2 signaling are thought not to suppress T cell-mediated immune responses (Non-Patent Document 22). Studies using anti-mouse CD25 antibodies have been reported. In recent years, the therapeutic effects of these antibodies in mouse tumor models have been reported, and results suggesting that these effects are due to the elimination of intratumoral Tregs and the activation of Teff have been reported (Non-Patent Document 22). It has been reported that among Tregs, Tregs with strong immunosuppressive activity are present within human tumors (Non-Patent Document 23). Because this cell population strongly expresses CD25, CD25 is a promising target for targeting intratumoral Tregs.

[0007] Antibody-drug conjugates (ADCs) are compounds that combine an antibody that specifically binds to a target antigen with a cytotoxic drug (Non-Patent Document 24). This characteristic allows them to kill cells that express the target antigen while minimizing the effects on cells that do not. ADCs have been developed as therapeutic agents in the field of cancer. For example, Enhertz™ (trastuzumab deruxtecan), an anti-HER2 monoclonal antibody conjugated to deruxtecan, is used to treat HER2-positive advanced or recurrent breast cancer. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are known mechanisms for specifically eliminating cells that express a target antigen using antibodies. Focusing on this mechanism, antibody technology with enhanced ADCC activity has been developed (Non-Patent Document 25). Poteligio™ (mogamulizumab), an anti-human CCR4 antibody with enhanced ADCC activity, has been shown to be effective in a limited range of T-cell lymphomas (Non-Patent Document 26). On the other hand, there are mixed opinions about the effectiveness of antibodies with enhanced ADCC activity in treating solid cancers (Non-Patent Document 27). One possible reason for the lack of progress in treating solid cancers is the low presence of NK cells, the main immune cells that exert ADCC activity (Non-Patent Document 28).

[0008] For these reasons, the application of CD25 as a cancer treatment drug aimed at eliminating Tregs by targeting CD25 has been considered. However, there have been no reports to date of the selective elimination of intratumoral Tregs in solid cancers. Clinical trials are currently being conducted in solid cancers for RG-6292, an ADCC-enhanced Ab that targets CD25, and ADCT-301, an ADC in which pyrrolobenzodiazepine (PBD) is conjugated to an anti-CD25 antibody.

[0009] Sakaguchi S, et al.,Annu Rev Immunol. 2004;22:531-562.Sakaguchi S,et al.,Annu Rev Immunol. 2020;38:541-566.Brunkow ME,et al., 2001 Nature Publishing Group http: / / genetics.nature.com. 2001;27(january):68-73.Sakaguchi S,et al., Cell. 2008;133(5):775-787.Bacchetta R,et al.,Ann N Y Acad Sci. 2016;1417(1):5-22.Sung H,et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-249.Hanahan D, et al., Cell. 2011;144(5):646-674.Chen DS,et al., Immunity. 2013;39(1):1-10.Huang AC,et al., Nat Immunol. Published online 2022;23(5),660-670.Larkin J,et al., N Engl J Med. 2015;373(1):23-34.Mok TSK,et al., Lancet. 2019;393(10183):1819-1830.O’Callaghan DS,et al., Eur Respir J. 2015;46(6):1762-1772.Rickert M,et al., Rickert M, Wang X, Boulanger MJ, Goriatcheva N, Garcia KC. The Structure of Interleukin-2 Complexed with Its Alpha Receptor. 2014;1477(June):1477-1481.Wang X,et al.,Science. 2005;310(5751):1159-1163.Spolski R,et al., Nat Rev Immunol. 2018;18(10):648-659.Tsudo M,et al., J Immunol. 1982;129(2):592-595.Brisslert M,et al., Immunology. 2006;117(4):548-557.Miyara M,et al., Immunity. 2009;30(6):899-911.Chinen T,et al., Nat Immunol. 2016;17(11):1322-1333.Flynn MJ,et al., Br J Haematol. 2017;179(1):20-35.Rubin LA.et al., A monoclonal antibody 7G7 / B6, binds to an epitope on the human interleukin-2 (IL-2) receptor that is distinct from that recognized by IL-2 or anti-Tac Hybridoma 1985;4(2): 91-102.Solomon I,et al., Nat Cancer. 2020;1(12):1153-1166.Saito T,et al., Nat Med. 2016;22(6):679-684.Chari RVJ,et al., Angew Chemie - Int Ed. 2014;53(15):3796-3827.Niwa R,et al., Cancer Res. 2004;64(6):2127-2133.Ishida T,et al., J Clin Oncol. 2012;30(8):837-842.Lo Nigro C,et al., Ann Transl Med. 2019;7(5):105-105.Marechal R,et al., BMC Cancer. 2010;10.;

[0010] An object of the present invention is to provide an antibody that binds to CD25, an antibody-drug conjugate containing the antibody and having antitumor activity, a pharmaceutical product using the antibody-drug conjugate and having a therapeutic effect against tumors, and a method for treating tumors using the antibody, antibody-drug conjugate, or pharmaceutical product.

[0011] As a result of extensive research to achieve the above object, the present inventors have found that a specific antibody disclosed in the present invention binds to CD25 and is useful as an antibody for antibody-drug conjugates. Furthermore, they have found that the anti-CD25 antibody-drug conjugates disclosed in the present invention are useful as pharmaceuticals.

[0012] The present invention encompasses the following inventions. [1] An anti-CD25 antibody or an antigen-binding fragment of the antibody, characterized by having the following properties: (1) No IL-2 blocking ability. (2) When conjugated with a cytotoxic compound, the antibody or antigen-binding fragment exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells. [2] The antibody or antigen-binding fragment of the antibody according to [1], which has the activity of being internalized into CD25-expressing cells by binding to CD25. [3] The antibody or antigen-binding fragment of the antibody according to [1] or [2], wherein CD25 is human CD25, cynomolgus monkey CD25, or mouse CD25. [4] The antibody or antigen-binding fragment of the antibody according to [3], wherein CD25 is human CD25 or cynomolgus monkey CD25. [4-2] The antibody or antigen-binding fragment of the antibody according to [3], wherein CD25 is human CD25. [5] The antibody or antigen-binding fragment of the antibody according to [3], wherein CD25 is mouse CD25. [5-2] The following (1) to (6) are effective in binding to CD25: (1) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 232 of SEQ ID NO: 26 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 27; (2) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15; (3) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16; (4) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 232 of SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence of positions 20 to 463 of SEQ ID NO: 31; (5) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 239 of SEQ ID NO: 32 and a heavy chain consisting of the amino acid sequence of positions 20 to 463 of SEQ ID NO: 33; (6) An antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 239 of SEQ ID NO: 54 and a heavy chain consisting of the amino acid sequence of positions 20 to 463 of SEQ ID NO: 55. The antibody or antigen-binding fragment of the antibody according to any one of [1] to [5], which has competitive inhibitory activity with at least one of antibodies selected from the group consisting of:[5-3] The antibody or antigen-binding fragment of any one of [1] to [5], which has competitive inhibitory activity against binding to CD25 with at least one of the antibodies selected from the group consisting of the following (1) to (3): (1) an antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 232 of SEQ ID NO: 26 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 27; (2) an antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 15; and (3) an antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 16. [5-4] The antibody or antigen-binding fragment of any one of [1] to [5], which has competitive inhibitory activity against binding to CD25 with at least one of the following (1) and (2): (1) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15, and (2) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16.[6] A CDRL1, CDRL2, and CDRL3 selected from the group consisting of the following (1) to (2): (1) a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 (KAS), and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (2) a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5 (FVS), and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; and (3) a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9; (4) a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 12, An antibody or an antigen-binding fragment thereof, or the antibody or antigen-binding fragment thereof according to any one of [1] to [5-4]. [7] The antibody or antigen-binding fragment of the antibody according to any one of [1] to [6], comprising CDRL1, CDRL2, and CDRL3, and CDRH1, CDRH2, and CDRH3 selected from the group consisting of: (1) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 (KAS), and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9; (2) CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5 (FVS), and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, and CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 12. [8] The antibody or antigen-binding fragment of any one of [1] to [7], which is humanized.[9] A light chain variable region according to any one of the following (1) to (4) selected from the group consisting of: (1) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13; (2) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14; (3) a light chain variable region consisting of an amino acid sequence having at least 95% sequence identity to the sequence of framework regions other than each CDR sequence in the amino acid sequences of (1) to (2); and (4) a light chain variable region consisting of the amino acid sequence of (1) to (2) in which one or several amino acids are deleted, substituted, or added in the sequence of framework regions other than each CDR sequence; and (5) a light chain variable region according to any one of the following (5) to (8): (5) a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15; (6) a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16; (7) A heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity to the framework region sequences other than each CDR sequence in the amino acid sequences of (5) to (6), and (8) a heavy chain variable region comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the framework region sequences other than each CDR sequence in the amino acid sequences of (5) to (6). The antibody or antigen-binding fragment of the antibody according to any one of [1] to [8],

[10] The antibody or antigen-binding fragment of the antibody according to any one of [1] to [9], comprising a light chain variable region and a heavy chain variable region selected from the following (1) to (2): (1) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15; or (2) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16.

[11] The antibody or antigen-binding fragment of the antibody according to any one of [1] to

[10] , comprising any of the following (1) to (2): (1) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15, or (2) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16.

[12] The antibody or antigen-binding fragment of the antibody according to

[11] , comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15.

[13] The antibody or antigen-binding fragment of the antibody according to

[11] , comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16.

[14] An antigen-binding fragment of the antibody according to any one of [1] to

[13] , wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', and Fv.

[15] An antigen-binding fragment of the antibody according to any one of [1] to

[13] , wherein the antigen-binding fragment is scFv. [15-2] A humanized antibody comprising a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15. [15-3] A humanized antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15. [15-4] A humanized antibody comprising a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16. [15-5] A humanized antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16. [15-6] An anti-CD25 antibody or an antigen-binding fragment of said antibody, characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; and (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells.(d) It has the activity of being internalized into CD25-expressing cells by binding to CD25. the CD25 is human CD25, cynomolgus monkey CD25, or mouse CD25; and the antibody or antigen-binding fragment thereof comprises CDRL1, CDRL2, and CDRL3 selected from the group consisting of the following (1) to (2): (1) a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 (KAS), and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (2) a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5 (FVS), and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; and the following (3) to (4): (3) a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9; (4) a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 12; [15-7] An anti-CD25 antibody or an antigen-binding fragment of the antibody, characterized in that it has any one or more of the following properties (a) to (d): (a) it specifically binds to CD25; (b) it does not have IL-2 blocking ability; (c) it exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells when conjugated with a cytotoxic compound; and (d) it has the activity of being internalized into CD25-expressing cells by binding to CD25.the CD25 is human CD25 or cynomolgus monkey CD25, and the antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of the following (1) to (4): (1) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13, (2) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14, (3) a light chain variable region consisting of an amino acid sequence having at least 95% sequence identity to the framework region sequence other than each CDR sequence in the amino acid sequence of (1) to (2), and (4) a light chain variable region consisting of the amino acid sequence in which one or several amino acids are deleted, substituted, or added in the framework region sequence other than each CDR sequence in the amino acid sequence of (1) to (2), and the following (5) to (8): (5) a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15, (6) a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 146 of SEQ ID NO: 16, (7) a heavy chain variable region consisting of an amino acid sequence having at least 95% sequence identity to the framework regions excluding each CDR sequence in the amino acid sequences of (5) to (6), and (8) a heavy chain variable region consisting of the amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the framework regions excluding each CDR sequence in the amino acid sequences of (5) to (6). [15-8] An anti-CD25 antibody or an antigen-binding fragment of the antibody, characterized in that it has any one or more of the following properties (a) to (d): (a) it specifically binds to CD25, (b) it does not have IL-2 blocking ability, and (c) when conjugated with a cytotoxic compound, it exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells. (d) It has the activity of being internalized into CD25-expressing cells by binding to CD25.The CD25 is human CD25 or cynomolgus monkey CD25, and the antibody or antigen-binding fragment thereof comprises any of the following (1) to (2): (1) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15, or (2) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16, or an antibody or antigen-binding fragment thereof.

[0013]

[16] A polynucleotide encoding the antibody or antigen-binding fragment of the antibody according to any one of [1] to [15-8].

[17] The polynucleotide according to

[16] , comprising any one of the polynucleotides selected from the group consisting of the following (1) to (2): (1) a polynucleotide encoding a light chain variable region comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 (KAS), and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9; (2) a polynucleotide encoding a light chain variable region comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5 (FVS), and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 12.

[18] The polynucleotide according to

[16] or

[17] , comprising a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15.

[19] The polynucleotide according to

[16] or

[17] , comprising a polynucleotide encoding a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16.

[20] An expression vector comprising the polynucleotide according to any one of

[16] to

[19] .

[21] A host cell transformed with the expression vector according to

[20] .

[22] The host cell according to

[21] , wherein the host cell is a eukaryotic cell.

[23] A method for producing the antibody or the antigen-binding fragment of the antibody, comprising the steps of culturing the host cell according to

[21] or

[22] , and collecting the antibody of interest or the antigen-binding fragment of the antibody from the culture obtained in the step.

[0014]

[24] The antibody or antigen-binding fragment of any one of [1] to [15-8], wherein the heavy chain or light chain has one or more modifications selected from the group consisting of addition of an N-linked sugar chain, addition of an O-linked sugar chain, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue at the N-terminus, amidation of a proline residue, pyroglutamic acid oxidation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus.

[25] The antibody of

[24] , wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

[26] The antibody of

[25] , wherein one amino acid is deleted at the carboxyl terminus of each of the two heavy chains. [26-2] The humanized antibody according to any one of [15-2] to [15-8], wherein one amino acid is deleted at the carboxyl terminus of each of the two heavy chains of the antibody. [26-3] The humanized antibody according to [26-2], wherein the deleted amino acid is lysine.

[27] The antibody according to

[24] , wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.

[28] The antibody or antigen-binding fragment of said antibody according to any one of [1] to [15-8] and

[24] to

[27] , which has a glycosylation modification that enhances antibody-dependent cellular cytotoxicity.

[0015]

[29] An antibody-drug conjugate in which a drug is bound to the antibody or antigen-binding fragment thereof according to any one of the groups consisting of [1] to [15-8] and

[24] to

[28] .

[30] The antibody-drug conjugate according to

[29] , wherein the drug is any one or more selected from the group consisting of substances with cytotoxic activity, cytotoxic compounds, substances with antitumor activity, chemotherapeutic agents, molecular targeted drugs, immune activators, immunosuppressants, toxins, photosensitizers, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, vitamins, hormones, substances effective against blood diseases, substances effective against autoimmune diseases, anti-inflammatory substances, antibacterial substances, antifungal substances, antiparasitic substances, antiviral substances, and antianesthetic substances.

[31] The antibody-drug conjugate according to

[30] , wherein the drug is a cytotoxic compound.

[32] The cytotoxic compound is represented by the following formula: [32-2] The antibody-drug conjugate according to

[31] , wherein the antibody and the drug are a cytotoxic compound represented by the following formula: -(Succinimid-3-yl-N)-(CH2)n １ The antibody-drug conjugate according to any one of

[29] to

[32] , wherein the antibody-drug conjugate is bound via a linker having a structure represented by -C(=O)-L-GGFG-NH-X-C(=O) - (wherein n １ represents an integer of 2 to 8, and L represents -NH-(CH2-CH2-O)n ２ -CH2-CH2- or a single bond, n ２ represents an integer from 1 to 6, and X represents a linear or branched alkylene group having 1 to 20 carbon atoms (one or more methylene groups in the chain may be replaced with an oxygen atom or a sulfur atom). The alkylene group may have a cyclic saturated hydrocarbon group having 1 to 6 carbon atoms as part of its structure. The antibody is bound at the end of -(Succinimid-3-yl-N). The drug is bound to the carbonyl group at the other end. In the above formula, GGFG represents an amino acid sequence connected by peptide bonds consisting of glycine-glycine-phenylalanine-glycine. -(Succinimid-3-yl-N)- represents the following formula: and is bonded to the antibody at position 3, and bonded to a methylene group in a linker structure containing the nitrogen atom at position 1.)

[33] An antibody and a drug have a structure represented by the following formulae (a) to (f): (a) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (b) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, (c) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (d) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, (e) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, and (f) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, The antibody-drug conjugate according to any one of

[29] to [32-2], wherein the antibody is bonded via a linker having any structure selected from the group consisting of: (wherein the antibody is bonded at the end of -(Succinimid-3-yl-N). The drug is bonded to the carbonyl group at the other end, and when present, the nitrogen atom of the amino group at position 1 serves as the bonding site. In the above formula, GGFG represents an amino acid sequence connected by peptide bonds consisting of glycine-glycine-phenylalanine-glycine. -(Succinimid-3-yl-N)- represents the following formula: wherein the linker is represented by a formula selected from the group consisting of (c), (d), and (e): (c) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (d) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, (e) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[35] The antibody-drug conjugate according to any one of

[29] to

[34] , wherein the linker is represented by the following formula (c) or (e): (c) -(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, (e) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[36] A linker represented by the following formula: The antibody-drug conjugate according to any one of

[29] to

[35] , having a structure represented by the following formula: (wherein AB represents an antibody or an antigen-binding fragment of the antibody, and n represents the average number of drug-linker structures bound to an antibody or an antigen-binding fragment of the antibody per antibody or antigen-binding fragment of the antibody. The antibody or antigen-binding fragment of the antibody and the linker are bound via a sulfhydryl group derived from the antibody or antigen-binding fragment of the antibody.)

[37] A compound represented by the following formula: The antibody-drug conjugate according to any one of

[29] to

[35] , having a structure represented by the following formula: (Here, AB represents an antibody or an antigen-binding fragment of the antibody. n represents the average number of drug-linker structures bound to an antibody or an antigen-binding fragment of the antibody per antibody or antigen-binding fragment of the antibody. The antibody or the antigen-binding fragment of the antibody and the linker are bound via a sulfhydryl group derived from the antibody or the antigen-binding fragment of the antibody.)

[38] The antibody-drug conjugate according to any one of

[29] to

[37] , wherein the antibody is an antibody or an antigen-binding fragment of the antibody comprising a light chain variable region and a heavy chain variable region selected from the group consisting of the following (1) to (2): (1) a light chain variable region consisting of the amino acid sequence of amino acids 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 146 of SEQ ID NO: 15, (2) a light chain variable region consisting of the amino acid sequence of amino acids 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 146 of SEQ ID NO: 16.

[39] The antibody-drug conjugate according to

[38] , wherein the antibody is an antibody or an antigen-binding fragment of the antibody, comprising a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15.

[40] The antibody-drug conjugate according to

[38] , wherein the antibody is an antibody or an antigen-binding fragment of the antibody, comprising a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16.

[41] The antibody-drug conjugate according to any one of

[29] to

[37] , wherein the antibody is an antibody comprising a light chain and a heavy chain selected from the group consisting of the following (1) to (2): (1) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15, or (2) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16, or an antigen-binding fragment of the antibody.

[42] The antibody-drug conjugate according to

[41] , wherein the antibody is an antibody or an antigen-binding fragment of the antibody, comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15.

[43] The antibody-drug conjugate according to

[41] , wherein the antibody is an antibody or an antigen-binding fragment of the antibody, comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16.

[44] The antibody-drug conjugate according to any one of

[29] to

[43] , wherein the heavy chain or light chain has one or more modifications selected from the group consisting of addition of an N-linked sugar chain, addition of an O-linked sugar chain, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of a proline residue, pyroglutamic oxidation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus.

[45] The antibody-drug conjugate according to

[44] , wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

[46] The antibody-drug conjugate according to

[45] , wherein one amino acid is deleted at the carboxyl terminus of each of the two heavy chains.

[47] The antibody-drug conjugate according to

[44] , wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.

[48] ​​The antibody-drug conjugate according to any one of

[29] to

[47] , which has a glycosylation modification for enhancing antibody-dependent cellular cytotoxicity.

[49] The antibody-drug conjugate according to any one of

[29] to

[48] , wherein the average number of drug-linker structures bound per antibody is in the range of 1 to 10.

[50] The antibody-drug conjugate according to

[49] , wherein the average number of drug-linker structures bound per antibody is in the range of 2 to 8.

[51] The antibody-drug conjugate according to

[50] , wherein the average number of drug-linker structures bound per antibody is in the range of 5 to 8.

[52] The antibody-drug conjugate according to

[51] , wherein the average number of drug-linker structures bound per antibody is 7 to 8. [52-2] A compound represented by the following formula: (Here, AB represents a humanized antibody comprising a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15. n represents the average number of drug-linker structures bound to the antibody per antibody, and the average number of bonds is in the range of 7 to 8. The antibody and linker are bound via a sulfhydryl group derived from the antibody.) [52-3] An antibody-drug conjugate having the following formula: (Here, AB represents a humanized antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 15. n represents the average number of drug-linker structures bound to the antibody per antibody, and the average number of bonds is in the range of 7 to 8. The antibody and linker are bound via a sulfhydryl group derived from the antibody.) [52-4] An antibody-drug conjugate having the structure represented by the following formula: (Here, AB represents a humanized antibody comprising a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16. n represents the average number of drug-linker structures bound to the antibody per antibody, and the average number of bonds is in the range of 7 to 8. The antibody and linker are bound via a sulfhydryl group derived from the antibody.) [52-5] An antibody-drug conjugate having the following formula: (Here, AB represents a humanized antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 16. n represents the average number of drug-linker structures bound to the antibody per antibody, and the average number of bonds is in the range of 7 to 8. The antibody and linker are bound via a sulfhydryl group derived from the antibody.) [52-6] An antibody-drug conjugate having the structure represented by the following formula: (Here, AB represents an antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 20. n represents the average number of drug-linker structures bound to the antibody per antibody, and the average number of bonds is in the range of 7 to 8. The antibody and linker are bound via a sulfhydryl group derived from the antibody.) [52-7] An antibody-drug conjugate having the structure represented by the following formula: An antibody-drug conjugate having the structure represented by the formula: (Here, AB represents an antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 232 of SEQ ID NO: 26 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 27. n represents the average number of drug-linker structures bound to the antibody per antibody, said average number of bonds being in the range of 7 to 8. The antibody and linker are bound via a sulfhydryl group derived from the antibody.) [52-8] The antibody-drug conjugate according to any one of [52-2] to [52-7], wherein one amino acid is deleted at the carboxyl terminus of each of the two heavy chains of the antibody. [52-9] The antibody-drug conjugate according to [52-8], wherein the deleted amino acid is lysine.

[53] A pharmaceutical composition comprising the antibody or antigen-binding fragment of the antibody according to any one of [1] to [15-8] and

[24] to

[28] , or the antibody-drug conjugate according to any one of

[29] to [52-9], its salt, or a hydrate of these.

[54] The pharmaceutical composition according to

[53] , which is an antitumor drug.

[55] The pharmaceutical composition according to

[54] , wherein tumor cells in the subject express CD25 or tumor immunity is suppressed by regulatory T cells.

[56] The pharmaceutical composition according to

[55] , wherein regulatory T cells are present in the tumor environment and / or lymph nodes in the subject.

[57] The pharmaceutical composition according to any one of

[53] to

[56] , wherein the tumor is blood cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, brain tumor, glioma, eye tumor, esophageal cancer, gastric cancer, colorectal cancer, colon cancer, liver cancer, pancreatic cancer, renal cancer, kidney cancer, bladder cancer, adrenocortical carcinoma, prostate cancer, cervical cancer, uterine corpus cancer, ovarian cancer, melanoma, or sarcoma.

[0016]

[58] A method for treating a tumor, comprising administering to an individual any one selected from the antibody or antigen-binding fragment of the antibody according to any one of [1] to [15-8] and

[24] to

[28] , or the antibody-drug conjugate according to any one of

[29] to [52-9], a salt thereof, or a hydrate thereof.

[59] The method for treating a tumor according to

[58] , wherein the tumor cells in the subject express CD25 or tumor immunity is suppressed by regulatory T cells.

[60] The method for treating a tumor according to

[58] or

[59] , wherein the subject has regulatory T cells present in the tumor environment and / or in the lymph nodes.

[61] The method for treating a tumor according to any one of

[58] to

[60] , wherein the tumor is blood cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, brain tumor, glioma, eye tumor, esophageal cancer, gastric cancer, colorectal cancer, colon cancer, liver cancer, pancreatic cancer, renal cancer, kidney cancer, bladder cancer, adrenocortical carcinoma, prostate cancer, cervical cancer, uterine corpus cancer, ovarian cancer, melanoma, or sarcoma.

[62] A method for treating a tumor, comprising simultaneously, separately, or sequentially administering to an individual a pharmaceutical composition comprising at least one selected from the group consisting of the antibody according to any one of [1] to [15-8] and

[24] to

[28] or an antigen-binding fragment thereof, or the antibody-drug conjugate according to any one of

[29] to [52-9], its salt, or a hydrate thereof, and at least one antitumor drug.

[0017]

[63] A method for producing an antibody-drug conjugate, comprising the steps of: culturing the host cell according to

[21] or

[22] ; collecting the antibody of interest or an antigen-binding fragment of the antibody from the culture obtained in the step; and reacting the antibody or the antigen-binding fragment of the antibody obtained in the step with a drug-linker intermediate compound.

[0018]

[64] A pharmaceutical composition in which the antibody-drug conjugate according to any one of

[29] to [52-9] and an immune checkpoint inhibitor are administered in combination.

[65] The pharmaceutical composition according to

[64] , in which the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations and administered simultaneously or at different times.

[66] The pharmaceutical composition according to

[64] , in which the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[67] The pharmaceutical composition according to any one of

[64] to

[66] , in which the immune checkpoint inhibitor is any one or more selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody.

[68] The pharmaceutical composition according to

[67] , in which the immune checkpoint inhibitor is an anti-PD-1 antibody.

[69] The pharmaceutical composition of

[68] , wherein the anti-PD-1 antibody is any one or more selected from the group consisting of nivolumab, pembrolizumab, sintilimab, spartalizumab, dostallimab, selplulimab, tislelizumab, penprimab, toripalimab, zimberelimab, camrelizumab, retifanlimab, cemiplimab, prolgolimab, geptanolimab, enlonstobart, QL-1604, pucotenlimab, and finotonlimab. [69-2] The pharmaceutical composition of

[68] , wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

[70] The pharmaceutical composition of

[67] , wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[71] The pharmaceutical composition of

[70] , wherein the anti-PD-L1 antibody is any one or more selected from the group consisting of atezolizumab, durvalumab, cosibelimab, adebrelimab, sugemalimab, avelumab, socazolimab, KL-A167, and envafolimab. [71-2] The pharmaceutical composition of

[70] , wherein the anti-PD-L1 antibody is any one or more selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[72] The pharmaceutical composition of

[67] , wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[73] The pharmaceutical composition according to

[72] , wherein the anti-CTLA-4 antibody is any one or more selected from the group consisting of ipilimumab, botensilimab, and tremelimumab. [73-2] The pharmaceutical composition according to

[72] , wherein the anti-CTLA-4 antibody is ipilimumab. [73-3] The pharmaceutical composition according to any one of

[64] to [73-2], for the treatment of cancer. [73-4] The pharmaceutical composition according to any one of

[64] to [73-2], for the treatment of at least one selected from the group consisting of blood cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, brain tumor, glioma, eye tumor, esophageal cancer, gastric cancer, colorectal cancer, colon cancer, liver cancer, pancreatic cancer, renal cancer, kidney cancer, bladder cancer, adrenocortical carcinoma, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma, and sarcoma.

[74] The pharmaceutical composition according to any one of

[64] to [73-2], for treating cancer resistant to immune checkpoint inhibitors.

[0019]

[75] A method for treating a disease, characterized in that an antibody-drug conjugate and an immune checkpoint inhibitor are administered in combination to an individual in need of treatment, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are as defined in any one of

[29] to [52-9],

[67] to [73-2].

[76] The method for treating a disease according to

[75] , wherein the disease is defined in any one of [73-3] to

[74] .

[77] The method for treating a disease according to

[75] or

[76] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations, and are administered simultaneously or at different times.

[78] The method for treating a disease according to

[75] or

[76] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[79] An antibody-drug conjugate for use in combination with an immune checkpoint inhibitor for the treatment of a disease, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are as defined in any one of

[29] to [52-9],

[67] to [73-2].

[80] The antibody-drug conjugate according to

[79] , wherein the disease is defined in any one of [73-3] to

[74] .

[81] The antibody-drug conjugate according to

[79] or

[80] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations and are administered simultaneously or at different times.

[82] The antibody-drug conjugate according to

[79] or

[80] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[83] Use of an antibody-drug conjugate in combination with an immune checkpoint inhibitor in the manufacture of a medicament for treating a disease, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are as defined in any one of

[29] to [52-9],

[67] to [73-2].

[84] The use according to

[83] , wherein the disease is defined in any one of [73-3] to

[74] .

[85] The use according to

[83] or

[84] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations and are administered simultaneously or at different times.

[86] The use according to

[83] or

[84] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[0020]

[87] A pharmaceutical product containing an antibody-drug conjugate and an immune checkpoint inhibitor for combined administration, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are as defined in any one of

[29] to [52-9],

[67] to [73-2].

[88] The pharmaceutical product according to

[87] , for the treatment of a disease defined in any one of [73-3] to

[74] .

[89] The pharmaceutical product according to

[87] or

[88] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations and are administered simultaneously or at different times.

[90] The pharmaceutical product according to

[87] or

[88] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[91] A combination pharmaceutical comprising an antibody-drug conjugate and an immune checkpoint inhibitor, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are as defined in any one of

[29] to [52-9],

[67] to [73-2].

[92] The combination pharmaceutical according to

[91] , for the treatment of a disease defined in any one of [73-3] to

[74] .

[93] The combination pharmaceutical according to

[91] or

[92] , characterized in that the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations and are administered simultaneously or at different times.

[94] The combination pharmaceutical according to

[91] or

[92] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[95] A pharmaceutical combination comprising an antibody-drug conjugate and an immune checkpoint inhibitor, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are as defined in any one of

[29] to [52-9],

[67] to [73-2].

[96] The pharmaceutical combination according to

[95] , for the treatment of a disease defined in any one of [73-3] to

[74] .

[97] The pharmaceutical combination according to

[95] or

[96] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations and administered simultaneously or at different times.

[98] The pharmaceutical combination according to

[95] or

[96] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[99] Use of an antibody-drug conjugate in combination with an immune checkpoint inhibitor for the treatment of a disease, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are as defined in any one of

[29] to [52-9],

[67] to [73-2].

[100] The use according to

[99] , wherein the disease is defined in any one of [73-3] to

[74] .

[101] The use according to

[99] or

[100] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations and administered simultaneously or at different times.

[102] The use according to

[99] or

[100] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[103] A pharmaceutical comprising the antibody-drug conjugate and the immune checkpoint inhibitor, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are administered in combination, and the antibody-drug conjugate and the immune checkpoint inhibitor are as defined in any one of

[29] to [52-9] or

[67] to [73-2].

[104] The pharmaceutical according to

[103] , for treating a disease defined in any one of [73-3] to

[74] .

[105] The pharmaceutical according to

[103] or

[104] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in different formulations and are administered simultaneously or at different times.

[106] The pharmaceutical according to

[103] or

[104] , wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

[0021] The present invention provides an antibody that binds to CD25, an antibody-drug conjugate containing the antibody and having antitumor activity, a pharmaceutical product using the antibody-drug conjugate and having a therapeutic effect against tumors, and a method for treating tumors using the antibody, antibody-drug conjugate, or pharmaceutical product.

[0022] This figure shows the binding activity of MAb1 to human, cynomolgus monkey, and mouse CD25-expressing cells. This figure shows the change in proliferation of human pan T cells in the presence of IL-2 treated with MAb1, a rat IgG2a control antibody, the IL-2-blocking anti-human CD25 antibody basiliximab, or the IL-2-nonblocking anti-human CD25 antibody 7G7B6. The error bars in the figure represent standard error (n = 2 to 24). This figure shows the change in phosphorylated STAT5 in human PBMC cells in the presence of IL-2 treated with MAb1, a rat IgG2a control antibody, basiliximab, or 7G7B6. The error bars in the figure represent standard error (n = 3). This figure shows the internalization activity of MAb1 or the rat IgG2a control antibody by measuring the change in viability of human pan T cells using a saporin-conjugated anti-rat IgG reagent (Fab-ZAP). The error bars in the figure represent standard error (n = 3). This figure shows the competitiveness of MAb1, a human IgG1 control antibody, or the anti-human CD25 antibody basiliximab with the CD25 antibody M-A251. The error bars in the figure indicate standard deviation (n = 3). This figure shows the binding activity of MAb2 to human, cynomolgus monkey, and mouse CD25-expressing cells. The error bars in the figure indicate standard error (n = 3). This figure shows the change in phosphorylated STAT5 in CTLL-2 cells treated with MAb2, a rat IgG2a control antibody, or the IL-2-blocking anti-mouse CD25 antibody PC61_hIgG1LALA (referred to as PC61 in the figure) in the presence of IL-2. The error bars in the figure indicate standard error (n = 3). This figure shows the internalization activity of MAb2, PC61, or the rat IgG2a control antibody, as measured by the change in viability of mouse pan T cells using a saporin-conjugated anti-rat IgG reagent (Fab-ZAP). The error bars in the figure indicate standard error (n=3). This figure shows the binding activity of cMAb1_hIgG1LALA to human, cynomolgus monkey, and mouse CD25-expressing cells. The error bars in the figure indicate standard error (n=3). This figure shows the variation in proliferation of human CD4-positive T cells treated with cMAb1_hIgG1LALA, a human IgG1 control antibody, or basiliximab in the presence of PMA / PHA. The error bars in the figure indicate standard error (n=3).This figure shows the changes in phosphorylated STAT5 in human PBMCs treated with cMAb1_hIgG1LALA, a human IgG1 control antibody, and basiliximab in the presence of IL-2. The error bars in the figure indicate standard error (n=3). This figure shows the in vitro cell growth inhibitory activity of cMAb1_hIgG1LALA-ADC or human IgG1LALA-ADC against the CD25-positive human tumor cell line Karpas-299 and the CD25-negative human tumor cell line Daudi. The error bars in the figure indicate standard deviation (n=3). This figure shows the changes in the proportions of Tregs, CD8-positive T cells, and GZMB-positive CD8-positive T cells per human immune cell in tumor tissues of MDA-MB-231 cell-bearing hematopoietic stem cell-transplanted NOG mice administered cMAb1_hIgG1LALA-ADC or human IgG1LALA-ADC (n=5). This figure shows the binding activity of cMAb2_hIgG1LALA to human, cynomolgus monkey, and mouse CD25-expressing cells. Error bars in the figure indicate standard error (n=3). This figure shows the in vitro cytostatic activity of cMAb2_hIgG1LALA, human IgG1, or PC61_hIgG1LALA against the CD25-positive mouse T cell line CTLL-2. Error bars in the figure indicate standard error (n=3). This figure shows the in vitro cell growth inhibitory activity of cMAb2_hIgG1LALA-ADC or human IgG1LALA-ADC against the CD25-positive murine T cell line CTLL-2. Error bars in the figure indicate standard deviation (n=3). This figure shows the change in tumor volume in CT26 cell tumor-bearing mice administered with cMAb2_hIgG1-ADC or human IgG1-ADC. Error bars in the figure indicate standard error (n=9). This figure shows the change in the number of Tregs, FoxP3-negative CD4-positive T cells, CD8-positive T cells, and granzyme-positive CD8-positive T cells per tissue weight in the tumors of CT26 cell tumor-bearing mice administered with cMAb2_hIgG1-ADC or human IgG1-ADC. The dots in the figure represent individuals, and the figures are presented as boxplots (n=10). 1 shows the binding activity of hMAb1A and hMAb1B to human, cynomolgus monkey, and mouse CD25 transiently expressing cells. Error bars in the figure indicate standard deviation (n=3).This figure shows the changes in human PBMC proliferation caused by hMAb1A, hMAb1B, a human IgG1 control antibody, or basiliximab in the presence of IL-2. The error bars in the figure indicate standard error (n=3). This figure shows the changes in phosphorylated STAT5 in human PBMC caused by hMAb1A, hMAb1B, a human IgG1 control antibody, and basiliximab in the presence of PMA / PHA. The error bars in the figure indicate standard error (n=3). This figure shows the in vitro cell growth inhibitory activity of hMAb1A-ADC, hMAb1A, or human IgG1-ADC against the CD25-positive human tumor cell line Karpas-299 and the CD25-negative human tumor cell line Daudi. The error bars in the figure indicate standard deviation (n=3 or 6). This figure shows the in vitro cell growth inhibitory activity of hMAb1B-ADC, hMAb1B, or human IgG1LALA-ADC against the CD25-positive human tumor cell line EoL-1 and the CD25-negative human tumor cell line Daudi. Error bars in the figure indicate standard deviation (n=3). This figure shows the changes in the proportions of Tregs, FoxP3-negative CD4-positive T cells, CD8-positive T cells, and GZMB-positive CD8-positive T cells among human CD45-positive cells in tumor tissues of NOG mice transplanted with hematopoietic stem cells bearing MDA-MB-231 cells and administered hMAb1A-ADC or ABS (n=5). This figure shows the changes in the proportion of Tregs and GZMB-positive CD8-positive T cells among human CD45-positive cells in tumor tissues of NOG mice transplanted with hematopoietic stem cells bearing MDA-MB-231 cells and administered hMAb1A-ADC, hMAb1B-ADC, or human IgG1LALA-ADC (n=4 or 5). This figure shows the ADCC activity of RG-6292 targeting iTregs. This figure compares the cytotoxic activity of hMAb1A-ADC and RG-6292 against iTregs at different ratios of iTregs to NK cells (n=3). This figure shows the changes in the number of GZMB-positive CD8-positive T cells per mg of tumor tissue in tumor tissues of NOG mice transplanted with hematopoietic stem cells bearing MDA-MB-231 cells and administered hMAb1A-ADC, ADCT-301, or ABS (n=5). The full-length amino acid sequence of the hMAb1A-L light chain (signal sequence and amino acid sequence of the hMAb1A light chain) (SEQ ID NO: 13) is shown.The full-length amino acid sequence of the hMAb1A-H heavy chain (signal sequence and amino acid sequence of the hMAb1A heavy chain) (SEQ ID NO: 15) is shown. The full-length amino acid sequence of the hMAb1B-L light chain (signal sequence and amino acid sequence of the hMAb1B light chain) (SEQ ID NO: 14) is shown. The full-length amino acid sequence of the hMAb1B-H heavy chain (signal sequence and amino acid sequence of the hMAb1B heavy chain) (SEQ ID NO: 16) is shown. This figure shows the changes in tumor volume in cancer-bearing mice administered cMAb2_hIgG1-ADC in combination with an anti-PD-1 antibody or a rat IgG2a control antibody. The results of administering human IgG1-ADC in combination with an anti-PD-1 antibody or a rat IgG2a control antibody are also shown. Error bars in the figure represent standard error (n = 3 to 10). CT26. Evaluation was performed using an animal model transplanted with WT cells, EMT6 cells, MC38 cells, RENCA cells, or B16F10 cells. This figure shows the change in tumor volume in 3LL cell tumor-bearing mice administered cMAb2_hIgG1-ADC in combination with an anti-PD-1 antibody or a rat IgG2a control antibody. The results of administering human IgG1-ADC in combination with an anti-PD-1 antibody or a rat IgG2a control antibody are also shown. The error bars in the figure represent standard error (n=8). This figure shows the change in tumor volume in MC38 cell tumor-bearing mice administered cMAb2_hIgG1-ADC in combination with an anti-CTLA-4 antibody or a rat IgG2b control antibody. The results of administering human IgG1-ADC in combination with an anti-CTLA-4 antibody or a rat IgG2b control antibody are also shown. The error bars in the figure represent standard error (n=7 or 8).

[0023] Preferred embodiments for carrying out the present invention will be described below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0024] As used herein, the terms "cancer" and "tumor" are used interchangeably. As used herein, the term "gene" includes not only DNA but also its mRNA, cDNA, and cRNA. As used herein, "polynucleotide" or "nucleotide" is used interchangeably with "nucleic acid," and includes DNA, RNA, probes, oligonucleotides, and primers. As used herein, "polynucleotide" and "nucleotide" may be used interchangeably unless otherwise specified. As used herein, "polypeptide" and "protein" may be used interchangeably. As used herein, "cell" includes cells within an animal and cultured cells. As used herein, "CD25" may be used interchangeably with CD25 protein. As used herein, human CD25 may be referred to as "hCD25." As used herein, "cytotoxic activity" refers to the initiation of pathological changes in cells in some form, including not only direct trauma but also any damage to the structure or function of cells, such as DNA breakage, base dimer formation, chromosome breakage, damage to the cell division apparatus, and decreased activity of various enzymes. As used herein, the phrase "exerts toxicity within cells" refers to exerting toxicity within cells in some form, including not only direct trauma but also any effect on the structure, function, or metabolism of cells, such as DNA cleavage, base dimer formation, chromosome cleavage, damage to the cell division apparatus, reduced enzyme activity, or inhibition of the action of cell growth factors. As used herein, the term "antibody" may refer to an antibody derived from any species, but preferred examples include antibodies derived from humans, monkeys, rats, mice, rabbits, camels, alpacas, or llamas. When derived from a species other than humans, the antibody is preferably chimerized or humanized using well-known techniques. Therefore, in one aspect of the present invention, the antibody is a human antibody, a chimerized antibody, or a humanized antibody, preferably a human antibody or a humanized antibody. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody, and may be a monospecific antibody or a multispecific antibody (bispecific antibody, trispecific antibody, etc.).As used herein, the term "antigen-binding fragment of an antibody" refers to a partial fragment of an antibody that has antigen-binding activity, or a fusion polypeptide containing the antigen-binding site of the antibody (such as the light and heavy chain variable regions or CDRs contained in the variable regions). Antigen-binding fragments of antibodies include Fab, F(ab')2, Fv, scFv, VHH (variable domain of heavy chain of heavy-chain antibody) antibodies, diabodies, linear antibodies, and fusion proteins in which antigen-binding ability is imparted by linking an scFv to a functional domain of another protein. Antigen-binding fragments of antibodies also include multispecific antibodies formed containing such fragments. Also included in the antigen-binding fragment of an antibody is Fab', a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions. However, antigen-binding fragments of antibodies are not limited to these molecules as long as they retain the ability to bind to antigens. These antigen-binding fragments include not only full-length antibody protein molecules treated with appropriate enzymes, but also genetically engineered antibodies or proteins produced in appropriate host cells using genes encoding their antigen-binding sites. As used herein, "epitope" refers to a partial peptide or partial three-dimensional structure of CD25 to which a specific anti-CD25 antibody binds. Epitopes, which are partial peptides of CD25, can be determined by methods well known to those skilled in the art, such as immunoassays. First, various partial structures of the antigen are prepared. These partial structures can be prepared using known oligonucleotide synthesis techniques. For example, a series of polypeptides sequentially shortened to an appropriate length from the C-terminus or N-terminus of CD25 can be prepared using genetic recombination techniques well known to those skilled in the art. The reactivity of antibodies against these polypeptides can then be examined to determine the approximate recognition site, and further shorter peptides can be synthesized and their reactivity examined to determine the epitope. Furthermore, when an antibody that binds to a membrane protein consisting of multiple extracellular domains has an epitope that is a three-dimensional structure consisting of multiple domains, it is possible to determine which domain the antibody will bind to by modifying the amino acid sequence of a specific extracellular domain and thereby altering the three-dimensional structure.An epitope, which is a partial three-dimensional structure of an antigen to which a specific antibody binds, can also be determined by identifying amino acid residues of the antigen adjacent to the antibody by X-ray structural analysis or the like. As used herein, "binding to the same epitope" refers to an antibody that binds to a common epitope. If a second antibody binds to a partial peptide or partial three-dimensional structure to which a first antibody binds, it can be determined that the first and second antibodies bind to the same epitope. Alternatively, by confirming that the second antibody competes with the binding of the first antibody to the antigen (i.e., the second antibody interferes with the binding of the first antibody to the antigen), it can be determined that the first and second antibodies bind to the same epitope, even if the specific sequence or structure of the epitope has not been determined. As used herein, "binding to the same epitope" refers to a case in which the first and second antibodies are determined to bind to a common epitope by one or both of the determination methods. If a first antibody and a second antibody bind to the same epitope and the first antibody has a special effect such as antitumor activity or internalization activity, the second antibody is expected to have similar activity. As used herein, "CDR" refers to a complementarity-determining region (CDR). It is known that the heavy and light chains of an antibody molecule each contain three CDRs. CDRs, also known as hypervariable regions, are located within the variable regions of the heavy and light chains of an antibody and are sites with particularly high variability in the primary structure. They are separated into three locations in the primary structure of the heavy and light chain polypeptide chains. Herein, with regard to antibody CDRs, heavy chain CDRs are designated as CDRH1, CDRH2, and CDRH3 from the amino-terminus of the heavy chain amino acid sequence, and light chain CDRs are designated as CDRL1, CDRL2, and CDRL3 from the amino-terminus of the light chain amino acid sequence. These sites are adjacent to each other in the three-dimensional structure and determine specificity for the antigen to which they bind.CDR sequences are defined according to definitions well known in the art, for example, schemes such as IMGT (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77), Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and Chothia (Al-Lazikani et al., 1997, J. Mol. Biol 273:927-948). In the present invention, CDR sequences are determined according to the definition of IMGT. As used herein, "several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2. As used herein, "A to B" indicates a range including A and B. The same applies to "A to B."

[0025] 1. CD25 CD25 (also known as the IL-2 receptor α chain or Tac antigen) is a type I transmembrane protein with two extracellular Sushi domains (Non-Patent Documents 13, 14). It is known as the IL-2 receptor α chain and plays a role in transmitting IL-2 signals into cells upon binding to IL-2 (Non-Patent Document 15). CD25 is expressed at low levels on limited immune cells, such as activated T cells and B cells (Non-Patent Documents 16, 17), but has been reported to be highly expressed on Tregs (Non-Patent Document 18). Furthermore, the immunosuppressive activity of Tregs has been shown to correlate with the strength of CD25 expression (Non-Patent Document 18). The role of CD25 in Tregs has been reported to include the essentiality of IL-2 signaling for Treg survival (Non-Patent Document 19), and its binding to IL-2, consuming IL-2 and thereby reducing the opportunity for other immune cells to bind IL-2 (Non-Patent Document 19). Furthermore, CD25 has also been reported to be expressed in blood cancer cells such as non-Hodgkin's lymphoma (Non-Patent Document 20).

[0026] The CD25 protein used in the present invention can be directly purified from CD25-expressing cells of humans or non-human mammals (e.g., rats, mice, monkeys), or prepared from cell membrane fractions of such cells. Alternatively, the CD25 protein can be synthesized in vitro or produced in host cells by genetic engineering. Specifically, genetic engineering can involve incorporating CD25 cDNA into an expression vector and then synthesizing the protein in a solution containing enzymes, substrates, and energy sources necessary for transcription and translation, or by transforming other prokaryotic or eukaryotic host cells to express CD25. Alternatively, the CD25 protein can be obtained from the genetically engineered CD25-expressing cells or cell lines expressing CD25. Alternatively, an expression vector incorporating CD25 cDNA can be directly administered to an immunized animal to express CD25 in the body of the immunized animal.

[0027] Furthermore, CD25 also includes proteins that have the same biological activity as the above-mentioned CD25 amino acid sequence, but in which one or more amino acids have been substituted, deleted, and / or added. Human CD25 protein has the amino acid sequence set forth in SEQ ID NO: 17.

[0028] 2. Anti-CD25 Antibodies An example of an anti-CD25 antibody of the present invention is an anti-CD25 antibody that does not have IL-2 blocking ability and has internalization activity. The anti-CD25 antibody of the present invention may be derived from any species, but preferably is derived from human, monkey, rat, mouse, or rabbit. When derived from a species other than human, it is preferably chimerized or humanized using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but monoclonal antibodies are preferred.

[0029] The anti-CD25 antibodies of the present invention are antibodies that can target blood cells and tumor cells, i.e., they have the properties of being able to recognize blood cells and tumor cells, being able to bind to blood cells and tumor cells, and / or being taken up and internalized into blood cells and tumor cells. Therefore, the anti-CD25 antibodies of the present invention can be linked to drugs via a linker to form antibody-drug conjugates. The binding ability of the antibodies to blood cells and tumor cells can be confirmed using flow cytometry. The internalization of antibodies into blood cells and tumor cells can be confirmed using (1) an assay in which antibodies taken up into cells are visualized under a fluorescence microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation, 2008, 15, 751-761), (2) an assay in which the amount of fluorescence taken up into cells is measured using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay in which an immunotoxin that binds to the therapeutic antibody is released upon its intracellular uptake, thereby suppressing cell proliferation (Bio Techniques 28:162-165, January 2000). As an immunotoxin, a recombinant conjugated protein of the catalytic domain of diphtheria toxin and protein G can also be used.

[0030] As used herein, the terms "not having IL-2 blocking ability" and "not inhibiting IL-2 signaling" mean that IL-2 signaling in the presence of an anti-CD25 antibody or an antigen-binding fragment thereof is maintained at least 70% or more compared to IL-2 signaling in the presence of a control antibody. IL-2 signaling can be evaluated, for example, using IL-2-dependent T cell proliferation or protein (e.g., STAT5) phosphorylation as an indicator. An antibody can be determined to "not have IL-2 blocking ability" and "not inhibit IL-2 signaling" (IL-2 non-blocking antibody) when IL-2-dependent T cell proliferation or protein phosphorylation in the presence of IL-2 and an antibody is at least 70% or more, preferably 80% or more, and more preferably 90% or more compared to cell proliferation or protein phosphorylation in the presence of IL-2 and a control antibody. The control antibody may be an antibody known to have no IL-2 blocking ability, and the antibodies used in the evaluation in this example can be used, for example, rat IgG2a or human IgG1. Depending on the measurement system, IL-2-dependent T cell proliferation or protein phosphorylation in the presence of IL-2 and an antibody may exceed 100% compared to cell proliferation or protein phosphorylation in the presence of IL-2 and a control antibody. Even in this case, the antibody may be determined to be an IL-2 non-blocking antibody. Anti-CD25 antibodies that inhibit IL-2 signaling have been reported to inhibit IL-2-dependent T cell proliferation (Non-Patent Document 21) or suppress T cell-mediated immune responses (Non-Patent Document 22). In a mouse tumor model in which anti-CD25 antibodies that do not inhibit IL-2 signaling exhibited a high anti-tumor effect, anti-CD25 antibodies that inhibit IL-2 signaling showed almost no anti-tumor effect. This suggests that the therapeutic effect of anti-CD25 antibodies on tumors is based on both Treg elimination and Teff activation within the tumor (Non-Patent Document 22). Therefore, it is expected that anti-CD25 antibodies that inhibit IL-2 signaling will inhibit IL-2-dependent Teff proliferation within tumors, resulting in insufficient therapeutic effects.In this regard, the antibody of the present invention, which does not inhibit IL-2 signaling, is expected to exhibit high antitumor activity without inhibiting Teff proliferation, and is considered to be advantageous as a therapeutic agent.

[0031] As used herein, "exhibiting the ability to eliminate human regulatory T cells" means that, for example, in the presence of an anti-CD25 antibody or antigen-binding fragment thereof conjugated to a cytotoxic compound of the present invention, the ratio of human regulatory T cells per human CD45 cells or the number of human regulatory T cells per tissue weight in the tumor environment, lymph nodes, and / or blood can be reduced by inducing cell death in human regulatory T cells, compared to a non-administered group, a vehicle-administered group, or the presence of an ADC of a control antibody. As used herein, "exhibiting the ability to promote the proliferation of human granzyme-positive CD8-positive cells" means, for example, that the ratio of human granzyme-positive CD8-positive cells per human CD45 cells or the number of human granzyme-positive CD8-positive cells per tissue weight can be increased in the presence of an anti-CD25 antibody or antigen-binding fragment thereof conjugated to a cytotoxic compound of the present invention, compared to a non-administered group, a vehicle-administered group, or the presence of an ADC of a control antibody. The vehicle and the ADC of the control antibody used for comparison can be, for example, those used in the evaluation in this example.

[0032] As used herein, the "activity of being internalized into CD25-expressing cells upon binding to CD25" can be evaluated, for example, by contacting CD25-expressing cells with a control antibody or test antibody as a primary antibody and with an antibody conjugated to a cytotoxic substance (e.g., saporin) that is capable of binding to the primary antibody as a secondary antibody, and detecting cell death of CD25-expressing cells induced by the cytotoxic substance. That is, when the cell viability of CD25-expressing cells contacted with a control antibody as a primary antibody is taken as 100%, the test antibody can be determined to have the activity of being internalized into CD25-expressing cells upon binding to CD25 if the cell viability of CD25-expressing cells contacted with a test antibody as a primary antibody is 90% or less, 80% or less, or 70% or less, more preferably 60% or less, 50% or less, or 40% or less, and even more preferably 30% or less, 20% or less, 10% or less. The control antibody may be any antibody that does not bind to CD25, and the antibody used in the evaluation of this example may be used, for example, rat IgG2a. The term "internalizing activity" is used interchangeably with "internalization activity" and "internalization effect."

[0033] Since the antibody-drug conjugate is bound to a compound that exerts a cytotoxic effect, it is preferable, but not essential, that the antibody itself have antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. For the purpose of specifically and / or selectively exerting the cytotoxic activity of the cytotoxic compound on blood cells and tumor cells, it is important and preferable that the antibody has the property of being internalized and transferred into blood cells and tumor cells.

[0034] Anti-CD25 antibodies can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibodies produced in the body using methods commonly used in this field, but it is preferable to use CD25 that retains its three-dimensional structure as the antigen.

[0035] The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to the heterologous antigen with human antigens. Alternatively, monoclonal antibodies can be obtained by fusing antibody-producing cells that produce antibodies against the antigen with myeloma cells to establish hybridomas, according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, 495-497; Kennet, R. ed., Monoclonal Antibodies, 365-367, Plenum Press, N.Y. (1980)).

[0036] The following describes specific methods for obtaining antibodies against CD25. (1) Preparation of Antigens Antigens can be obtained by genetically engineering a gene encoding an antigen protein in a host cell to produce it. Specifically, a vector capable of expressing the antigen gene is prepared, introduced into a host cell to express the gene, and the expressed antigen is purified. Antibodies can also be obtained by immunizing an animal with the above-mentioned genetically engineered antigen-expressing cells or a cell line expressing the antigen. Alternatively, antibodies can be obtained without using an antigen protein by incorporating the cDNA of the antigen protein into an expression vector and administering it to an immunized animal, thereby expressing the antigen protein in the body of the immunized animal and producing antibodies against the antigen protein.

[0037] (2) Production of Anti-CD25 Monoclonal Antibodies The anti-CD25 antibody or antigen-binding fragment thereof used in the present invention is not particularly limited, and for example, an antibody specified by the amino acid sequence shown in the Sequence Listing of the present application can be preferably used. The anti-CD25 antibody or antigen-binding fragment thereof used in the present invention preferably has the following properties. (1) An antibody or antigen-binding fragment thereof (preferably an antibody) characterized by having one or more of the following properties: (a) Specific binding to CD25. (b) No IL-2 blocking ability. (c) When conjugated with a cytotoxic compound, it exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells. (d) Has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d). (2) The antibody according to the above (1), wherein CD25 is human CD25, cynomolgus monkey CD25 or mouse CD25 (preferably human CD25 or mouse CD25, more preferably human CD25). The method for obtaining the antibody against CD25 of the present invention is not particularly limited as long as an anti-CD25 antibody can be obtained, but it is preferable to use CD25 retaining its higher-order structure as the antigen.

[0038] The anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention preferably has the following properties (1) to (6) for binding to CD25: (1) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 232 of SEQ ID NO: 26 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 27; (2) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15; (3) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16; (4) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 232 of SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence of positions 20 to 463 of SEQ ID NO: 31; (5) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 239 of SEQ ID NO: 32 and a heavy chain consisting of the amino acid sequence of positions 20 to 463 of SEQ ID NO: 33; (6) an antibody having competitive inhibitory activity with at least one of the antibodies selected from the group consisting of: an antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 239 of SEQ ID NO: 54 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 463 of SEQ ID NO: 55; and more preferably an antibody or an antigen-binding fragment of the antibody having competitive inhibitory activity with at least one of the antibodies selected from the group consisting of the following (1) to (3) for binding to CD25: (1) an antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 232 of SEQ ID NO: 26 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 27; (2) an antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 15; (3) an antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 16; Even more preferably, the antibody has the following (1) and (2) for binding to CD25: (1) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15; (2) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16;The antibody or antigen-binding fragment thereof has competitive inhibitory activity with at least one of the above.

[0039] The anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention preferably comprises a CDRL1, CDRL2, and CDRL3 selected from the group consisting of the following (1) to (2): (1) a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 (KAS), and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; (2) a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5 (FVS), and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; and the following (3) to (4): (3) a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9; (4) a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 12; and more preferably, the antibody or antigen-binding fragment thereof (preferably an antibody) comprising a CDRH1, a CDRH2, and a CDRH3 selected from the group consisting of: (1) a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 2 (KAS), and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; and a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9; (2) a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5 (FVS), and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; and a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 12; an antibody or an antigen-binding fragment thereof (preferably an antibody), comprising CDRL1, CDRL2, and CDRL3, and CDRH1, CDRH2, and CDRH3 selected from the group consisting of:Even more preferably, it is an antibody or an antigen-binding fragment of the antibody (preferably an antibody) comprising: a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1; a CDRL2 consisting of the amino acid sequence (KAS) set forth in SEQ ID NO: 2; and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; and a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7; a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8; and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9.

[0040] The anti-CD25 antibody or antigen-binding fragment of said antibody used in the present invention preferably comprises any one of the following light chain variable regions selected from the group consisting of (1) to (4): (1) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13, (2) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14, (3) a light chain variable region consisting of an amino acid sequence having at least 95% sequence identity to the framework region sequence other than each CDR sequence in the amino acid sequence of (1) to (2), and (4) a light chain variable region consisting of the amino acid sequence of (1) to (2) in which one or several amino acids have been deleted, substituted or added in the framework region sequence other than each CDR sequence, and (5) to (8) below: (5) a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15, (6) a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16, (7) a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity to the framework region sequences other than each CDR sequence in the amino acid sequences of (5) to (6), and (8) a heavy chain variable region comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the framework region sequences other than each CDR sequence in the amino acid sequences of (5) to (6), or an antigen-binding fragment of the antibody (preferably, an antibody), more preferably an antibody or an antigen-binding fragment of the antibody (preferably, an antibody) comprising: (1) a light chain variable region comprising the amino acid sequence of amino acids 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region comprising the amino acid sequence of amino acids 20 to 146 of SEQ ID NO: 15, or (2) a light chain variable region comprising the amino acid sequence of amino acids 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region comprising the amino acid sequence of amino acids 20 to 146 of SEQ ID NO: 16.

[0041] The anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is preferably an antibody or antigen-binding fragment of the antibody (preferably an antibody) comprising: (1) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15; or (2) a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16; and more preferably an antibody or antigen-binding fragment of the antibody (preferably an antibody) in which the heavy chain or the light chain has one or more modifications selected from the group consisting of addition of an N-linked sugar chain, addition of an O-linked sugar chain, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, addition of a methionine residue to the N-terminus, amidation of proline residues, pyroglutamic acid oxidation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus. Even more preferably, the antibody has one or two amino acids deleted at the carboxyl terminus of the heavy chain, even more preferably, one amino acid deleted at the carboxyl terminus of each of the two heavy chains, and even more preferably, the amino acid deleted in the heavy chain is lysine.

[0042] In one embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody or antigen-binding fragment of the antibody (preferably an antibody) characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), still more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25, and The antibody or antigen-binding fragment of the antibody comprises a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRL2 consisting of the amino acid sequence (KAS) set forth in SEQ ID NO: 2, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9, or an antigen-binding fragment of the antibody.

[0043] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody or antigen-binding fragment of the antibody (preferably an antibody) characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody or antigen-binding fragment of the antibody is an antibody or antigen-binding fragment of the antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 146 of SEQ ID NO: 15.

[0044] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody or antigen-binding fragment of the antibody (preferably an antibody) characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody or antigen-binding fragment of the antibody is an antibody or antigen-binding fragment of the antibody comprising a light chain variable region consisting of the amino acid sequence of amino acids 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 146 of SEQ ID NO: 16.

[0045] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody or antigen-binding fragment of the antibody (preferably an antibody) characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody or antigen-binding fragment of the antibody is an antibody or antigen-binding fragment of the antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 15.

[0046] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody or antigen-binding fragment of the antibody (preferably an antibody) characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody or antigen-binding fragment of the antibody is an antibody or antigen-binding fragment of the antibody comprising a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 16.

[0047] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody comprises a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 15, and one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

[0048] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody comprises a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 15, and one amino acid is deleted at the carboxyl terminus of each of the two heavy chains.

[0049] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody comprises a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 15, and one lysine is deleted at the carboxyl terminus of each of the two heavy chains.

[0050] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody comprises a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 16, and one or two amino acids are deleted at the carboxyl terminus of the heavy chain.

[0051] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody comprises a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 16, and one amino acid is deleted at the carboxyl terminus of each of the two heavy chains.

[0052] In another embodiment, the anti-CD25 antibody or antigen-binding fragment of the antibody used in the present invention is an anti-CD25 antibody characterized by having at least one of the following properties (a) to (d): (a) specifically binds to CD25; (b) does not have IL-2 blocking ability; (c) when conjugated with a cytotoxic compound, exhibits the ability to eliminate human regulatory T cells and / or the ability to promote the proliferation of human granzyme-positive CD8-positive cells; and (d) has the activity of being internalized into CD25-expressing cells by binding to CD25. (Among the above (a) to (d), preferably (a), (b), (c) or (d), more preferably (a) and (b), (a) and (c), (a) and (d), (b) and (c), (b) and (d), or (c) and (d), even more preferably (a), (b) and (c), (a), (b) and (d), (a), (c) and (d), or (b), (c) and (d), and most preferably (a), (b), (c) and (d)); the CD25 is human CD25; and the antibody comprises a light chain consisting of the amino acid sequence of amino acids 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of amino acids 20 to 476 of SEQ ID NO: 16, and one lysine is deleted at the carboxyl terminus of each of the two heavy chains.

[0053] The antibody-drug conjugate used in the present invention may comprise any of the anti-CD25 antibodies or antigen-binding fragments described above. Anti-CD25 antibody-drug conjugates in which a drug that exerts intracellular toxicity is bound to the anti-CD25 antibody or the like via a linker of a specific structure may exhibit stronger Treg depletion ability and / or granzyme (GZMB)-positive CD8-positive T cell induction activity than conventional anti-CD25 antibodies and anti-CD25 antibody-drug complexes.

[0054] Specific examples of obtaining monoclonal antibodies include the following: (a) purifying a biopolymer to be used as an antigen; and (b) inducing an immune response by directly administering recombinant CD25 and an adjuvant to an animal to be immunized (e.g., a rat or a mouse). These administrations may be performed once or multiple times as necessary to increase the antibody titer. (c) collecting tissues (e.g., lymph nodes) containing antibody-producing cells from the above-mentioned animal in which an immune response has been induced; (d) preparing myeloma cells (hereinafter referred to as "myeloma") (e.g., mouse myeloma SP2 / 0-ag14 cells); (e) cell fusion between antibody-producing cells and myeloma; (f) selecting a group of hybridomas producing the desired antibody; (g) dividing into single-cell clones (cloning); (h) optionally culturing the hybridomas to produce monoclonal antibodies in large quantities, or raising animals transplanted with hybridomas; and / or (i) examining the physiological activity (internalization activity) and binding specificity of the monoclonal antibodies produced in this manner, or assaying their properties as a labeling reagent. Methods for measuring antibody titers used herein include, but are not limited to, flow cytometry or Cell-ELISA.

[0055] Examples of hybridoma strains established in this manner include anti-CD25 antibody-producing hybridomas MAb1 and MAb2. In this specification, the antibody produced by the anti-CD25 antibody-producing hybridoma MAb1 will be referred to as the "MAb1 antibody" or simply "MAb1," and the antibody produced by the hybridoma MAb2 will be referred to as the "MAb2 antibody" or simply "MAb2."

[0056] The light chain variable region of the MAb1 antibody consists of the amino acid sequence set forth in SEQ ID NO: 18. The amino acid sequence of the light chain variable region of the MAb1 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 19. The light chain variable region of the MAb1 antibody has a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3. The heavy chain variable region of the MAb1 antibody consists of the amino acid sequence set forth in SEQ ID NO: 20. The amino acid sequence of the heavy chain variable region of the MAb1 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 21. The heavy chain variable region of the MAb1 antibody has a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9. The sequence of the MAb1 antibody is shown in Table 1.

[0057]

[0058] The light chain variable region of the MAb2 antibody consists of the amino acid sequence set forth in SEQ ID NO: 22. The amino acid sequence of the light chain variable region of the MAb2 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 23. The light chain variable region of the MAb2 antibody has a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6. The heavy chain variable region of the MAb2 antibody consists of the amino acid sequence set forth in SEQ ID NO: 24. The amino acid sequence of the heavy chain variable region of the MAb2 antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 25. The heavy chain variable region of the MAb2 antibody has a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 12. The sequence of the MAb2 antibody is shown in Table 2.

[0059]

[0060] Furthermore, even if a monoclonal antibody is obtained independently by repeating steps (a) to (h) of "2. Production of anti-CD25 antibodies" or by other methods, it is possible to obtain an antibody with internalization activity equivalent to that of MAb1 or MAb2. An example of such an antibody is an antibody that binds to the same epitope as MAb1 or MAb2. If a newly produced monoclonal antibody binds to a partial peptide or partial three-dimensional structure to which MAb1 or MAb2 binds, it can be determined that the monoclonal antibody binds to the same epitope as MAb1 or MAb2. Furthermore, by confirming that the monoclonal antibody competes with the binding of MAb1 or MAb2 to CD25 (i.e., that the monoclonal antibody interferes with the binding of MAb1 or MAb2 to CD25), it can be determined that the monoclonal antibody binds to the same epitope as the anti-CD25 antibody, even if the specific sequence or structure of the epitope has not been determined. If it is confirmed that the epitope is the same, it is strongly expected that the monoclonal antibody will have antigen-binding ability, biological activity and / or internalization activity equivalent to those of the MAb1 or MAb2 antibody.

[0061] (3) Other Antibodies In addition to the above-mentioned monoclonal antibodies against CD25, the antibodies of the present invention also include genetically engineered antibodies that have been artificially modified for the purpose of reducing heterologous antigenicity to humans, such as chimeric antibodies, humanized antibodies, or human antibodies. These antibodies can be produced using known methods.

[0062] Chimeric antibodies include antibodies whose variable and constant regions are heterologous, such as chimeric antibodies in which the variable region of a mouse- or rat-derived antibody is joined to a human-derived constant region (see Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)). Examples of chimeric antibodies derived from rat anti-human CD25 antibodies include antibodies consisting of a light chain comprising the light chain variable region of a rat anti-human CD25 antibody (e.g., MAb1 antibody) described herein and a human-derived constant region, and a heavy chain comprising the heavy chain variable region of the rat anti-human CD25 antibody and a human-derived constant region.

[0063] Another example of a chimeric antibody derived from a rat anti-human CD25 antibody includes an antibody consisting of a light chain comprising a light chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in the light chain variable region of a rat anti-human CD25 antibody described herein (e.g., MAb1 antibody) has been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in the heavy chain variable region has been substituted with another amino acid residue, and the antibody may have a constant region derived from any human.

[0064] Another example of a chimeric antibody derived from a rat anti-human CD25 antibody includes an antibody consisting of a light chain comprising a light chain variable region in which one to two amino acid residues, preferably one amino acid residue, in any one to three CDRs in the light chain variable region of a rat anti-human CD25 antibody described herein (e.g., MAb1 antibody) have been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one to two amino acid residues, preferably one amino acid residue, in any one to three CDRs in the heavy chain variable region have been substituted with another amino acid residue, and the antibody may have a constant region derived from any human.

[0065] Examples of chimeric antibodies derived from MAb1 antibody include antibodies consisting of a light chain comprising a light chain variable region consisting of the amino acid sequence of amino acids 21 to 126 of SEQ ID NO: 26, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 146 of SEQ ID NO: 27, and the antibody may have a constant region derived from any human.

[0066] Other examples of chimeric antibodies derived from MAb1 antibody include antibodies comprising a light chain comprising a light chain variable region consisting of the amino acid sequence of positions 21 to 126 of SEQ ID NO: 26, in which one to several, one to three, one to two, and preferably one amino acid residue has been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 27, in which one to several, one to three, one to two, and preferably one amino acid residue has been substituted with another amino acid residue, and such antibodies may have constant regions derived from any human.

[0067] Other examples of chimeric antibodies derived from MAb1 antibody include antibodies comprising a light chain comprising a light chain variable region in which one or two amino acid residues (preferably one residue) in any one to three CDRs in the light chain variable region consisting of the amino acid sequence of positions 21 to 126 of SEQ ID NO: 26 have been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one or two amino acid residues (preferably one residue) in any one to three CDRs in the heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 27 have been substituted with another amino acid residue, and such antibodies may have constant regions derived from any human.

[0068] A specific example of a chimeric antibody derived from the MAb1 antibody is an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 232 of SEQ ID NO: 26 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 27. This chimeric anti-human CD25 antibody is referred to herein as "cMAb1_hIgG1LALA antibody" or "cMAb1_hIgG1LALA." The light chain amino acid sequence of the cMAb1_hIgG1LALA antibody is encoded by the nucleotide sequence of positions 61 to 696 of SEQ ID NO: 28, and the heavy chain amino acid sequence of the cMAb1_hIgG1LALA antibody is encoded by the nucleotide sequence of positions 58 to 1428 of SEQ ID NO: 29.

[0069] The amino acid sequence of the light chain variable region of the cMAb1_hIgG1LALA antibody is identical to that of the MAb1 antibody and consists of the amino acid sequence set forth in SEQ ID NO: 18. The light chain of the cMAb1_hIgG1LALA antibody has CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, which are identical to the CDRL1, CDRL2, and CDRL3 of the light chain of MAb1, respectively. The amino acid sequence of the light chain variable region of the cMAb1_hIgG1LALA antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 19.

[0070] The heavy chain variable region amino acid sequence of the cMAb1_hIgG1LALA antibody is identical to that of the MAb1 antibody and consists of the amino acid sequence set forth in SEQ ID NO: 20. The heavy chain of the cMAb1_hIgG1LALA antibody has a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9, which are identical to the CDRH1, CDRH2, and CDRH3 of the heavy chain of MAb1, respectively. The heavy chain variable region amino acid sequence of the cMAb1_hIgG1LALA antibody is encoded by the nucleotide sequence set forth in SEQ ID NO: 21. The sequence of the cMAb1_hIgG1LALA antibody is shown in Table 3.

[0071]

[0072] Examples of chimeric antibodies derived from rat anti-mouse CD25 antibodies include antibodies consisting of a light chain comprising the light chain variable region of a rat anti-mouse CD25 antibody described herein (e.g., MAb2 antibody) and a human-derived constant region, and a heavy chain comprising the heavy chain variable region of the rat anti-mouse CD25 antibody and a human-derived constant region.

[0073] Other examples of chimeric antibodies derived from rat anti-mouse CD25 antibodies include antibodies consisting of a light chain comprising a light chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in the light chain variable region of a rat anti-mouse CD25 antibody described herein (e.g., MAb2 antibody) has been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in the heavy chain variable region has been substituted with another amino acid residue, and such antibodies may have constant regions derived from any human.

[0074] Another example of a chimeric antibody derived from a rat anti-mouse CD25 antibody includes an antibody consisting of a light chain comprising a light chain variable region in which one to two amino acid residues, preferably one amino acid residue, in any one to three CDRs in the light chain variable region of the rat anti-mouse CD25 antibody described herein (e.g., MAb2 antibody) have been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one to two amino acid residues, preferably one amino acid residue, in any one to three CDRs in the heavy chain variable region have been substituted with another amino acid residue, and the antibody may have a constant region derived from any human.

[0075] Examples of chimeric antibodies derived from MAb2 antibody include antibodies consisting of a light chain comprising a light chain variable region consisting of the amino acid sequence of amino acids 21 to 133 of SEQ ID NO: 30, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 133 of SEQ ID NO: 31, and the antibody may have a constant region derived from any human.

[0076] Other examples of chimeric antibodies derived from MAb2 antibody include antibodies comprising a light chain comprising a light chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in the light chain variable region consisting of the amino acid sequence of positions 21 to 133 of SEQ ID NO: 30 has been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in the heavy chain variable region consisting of the amino acid sequence of positions 20 to 133 of SEQ ID NO: 31 has been substituted with another amino acid residue, and these antibodies may have constant regions derived from any human.

[0077] Other examples of chimeric antibodies derived from MAb2 antibody include antibodies comprising a light chain comprising a light chain variable region in which one or two amino acid residues (preferably one residue) in any one to three CDRs in the light chain variable region consisting of the amino acid sequence of positions 21 to 133 of SEQ ID NO: 30 have been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one or two amino acid residues (preferably one residue) in any one to three CDRs in the heavy chain variable region consisting of the amino acid sequence of positions 20 to 133 of SEQ ID NO: 31 have been substituted with another amino acid residue, and such antibodies may have constant regions derived from any human.

[0078] A specific example of a chimeric antibody derived from the MAb2 antibody is an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 232 of SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence of positions 20 to 463 of SEQ ID NO: 31. This chimeric anti-mouse CD25 antibody is referred to herein as "cMAb2_hIgG1LALA antibody" or "cMAb2_hIgG1LALA." The light chain amino acid sequence of the cMAb2_hIgG1LALA antibody is encoded by the nucleotide sequence of positions 61 to 717 of SEQ ID NO: 34, and the heavy chain amino acid sequence of the cMAb2_hIgG1LALA antibody is encoded by the nucleotide sequence of positions 58 to 1389 of SEQ ID NO: 35.

[0079] Another specific example of a chimeric antibody derived from the MAb2 antibody is an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 239 of SEQ ID NO: 32 and a heavy chain consisting of the amino acid sequence of positions 20 to 463 of SEQ ID NO: 33, and this chimeric anti-mouse CD25 antibody is referred to herein as "cMAb2_hIgG1 antibody" or "cMAb2_hIgG1." The light chain amino acid sequence of the cMAb2_hIgG1 antibody is encoded by the nucleotide sequence of positions 61 to 717 of SEQ ID NO: 36, and the heavy chain amino acid sequence of the cMAb2_hIgG1 antibody is encoded by the nucleotide sequence of positions 58 to 1389 of SEQ ID NO: 37.

[0080] Another example of a chimeric antibody derived from the MAb2 antibody is an antibody consisting of a light chain comprising a light chain variable region consisting of the amino acid sequence of amino acids 21 to 133 of SEQ ID NO: 54, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 20 to 133 of SEQ ID NO: 55, and the antibody may have a constant region derived from any human.

[0081] Other examples of chimeric antibodies derived from MAb2 antibody include antibodies comprising a light chain comprising a light chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in the light chain variable region consisting of the amino acid sequence of positions 21 to 133 of SEQ ID NO: 54 has been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one to several, one to three, one to two, and preferably one amino acid residue in the heavy chain variable region consisting of the amino acid sequence of positions 20 to 133 of SEQ ID NO: 55 has been substituted with another amino acid residue, and these antibodies may have constant regions derived from any human.

[0082] Other examples of chimeric antibodies derived from MAb2 antibody include antibodies comprising a light chain comprising a light chain variable region in which one or two amino acid residues (preferably one residue) in any one to three CDRs in the light chain variable region consisting of the amino acid sequence of positions 21 to 133 of SEQ ID NO: 54 have been substituted with another amino acid residue, and a heavy chain comprising a heavy chain variable region in which one or two amino acid residues (preferably one residue) in any one to three CDRs in the heavy chain variable region consisting of the amino acid sequence of positions 20 to 133 of SEQ ID NO: 55 have been substituted with another amino acid residue, and such antibodies may have constant regions derived from any human.

[0083] A specific example of a chimeric antibody derived from the MAb2 antibody is an antibody consisting of a light chain consisting of the amino acid sequence of positions 21 to 239 of SEQ ID NO: 54 and a heavy chain consisting of the amino acid sequence of positions 20 to 463 of SEQ ID NO: 55. This chimeric anti-mouse CD25 antibody is referred to herein as "cMAb2_hIgG1LALA-PA antibody" or "cMAb2_hIgG1LALA-PA." The light chain amino acid sequence of the cMAb2_hIgG1LALA-PA antibody is encoded by the nucleotide sequence of positions 61 to 717 of SEQ ID NO: 56, and the heavy chain amino acid sequence of the cMAb2_hIgG1LALA-PA antibody is encoded by the nucleotide sequence of positions 58 to 1389 of SEQ ID NO: 57.

[0084] The amino acid sequence of the light chain variable region of the cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, and cMAb2_hIgG1LALA-PA antibody is identical to that of the light chain variable region of the MAb2 antibody, and consists of the amino acid sequence set forth in SEQ ID NO: 22. The light chains of the cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, and cMAb2_hIgG1LALA-PA antibody have CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, which are identical to the CDRL1, CDRL2, and CDRL3, respectively, of the light chain of MAb2. The light chain variable region amino acids of the cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, and cMAb2_hIgG1LALA-PA antibody are encoded by the nucleotide sequence shown in SEQ ID NO:23.

[0085] The amino acid sequence of the heavy chain variable region of the cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, and cMAb2_hIgG1LALA-PA antibody is identical to that of the MAb2 antibody, and consists of the amino acid sequence set forth in SEQ ID NO: 24. The heavy chains of the cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, and cMAb2_hIgG1LALA-PA antibody have a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 12, which are identical to the CDRH1, CDRH2, and CDRH3, respectively, of the heavy chain of MAb2. The amino acid sequences of the heavy chain variable regions of the cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, and cMAb2_hIgG1LALA-PA antibody are encoded by the nucleotide sequence shown in SEQ ID NO: 25. The sequences of the cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, and cMAb2_hIgG1LALA-PA antibody are shown in Table 4.

[0086]

[0087] Examples of humanized antibodies include antibodies in which only the complementarity-determining region (CDR) has been incorporated into a human-derived antibody (see Nature (1986) 321, pp. 522-525); antibodies in which not only the CDR sequences but also some framework amino acid residues have been grafted onto a human antibody by CDR grafting (WO 90 / 07861); and antibodies in which the amino acid sequences of some CDRs have been modified while maintaining antigen-binding ability. Herein, a humanized antibody derived from the MAb1 antibody is not limited to a specific humanized antibody as long as it retains all six CDR sequences unique to the MAb1 antibody and has internalization activity. As long as the humanized antibody has internalization activity, the amino acid sequences of some CDRs may also be modified.

[0088] Examples of humanized antibodies of MAb1 antibody include: (1) the amino acid sequence of 21 to 127 of SEQ ID NO: 13 or 21 to 127 of SEQ ID NO: 14 (hMAb1A-L, hMAb1B-L), (2) an amino acid sequence having at least 95% or more sequence identity to the amino acid sequence of (1) above (preferably, an amino acid sequence having at least 95% or more sequence identity to the framework region sequence other than each CDR sequence), and (3) an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of (1) above; and heavy chains comprising a heavy chain variable region having at least one of the following selected from the group consisting of: (4) the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15 or positions 20 to 146 of SEQ ID NO: 16 (hMAb1A-H, hMAb1B-H), (5) an amino acid sequence having at least 95% or more sequence identity to the amino acid sequence of (4) above (preferably, an amino acid sequence having at least 95% or more sequence identity to the framework region sequence other than each CDR sequence), and (6) an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of (4) above.

[0089] Conservative amino acid substitutions are preferred for amino acid substitutions herein. Conservative amino acid substitutions are substitutions that occur within amino acid groups that are related in their side chains. Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar group = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine, and isoleucine; and aromatic group = phenylalanine, tryptophan, and tyrosine. Such amino acid substitutions are preferably made within a range that does not impair the properties of the substance having the original amino acid sequence.

[0090] Antibodies with suitable combinations of the above-mentioned light chain and heavy chain include antibodies consisting of a light chain having the light chain variable region amino acid sequence of positions 21 to 127 of SEQ ID NO: 13 (also referred to herein as the hMAb1A-L light chain variable region amino acid sequence), or a light chain having the light chain variable region amino acid sequence of positions 21 to 127 of SEQ ID NO: 14 (also referred to herein as the hMAb1B-L light chain variable region amino acid sequence), and a heavy chain having the heavy chain variable region amino acid sequence of positions 20 to 146 of SEQ ID NO: 15 (also referred to herein as the hMAb1A-H heavy chain variable region amino acid sequence), or a heavy chain having the heavy chain variable region amino acid sequence of positions 20 to 146 of SEQ ID NO: 16 (also referred to herein as the hMAb1B-H heavy chain variable region amino acid sequence). Preferred examples include antibodies consisting of a light chain having the light chain variable region amino acid sequence of positions 21 to 127 of SEQ ID NO: 13 and a heavy chain having the heavy chain variable region amino acid sequence of positions 20 to 146 of SEQ ID NO: 15; and antibodies consisting of a light chain having the light chain variable region amino acid sequence of positions 21 to 127 of SEQ ID NO: 14 and a heavy chain having the heavy chain variable region amino acid sequence of positions 20 to 146 of SEQ ID NO: 16.

[0091] Other examples of antibodies with suitable combinations of light chains and heavy chains include antibodies comprising a light chain consisting of the amino acid sequence from 21 to 233 of the amino acid sequence shown in SEQ ID NO: 13 (also referred to herein as the full-length amino acid sequence of hMAb1A-L light chain), or a light chain consisting of the amino acid sequence from 21 to 233 of the amino acid sequence shown in SEQ ID NO: 14 (also referred to herein as the full-length amino acid sequence of hMAb1B-L light chain), a heavy chain consisting of the amino acid sequence from 20 to 476 of the amino acid sequence shown in SEQ ID NO: 15 (also referred to herein as the full-length amino acid sequence of hMAb1A-H heavy chain), or a heavy chain consisting of the amino acid sequence from 20 to 476 of the amino acid sequence shown in SEQ ID NO: 16 (also referred to herein as the full-length amino acid sequence of hMAb1B-H heavy chain). Preferred examples include antibodies comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of the amino acid sequence shown in SEQ ID NO: 15; and antibodies comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of the amino acid sequence shown in SEQ ID NO: 16 (also referred to herein as "hMAb1A antibody" or "hMAb1B antibody"). More preferred examples include antibodies comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of the amino acid sequence shown in SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of the amino acid sequence shown in SEQ ID NO: 15. The sequences of hMAb1A antibody and hMAb1B antibody are shown in Tables 5-1 to 5-4.

[0092]

[0093] By combining sequences that show high sequence identity with the above heavy chain and light chain amino acid sequences, it is possible to select antibodies with biological activity equivalent to each of the above antibodies. Such sequence identity is generally 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more. Furthermore, by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, or added to the heavy chain or light chain amino acid sequences, it is also possible to select antibodies with biological activity equivalent to each of the above antibodies.

[0094] In one embodiment of the present invention, the antibody or antigen-binding fragment of the antibody comprises a CDRL1, CDRL2, and CDRL3, and a CDRH1, CDRH2, and CDRH3 of the following (a) or (b): (a) a CDRL1 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, identity to the amino acid sequence set forth in SEQ ID NO: 1; CDRL2 consisting of an amino acid sequence having 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:3; and CDRL3 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:3. and CDRH1 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; CDRH2 consisting of an amino acid sequence having 4% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the amino acid sequence set forth in SEQ ID NO:9, and CDRH3 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the amino acid sequence set forth in SEQ ID NO:9.(b) a CDRL1 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4; CDRL2 consisting of an amino acid sequence having 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:6; and CDRL3 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:6. and a CDRH1 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 10; a CDRH2 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. CDRH2 consisting of an amino acid sequence having 4% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the amino acid sequence set forth in SEQ ID NO: 12, and CDRH3 consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the amino acid sequence set forth in SEQ ID NO: 12.

[0095] In one embodiment of the present invention, the antibody or antigen-binding fragment of the antibody comprises a heavy chain variable region and a light chain variable region of the following (a) or (b): (a) a light chain variable region consisting of an amino acid sequence that has 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, identity to the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13, and a heavy chain variable region consisting of an amino acid sequence that has 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, identity to the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15. (b) a light chain variable region consisting of an amino acid sequence that has 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14; and a heavy chain variable region consisting of an amino acid sequence that has 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97% or 98% or more, and even more preferably 99% or more, sequence identity to the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16.

[0096] In another aspect of the present invention, the antibody or antigen-binding fragment of the antibody comprises a heavy chain variable region and a light chain variable region of the following (a) or (b): (a) a light chain variable region comprising CDRs having 100% sequence identity with each CDR sequence in the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13, and consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the sequences of the framework regions other than each CDR sequence; and CDRs having 100% sequence identity with each CDR sequence in the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15, and a heavy chain variable region consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the sequence of the framework region other than each CDR sequence. (b) a light chain variable region comprising an amino acid sequence having 100% sequence identity with each CDR sequence in the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14, and having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% or more, more preferably 95%, 96%, 97% or more, and even more preferably 99% or more sequence identity with the sequence of the framework region other than each CDR sequence; and CDRs having 100% sequence identity with each CDR sequence in the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16, and a heavy chain variable region consisting of an amino acid sequence having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the sequence of the framework region other than each CDR sequence.

[0097] In another aspect of the present invention, the antibody or antigen-binding fragment of the antibody comprises a heavy chain and a light chain of the following (a) or (b): (a) a light chain consisting of an amino acid sequence that has 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, identity to the sequence of amino acids 21 to 233 of SEQ ID NO: 13, and a heavy chain consisting of an amino acid sequence that has 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97% or 98% or more, and even more preferably 99% or more, identity to the sequence of amino acids 20 to 476 of SEQ ID NO: 15. (b) a light chain consisting of an amino acid sequence that has 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity to the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14; and a heavy chain consisting of an amino acid sequence that has 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97% or 98% or more, and even more preferably 99% or more, sequence identity to the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16.

[0098] In another aspect of the present invention, the antibody or antigen-binding fragment of the antibody of the present invention comprises a heavy chain and a light chain of (a) or (b) below: (a) a light chain comprising an amino acid sequence having 100% sequence identity with each CDR sequence in the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13, and having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity with sequences other than each CDR sequence; and a heavy chain comprising 100% sequence identity with each CDR sequence in the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15, and having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97% or 98% or more, and even more preferably 99% or more, sequence identity with sequences other than each CDR sequence. (b) a light chain comprising an amino acid sequence having 100% sequence identity with each CDR sequence in the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14, and having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97%, or 98% or more, and even more preferably 99% or more, sequence identity with sequences other than each CDR sequence; and a heavy chain comprising 100% sequence identity with each CDR sequence in the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16, and having 80% or more, preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% or more, more preferably 95%, 96%, 97% or 98% or more, and even more preferably 99% or more, sequence identity with sequences other than each CDR sequence.

[0099] The sequence identity between two amino acid sequences can be determined, for example, by aligning the sequences using the default parameters of ClustalW version 2 (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ and Higgins DG (2007), "Clustal W and Clustal X version 2.0", Bioinformatics. 23(21):2947-2948), but is not limited to this as long as it is used by those skilled in the art. Here, the "identity" of sequences refers to, in the case of amino acid sequences, aligning the two amino acid sequences to be compared so that as many residues as possible are identical, and dividing the number of identical residues by the total number of residues, expressed as a percentage. During the alignment, gaps may be inserted as needed into one or both of the two sequences being compared. Such sequence alignment can be performed using well-known programs such as BLAST, FASTA, and CLUSTALW. When gaps are inserted, the total number of residues is calculated by counting each gap as one residue. If the total number of residues counted in this way differs between the two sequences being compared, the identity (%) is calculated by dividing the number of identical residues by the total number of residues in the longer sequence. The same applies to the identity of nucleotide sequences. The amino acid sequence identity herein is calculated using the sequence analysis software GENETYX-SV / RC (manufactured by Genetyx Corporation), an algorithm commonly used in the art.

[0100] In the full-length amino acid sequence of the hMAb1A-L light chain shown in SEQ ID NO: 13, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, the amino acid sequence consisting of amino acid residues 21 to 127 is a variable region, and the amino acid sequence consisting of amino acid residues 128 to 233 is a constant region. In the full-length nucleotide sequence of the hMAb1A-L light chain shown in SEQ ID NO: 38, the nucleotide sequence consisting of nucleotides 1 to 60 encodes a signal sequence, the nucleotide sequence consisting of nucleotides 61 to 381 encodes a variable region, and the nucleotide sequence consisting of nucleotides 382 to 699 encodes a constant region. In the full-length amino acid sequence of the hMAb1B-L light chain shown in SEQ ID NO: 14, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, the amino acid sequence consisting of amino acid residues 21 to 127 is a variable region, and the amino acid sequence consisting of amino acid residues 128 to 233 is a constant region. In the full-length nucleotide sequence of the hMAb1B-L light chain shown in SEQ ID NO: 39, the nucleotide sequence consisting of nucleotides 1 to 60 encodes a signal sequence, the nucleotide sequence consisting of nucleotides 61 to 381 encodes a variable region, and the nucleotide sequence consisting of nucleotides 382 to 699 encodes a constant region. In the full-length amino acid sequence of the hMAb1A-H heavy chain shown in SEQ ID NO: 15, the amino acid sequence consisting of amino acid residues 1 to 19 encodes a signal sequence, the amino acid sequence consisting of amino acid residues 20 to 146 encodes a variable region, and the amino acid sequence consisting of amino acid residues 147 to 476 encodes a constant region. In the full-length nucleotide sequence of the hMAb1A-H heavy chain shown in SEQ ID NO: 40, the nucleotide sequence consisting of nucleotides 1 to 57 encodes a signal sequence, the nucleotide sequence consisting of nucleotides 58 to 438 encodes a variable region, and the nucleotide sequence consisting of nucleotides 439 to 1428 encodes a constant region. In the full-length amino acid sequence of the heavy chain of hMAb1B-H shown in SEQ ID NO: 16, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues 20 to 146 is a variable region, and the amino acid sequence consisting of amino acid residues 147 to 476 is a constant region.In the full-length nucleotide sequence of the heavy chain of hMAb1B-H shown in SEQ ID NO:41, the nucleotide sequence consisting of nucleotides 1 to 57 encodes a signal sequence, the nucleotide sequence consisting of nucleotides 58 to 438 encodes a variable region, and the nucleotide sequence consisting of nucleotides 439 to 1428 encodes a constant region.

[0101] The antibody of the present invention further includes a human antibody that binds to CD25. An anti-CD25 human antibody means a human antibody that has only the gene sequence of an antibody derived from a human chromosome. The anti-CD25 human antibody was produced by a method using a human antibody-producing mouse carrying a human chromosome fragment containing the heavy and light chain genes of a human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.

[0102] Specifically, such human antibody-producing mice can be produced by producing knockout animals and transgenic animals as genetically modified animals in which the endogenous immunoglobulin heavy chain and light chain gene loci have been destroyed and instead human immunoglobulin heavy chain and light chain gene loci have been introduced via yeast artificial chromosome (YAC) vectors or the like, and by crossing these animals with each other.

[0103] Alternatively, eukaryotic cells can be transformed by recombinant DNA technology with cDNAs encoding the heavy and light chains of such human antibodies, preferably vectors containing the cDNAs, and the transformed cells can be cultured to produce recombinant human monoclonal antibodies, thereby obtaining the antibodies from the culture supernatant. The host can be, for example, eukaryotic cells, preferably CHO cells, or mammalian cells such as lymphocytes and myeloma cells.

[0104] Also known are methods for obtaining phage-display-derived human antibodies selected from a human antibody library (see, for example, Wormstone, I.M. et al., Investigative Ophthalmology & Visual Science. (2002) 43(7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1(2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109(3), pp. 427-431). For example, a phage display method (Nature Biotechnology (2005), 23, (9), pp. 1105-1116) can be used, in which the variable regions of human antibodies are expressed on the surface of phages as single-chain fragments (scFv), and phages that bind to the antigen are selected. By analyzing the genes of phages selected by binding to the antigen, the DNA sequence encoding the variable regions of human antibodies that bind to the antigen can be determined. Once the DNA sequence of an scFv that binds to an antigen is clarified, an expression vector containing that sequence can be prepared and introduced into an appropriate host for expression to obtain a human antibody (WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, WO 95 / 15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23(9), pp. 1105-1116).

[0105] If a newly created human antibody binds to a partial peptide or partial three-dimensional structure to which any one of the rat anti-CD25 antibodies, chimeric anti-CD25 antibodies, or humanized anti-human CD25 antibodies described herein (e.g., MAb1 antibody, MAb2 antibody, cMAb1_hIgG1LALA antibody, cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, cMAb2_hIgG1LALA-PA antibody, hMAb1A antibody, and hMAb1B antibody) binds, it can be determined that the human antibody binds to the same epitope as the rat anti-CD25 antibody, chimeric anti-CD25 antibody, or humanized anti-human CD25 antibody. Alternatively, the human antibody competes for binding to CD25 with a rat anti-CD25 antibody, chimeric anti-CD25 antibody, or humanized anti-human CD25 antibody described herein (e.g., MAb1 antibody, MAb2 antibody, cMAb1_hIgG1LALA antibody, cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, cMAb2_hIgG1LALA-PA antibody, hMAb1A antibody, hMAb1B antibody). By confirming that the human antibody "interferes with the binding of the rat anti-CD25 antibody, chimeric anti-CD25 antibody, or humanized anti-human CD25 antibody" to CD25 (cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, cMAb2_hIgG1LALA-PA antibody, hMAb1A antibody, or hMAb1B antibody to CD25), it can be determined that the human antibody binds to the same epitope as the rat anti-CD25 antibody, chimeric anti-CD25 antibody, or humanized anti-human CD25 antibody described herein, even if the specific sequence or structure of the epitope has not been determined. Herein, when a newly created human antibody is determined to "bind to the same epitope" by at least one of these determination methods, it can be said that the newly created human antibody "binds to the same epitope" as the rat anti-CD25 antibody, chimeric anti-CD25 antibody, or humanized anti-human CD25 antibody described herein.When it is confirmed that the epitope is the same, the human antibody is expected to have biological activity equivalent to that of a rat anti-CD25 antibody, a chimeric anti-CD25 antibody, or a humanized anti-human CD25 antibody (e.g., MAb1 antibody, MAb2 antibody, cMAb1_hIgG1LALA antibody, cMAb2_hIgG1LALA antibody, cMAb2_hIgG1 antibody, cMAb2_hIgG1LALA-PA antibody, hMAb1A antibody, or hMAb1B antibody). The chimeric, humanized, or human antibody obtained by the above method can be evaluated for its antigen-binding ability by known methods, etc., and a suitable antibody can be selected.

[0106] The antibodies of the present invention also include modified antibodies. The term "modified antibodies" refers to antibodies of the present invention that have been chemically or biologically modified. Chemical modifications include attachment of a chemical moiety to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like. Biological modifications include those that have undergone post-translational modifications (e.g., addition of N-linked or O-linked sugar chains, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, or pyroglutamation of N-terminal glutamine or N-terminal glutamic acid), and those in which a methionine residue has been added to the N-terminus by expression in a prokaryotic host cell. Also included within the meaning of such modifications are those labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies. Such modified antibodies of the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and detecting or isolating antibodies or antigens, etc.

[0107] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the glycosylation (glycosylation, defucosylation, etc.) of the antibodies of the present invention. Techniques for modulating antibody glycosylation are known, including, but not limited to, those described in International Publication Nos. 1999 / 54342, 2000 / 61739, 2002 / 31140, and 2013 / 120066. The antibodies of the present invention also include antibodies with modified glycosylation. When antibody genes are isolated and then introduced into a suitable host to produce the antibody, a suitable combination of host and expression vector can be used. A specific example of an antibody gene is a combination of a gene encoding the heavy chain sequence and a gene encoding the light chain sequence of an antibody described herein. When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector or into separate expression vectors.

[0108] When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Particularly, examples of animal cells include mammalian cells, such as monkey COS cells (Gluzman, Y. Cell (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, L.A. Proc. Natl. Acad. Sci. U.S.A. (1980) 77, pp. 4126-4220), and FreeStyle 293F cells (Invitrogen). When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis.

[0109] Antibodies can be obtained by introducing the desired antibody gene into these cells by transformation and culturing the transformed cells in vitro. The yield of the antibody produced during this culture may vary depending on the antibody sequence, and antibodies with equivalent binding activity can be selected based on the yield as an indicator to determine which antibodies are easy to produce as pharmaceuticals. Therefore, the antibodies of the present invention also include antibodies obtained by a method for producing the antibody, which method comprises the steps of culturing the transformed host cells and recovering the desired antibody or antigen-binding fragment of the antibody from the culture obtained in this step.

[0110] It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705:129-134 (1995)), and that two amino acid residues, glycine and lysine, are deleted at the carboxyl terminus of the heavy chain, and a proline residue newly positioned at the carboxyl terminus is amidated (Analytical Biochemistry, 360:75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibodies of the present invention also include antibodies and antigen-binding fragments of such antibodies that have been modified in this manner, as well as deletions in which one or two amino acids have been deleted from the carboxyl terminus of the heavy chain, and deletions in which the heavy chain has been amidated (e.g., a heavy chain in which the proline residue at the carboxyl terminus has been amidated). However, as long as the antigen-binding ability and effector function are maintained, the deletions in the carboxyl termini of the heavy chains of the antibodies of the present invention are not limited to the above types. The two heavy chains constituting the antibodies of the present invention may be any one type of heavy chain selected from the group consisting of full-length heavy chains and the above deletions, or a combination of two types of heavy chains. The quantitative ratio of each deletion may be affected by the type and culture conditions of the cultured mammalian cells that produce the antibodies of the present invention, but an example of a major component of the antibodies of the present invention is one in which one amino acid residue has been deleted from the carboxyl terminus of each heavy chain.

[0111] Examples of the isotype of the antibody of the present invention include IgG (IgG1, IgG2, IgG3, IgG4), with IgG1 or IgG4 being preferred. When the antibody of the present invention is of the IgG1 isotype, the IgG1 antibody may have mutations, and effector function can be adjusted by substituting a portion of the amino acid residues in the constant region (see WO88 / 007089, WO94 / 28027, and WO94 / 29351). Examples of IgG1 mutants with attenuated effector function include IgG1 LALA (IgG1-L234A, L235A) and IgG1 LALA-PA (IgG1-L234A, L235A, P329A). The biological activities of an antibody generally include antigen-binding activity, the activity of internalizing into cells expressing the antigen by binding to the antigen, the activity of neutralizing antigen activity, the activity of enhancing antigen activity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). However, the function of the antibody of the present invention is binding activity to CD25, preferably an activity of internalizing into CD25-expressing cells by binding to CD25, without having IL-2 blocking ability. Furthermore, the antibody of the present invention may have ADCC activity, CDC activity, and / or ADCP activity in addition to the cell-internalizing activity.

[0112] The resulting antibodies can be purified to homogeneity. Antibody isolation and purification can be performed using isolation and purification methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectric focusing (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press (1996)). Laboratory (1988), but is not limited thereto. Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography. These chromatographies can be performed using liquid chromatography such as HPLC and FPLC. Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, columns using protein A columns include Hyper D, POROS, and Sepharose F.F. (Pharmacia). It is also possible to purify antibodies by utilizing their binding to antigens using a carrier on which an antigen is immobilized.

[0113] 3. Anti-CD25 Antibody-Drug Conjugates (1) Drugs The anti-CD25 antibodies obtained in "2. Production of Anti-CD25 Antibodies" above can be converted into anti-CD25 antibody-drug conjugates by conjugating a drug via a linker structure. The drug is not particularly limited as long as it has a substituent or partial structure that can be conjugated to the linker structure. Anti-CD25 antibody-drug conjugates can be used for a variety of purposes depending on the drug to be conjugated. Examples of such drugs include substances with cytotoxic activity (including cytotoxic compounds), substances with antitumor activity, chemotherapeutic agents, molecular targeted drugs, immune activators, immunosuppressants, toxins, photosensitizers, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, vitamins, hormones, substances effective against blood diseases, substances effective against autoimmune diseases, anti-inflammatory substances, antibacterial substances, antifungal substances, antiparasitic substances, antiviral substances, and antianesthetic substances.

[0114] (1)-1 Cytotoxic Compounds Examples of using a cytotoxic compound as the compound bound to the anti-CD25 antibody-drug conjugate of the present invention are described below. Anti-CD25 antibody-drug conjugates in which a drug that exerts its toxicity intracellularly is bound to the anti-CD25 antibody of the present invention via a linker of a specific structure can exhibit the effect of directly killing cancer cells expressing CD25. The cytotoxic compound is not particularly limited as long as it is a compound that has a cytotoxic effect and has a substituent or partial structure that can be bound to the linker structure. The cytotoxic effect of the cytotoxic compound is exerted when part or all of the linker is cleaved in blood cells and tumor cells, releasing the cytotoxic compound (which may include part of the linker). Cleavage of the linker at the bond to the drug is preferred because it releases the cytotoxic compound in its original structure and allows its original cytotoxic effect to be exerted.

[0115] The anti-CD25 antibody obtained in the above "2. Production of anti-CD25 antibody" can be converted into an anti-CD25 antibody-drug conjugate by binding a cytotoxic compound via a linker structure. One example of the cytotoxic compound used in the present invention is the camptothecin derivative exatecan ((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione; the following formula:

[0116] can be preferably used. This compound can be easily obtained, for example, by the method described in U.S. Patent Publication No. US 2016 / 0297890 or other known methods, and the amino group at position 1 can be preferably used as the binding site to the linker structure. Furthermore, exatecan may be released intracellularly with a portion of the linker still attached, but even in such a state, it is a compound that exhibits excellent cytotoxic effects. Because exatecan has a camptothecin structure, it is known that in an acidic aqueous medium (e.g., about pH 3), the equilibrium shifts toward a structure in which the lactone ring is formed (closed ring form), while in a basic aqueous medium (e.g., about pH 10), the equilibrium shifts toward a structure in which the lactone ring is opened (open ring form). Drug conjugates incorporating exatecan residues corresponding to such closed ring and open ring structures are expected to have equivalent cytotoxic effects, and it goes without saying that both are encompassed within the scope of the present invention.

[0117] Other cytotoxic active compounds include, for example, antitumor compounds described in the literature (Pharmacological Reviews, 68, pp. 3-19, 2016), and examples thereof include auristatins such as doxorubicin, calchemicin, dorastatin 10, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, and pyrrolobenzodiazepines (Py Examples of suitable antitumor agents include SG2000 (SJG-136), a dimer of benzodiazepine (benzotriazole), SN-38 which is a camptothecin derivative, duocarmycins such as CC-1065, amanitin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin or a derivative thereof), taxol or a derivative thereof, and the like.

[0118] In antibody-drug conjugates, the number of drugs bound to one antibody molecule is an important factor affecting their efficacy and safety. Antibody-drug conjugates are produced by specifying reaction conditions, such as the amounts of raw materials and reagents used, so that a certain number of drugs are bound. However, unlike chemical reactions of low-molecular-weight compounds, they are usually obtained as mixtures in which different numbers of drugs are bound. The number of drugs bound to one antibody molecule is specified and expressed as an average value, i.e., the average drug binding number. In the present invention, as a general rule, unless otherwise specified, i.e., except when referring to antibody-drug conjugates having a specific drug binding number contained in a mixture of antibody-drug conjugates having different drug binding numbers, the drug binding number refers to the average value. The number of exatecans bound to an antibody molecule can be controlled, and the average number of drugs bound per antibody can be about 1 to 10, preferably 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, or 7 to 8, more preferably 5 to 8, even more preferably 7 to 8, and still more preferably 8. Those skilled in the art will be able to design a reaction for binding the required number of drugs to an antibody from the description of the Examples of the present application, and will be able to obtain an antibody-drug conjugate in which the number of exatecans bound is controlled.

[0119] (2) Linker Structure The linker structure that binds the drug to the anti-CD25 antibody in the anti-CD25 antibody-drug conjugate of the present invention will be described.

[0120] In the antibody-drug conjugate of the present application, the linker structure linking the anti-CD25 antibody and the drug is not particularly limited as long as it can be used as an antibody-drug conjugate, and can be appropriately selected depending on the purpose of use. Examples of linker structures include linkers described in publicly known literature (Pharmacol Rev 68:3-19, January 2016, Protein Cell DOI 10.1007 / s13238-016-0323-0, etc.), and more specific examples include VC (valine-citrulline), MC (maleimidocaproyl), SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and SPP (N-succinimidyl Examples of the hydrazone include 4-(2-pyridyldithio)pentanoic acid (N-succinimidyl 4-(2-pyridyldithio)pentanoate), SS (disulfide), SPDB (N-succinimidyl 4-(2-pyridyldithio)butyrate), SS / hydrazone, and carbonate.

[0121] Other examples include the linker structure described in U.S. Patent Publication US2016 / 0297890 (for example, those described in paragraphs

[0260] to

[0289] ), and the following structure can be suitably used. In the structure shown below, the left end is the binding site with the antibody, and the right end is the binding site with the drug. In addition, GGFG in the following linker structure represents an amino acid sequence connected by a peptide bond consisting of glycine-glycine-phenylalanine-glycine (GGFG). -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0122] More preferred examples include the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0123] Even more preferred are -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0124] The antibody is bound to the -(Succinimid-3-yl-N) end (for example, in "-(Succinimid-3-yl-N)-CHCHCHCHCHCH-C(=O)-GGFG-NH-CH-O-CH-C(=O)-", the end opposite to where (-CHCHCHCHCHCHCH-) is bound (the left end)), and the cytotoxic compound is bound to the end opposite to -(Succinimid-3-yl-N) (the right end in the above example, via the carbonyl group of CH-O-CH-C(=O)-). "-(Succinimid-3-yl-N)-" is represented by the following formula:

[0125] The partial structure has a structure shown in the following formula: Position 3 of this partial structure is the binding site for the anti-CD25 antibody. The binding to the antibody at position 3 is characterized by the formation of a thioether bond. The nitrogen atom at position 1 of this structural portion is bonded to the carbon atom of a methylene group present in a linker containing this structure.

[0126] In the antibody-drug conjugate of the present invention in which the drug is exatecan, a drug-linker structure having the following structure is preferably conjugated to an antibody or an antigen-binding fragment of the antibody (preferably an antibody). The average number of these drug-linker structures conjugated per antibody or antigen-binding fragment of the antibody may be 1 to 10, preferably 2 to 8, more preferably 5 to 8, even more preferably 7 to 8, and even more preferably 8. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0127] More preferred are the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0128] More preferred examples include the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX). In addition, -(NH-DX) is a group represented by the following formula:

[0129] This represents the group generated by removing one hydrogen atom from the amino group at position 1 of exatecan.

[0130] In one embodiment, the linker structure connecting the anti-CD25 antibody and the drug in the antibody-drug conjugate of the present application may have the following structure: -(Succinimid-3-yl-N)-(CH) １ -C(=O)-L-GGGFG-NH-X-C(=O)-, where n １ represents an integer of 2 to 8, and L represents -NH-(CH2-CH2-O)n ２ represents —CH2—CH2— or a single bond, ２represents an integer of 1 to 6, and X represents a linear or branched alkylene group having 1 to 20 carbon atoms (preferably 1 to 10 carbon atoms) (one or more methylene groups in the chain may be replaced with an oxygen atom or a sulfur atom). The alkylene group may have a cyclic saturated hydrocarbon group having 1 to 6 carbon atoms as part of its structure (e.g., a cyclopropyl group, a cyclobutyl group, a cyclopentanyl group, a cyclohexanyl group, a cis-1,3-cyclobutylene group, a trans-1,3-cyclobutylene group, a cis-1,2-cyclopropylene group, a trans-1,2-cyclopropylene group, a cis-1,4-cyclohexylene group, a trans-1,4-cyclohexylene group, etc.). The antibody-binding site, the drug-binding site, GGFG, and "-(Succinimid-3-yl-N)-" are as defined above.

[0131] In one embodiment, the average number of drug linkers bound per antibody molecule in the anti-CD25 antibody-drug conjugate used in the present invention is preferably 1 to 10, more preferably 2 to 8, even more preferably 5 to 8, even more preferably 7 to 8, even more preferably 7.5 to 8, and even more preferably about 8. In another embodiment, the number of drugs or drug linkers bound per antibody molecule in the anti-CD25 antibody-drug conjugate used in the present invention is preferably an integer within the range of 2 to 8, more preferably 2, 4, 6, or 8, and even more preferably 8.

[0132] (3) Method for producing antibody-drug conjugate There are no particular limitations on the antibody that can be used for the antibody-drug conjugate of the present invention, as long as it is an anti-CD25 antibody or an antigen-binding fragment of the antibody that has internalization activity as described in the above section "2. Production of anti-CD25 antibody" and the Examples.

[0133] Next, a representative method for producing the antibody-drug conjugate of the present invention will be described. In the following, the compound numbers shown in each reaction scheme will be used to indicate the compounds. That is, they will be referred to as "compound of formula (1)," "compound (1)," etc. Compounds with other numbers will also be referred to in the same manner.

[0134] (3)-1 Production Method 1 Among the antibody-drug conjugates represented by the following formula (1), those in which an anti-CD25 antibody and a linker structure are linked via a thioether can be produced by reacting an anti-CD25 antibody obtained by reducing the anti-CD25 antibody to convert the disulfide bonds to sulfhydryl groups with compound (2) available by a known method (for example, available by the method described in US Patent Publication No. 2016 / 297890 (for example, the method described in paragraphs

[0336] to

[0374] )). For example, it can be produced by the following method.

[0135] [wherein AB represents an antibody having a sulfhydryl group.]

[0136] Here, L 1 is represented by the structure -(Succinimid-3-yl-N)-. 1 ' represents a maleimidyl group represented by the following formula:

[0137]

[0138] -L 1 -L Xhas any of the structures represented by the following formulae: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0139] Among these, the following are more preferred: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0140] Further preferred examples include the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-.

[0141] Furthermore, in the above reaction scheme, antibody-drug conjugate (1) is depicted as having a structure in which one structural moiety from the drug to the linker terminal is bound to one antibody. However, this is a description for convenience of explanation, and in reality, multiple structural moieties are often bound to one antibody molecule. This situation also applies to the following description of the production method. That is, antibody-drug conjugate (1) can be produced by reacting compound (2) obtainable by a known method (e.g., obtainable by the method described in US 2016 / 297890 (e.g., the method described in paragraphs

[0336] to

[0374] )) with antibody (3a) having a sulfhydryl group.

[0142] The antibody (3a) having a sulfhydryl group can be obtained by a method known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). Examples of such methods include, but are not limited to, reacting Traut's reagent with the amino groups of an antibody; reacting N-succinimidyl S-acetylthioalkanoates with the amino groups of an antibody, followed by reaction with hydroxylamine; reacting N-succinimidyl 3-(pyridyldithio)propionate, followed by reaction with a reducing agent; and reacting an antibody with a reducing agent such as dithiothreitol, 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce disulfide bonds in the intra-chain regions of the antibody and generate sulfhydryl groups.

[0143] Specifically, an antibody with partially or completely reduced intra-chain disulfides can be obtained by reacting the antibody with TCEP as a reducing agent in an amount of 0.3 to 3 molar equivalents per antibody intra-chain disulfide in a buffer containing a chelating agent. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). These can be used at concentrations of 1 mM to 20 mM. Examples of buffer solutions that can be used include sodium phosphate, sodium borate, and sodium acetate solutions. In a specific example, an antibody (3a) with partially or completely reduced sulfhydryl groups can be obtained by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours. Furthermore, a reaction to add the sulfhydryl groups to the drug-linker moiety can be carried out here, thereby linking the drug-linker moiety via a thioether bond.

[0144] Next, 2 to 20 molar equivalents of compound (2) can be used per antibody (3a) having a sulfhydryl group to produce an antibody-drug conjugate (1) in which 2 to 8 drugs are bound per antibody. Specifically, a solution of compound (2) can be added to a buffer solution containing antibody (3a) having a sulfhydryl group, followed by reaction. Examples of the buffer solution include sodium acetate, sodium phosphate, and sodium borate. The pH during the reaction is 5 to 9, preferably around pH 7. Examples of solvents that can be used to dissolve compound (2) include organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyridone (NMP). The organic solvent solution containing compound (2) can be added to a buffer solution containing antibody (3a) having a sulfhydryl group at a concentration of 1 to 20% v / v, followed by reaction. The reaction temperature is 0 to 37°C, more preferably 10 to 25°C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by deactivating the reactivity of unreacted compound (2) with a thiol-containing reagent. Examples of the thiol-containing reagent include cysteine ​​or N-acetyl-L-cysteine ​​(NAC). More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of NAC relative to the compound (2) used and incubating at room temperature for 10 to 30 minutes.

[0145] (4) Identification of Antibody-Drug Conjugate The produced antibody-drug conjugate (1) can be identified by concentrating, buffer exchanging, purifying, measuring the antibody concentration, and measuring the average number of drugs bound per antibody molecule, according to the following common procedures.

[0146] (4)-1 Common Procedure A: Concentration of Aqueous Antibody or Antibody-Drug Conjugate Solution An antibody or antibody-drug conjugate solution is placed in an Amicon Ultra (50,000 MWCO, Millipore Corporation) container, and the antibody or antibody-drug conjugate solution is concentrated by centrifugation (centrifugation at 2000 G to 3800 G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

[0147] (4)-2 Common Procedure B: Antibody Concentration Measurement Antibody concentrations are measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. -1 cm -1 ~1.8mLmg -1 cm -1 ) is used.

[0148] (4)-3 Common Procedure C: Antibody Buffer Exchange A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 carrier was equilibrated with phosphate buffer (50 mM, pH 6.0) containing sodium chloride (50 mM) and EDTA (2 mM) (referred to herein as PBS 6.0 / EDTA) according to the manufacturer's instructions. 2.5 mL of antibody aqueous solution was loaded onto each NAP-25 column, and a fraction (3.5 mL) eluted with 3.5 mL of PBS 6.0 / EDTA was collected. This fraction was concentrated using Common Procedure A, and the antibody concentration was measured using Common Procedure B. The antibody concentration was then adjusted to 20 mg / mL using PBS 6.0 / EDTA.

[0149] (4)-4 Common Procedure D: Purification of Antibody-Drug Conjugates A NAP-25 column was equilibrated with one of the commercially available acetate buffers (10 mM, pH 5.5; referred to herein as ABS) containing sorbitol (5%). The antibody-drug conjugate reaction solution (approximately 2.5 mL) was loaded onto this NAP-25 column and eluted with the amount of buffer specified by the manufacturer to separate the antibody fraction. This gel filtration purification procedure, in which the separated fraction was loaded onto the NAP-25 column again and eluted with buffer, was repeated two or three times to obtain the antibody-drug conjugate from which unbound drug linkers and low-molecular-weight compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine ​​(NAC), dimethyl sulfoxide) had been removed.

[0150] (4)-5 Common Procedure E: Measurement of antibody concentration in antibody-drug conjugate and average number of drugs bound per antibody molecule The bound drug concentration in an antibody-drug conjugate can be calculated by measuring the UV absorbance of an aqueous antibody-drug conjugate solution at two wavelengths, 280 nm and 370 nm, and then performing the following calculation. Since the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in the system [additivity of absorbance], assuming that there is no change in the molar extinction coefficients of the antibody and drug before and after conjugation of the antibody and drug, the antibody concentration and drug concentration in the antibody-drug conjugate are expressed by the following relationship:

[0151] A 280 = A D,280 +A A,280 = ε D,280 C D +ε A,280 C A Formula (1) A 370 = A D,370 +A A,370 = ε D,370 C D +ε A,370 C A Formula (2)

[0152] Here, A 280 indicates the absorbance of an aqueous solution of an antibody-drug conjugate at 280 nm, and A 370indicates the absorbance of an aqueous solution of an antibody-drug conjugate at 370 nm, and A A,280 indicates the absorbance of the antibody at 280 nm, and A A,370 indicates the absorbance of the antibody at 370 nm, and A D,280 denotes the absorbance of the conjugate precursor at 280 nm, and A D,370 denotes the absorbance of the conjugate precursor at 370 nm, and ε A,280 denotes the molar extinction coefficient of the antibody at 280 nm, and ε A,370 denotes the molar extinction coefficient of the antibody at 370 nm, and ε D,280 denotes the molar extinction coefficient of the conjugate precursor at 280 nm, and ε D,370 denotes the molar extinction coefficient of the conjugate precursor at 370 nm, and C A indicates the antibody concentration in the antibody-drug conjugate, and C D indicates the drug concentration in the antibody-drug conjugate.

[0153] where ε A,280 , ε A,370 , ε D,280 , ε D,370 A value prepared in advance (a calculated estimated value or an actual measured value obtained from UV measurement of the compound) is used for ε. For example, ε A,280 can be estimated from the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). A,370 is usually zero. D,280 and ε D,370 can be obtained by measuring the absorbance of a solution in which the conjugate precursor used is dissolved at a certain molar concentration, according to the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length). 280 and A 370 By measuring these values ​​and substituting them into equations (1) and (2) to solve the simultaneous equations, C A and C D Furthermore, C D C A The average number of drugs bound per antibody can be calculated by dividing by .

[0154] (4)-6 Common Procedure F: Measurement of the Average Number of Drugs Bonded per Antibody Molecule in an Antibody-Drug Conjugate (2) The average number of drugs bonded per antibody molecule in an antibody-drug conjugate can also be determined by high-performance liquid chromatography (HPLC) analysis using the following method, in addition to the aforementioned "(4)-5 Common Procedure E." The following describes a method for measuring the average number of drugs bonded by HPLC when the antibody and the drug linker are disulfide-bonded. Those skilled in the art can measure the average number of drugs bonded by HPLC as appropriate, depending on the type of bond between the antibody and the drug linker, with reference to this method.

[0155] F-1. Preparation of sample for HPLC analysis (reduction of antibody-drug conjugate) The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) is mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). The mixture is incubated at 37°C for 30 minutes to cleave the disulfide bond between the light and heavy chains of the antibody-drug conjugate, and the resulting sample is used for HPLC analysis.

[0156] F-2. HPLC Analysis HPLC analysis is performed under the following measurement conditions. HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm) Column: ACQUITY UPLC BEH Phenyl (2.1 x 50 mm, 1.7 μm, 130 Å; Waters, P / N 186002884) Column temperature: 80°C Mobile phase A: Aqueous solution containing 0.10% trifluoroacetic acid (TFA) and 15% 2-propanol Mobile phase B: Acetonitrile solution containing 0.075% TFA and 15% 2-propanol Gradient program: 14%-36% (0 min-15 min), 36%-80% (15 min-17 min), 80%-14% (17 min-17.01 min), 14% (17.01 min-25 min) Sample injection volume: 10 μL

[0157] F-3. Data analysis F-3-1 Drug-bound light chains (L0) and heavy chains (H0) of an antibody to which no drug is bound are compared. i) and heavy chain (heavy chain with i drugs bound: H i ) increases in hydrophobicity and retention time in proportion to the number of drugs bound, and are therefore eluted in the order of, for example, L0, L1, H0, H1, H2, and H3. By comparing the retention times with L0 and H0, the detected peak can be assigned to any of L0, L1, H0, H1, H2, and H3. The number of drugs bound can be defined by those skilled in the art, but is preferably L0, L1, H0, H1, H2, or H3. F-3-2 Because the drug linker has UV absorption, the peak area value is corrected according to the number of drug linkers bound using the molar extinction coefficients of the light chain, heavy chain, and drug linker according to the following formula:

[0158]

[0159]

[0160] Here, the molar extinction coefficients (280 nm) of the light and heavy chains of each antibody can be estimated from the amino acid sequences of the light and heavy chains of each antibody using a known calculation method (Protein Science, 1995, vol. 4, pp. 2411-2423). In the case of cMAb1_hIGg1LALA, the molar extinction coefficient of the light chain can be estimated as 32742, and the molar extinction coefficient of the heavy chain can be estimated as 84458, based on the amino acid sequence. Furthermore, the molar extinction coefficient (280 nm) of the drug linker can be determined by reacting each drug linker with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to a succinimide thioether. The wavelength at which absorbance is measured can be determined appropriately by those skilled in the art, but is preferably the wavelength at which the antibody peak can be measured, more preferably 280 nm. F-3-3 Calculate the peak area ratio (%) of each chain to the total corrected peak area according to the following formula.

[0161]

[0162] F-3-4 Calculate the average number of drugs bound per antibody molecule in an antibody-drug conjugate according to the following formula: Average number of drugs bound = (L0 peak area ratio × 0 + L1 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3) / 100 × 2. Note that, in order to ensure the amount of antibody-drug conjugate, multiple antibody-drug conjugates prepared under similar conditions and having similar average drug binding numbers (e.g., approximately ±1) can be mixed to create a new lot. In this case, the average drug binding number will fall within the average drug binding numbers before mixing.

[0163] One specific example of the antibody-drug conjugate of the present invention is a conjugate of the following formula:

[0164] or the following formula:

[0165] Examples of the compound include those having the structure shown below.

[0166] Here, AB represents an anti-CD25 antibody disclosed herein, which is conjugated to a conjugated linker via an antibody-derived sulfhydryl group. Here, n is synonymous with the so-called drug-to-antibody ratio (DAR) and represents the drug-antibody ratio per antibody. That is, it represents the number of drugs conjugated to one antibody molecule, and this is a numerical value specified and expressed as the average value, i.e., the average number of drugs conjugated. AB may be an antigen-binding fragment of the antibody, and in this case, n represents the average number of drug-linker structures conjugated to the antigen-binding fragment of the antibody per antigen-binding fragment of the antibody. In the case of the antibody-drug conjugates represented by [Chemical Formula 16] and [Chemical Formula 17] of the present invention, n may be 2 to 8, preferably 5 to 8, more preferably 7 to 8, and even more preferably 8, as measured by common procedure F.

[0167] An example of the antibody-drug conjugate of the present invention is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, in which the antibody represented by AB in the structure shown in the above formula [Chemical Formula 16] or [Chemical Formula 17] comprises an antibody having a heavy chain and a light chain, or an antigen-binding fragment thereof, selected from the group consisting of (a) to (c) below: (a) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 15; (b) an antibody comprising a light chain consisting of the amino acid sequence of positions 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of the amino acid sequence of positions 20 to 476 of SEQ ID NO: 16; or (c) The antibody according to (a) or (b), wherein the heavy or light chain contains one or more modifications selected from the group consisting of post-translational modifications typified by the addition of an N-linked sugar chain, the addition of an O-linked sugar chain, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of proline residues, pyroglutamylation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus.

[0168] Another example of the antibody-drug conjugate of the present invention is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody represented by AB in the structure shown in the above formula [Chemical Formula 16] or [Chemical Formula 17] comprises an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region selected from the group consisting of the following (a) to (c): (a) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 15; (b) a light chain variable region consisting of the amino acid sequence of positions 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region consisting of the amino acid sequence of positions 20 to 146 of SEQ ID NO: 16; or (c) The antibody according to (a) or (b), wherein the heavy or light chain contains one or more modifications selected from the group consisting of post-translational modifications typified by the addition of an N-linked sugar chain, the addition of an O-linked sugar chain, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of proline residues, pyroglutamylation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus.

[0169] 4. Pharmaceuticals The anti-CD25 antibodies and antigen-binding fragments of the antibodies of the present invention described in the above section "2. Production of anti-CD25 antibodies" and in the Examples bind to CD25 on the cell surface and have internalization activity, and therefore can be used alone or in combination with other drugs as pharmaceuticals, particularly as therapeutic agents for cancer. They can also be used to detect cells expressing CD25. Furthermore, because the anti-CD25 antibodies and antigen-binding fragments of the antibodies of the present invention have internalization activity, they can be used as antibodies for antibody-drug conjugates.

[0170] Among the anti-CD25 antibody-drug conjugates of the present invention described in the above section "3. Anti-CD25 Antibody-Drug Conjugates" and in the Examples, those that use a drug having cytotoxic activity as the drug are conjugates of an anti-CD25 antibody having internalization activity and / or an antigen-binding fragment of the antibody with a drug having cytotoxic activity, and because they exhibit cytotoxic activity against cancer cells expressing Tregs and CD25, they can be used as pharmaceuticals, particularly as therapeutic and / or preventive agents for cancer. These anti-CD25 antibody-drug conjugates can exhibit the effect of directly killing cancer cells expressing CD25. In addition, these anti-CD25 antibody-drug conjugates can exhibit stronger Treg-eliminating ability and / or granzyme (GZMB)-positive CD8-positive T cell-inducing activity than conventional anti-CD25 antibodies and anti-CD25 antibody-drug complexes. Therefore, the anti-CD25 antibody-drug conjugate is expected to exhibit anti-tumor activity due to its Treg-suppressing effect, and when administered to patients with cancer cells expressing CD25 and cancer patients whose tumor immunity is suppressed by Treg, it is expected to achieve excellent anti-tumor effects and safety.

[0171] The anti-CD25 antibody-drug conjugate of the present invention may become a hydrate by absorbing moisture or by adsorbed water being attached thereto when left in the air or when subjected to recrystallization or purification procedures. Such water-containing compounds or pharmaceutically acceptable salts are also encompassed in the present invention as embodiments of the present invention.

[0172] When the anti-CD25 antibody-drug conjugate of the present invention has a basic group such as an amino group, it can form a pharmaceutically acceptable acid addition salt, if desired. Examples of such acid addition salts include hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formate, acetate, trifluoroacetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0173] When the anti-CD25 antibody-drug conjugate of the present invention has an acidic group such as a carboxy group, it can form a pharmaceutically acceptable base addition salt, if desired. Examples of such base addition salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; and organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt.

[0174] The present invention also encompasses anti-CD25 antibodies or antigen-binding fragments thereof, or anti-CD25 antibody-drug conjugates of the present invention, in which one or more atoms constituting the antibody or antibody-drug conjugate are substituted with an isotope of that atom. There are two types of isotopes: radioactive isotopes and stable isotopes. Examples of isotopes include hydrogen isotopes (H and H), carbon isotopes (C, C, and C), nitrogen isotopes (N and N), oxygen isotopes (O, O, and O), and fluorine isotope (F). Compositions containing isotope-labeled anti-CD25 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates, are useful, for example, as therapeutic agents, preventive agents, research reagents, assay reagents, diagnostic agents, and in vivo diagnostic imaging agents. The present invention also encompasses isotope-labeled anti-CD25 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates, as well as mixtures of isotope-labeled anti-CD25 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates in any ratio. Isotopically labeled anti-CD25 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates, can be produced by methods known in the art, for example, by using isotope-labeled starting materials instead of the starting materials in the production methods of the present invention described below.

[0175] In vitro cytocidal activity can be measured, for example, by measuring cell proliferation inhibitory activity. For example, cancer cell lines overexpressing CD25 are cultured, and various concentrations of the anti-CD25 antibody of the present invention or its antigen-binding fragment, or an anti-CD25 antibody-drug conjugate, are added to the culture system, and the inhibitory activity against focus activity, colony formation, and spheroid growth can be measured. Here, by using a non-Hodgkin's lymphoma-derived cancer cell line and an acute myeloid leukemia-derived cancer cell line, the cytostatic activity against non-Hodgkin's lymphoma and acute myeloid leukemia can be examined. Furthermore, by using Tregs isolated from healthy human donors and / or patients or Tregs induced in vitro (iTregs), the cytostatic activity against Tregs can be examined. The therapeutic effect against cancer in vivo using experimental animals can be evaluated, for example, by administering an anti-CD25 antibody or an antigen-binding fragment of the antibody, or an anti-CD25 antibody-drug conjugate of the present invention to a syngenic mouse transplanted with a tumor cell line, and measuring changes in cancer cells (e.g., changes in tumor volume). Fluctuations in immune cells within the tumor (e.g., changes in immune cell count) can also be measured. Furthermore, the anti-CD25 antibody or an antigen-binding fragment of the antibody, or an anti-CD25 antibody-drug conjugate of the present invention can be administered to a NOG mouse (huNOG mouse) transplanted with a tumor cell line and transfused with human umbilical cord blood-derived CD34+ hematopoietic stem cells, and fluctuations in immune cells within the tumor (e.g., changes in immune cell count) can also be measured. These methods allow the therapeutic effect against cancer to be measured.

[0176] The type of cancer to which the anti-CD25 antibody of the present invention or an antigen-binding fragment of the antibody, or the anti-CD25 antibody-drug conjugate of the present invention can be applied is not particularly limited, as long as the cancer expresses CD25 in the cancer cells to be treated or tumor immunity is suppressed by Treg. Examples of such cancers include blood cancers (B-cell lymphoma, T / NK-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, chronic myeloid leukemia), solid tumors (breast cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, brain tumor, glioma, eye tumor, esophageal cancer, gastric cancer, colorectal cancer, colon cancer, liver cancer, pancreatic cancer, renal cancer, kidney cancer, bladder cancer, adrenocortical carcinoma, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, melanoma, and sarcoma). Further examples include cancers identified as having biomarkers such as high microsatellite instability (MSI-H), mismatch repair deficiency (dMMR), or high tumor mutation burden (TMB-H). More preferred examples of cancers include non-small cell lung cancer, head and neck cancer, esophageal cancer, gastric cancer, and melanoma.

[0177] The antibody of the present invention, the antigen-binding fragment of the antibody, or the anti-CD25 antibody-drug conjugate of the present invention (this paragraph also includes a drug released by cleavage of the linker of the anti-CD25 antibody-drug conjugate) has excellent properties in one or more aspects such as binding activity to CD25, IL-2 blocking ability, effector function, ability to remove regulatory T cells, ability to promote the proliferation of FoxP3-negative CD4-positive cells, CD8-positive cells and / or granzyme-positive CD8-positive cells, binding specificity, antigen-binding ability, internalization activity, antitumor activity, solubility, cell membrane permeability, blood concentration, metabolic stability, tissue delivery, bioavailability, in vitro activity, in vivo activity, speed of onset of therapeutic effect, duration of therapeutic effect, physical stability, drug interactions, toxicity, etc., and is useful as a pharmaceutical. In this paragraph, "useful as a pharmaceutical" does not necessarily mean that the above-mentioned various abilities, effects, actions, and properties have excellent properties, such as "present," "high," "large," "large," "positive," etc., making the substance useful as a pharmaceutical; it also includes cases where the above-mentioned abilities, effects, actions, and properties have excellent properties, such as "absent," "low," "small," "few," "negative," etc., making the substance useful as a pharmaceutical (for example, low toxicity, etc.).

[0178] The antibody of the present invention or the antigen-binding fragment of the antibody, or the anti-CD25 antibody-drug conjugate of the present invention can be suitably administered to mammals, more preferably humans.

[0179] Substances used in pharmaceutical compositions containing the antibody of the present invention or the antigen-binding fragment of the antibody, or the anti-CD25 antibody-drug conjugate of the present invention can be appropriately selected from pharmaceutical additives commonly used in this field and others, depending on the dosage and administration concentration.

[0180] The antibody of the present invention or an antigen-binding fragment thereof, or the anti-CD25 antibody-drug conjugate of the present invention can be administered as a pharmaceutical composition containing one or more pharmaceutically compatible ingredients. For example, the pharmaceutical compositions typically include one or more pharmaceutical carriers, such as sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a more typical carrier when the pharmaceutical compositions are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. The formulation will be dependent on the mode of administration.

[0181] Various delivery systems are known and can be used to administer the anti-CD25 antibodies or antigen-binding fragments thereof, or the anti-CD25 antibody-drug conjugates of the present invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be by, for example, infusion or bolus injection. In certain preferred embodiments, the anti-CD25 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates are administered by infusion. Parenteral administration is a preferred route of administration.

[0182] In a representative embodiment, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the medicament may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form (e.g., as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent). Where the medicament is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the medicament is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration.

[0183] The pharmaceutical compositions of the present invention may be pharmaceutical compositions containing only the anti-CD25 antibody or antigen-binding fragment thereof, or the anti-CD25 antibody-drug conjugate of the present invention, or they may be pharmaceutical compositions containing the anti-CD25 antibody or antigen-binding fragment thereof, or the anti-CD25 antibody-drug conjugate and at least one other cancer therapeutic agent. The anti-CD25 antibody or antigen-binding fragment thereof, or the anti-CD25 antibody-drug conjugate of the present invention can also be administered together with other cancer therapeutic agents, thereby enhancing the anti-cancer effect. The other anti-cancer agent used for such a purpose may be administered to an individual simultaneously, separately, or sequentially with the anti-CD25 antibody or antigen-binding fragment thereof, or antibody-drug conjugate of the present invention, or the respective agents may be administered at different administration intervals. Examples of such cancer therapeutic agents include platinum preparations such as cisplatin, oxaliplatin, and carboplatin; taxane compounds such as paclitaxel and docetaxel; metabolic antagonists such as 5-fluorouracil and gemcitabine; antiestrogens such as tamoxifen; aromatase inhibitors such as letrozole; androgen receptor antagonists such as enzalutamide; tyrosine kinase inhibitors such as imatinib, sunitinib, and lenvatinib; CDK4 / 6 inhibitors such as palbociclib; Examples of such antitumor agents include PARP inhibitors including rib and the like, HSP90 inhibitors including pimitespib and the like, MEK inhibitors including cabozantinib and the like, BRAF inhibitors including dabrafenib and the like, KRAS inhibitors including adagrasib and the like, FGFR inhibitors including futibatinib and the like, cytokines including IL-2, IFN-γ, G-CSF and the like, angiogenesis inhibitors including bevacizumab and the like, monoclonal antibodies targeting tumor antigens or marker antigens including trastuzumab, rituximab, panitumumab and the like, antibody-drug conjugates including trastuzumab deruxtecan and the like, immune checkpoint inhibitors including ipilimumab, leratolimab and the like, but are not limited thereto as long as they are drugs having antitumor activity.

[0184] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as a lyophilized preparation, it may be a preparation containing appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0185] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the anti-CD25 antibody or antigen-binding fragment thereof, or anti-CD25 antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exert its medicinal effect at a lower dose, as the affinity of the anti-CD25 antibody or antigen-binding fragment thereof, or antibody-drug conjugate for the antigen, i.e., the dissociation constant (Kd value), of the anti-CD25 antibody or antigen-binding fragment thereof, or antibody-drug conjugate for the antigen increases (i.e., the lower the Kd value). Therefore, when determining the dose of the anti-CD25 antibody or antigen-binding fragment thereof, or antibody-drug conjugate of the present application, the dose can be set based on the affinity between the anti-CD25 antibody or antigen-binding fragment thereof, or antibody-drug conjugate and the antigen. When the anti-CD25 antibody or antigen-binding fragment thereof, or antibody-drug conjugate of the present application is administered to a human, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of once every 1 to 180 days.

[0186] Other cancer therapeutic agents that can be administered together with the anti-CD25 antibody or antigen-binding fragment thereof, or the anti-CD25 antibody-drug conjugate of the present invention are preferably immune checkpoint inhibitors. In one aspect of the present invention, the anti-CD25 antibody or antigen-binding fragment thereof, or the anti-CD25 antibody-drug conjugate of the present invention, and the immune checkpoint inhibitor may be contained as active ingredients in separate formulations and administered simultaneously or at different times, or the antibody-drug conjugate and the immune checkpoint inhibitor may be contained as active ingredients in a single formulation and administered. In one aspect of the present invention, the anti-CD25 antibody or antigen-binding fragment thereof, or the anti-CD25 antibody-drug conjugate of the present invention, and the immune checkpoint inhibitor may each be administered via different routes. Furthermore, the anti-CD25 antibody or antigen-binding fragment thereof, or the anti-CD25 antibody-drug conjugate of the present invention may be contained as active ingredients in a single formulation and administered for the treatment of a disease that is ameliorated by the activity of activating anti-tumor immunity, and administered.

[0187] In the present invention, the term "immune checkpoint inhibitor" refers to a drug that binds to an immune checkpoint molecule or its ligand, inhibits immunosuppressive signal transduction, and thereby releases the inhibitory signal for T cell activation, thereby activating tumor immunity. The immune checkpoint inhibitor is not particularly limited as long as it is an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or the like, whose clinical efficacy and safety have been confirmed, or which has the potential for clinical application due to an action mechanism equivalent to these, but preferred examples include anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody. These can be used alone or in combination of two or more.

[0188] In the present invention, the term "anti-PD-1 antibody" refers to an antibody that specifically binds to PD-1 (programmed cell death-1; CD279; PDCD1), thereby reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between PD-1 and its binding partners, PD-L1 and PD-L2. The anti-PD-1 antibody is not particularly limited as long as its clinical efficacy and safety have been confirmed, but preferred examples include nivolumab (WO 2006 / 121168, etc.), pembrolizumab (WO 2008 / 156712, etc.), sintilimab, spartalizumab, dostarlimab, serplulimab, and tislelizumab. Examples of suitable anti-PD-1 antibodies include pembrolizumab, ...

[0189] In the present invention, the term "anti-PD-L1 antibody" refers to an antibody that specifically binds to PD-L1 (programmed cell death ligand 1; CD274; B7-H1), thereby reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between PD-L1 and its binding partners, PD-1 and B7.1 (CD80). The anti-PD-L1 antibody is not particularly limited as long as its clinical efficacy and safety have been confirmed, but preferred examples include atezolizumab (WO 2010 / 077634, etc.), durvalumab (WO 2011 / 066389, etc.), cosibelimab, adebrelimab, sugemalimab, avelumab (WO 2013 / 079174, etc.), socazolimab, KL-A167, and envafolimab, and more preferred examples include atezolizumab, durvalumab, and avelumab. These can be used alone or in combination of two or more. Furthermore, for the purpose of confirming the combined effect of the antibody-drug conjugate used in the present invention in preclinical studies, commercially available anti-PD-L1 antibodies for research use (e.g., clone 10F.9G2) and the like can also be used.

[0190] In the present invention, the term "anti-CTLA-4 antibody" refers to an antibody that specifically binds to CTLA-4 (cytotoxic T-lymphocyte-associated protein 4; CD152), and thereby has the effect of reducing, inhibiting, and / or interfering with signal transduction resulting from the interaction between CTLA-4 and its binding partners, B7.1 (CD80) and B7.2 (CD86). The anti-CTLA-4 antibody is not particularly limited as long as its clinical efficacy and safety have been confirmed, but preferred examples include ipilimumab (WO 2001 / 014424, etc.), botensilimab, and tremelimumab (WO 2000 / 037504, etc.), and more preferred examples include ipilimumab. These can be used alone or in combination of two or more. Furthermore, for the purpose of confirming the combined effect of the antibody-drug conjugate used in the present invention in preclinical studies, commercially available anti-CTLA-4 antibodies for research use (e.g., clone 9H10) and the like can also be used.

[0191] Immune checkpoint inhibitors include multispecific molecules. Examples of multispecific molecules include, but are not limited to, IgG-type multispecific molecules, multispecific molecules having two or more types of variable regions, such as antibody fragments such as tandem scFvs, single-chain diabodies, diabodies, and triabodies, and antibody fragments linked by covalent or non-covalent bonds. Multispecific molecules may also contain an Fc. Specific examples of multispecific molecules include, but are not limited to, multispecific antibodies (also referred to as multiantibodies) comprising one or more antibodies or antigen-binding fragments thereof selected from the group consisting of an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-PD-L2 antibody or an antigen-binding fragment thereof, and an anti-CTLA-4 antibody or an antigen-binding fragment thereof, and more preferably, bispecific antibodies (also referred to as diabodies) comprising an anti-PD-1 antibody or an antigen-binding fragment thereof or an anti-CTLA4 antibody or an antigen-binding fragment thereof. Such multispecific molecules are not particularly limited as long as their clinical efficacy and safety have been confirmed, but preferred examples include cadonilimab and ivonescimab.

[0192] The present invention includes a method for treating a disease, comprising administering an anti-CD25 antibody or antigen-binding fragment of the antibody, or an anti-CD25 antibody-drug conjugate of the present application to an individual in need of treatment. The present invention includes an anti-CD25 antibody or antigen-binding fragment of the antibody, or an anti-CD25 antibody-drug conjugate of the present application for use in treating a disease. The present invention includes use of an anti-CD25 antibody or antigen-binding fragment of the antibody, or an anti-CD25 antibody-drug conjugate of the present application for the manufacture of a medicament for treating a disease. The anti-CD25 antibody or antigen-binding fragment of the antibody, or the anti-CD25 antibody-drug conjugate of the present application can also be administered together with other cancer therapeutic agents, thereby enhancing the anti-cancer effect.

[0193] In the present invention, "cancer resistant to immune checkpoint inhibitors" refers to a cancer that has been confirmed to be resistant to immune checkpoint inhibitors, or a cancer that can be reasonably recognized or predicted to be resistant to immune checkpoint inhibitors. "Cancer resistant to immune checkpoint inhibitors" is preferably "cancer resistant to existing immune checkpoint inhibitors." In the present invention, "cancer resistant to existing immune checkpoint inhibitors" refers to a cancer that has been confirmed to be resistant to existing immune checkpoint inhibitors, or a cancer that can be reasonably recognized or predicted to be resistant to existing immune checkpoint inhibitors. In the present invention, "existing immune checkpoint inhibitors" refers to immune checkpoint inhibitors that are used clinically. In the present invention, "resistance" refers to the property of being unresponsive to treatment with anticancer drugs, and can also be expressed as "refractory," "unresponsive," or "refractory." Furthermore, because unresponsiveness makes it impossible to prevent tumor growth, it can also be expressed as "intolerant." Furthermore, in the present invention, "resistance" includes cases where, after treatment with an anticancer drug, the patient's cancer is low sensitive to treatment with the anticancer drug, cancer cells do not disappear or shrink, a complete response (CR) or partial response (PR) is not obtained, and / or the cancer progresses early (for example, within 6 months or less in the case of ovarian cancer). The "resistance" in the present invention may be "resistance acquired by treatment with an existing immune checkpoint inhibitor" or "intrinsic resistance not due to treatment with an existing immune checkpoint inhibitor."

[0194] The present invention will be specifically described using the following examples, but the present invention is not limited thereto. Furthermore, these examples should not be construed as limiting in any way. In the following examples, unless otherwise specified, each genetic manipulation procedure was performed according to the method described in "Molecular Cloning" (Sambrook, J., Fritsch, E.F., and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989) or other experimental manuals used by those skilled in the art. Alternatively, when using commercially available reagents or kits, the procedures were performed according to the instructions provided with the commercially available products. Furthermore, reagents, solvents, and starting materials not specifically described herein are readily available from commercial sources.

[0195] (Example 1) Preparation of Expression Vector 1)-1 Human CD25 Expression Vector The human CD25 expression vector (hereinafter referred to as "pCMV3-hCD25") used was CD25 / IL2R alpha cDNA ORF Clone, Human, untagged (Sino Biological Co., Ltd.). The polynucleotide sequence of the human CD25 gene cloned into this vector is shown in SEQ ID NO: 42 in the Sequence Listing, and the amino acid sequence of human CD25 is shown in SEQ ID NO: 17 in the Sequence Listing.

[0196] 1)-2 Mouse CD25 Expression Vector The mouse CD25 expression vector (hereinafter referred to as "pCMV3-mCD25") used was CD25 / IL2R alpha cDNA ORF Clone, Mouse, untagged (Sino Biological Co., Ltd.). The polynucleotide sequence of the ORF portion of the mouse CD25 gene cloned into this vector is shown in SEQ ID NO: 43 of the Sequence Listing, and the amino acid sequence of mouse CD25 is shown in SEQ ID NO: 44 of the Sequence Listing.

[0197] 1)-3 Preparation of Cynomolgus Macaque CD25 Expression Vector Using cDNA prepared from mRNA prepared from cynomolgus macaque PBMCs using an oligo-dT primer as a template, PCR was performed using the following primer set: Primer 1F: 5'-ggtaccgccatggatccatacctgctcatgtggg-3' (SEQ ID NO: 45) and Primer 1R: 5'-ctcgagctagattgttcttctattcttcctctg-3' (SEQ ID NO: 46). The resulting PCR product was inserted into the pCR4 Blunt-TOPO vector using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Thermo Fisher Scientific) to generate a vector encoding cynomolgus macaque CD25. A cloning vector containing the cDNA was constructed (the step of introducing the mutation is omitted). Cynomolgus monkey CD25 cDNA was excised from the cloning vector with Kpn I and Xho I, and inserted into the pCI vector between the Kpn I and Xho I sites to construct a cynomolgus monkey CD25 expression vector (hereinafter referred to as "pCI-cCD25"). The polynucleotide sequence of the ORF portion of the cynomolgus monkey CD25 gene cloned into this vector is shown in SEQ ID NO: 47 of the Sequence Listing, and the amino acid sequence of cynomolgus monkey CD25 is shown in SEQ ID NO: 48 of the Sequence Listing.

[0198] (Example 2) Production and screening of monoclonal antibodies 2)-1 Immunization Female WKY / Izm rats (Japan SLC) were used for immunization. Recombinant Human IL-2 Receptor Subunit α / IL-2RA / CD25(C-6His) (Novoprotein Scientific) or Recombinant Mouse IL-2R α / IL-2RA / CD25(C-6His) (Novoprotein Scientific) and Freund's Complete Adjuvant (Wako Pure Chemical Industries) were administered to the base of the tail of the rats, and the lymph nodes and spleens of the rats were collected and used to produce hybridomas.

[0199] 2)-2 Preparation of Hybridomas Lymph node cells or spleen cells were electrofused with mouse myeloma SP2 / 0-ag14 cells (ATCC: CRL-1581) using an LF301-Cell Fusion Unit (BEX), and the resulting mixture was diluted in ClonaCell-HY Selection Medium D (StemCell Technologies) and cultured. Monoclonal hybridomas were prepared by recovering the hybridoma colonies that emerged. Each recovered hybridoma colony was cultured, and anti-CD25 antibody-producing hybridomas were screened using the resulting hybridoma culture supernatant or antibodies purified from the hybridoma culture.

[0200] 2)-3 Antibody Screening 2)-3-1 Measurement of Antigen Binding 2)-3-1-1 Preparation of Antigen Gene-Expressing Cells CHO-K1 cells subcultured in 10% FBS-containing Ham's F-12K (Kaighn's) medium (Thermo Fisher Scientific) were cultured at a density of 4.5 × 10 in 10% FBS-containing Ham's F-12K (Kaighn's) medium. 5 The cells were prepared to a concentration of 100 μg / mL. 15 μg of pCMV3-hCD25, pCMV3-mCD25, or pCI-cCD25 was transfected into 10 mL of the cell suspension using FuGene 6 Transfection Reagent (Promega), and 100 μL of each was seeded into each well of a Collagen Type I-coated 96-well plate (AGC TECHNO GLASS) and cultured overnight at 37°C in 5% CO2. The resulting transfected cells were used in Cell-ELISA while still in an adherent state.

[0201] 2)-3-1-2 Cell-ELISA After removing the supernatant from the expression vector-transfected CHO-K1 cells prepared in 2)-3-1-1, 50 μL of hybridoma culture supernatant or purified antibody was added to each of the CHO-K1 cells transfected with pCMV3-hCD25, pCMV3-mCD25, or pCI-cCD25, and the mixture was incubated for 1 hour at 4° C. After washing the cells in the wells with PBS containing 5% FBS, 50 μL of ECL anti-Rat IgG, HRP-linked (GE Healthcare Bio-Sciences) diluted 500-fold with PBS containing 5% FBS was added, and the mixture was incubated for 1 hour at 4° C. After washing the cells in the wells with 5% FBS-containing PBS, 100 μL of TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories) was added. The color reaction was allowed to proceed for 10 minutes at room temperature with stirring in the dark. The color reaction was stopped by adding 100 μL / well of STOP Solution (Kirkegaard & Perry Laboratories). The absorbance at 450 nm was then measured using a plate reader (SpectraMax: Molecular Devices) (in the figures, A450 indicates the absorbance at 450 nm). To select hybridomas that produce antibodies that specifically bind to CD25 expressed on the cell membrane surface, hybridomas that produce culture supernatants whose absorbance increases depending on the concentration of added antibody were selected as being positive for anti-CD25 antibody production.

[0202] 2)-3-2 Purification of anti-CD25 antibodies Clones that showed binding to human and monkey CD25, and clones that showed binding to mouse CD25 were selected from the rat anti-CD25 antibody-producing hybridomas selected in 2)-3-1, and antibodies were purified from the hybridoma culture supernatant using a Spin column-based Antibody Purification Kit (COSMOBIO). 500 μL of binding buffer was added to a monolithic silica-based spin column, and the mixture was centrifuged at 6,000 rpm, 30 seconds, and 4°C using a centrifuge MX-160 (TOMY), and the flow-through was removed. 500 μL of hybridoma culture supernatant was added, and the mixture was centrifuged at 6,000 rpm, 30 seconds, and 4°C using a centrifuge MX-160 (TOMY Corporation), and the flow-through was removed. 500 μL of PBS (Wako Pure Chemical Industries, Ltd.) was added, and the mixture was centrifuged at 6,000 rpm, 30 seconds, and 4°C using a centrifuge MX-160 (TOMY Corporation), and the flow-through was removed. 20 μL of neutralization buffer was added to a 1.5 mL microtube, and a spin column was placed in the tube. 200 μL of elution buffer was added to the spin column, and the mixture was centrifuged at 6,000 rpm, 30 seconds, and 4°C using a centrifuge MX-160 (TOMY Corporation) to obtain purified antibodies.

[0203] 2)-3-3 Measurement of antibody concentration 2)-3-3-1 Identification of antibody isotype Clones that showed binding to human and monkey CD25, and clones that showed binding to mouse CD25 were selected from the rat anti-CD25 antibody-producing hybridomas selected in 2)-3-1, and the isotype of each antibody was identified. The heavy chain subclass and light chain type of the antibody were identified using Rat Ig Isotyping Ready-Set-Go (Thermo Fischer Scientific).

[0204] 100 μL / well of Capture Antibody, diluted 250-fold with PBS, was added to a MaxiSorp flat-bottom 96-well plate (Thermo Fischer Scientific) and allowed to stand at 4°C for 3 days for immobilization. After washing twice with Wash Buffer (20x Wash Buffer from BD OptEIA Set A (BD Biosciences Pharmingen) diluted 20-fold with MilliQ), the plate was blocked with Blocking Buffer (PBS containing 0.1% Tween-20 (BIO-RAD) and 1% BSA (Wako Pure Chemical Industries)). After washing twice with wash buffer, 50 μL / well of assay buffer (PBS containing 0.05% Tween-20 (BIO-RAD) and 0.5% BSA (Wako Pure Chemical Industries)) was added. Furthermore, 50 μL / well of culture supernatant diluted with PBS was added, and the mixture was incubated at room temperature for 2 hours. After washing four times with wash buffer, a detection antibody (diluted 500-fold with assay buffer) was added. After washing four times with Wash Buffer, 100 μL / well of TMB Substrate Solution (a mixture of equal amounts of Substrate Reagents A and B from BD OptEIA Set A (BD Biosciences Pharmingen)) was added, and the plate was incubated for 10 minutes at room temperature with stirring in the dark. 100 μL / well of Stop Solution (BD OptEIA Set A (BD Biosciences Pharmingen)) was added, and the absorbance at 450 nm and 570 nm was measured using a plate reader (Spectra Max, Molecular Devices).

[0205] 2)-3-3-2 Concentration Measurement Antibody concentrations were measured using a Rat IgG ELISA Quantitation Set (Bethyl Laboratories). Standards were set for each clone based on the isotype information determined in 2)-3-3-1, and Rat IgG1 Isotype Control (R&D Systems), Rat IgG2A Isotype Control (R&D Systems), or Rat IgG2B Isotype Control (R&D Systems) were used.

[0206] 2)-3-4 Effect on IL-2-dependent cell proliferation using human pan T cells 2)-3-4-1 Preparation of human pan T cells and serum starvation Frozen human PBMCs (Cellular Technology Limited) were thawed according to the C.T.L. protocol. Then, pan T cells were isolated from the PBMCs using a Pan T Cell Isolation Kit (Miltenyi BIOTEC). The isolated pan T cells were cultured at a density of 5.0 × 10 in RPMI (Life Technologies) medium supplemented with 10% FBS (Hyclone, Global Life Science Technologies Japan Co., Ltd.) and 1% Penicillin-Streptomycin (Life Technologies). 5 After suspending the cells to 1.0 × 10 cells / mL, PHA (Sigma-Aldrich) was added to the cell suspension at 3 μg / mL and the cells were cultured for 3 days at 37°C and 5% CO. After culturing, the collected cells were washed twice with PBS, counted, and then cultured at 2.0 × 10 cells / mL in RPMI medium supplemented with 1% FBS and 1% Penicillin-Streptomycin. 5 The cells were suspended at 1000 cells / mL and added to a culture flask. The cells were further cultured at 37°C under 5% CO for 24 hours to perform serum starvation.

[0207] 2)-3-4-2 Preparation and addition of antibody solutions The anti-human CD25 antibodies basiliximab (Novartis Pharma) and 7G7B6 (Bio X Cell), as well as the control antibody InVivoMab Rat IgG2a Isotype control (rat IgG2a) (Bio X Cell), were diluted with ClonaCell-HY Cloning Medium E (Medium E) (STEM CELL) to 10 μg / mL, 25 μg / mL, or 50 μg / mL before use. 5 μL of each concentration of basiliximab, 7G7B6, control antibody, or hybridoma culture supernatant was added to each well of a 96-well plate, and 15 μL of RPMI medium supplemented with 1% FBS and 1% Penicillin-Streptomycin was further added to each well.

[0208] 2)-3-4-3 Addition of human Pan T cells and IL-2 solution Pan T cells were collected after serum starvation and added to a 1.25 x 10 5 RPMI medium supplemented with 1% FBS and 1% penicillin-streptomycin was added to the wells to achieve a concentration of 100 μg / mL. The cell suspension was added at 40 μL / well to the wells to which the solution in 2)-3-5-2 had been added. The wells were then incubated for 15 minutes at 5% CO2 and 37°C. Recombinant human IL-2 (PeproTech) was dissolved in 50 μL of 0.1 M acetic acid and then diluted with 0.45 mL of PBS containing 0.1% BSA to prepare a 100 μg / mL solution. This was then diluted to 3.75 ng / mL with RPMI medium containing 1% FBS and 1% penicillin-streptomycin. 40 μL / well of the IL-2-added wells was added. To the wells of the IL-2 non-added group, 40 μL / well of RPMI medium containing 1% FBS / 1% Penicillin-Streptomycin was added, followed by incubation under conditions of 5% CO and 37°C for 48 hours.

[0209] 2)-3-4-4 Measurement of cell proliferation After incubation, the plate was centrifuged at 300 × g for 5 minutes, and 50 μL of supernatant was removed from each well. 50 μL of Celltiter-Glo Reagent from the CellTiter-Glo Luminescent Cell Viability Assay (Promega) kit was added to the wells and shaken for 2 minutes on a plate shaker. Fifty μL of the medium per well was transferred to an OptiPlate-96 (PerkinElmer), and the luminescence intensity (relative light unit, RLU) was measured using a plate reader (Ensight: PerkinElmer) and used as an index of cell number.

[0210] 2)-3-5 Effect on phosphorylated STAT5 (pSTAT5) increased by IL-2 stimulation in human pan T cells 2)-3-5-1 Preparation of human pan T cells and serum starvation Frozen human PBMCs were thawed according to the C.T.L. protocol. Pan T cells were then isolated from the PBMCs using a Pan T Cell Isolation Kit (Miltenyi BIOTEC). The isolated pan T cells were cultured at 5.0 x 10 in RPMI (Life Technologies) medium supplemented with 10% FBS (Hyclone, Global Life Science Technologies Japan) and 1% Penicillin-Streptomycin (Life Technologies). 5 After suspending the cells to 1.0 × 10 cells / mL, PHA (Sigma-Aldrich) was added to the cell suspension at 3 μg / mL and the cells were cultured for 3 days at 37°C and 5% CO. After culturing, the collected cells were washed twice with PBS, counted, and then cultured at 2.0 × 10 cells / mL in RPMI medium supplemented with 1% FBS and 1% Penicillin-Streptomycin. 5 The cells were suspended at 1000 cells / mL and added to a culture flask. The cells were further cultured at 37°C under 5% CO for 24 hours to perform serum starvation.

[0211] 2)-3-5-2 Preparation and addition of antibody solutions The anti-human CD25 antibodies basiliximab (Novartis Pharma) and 7G7B6 (Bio X Cell), or the control antibody InVivoMab Rat IgG2a Isotype Control (rat IgG2a) (Bio X Cell) were diluted to 10 μg / mL, 25 μg / mL, or 50 μg / mL in ClonaCell-HY Cloning Medium E (Medium E) (STEM CELL). 20 μL of each concentration of basiliximab, 7G7B6, control antibody, or hybridoma culture supernatant was added to each well of a 96-well plate.

[0212] 2)-3-5-3 Addition of human pan T cells and IL-2 solution The pan T cells were collected after serum starvation and added to a 1.25 x 10 5 RPMI medium supplemented with 1% FBS and 1% penicillin-streptomycin was added to the wells to obtain a concentration of 100 μg / mL cells. The cell suspension was added at 40 μL / well to the wells to which the solution in 2)-3-5-2 had been added. The cells were then incubated for 15 minutes at 37°C in 5% CO2. Recombinant human IL-2 (PeproTech) was dissolved in 50 μL of 0.1 M acetic acid and diluted with 0.45 mL of PBS containing 0.1% BSA to prepare a 100 μg / mL solution. This solution was then diluted to 3.75 ng / mL with RPMI medium containing 1% FBS and 1% penicillin-streptomycin, and added at 40 μL / well to the wells containing IL-2. To the wells of the IL-2 non-added group, 40 μL / well of RPMI medium containing 1% FBS / 1% Penicillin-Streptomycin was added, followed by incubation for 15 minutes under conditions of 5% CO and 37°C.

[0213] 2)-3-5-4 pSTAT5 Measurement The 5x lysis buffer included with the AlphaLISA SureFire Ultra phosho-STAT5 (Tyr694 / 699) kit (PerkinElmer) was diluted with HO to a 1x lysis buffer. After 15 minutes of incubation, the plate was centrifuged at 300 x g for 5 minutes, and the supernatant from each well was removed by suction. 50 μL of 1x lysis buffer was then added to each well. The plate was then agitated on a plate shaker for 10 minutes to prepare a cell lysate. The cell lysate from each well was added to a 384-well AlphaLISA SW plate (PerkinElmer) at 5 μL / well, and 2.5 μL / well of Acceptor Mix was added. The plate was then shaken for 1 hour on a plate shaker. After overnight incubation at 4°C in the dark, 2.5 μL / well of Donor Mix was added, and the plate was shaken for 2 minutes on a shaker. The plate was then further incubated at room temperature in the dark for 2 hours. The Alpha signal was then measured using a plate reader (Ensight: PerkinElmer).

[0214] 2)-3-6 Measurement of cellular internalization activity using human pan T cells (Fab-ZAP method) 2)-3-6-1 Immobilization of anti-human CD3 antibody and anti-human CD28 antibody on 6-well plate wells PBS containing 20 μg / mL anti-human CD3 antibody (EXBIO Praha) and anti-human CD28 antibody (BECTON DICKINSON) was added to each well of a 6-well plate (Corning) at 2 mL / well, and the plate was mixed using a plate mixer and incubated at 37°C for 2 hours. After incubation, the supernatant was removed, and the wells were washed once with PBS before use.

[0215] 2)-3-6-2 Isolation of pan T cells from frozen human PBMCs and culture of pan T cells Frozen PBMCs (Cellular Technology) were thawed in a 37°C water bath and added to RPMI (Life Technologies) medium containing 10% FBS, 1% Penicillin-streptomycin, and Anti-Aggregate Wash (1 / 20 added), followed by centrifugation at 310 x g for 10 minutes. The supernatant was removed and the cells were resuspended in fresh medium. Pan T cells were isolated from the resuspended PBMCs using a Pan T Cell Isolation Kit (Miltenyi BIOTEC). The isolated Pan T cells were cultured at a density of 1.07 × 10 in IMDM (Thermo Fisher Scientific) medium containing 10% CTS Immune Cell SR (Thermo Fisher Scientific). 6 A cell suspension of 1000 cells / mL was prepared. The cell suspension was seeded at 4 mL / well into the 6-well plate prepared in 2)-3-5-1 and cultured for 4 days under conditions of 5% CO2 and 37°C. On the second day of culture, IMDM medium containing 1.5 ng / mL Recombinant Human IL-2 (PeproTech, Inc.) and 10% CTS Immune Cell SR was added at 2 mL / well.

[0216] 2)-3-6-3 ZAP Assay Fab-ZAP rat (ADVANCED TARGETING SYSTEMS) was diluted with RPMI medium supplemented with 1% penicillin-streptomycin to prepare Fab-ZAP rat solutions at 40 μg / mL, 8 μg / mL, 1.6 μg / mL, and 0.32 μg / mL. Control antibody rat IgG2a and rat hybridoma culture supernatant or purified antibody were diluted with RPMI medium supplemented with 1% penicillin-streptomycin to give rat IgG concentrations of 3000 ng / mL, 600 ng / mL, 120 ng / mL, and 24 ng / mL. Each solution was added to a 96-well plate so that the concentration ratio of Fab-ZAP rat to anti-CD25 antibody in the well was 2.67:1. RPMI medium supplemented with 1% penicillin-streptomycin was added to wells lacking Fab-ZAP or anti-CD25 antibody (antibody concentration: 0 ng / mL). To obtain results in the absence of Fab-ZAP rat, 96-well plates were also prepared to which rat hybridoma culture supernatant or purified antibody solution alone was added. Pan T cells cultured in a 6-well plate were collected and centrifuged at 300 × g for 10 minutes. After removing the supernatant, 2.5 × 10 cells were added. 5 RPMI medium supplemented with 1% penicillin-streptomycin was added to give a concentration of 100 cells / mL. This was added to a 96-well plate at 40 μL / well. After culturing for 3 days under conditions of 5% CO2 and 37°C, the cell number was measured using a CellTiter-Glo luminescent Cell Viability Assay (Promega). The luminescence intensity of the well with an antibody concentration of 0 ng / mL was set as 100%, and the luminescence intensity percentage of each well was calculated.

[0217] 2)-3-7 Cross-competition assay between anti-human CD25 antibody and M-A251 2)-3-7-1 Preparation of human CD25-expressing CHO-1 cells CHO-K1 cells subcultured in Ham's F-12K (Kaighn's) medium (Thermo Fisher Scientific) containing 10% FBS and 1% Penicillin-streptomycin were cultured at a concentration of 4.5 × 10 in Ham's F-12K (Kaighn's) medium containing 10% FBS and 1% Penicillin-streptomycin. 5 The cell suspension was adjusted to a concentration of 100 μg of pCMV3-hCD25 cells / mL. 15 μg of pCMV3-hCD25 was transfected into 10 mL of the cell suspension using FuGene 6 Transfection Reagent (Promega), and 100 μL of the solution was seeded into each well of a Collagen Type I-coated 96-well plate (AGC TECHNO GLASS) and cultured overnight to 4 days at 37°C in 5% CO2. The resulting transfected cells were used in the assay while still adherent.

[0218] Biotin anti-human CD25 antibody (clone; M-A251, Biolegend) (0.5 mg / mL) was diluted to 0.1 mg / mL with 5% FBS-containing PBS. Basiliximab (Novartis Pharma), M-A251 (BD Biosciences), the antibody used, or IgG1,κ human myeloma plasma-derived antibody (hIgG1, Merck KGaA) used as a control antibody were diluted to 1 μg / mL, 3 μg / mL, or 10 μg / mL with 5% FBS-containing PBS and then mixed 1:1 with the biotinylated M-A251 antibody solution (mixture 1). For the antibody-free group (0 μg / mL), 5% FBS-containing PBS and biotinylated M-A251 antibody solution were mixed at a 1:1 ratio (mixture solution 2). HRP Streptavidin (Biolegend) 0.5 mg / mL was diluted 1000-fold with 5% FBS-containing PBS. TMB Peroxidase Substrate and Peroxidase Substrate Solution B from the TMB Microwell Peroxidase Substrate (2-Component System) (Sera Care Life Sciences) kit were mixed at a 1:1 ratio to prepare the substrate solution. The culture supernatant was removed from the wells of a 96-well plate, and the wells were washed once with 0.2 mL of 5% FBS-containing PBS. After removing the 5% FBS-containing PBS from each well, mixed solutions 1 and 2 were added at 50 μL / well and incubated at 4°C for 1 hour. After removing the mixed solution from the wells, each well was washed three times with 0.2 mL of 5% FBS-containing PBS. After removing the 5% FBS-containing PBS from each well, substrate solution was added to each well at 0.1 mL / well. The color reaction was carried out for 5 minutes at room temperature under light-shielded stirring, and the color reaction was stopped by adding 0.1 mL / well of STOP Solution (Sera Care Life Sciences, Inc.). The absorbance at 450 nm was then measured using a plate reader (SpectraMax: Molecular Devices).

[0219] 2)-3-8 Preparation of anti-mouse CD25 antibodies, PC61_hIgG1LALA and PC61G_hIgG1LALA The anti-mouse CD25 antibodies, PC61_hIgG1LALA and PC61G_hIgG1LALA, were prepared according to known methods described in the literature (Huss et al., 2016. Immunology. 148(3):276-286) and patent (WO2017 / 174331).

[0220] 2)-3-9 Effect of IL-2 stimulation on phosphorylated STAT5 (pSTAT5) in mouse T cell line CTLL-2 cells 2)-3-9-1 Culture of CTLL-2 cells and serum starvation The CD25-positive mouse tumor cell line CTLL-2 was cultured in RPMI1640 (Thermo Fisher Scientific) medium containing 1 mM Sodium Pyruvate (Thermo Fisher Scientific), 1x MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific), 10% T-STIM with ConA at 37°C, 5% CO₂. The cultured CTLL-2 cells were collected, centrifuged at 300 × g for 5 minutes, and then diluted to 2.0 × 10 cells in RPMI 1640 medium. 6 The cell suspension was added to a 96-well plate at 50 μL / well and cultured at 37°C and 5% CO for 5 hours.

[0221] 2)-3-9-2 Preparation and addition of antibody solution and mouse IL-2 solution. Purified antibody, PC61G_hIgGLALA, an anti-mouse CD25 antibody that inhibits the binding of IL-2 to mouse CD25, or rat IgG2a, a control antibody, were diluted to 20 μg / mL in RPMI 1640 medium. After adding these antibody solutions to the wells containing the cells, the plate was incubated at 37°C and 5% CO for 15 minutes.

[0222] 2)-3-9-3 pSTAT5 Measurement This was carried out according to the method in 2)-3-5-4.

[0223] 2)-3-10 Measurement of Cellular Internalization Activity Using Mouse Pan T Cells (Fab-ZAP Method) 2)-3-10-1 Immobilization of Anti-Mouse CD3 Antibody and Anti-Mouse CD28 Antibody to Wells 1 mg / mL solutions of Ultra-LEAF Purified anti-mouse CD3 Antibody (anti-mouse CD3 antibody: Biolegend) and LEAF Purified anti-mouse CD28 Antibody (anti-mouse CD28 antibody: Biolegend) were mixed and diluted with PBS to prepare a 10 μg / mL mixed solution. 2 mL of this mixed solution was added to the wells of a 6-well plate, mixed using a plate mixer, and then incubated at 37°C for 2 hours. The plate was washed three times with PBS and used in the experiment.

[0224] 2)-3-10-2 Preparation and activation of mouse pan T cells from mouse splenocytes Mouse pan T cells were isolated and recovered from splenocytes prepared from Balb / c mice using Pan T Cell Isolation Kit II, mouse (Miltenyi Biotec). Eight milliliters of RPMI medium containing 0.5% penicillin / streptomycin, 5 mM HEPES, 1 mM sodium pyruvate, 1x MEM-NEAA, and 10% FBS was added to the isolated and recovered mouse pan T cells, and a total of 1.31 x 10 6 A cell suspension of 50 μM 2-mercaptoethanol and 10 ng / mL mouse IL-2 was added to the cell suspension, and 4 mL of the suspension was seeded into wells of a 6-well plate solid-phased with 2)-3-10-1 and incubated at 37°C for 3 days.

[0225] 2)-3-10-3 ZAP Assay Fab-ZAP rat (ADVANCED TARGETING SYSTEMS) was diluted with RPMI medium supplemented with 1% Penicillin-Streptomycin to prepare 40 μg / mL, 8 μg / mL, 1.6 μg / mL, and 0.32 μg / mL Fab-ZAP rat solutions. Furthermore, the control antibody rat IgG2a, Purified NA / LE Rat Anti-Mouse CD25 (PC61 antibody) (Becton Dickinson), and rat hybridoma culture supernatant or purified antibody were diluted with RPMI medium supplemented with 1% Penicillin-Streptomycin to give rat IgG concentrations of 1500 ng / mL, 300 ng / mL, 60 ng / mL, and 12 ng / mL. Each solution was added to a 96-well plate so that the concentration ratio of Fab-ZAP rat to anti-CD25 antibody in the well was 0.53:1. RPMI medium supplemented with 1% penicillin-streptomycin was added to wells lacking Fab-ZAP rat and anti-CD25 antibody (antibody concentration: 0 ng / mL). To obtain results in the absence of Fab-ZAP rat, 96-well plates were also prepared to which rat hybridoma culture supernatant or purified antibody solution alone was added. Pan T cells cultured in a 6-well plate were collected and centrifuged at 310 × g for 10 minutes. After removing the supernatant, 2.5 × 10 cells were added to the wells. 5 RPMI medium supplemented with 1% penicillin-streptomycin was added to give a concentration of 100 cells / mL. This was added to a 96-well plate at 40 μL / well. After culturing for 3 days under conditions of 5% CO2 and 37°C, the cell number was measured using a CellTiter-Glo luminescent Cell Viability Assay (Promega). The luminescence intensity of the well with an antibody concentration of 0 ng / mL was set as 100%, and the luminescence intensity percentage of each well was calculated.

[0226] Approximately 2,000 clones prepared in 2)-2 were evaluated, and selected MAb1 and MAb2 were further analyzed.

[0227] (Example 3) Properties of MAb1 3)-1 Antigen binding activity This was carried out according to the method described in 2)-3-1. MAb1 was a purified antibody, and was added to the wells so that the final antibody concentrations were 0.02 μg / mL, 0.16 μg / mL, 1.25 μg / mL, and 10 μg / mL. 5% FBS / PBS was added to wells without antibody (0 μg / mL). As a result, MAb1 bound to CHO-K1 cells transfected with pCMV3-hCD25 or pCI-cCD25 in an antibody concentration-dependent manner, but did not bind to CHO-K1 cells transfected with pCMV3-mCD25 (Figure 1). This demonstrated that MAb1 binds to human CD25 and cynomolgus monkey CD25.

[0228] 3)-2 Effect on IL-2-dependent human pan T cell proliferation. This study was carried out according to the method described in 2)-3-4. MAb1 was added to hybridoma culture supernatant at a final antibody concentration of 11.1 μg / mL (n = 2). The results showed that, when the number of T cells in the group treated with IL-2 and the control antibody rat IgG2a (2.5 μg / mL) was taken as 100%, the number of T cells in the group treated with IL-2 and the IL-2 blocking antibody basiliximab (2.5 μg / mL) was 52%, the number of T cells in the group treated with IL-2 and the IL-2 non-blocking antibody 7G7B6 (2.5 μg / mL) was 94%, and the number of T cells in the group treated with IL-2 and MAb1 was 109% (Figure 2). The number of T cells increased by the addition of IL-2 in the MAb1-added group was similar to that in the control antibody rat IgG2a-added group, whereas the number of T cells increased by the addition of IL-2 in the IL-2-blocking antibody basiliximab-added group was reduced compared to the rat IgG2a-added group, but not in the IL-2 non-blocking antibody 7G7B6-added group. These findings demonstrate that MAb1 is an IL-2 non-blocking antibody that does not affect the promotion of IL-2-dependent human T cell proliferation.

[0229] 3)-3 Effect on phosphorylated STAT5 (pSTAT5) increased by IL-2 stimulation 3)-3-1 Immobilization of anti-human CD3 antibody to 6-well plate wells PBS containing 10 μg / mL anti-human CD3 antibody (EXBIO Praha) was added to each well of a 6-well plate (Corning) at 2 mL / well, and the plate was mixed using a plate mixer and incubated at 4°C. After incubation, the supernatant was removed, and the wells were washed three times with PBS before use.

[0230] 3)-3-2 Preparation of human PBMCs and serum starvation. Frozen human PBMCs were thawed according to the C.T.L. protocol. The thawed PBMCs were suspended in IMDM (Thermo Fisher Scientific) medium containing 10% CTS Immune Cell SR (Thermo Fisher Scientific) and seeded at 4 mL / well into the 6-well plate prepared in 3)-3-1. The plate was incubated at 37°C in 5% CO2 for 5 days.

[0231] 3)-3-3 Preparation and addition of antibody solution A purified antibody was used for MAb1, and it was added so that the final antibody concentration was 20 μg / mL (n=3). In addition, rat IgG2a was used as a control antibody, basiliximab as an IL-2 blocking antibody, and 7G7B6 as an IL-2 non-blocking antibody, and they were added so that the final concentration was 20 μg / mL (n=3).

[0232] 3)-3-4 Addition of human PBMC and IL-2 solution PBMC were collected after serum starvation and diluted to 2 × 10 6The antibody solution was prepared in IMDM medium (Thermo Fisher Scientific) to a concentration of 100 μg / mL. The cell suspension was added at 50 μL / well to the plate containing the antibody solution in 7)-3-2. The plate was then incubated for 15 minutes at 37°C with 5% CO2. Recombinant human IL-2 (PeproTech) was dissolved in 50 μL of 0.1 M acetic acid and diluted with 0.45 mL of PBS containing 0.1% BSA to prepare a 100 μg / mL solution. This solution was then diluted to 4 ng / mL with IMDM medium (Thermo Fisher Scientific) and added to the IL-2-treated wells at 25 μL / well. Furthermore, IMDM medium (Thermo Fisher Scientific) was added to wells in the IL-2 non-added group at 25 μL / well, followed by incubation for 15 minutes under conditions of 5% CO and 37°C.

[0233] 3)-3-5 pSTAT5 Measurement The 5x lysis buffer included with the AlphaLISA SureFire Ultra phosho-STAT5 (Tyr694 / 699) kit (PerkinElmer) was diluted with HO to a 1x lysis buffer. After 15 minutes of incubation, the plate was centrifuged at 377 g for 5 minutes, and the supernatant from each well was aspirated. 50 μL of 1x lysis buffer was added to each well. The plate was agitated on a plate shaker for 10 minutes to prepare a cell lysate. The cell lysate from each well was added to a 384-well AlphaLISA SW plate (PerkinElmer) at 5 μL / well, and after adding 4 μL / well of Acceptor Mix, the plate was shaken for 2 minutes on a plate shaker. This was then incubated for 1 hour in the dark. 2.5 μL / well of Donor Mix was added, and the plate was shaken for 2 minutes on a plate shaker, and then further incubated overnight in the dark. The Alpha signal was then measured using a plate reader (Ensight: PerkinElmer).

[0234] As a result, when the pSTAT5 level in the group treated with IL-2 and the control antibody rat IgG2a was taken as 100%, the level in the group treated with IL-2 and the IL-2 blocking antibody basiliximab was 1%, the level in the group treated with IL-2 and the IL-2 nonblocking antibody 7G7B6 was 103%, and the level in the group treated with IL-2 and MAb1 was 108% (Fig. 3). The pSTAT5 level increased with IL-2, but was reduced by the addition of basiliximab, but not by the addition of 7G7B6, compared with the group treated with rat IgG2a, demonstrating that MAb1 is an IL-2 nonblocking antibody that does not inhibit IL-2 signaling in T cells.

[0235] 3)-4 Measurement of cellular internalization activity using human pan T cells (Fab-ZAP method) This was performed according to the method described in 2)-3-6. MAb1 was a purified antibody, and was added to each well at concentrations of 1500 ng / mL, 300 ng / mL, 60 ng / mL, and 12 ng / mL. When the cell viability of the control antibody at each concentration was defined as 100%, the cell viability of MAb1 was 58% in the 12 ng / mL group, 53% in the 60 ng / mL group, 44% in the 300 ng / mL group, and 47% in the 1500 ng / mL group (n = 3) (Figure 4). In the Fab-ZAP-free group, no change in viability was observed in the control antibody rat IgG2a or MAb1 groups at any concentration. This demonstrates that MAb1 is an antibody internalized by CD25-positive cells.

[0236] 3)-5 Cross-competitive assay between anti-human CD25 antibodies and M-A251 This was performed according to the method described in 2)-3-7. As a result, the binding of biotinylated M-A251 to human CD25-expressing CHO-1 cells was inhibited by the addition of M-A251, but not by the addition of human IgG1, confirming the validity of the assay system (Figure 5). In this assay system, the binding of biotinylated M-A251 to human CD25-expressing CHO-1 cells was not inhibited by the addition of MAb1. This demonstrates that MAb1 and M-A251 bind to different sites on human CD25 (Figure 5).

[0237] Example 4 Properties of MAb2 4)-1 Antigen Binding Ability This was carried out according to the method described in 2)-3-1. MAb2 was a purified antibody, and was added to the wells so that the final antibody concentrations were 0.02 μg / mL, 0.16 μg / mL, 1.25 μg / mL, or 10 μg / mL. To wells without antibody (0 μg / mL), 5% FBS-containing PBS was added instead of the antibody solution. As a result, MAb2 bound to CHO-K1 cells transfected with pCMV3-mCD25, but did not bind to CHO-K1 cells transfected with pCMV3-hCD25 or pCI-cCD25 (Figure 6). This demonstrated that MAb2 binds to mouse CD25.

[0238] 4)-2 Effect of IL-2 stimulation on phosphorylated STAT5 (pSTAT5) levels in mouse CTLL-2 cells. This study was performed according to the method described in 2)-3-9. When the pSTAT5 level in the group treated with IL-2 and the control antibody rat IgG2a was defined as 100%, the level in the group treated with IL-2 and PC61_hIgG1LALA was 48% and the level in the group treated with IL-2 and MAb2 was 96.5% (Figure 7). The pSTAT5 level increased with IL-2 treatment, but was reduced by the addition of the IL-2-blocking antibody PC61_hIgG1LALA, demonstrating that MAb2 does not block IL-2 signaling.

[0239] 4)-3 Measurement of Internalization Activity This was performed according to the method described in 2)-3-10. MAb2 was a purified antibody, and was added to each well at concentrations of 7500 ng / mL, 1500 ng / mL, 300 ng / mL, and 60 ng / mL (n = 3). When the cell viability of the control antibody at each concentration was taken as 100%, the cell viabilities of MAb2 and PC61 were 6% and 59% at 60 ng / mL, 4% and 39% at 300 ng / mL, 4% and 31% at 1500 ng / mL, and 5% and 29% at 7500 ng / mL, respectively (Figure 8). In the Fab-ZAP-free group, no change in viability was observed in the control antibody rat IgG2a, PC61, or MAb2 groups. These results demonstrate that MAb2 is an antibody internalized by CD25-positive cells.

[0240] Example 5 Determination of the Amino Acid Sequences of MAb1 and MAb2 5)-1 Preparation of Total RNA from MAb1- and MAb2-Producing Hybridomas In order to amplify the cDNAs of the variable regions of MAb1 and MAb2, total RNA was prepared from the MAb1- and MAb2-producing hybridomas using an RNA purification kit, Direct-zol RNA Miniprep Kit (ZYMO RESEARCH).

[0241] 5)-2 Amplification and Sequencing of cDNA Containing the Light Chain Variable Regions of MAb1 and MAb2 by 5'-RACE PCR. Amplification of the light chain variable region cDNA was performed using approximately 1 μg of the total RNA prepared in 5)-1 and the SMARTer RACE 5' / 3' Kit (Thermo Scientific). Primers used to amplify the light chain variable region cDNAs of MAb1 and MAb2 by PCR included UPM (Universal Primer A Mix, included in the SMARTer RACE 5' / 3' Kit) and primers designed from known rat light chain constant region sequences. The light chain variable region cDNAs amplified by 5'-RACE PCR were cloned into a plasmid, and the nucleotide sequences of the light chain variable region cDNAs were then sequenced. The determined nucleotide sequences of the cDNAs of the variable regions of the light chains of MAb1 and MAb2 are shown in SEQ ID NOs: 19 and 23, and the amino acid sequences are shown in SEQ ID NOs: 18 and 22.

[0242] 5)-3 Amplification and Sequencing of cDNA Containing the Heavy Chain Variable Regions of MAb1 and MAb2 by 5'-RACE PCR. Amplification of the heavy chain variable region cDNA was performed using approximately 1 μg of the total RNA prepared in 5)-1 and the SMARTer RACE 5' / 3' Kit (Thermo Scientific). Primers used to amplify the heavy chain variable region cDNAs of MAb1 and MAb2 by PCR included UPM (Universal Primer A Mix: included in the SMARTer RACE 5' / 3' Kit) and primers designed from known rat heavy chain constant region sequences. The heavy chain variable region cDNAs amplified by 5'-RACE PCR were cloned into a plasmid, and the nucleotide sequences of the heavy chain variable region cDNAs were then sequenced. The determined nucleotide sequences of the cDNAs of the heavy chain variable regions of MAb1 and MAb2 are shown in SEQ ID NOs: 21 and 25, and the amino acid sequences are shown in SEQ ID NOs: 20 and 24.

[0243] Example 6 Preparation of human IgG1LALA chimeric MAb1 (cMAb1_hIgG1LALA) and human IgG1LALA chimeric MAb2 (cMAb2_hIgG1LALA) 6)-1 Construction of light chain expression vector pCMA-LK An approximately 5.4 kb fragment obtained by digesting the plasmid pcDNA3.3-TOPO / LacZ (Thermo Fisher Scientific) with the restriction enzymes XbaI and PmeI was ligated with a DNA fragment containing the nucleotide sequence encoding the human light chain signal sequence and human κ chain constant region shown in SEQ ID NO:51 using an In-Fusion HD PCR cloning kit (Thermo Fisher Scientific) to prepare pcDNA3.3 / LK. pCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.

[0244] 6)-2 Construction of heavy chain expression vector pCMA-G1LALA A DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the light chain signal sequence and human κ chain constant region was ligated with a DNA fragment containing the nucleotide sequence encoding the human heavy chain signal sequence and human IgG1LALA constant region shown in SEQ ID NO:52 using an In-Fusion HD PCR cloning kit (Thermo Fisher Scientific) to construct pCMA-G1LALA.

[0245] A DNA fragment represented by nucleotide positions 61 to 381 in the nucleotide sequence of the cMAb1_hIgG1LALA light chain shown in SEQ ID NO: 28, and a DNA fragment represented by nucleotide positions 61 to 402 in the nucleotide sequence of the cMAb2_hIgG1LALA light chain shown in SEQ ID NO: 34 were synthesized (manufactured by Thermo Scientific). Using an In-Fusion HD PCR cloning kit (manufactured by Thermo Scientific), the cMAb1_hIgG1LALA and cMAb2_hIgG1LALA light chain expression vectors were constructed by inserting the synthesized DNA fragments into pCMA-LK cleaved with the restriction enzyme BsiWI. The amino acid sequence of the cMAb1_hIgG1LALA light chain is shown in SEQ ID NO: 26, and the amino acid sequence of the cMAb2_hIgG1LALA light chain is shown in SEQ ID NO: 30.

[0246] A DNA fragment represented by nucleotide positions 58 to 438 in the nucleotide sequence of the cMAb1_hIgG1LALA heavy chain shown in SEQ ID NO: 29, and a DNA fragment represented by nucleotide positions 58 to 399 in the nucleotide sequence of the cMAb2_hIgG1LALA heavy chain shown in SEQ ID NO: 35 were synthesized (manufactured by Thermo Scientific). Using an In-Fusion HD PCR cloning kit (manufactured by Thermo Scientific), the cMAb1_hIgG1LALA and cMAb2_hIgG1LALA heavy chain expression vectors were constructed by inserting the synthesized DNA fragments into pCMA-G1LALA cleaved with the restriction enzyme BlpI. The amino acid sequence of the cMAb1_hIgG1LALA heavy chain is shown in SEQ ID NO: 27, and the amino acid sequence of the cMAb2_hIgG1LALA heavy chain is shown in SEQ ID NO: 31.

[0247] 6)-5 Preparation of cMAb1_hIgG1LALA and cMAb2_hIgG1LALA 6)-5-1 Production of cMAb1_hIgG1LALA and cMAb2_hIgG1LALA FreeStyle 293F cells (Thermo Scientific) were passaged and cultured according to the manual. 1.2 × 10 cells in the logarithmic growth phase were cultured. 9 FreeStyle 293F cells (manufactured by Thermo Scientific) were seeded in a 3 L Fernbach Erlenmeyer Flask (manufactured by Corning) and diluted with FreeStyle 293 expression medium (manufactured by Thermo Scientific) to a concentration of 2.0 × 10 6The concentration was adjusted to 100 cells / mL. 1.8 mg of polyethyleneimine (Polyscience) was added to 20 mL of Opti-Pro SFM medium (Thermo Scientific). Next, 0.24 mg of the heavy chain expression vector and 0.36 mg of the light chain expression vector were added to 20 mL of Opti-Pro SFM medium (Thermo Scientific). The expression vector / Opti-Pro SFM mixture was added to the polyethyleneimine / Opti-Pro SFM mixture, gently stirred, and then allowed to stand for another 5 minutes before being added to FreeStyle 293F cells. After culturing for 4 hours in an incubator at 37°C and 8% CO with shaking at 90 rpm, 600 mL of EX-CELL VPRO medium (SAFC Biosciences), 18 mL of GlutaMAX I (GIBCO), and 30 mL of Yeastolate Ultrafiltrate (GIBCO) were added, and the cells were cultured for 7 days in an incubator at 37°C and 8% CO with shaking at 90 rpm. The resulting culture supernatant was filtered through a Disposable Capsule Filter (Advantec).

[0248] 6)-5-2 Purification of cMAb1_hIgG1LALA The antibody was purified from the culture supernatant obtained in 6)-5-1 using a two-step process: rProtein A affinity chromatography and ceramic hydroxyapatite. The culture supernatant was applied to a column (Cytiva) packed with MabSelectSuRe equilibrated with PBS, and the column was washed with at least two column volumes of PBS. The antibody was then eluted with 2 M arginine hydrochloride solution (pH 4.0). Fractions containing the antibody were subjected to buffer exchange with PBS by dialysis (Slide-A-Lyzer Dialysis Cassette, Thermo Scientific), diluted 5-fold with 5 mM sodium phosphate / 50 mM MES / pH 7.0 buffer, and then applied to a ceramic hydroxyapatite column (Bio-Scale CHT Type-1 Hydroxyapatite Column, Japan Bio-Rad) equilibrated with 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0 buffer. Linear gradient elution with sodium chloride was performed, and fractions containing the antibody were collected. The fraction was subjected to dialysis (Slide-A-Lyzer Dialysis Cassette, Thermo Scientific) to exchange the buffer with HBSor (25 mM histidine / 5% sorbitol, pH 6.0). Finally, the fraction was filtered through a Minisart-Plus filter (Sartorius Stedim Biotech) to obtain a purified sample.

[0249] 6)-5-3 Purification of cMAb2_hIgG1LALA The antibody was purified from the culture supernatant obtained in 6)-5-1 by rProtein A affinity chromatography. The culture supernatant was applied to a column (Cytiva) packed with MabSelectSuRe equilibrated with PBS, and the column was washed with at least two column volumes of PBS. The antibody was then eluted with 2 M arginine hydrochloride solution (pH 4.0). The eluted fraction was dialyzed (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) to replace the buffer with HBSor (25 mM histidine / 5% sorbitol, pH 6.0) (concentration unadjusted). Finally, the mixture was filtered through a Minisart-Plus filter (manufactured by Sartorius Stedim Biotech) to obtain a purified sample.

[0250] (Example 7) Properties of cMAb1_hIgG1LALA 7)-1 Measurement of antigen binding This was carried out according to the method in 2)-3-1. The cMAb1_hIgG1LALA added to CHO-K1 cells was added at a concentration ranging from 80 to 0.001 μg / mL. The secondary antibody used was Peroxidase-AffiniPure F(ab')2 Fragment Goat Anti-Human IgG(H+L) (Jackson ImmunoResearch Laboratories), diluted 1000-fold with 5% FBS-containing PBS. Furthermore, TMB Microwell Peroxidase Substrate (2-Component System) (Sera Care Life Sciences) and TMB Stop Solution (Sera Care Life Sciences) were used for color development and color stop, respectively. As a result, cMAb1_hIgG1LALA bound to CHO-K1 cells transfected with pCMV3-hCD25 and pCI-cCD25 in an antibody concentration-dependent manner, but did not bind to CHO-K1 cells transfected with pCMV3-mCD25 (Figure 9). Therefore, it was demonstrated that cMAb1_hIgG1LALA binds to human CD25 and cynomolgus monkey CD25.

[0251] 7)-2 Effect on IL-2-dependent cell proliferation using human CD4+ T cells 7)-2-1 Preparation of human CD4+ T cells and serum starvation Frozen human PBMCs (Cellular Technology Limited) were thawed according to the C.T.L. protocol. CD4+ T cells were then isolated from the PBMCs using a CD4+ T Cell Isolation Kit, human (MILTENYI BIOTEC). The isolated CD4+ T cells were cultured at 2 x 10 in IMDM (Thermo Fisher Scientific) medium containing 10% CTS Immune Cell SR (Thermo Fisher Scientific). 5A cell suspension was prepared to a final concentration of 15 μg / mL. 25 μL of the cell suspension was seeded into a 96-well plate (U-bottom), and PHA (SIGMA-ALDRICH) was added at 2 μg / mL and Phorbol 12-myristate 13-acetate (PMA) (SIGMA-ALDRICH) at 0.2 μg / mL. Furthermore, human IgG1, basiliximab, and cMAb1_hIgG1LALA were added to a final concentration of 15 μg / mL. The plate was incubated at 37°C and 5% CO2 for 5 days. After incubation, the plate was returned to room temperature. 100 μL of CellTiter-Glo Reagent from the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) was added and the mixture was shaken for 10 minutes on a plate shaker. 100 μL / well of the reaction solution was transferred to a 96-well white flat bottom plate, and the luminescence intensity (relative light unit, RLU) was measured using a plate reader (Ensight: PerkinElmer) and used as an index of cell number. As a result, when the cell count in the human IgG1-added group was taken as 100%, the count in the IL-2 blocking antibody basiliximab-added group was 34%, and in the cMAb1_hIgG1LALA-added group was 92% (Figure 10). The T cell count was reduced in the IL-2 blocking antibody basiliximab-added group compared to the human IgG1-added group, but remained unchanged in the cMAb1_hIgG1LALA-added group. These results demonstrate that cMAb1_hIgG1LALA is an IL-2 non-blocking antibody that does not affect the proliferation of human CD4+ T cells.

[0252] 7)-3 Effect on phosphorylated STAT5 (pSTAT5) increased by IL-2 stimulation in human PBMCs 7)-3-1 Immobilization of anti-human CD3 antibody to 6-well plate wells PBS containing 10 μg / mL anti-human CD3 antibody (EXBIO Praha) was added to each well of a 6-well plate (Corning) at 2 mL / well, and the plate was mixed using a plate mixer and incubated at 4°C. After incubation, the supernatant was removed, and the wells were washed three times with PBS before use.

[0253] 7)-3-2 Preparation of human PBMCs and serum starvation. Frozen human PBMCs were thawed according to the C.T.L. protocol. The thawed PBMCs were suspended in IMDM (Thermo Fisher Scientific) medium containing 10% CTS Immune Cell SR (Thermo Fisher Scientific) and seeded at 4 mL / well into the 6-well plate prepared in 7)-3-1. The plate was incubated at 37°C in 5% CO2 for 3 days.

[0254] 7)-3-3 Preparation and addition of antibody solution The control antibody human IgG1 and the anti-human CD25 antibody basiliximab (Novartis Pharma) or cMAb1_hIgG1LALA were diluted to 80 μg / mL with IMDM medium (Thermo Fisher Scientific), and 25 μL was added to a V-bottom 96-well plate (Corning). 25 μL of IMDM medium (Thermo Fisher Scientific) was added to wells to which no antibody was added.

[0255] 7)-3-4 Addition of human PBMC and IL-2 solution PBMC were collected after serum starvation and diluted to 2 × 10 6The antibody solution was prepared in IMDM medium (Thermo Fisher Scientific) to a concentration of 100 μg / mL. The cell suspension was added at 50 μL / well to the plate containing the antibody solution in step 7)-3-3. The plate was then incubated for 15 minutes at 37°C with 5% CO2. Recombinant human IL-2 (PeproTech) was dissolved in 50 μL of 0.1 M acetic acid and diluted with 0.45 mL of PBS containing 0.1% BSA to prepare a 100 μg / mL solution. This solution was then diluted to 4 ng / mL with IMDM medium (Thermo Fisher Scientific) and added to the IL-2-treated wells at 25 μL / well. Furthermore, IMDM medium (Thermo Fisher Scientific) was added to wells in the IL-2 non-added group at 25 μL / well, followed by incubation for 15 minutes under conditions of 5% CO and 37°C.

[0256] 7)-3-5 pSTAT5 Measurement The 5x lysis buffer included with the AlphaLISA SureFire Ultra phosho-STAT5 (Tyr694 / 699) kit (PerkinElmer) was adjusted to 1x lysis buffer with HO. After 15 minutes of incubation, the plate was centrifuged at 340 x g for 5 minutes, and the supernatant from each well was aspirated. PBS was then added and centrifuged in the same manner, after which the supernatant was aspirated. 50 μL of 1x lysis buffer was added to each well. The plate was agitated on a plate shaker for 10 minutes to prepare a cell lysate. The cell lysate from each well was added to a 384-well AlphaLISA SW plate (PerkinElmer) at 8 μL / well, and after adding 4 μL / well of Acceptor Mix, the plate was shaken for 2 minutes on a plate shaker. This was then incubated for 1 hour in the dark. 4 μL / well of Donor Mix was added, and the plate was shaken for 2 minutes on a plate shaker, and then further incubated overnight in the dark. The Alpha signal was then measured using a plate reader (Ensight: PerkinElmer). As a result, when the pSTAT5 level in the group treated with IL-2 and the control antibody human IgG1 was taken as 100%, the level in the group treated with IL-2 and the IL-2 blocking antibody basiliximab was 1.2%, and the level in the group treated with IL-2 and cMAb1_hIgG1LALA was 83.2% (Figure 11). The pSTAT5 level, which increased with the addition of IL-2, decreased in the group treated with the IL-2 blocking antibody basiliximab, but did not change with the addition of cMAb1_hIgG1LALA. This demonstrated that cMAb1_hIgG1LALA is an antibody that does not block IL-2 signaling.

[0257] (Example 8) Preparation of cMAb1_hIgG1LALA-ADC, cMAb1_hIgG1LALA-ADC2, cMAb2_hIgG1LALA-ADC, and cMAb2_hIgG1LALA-ADC2 Using cMAb1_hIgG1LALA and cMAb2_hIgG1LALA prepared in Example 6, cMAb1_hIgG1LALA-ADC, cMAb1_hIgG1LALA-ADC2, cMAb2_hIgG1LALA-ADC, and cMAb2_hIgG1LALA-ADC2 were prepared by known methods such as those described in WO2014 / 057687. The cMAb1_hIgG1LALA antibody comprises a heavy chain comprising the amino acid sequence of positions 20 to 146 of SEQ ID NO: 27 and a light chain comprising the amino acid sequence of positions 21 to 126 of SEQ ID NO: 26. The cMAb2_hIgG1LALA antibody comprises a heavy chain comprising the amino acid sequence of positions 20 to 133 of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of positions 21 to 133 of SEQ ID NO: 30. The cMAb1_hIgG1LALA antibody and the cMAb2_hIgG1LALA antibody were each linked to a compound represented by the following formula via a linker.

[0258]

[0259] Such cMAb1_hIgG1LALA-ADC and cMAb2_hIgG1LALA-ADC have the structure shown in the following formula (n: the number of drug molecules conjugated per antibody molecule is 4 to 8, i.e., the average number of drug molecules conjugated per antibody (n): approximately 8), and AB represents cMAb1_hIgG1LALA or cMAb2_hIgG1LALA.

[0260]

[0261] cMAb1_hIgG1LALA-ADC2 and cMAb2_hIgG1LALA-ADC2 have the structure shown in the following formula (n: the number of drug molecules conjugated per antibody molecule is 4 to 8, i.e., the average number of drug molecules conjugated per antibody (n): approximately 8), and AB represents cMAb1_hIgG1LALA or cMAb2_hIgG1LALA.

[0262]

[0263] The control hIgG-ADC was constructed with the same drug-linker as cMAb1_hIgG1LALA-ADC and cMAb2_hIgG1LALA-ADC, using a humanized IgG1 isotype control monoclonal antibody that does not bind to mammalian cells. The drug-linker was prepared by known methods, such as those described in WO2015 / 115091.

[0264] 8)-1 Preparation of antibody-drug conjugate cMAb1_hIgG1LALA-ADC

[0265] Antibody reduction: cMAb1_hIgG1LALA prepared in Example 6 was reduced to 1.60 mL mg as an extinction coefficient at 280 nm using the common procedure B described in Production Method 1. －１ cm －１Using 10.3 mg / mL of TCEP (Tokyo Chemical Industry Co., Ltd.) and C, a 10 mM TCEP solution (0.169 mL; 6.0 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate solution (Nacalai Tesque, Inc.; 0.0600 mL) were added to this solution (4.0 mL). After confirming that the pH of this solution was within 7.0±0.1, the solution was incubated at 37°C for 2 hours to reduce the intra-chain disulfide bond between the antibody and the drug linker. Conjugation of the antibody and the drug linker: The above solution was incubated at 15°C for 10 minutes. Then, 10 mM of N-{3-[2-(2-{[3-(2,5-dioxo2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide (WO2015 / 115091) was added. A dimethyl sulfoxide solution (0.282 mL; 10 equivalents per antibody molecule) was added, and the mixture was incubated at 15°C for 1 hour to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0282 mL; 10 equivalents per antibody molecule) was added, and the mixture was stirred at room temperature for an additional 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 15.0 mL of a solution containing the title antibody-drug conjugate "cMAb1_hIgG1LALA-ADC". Characterization: The antibody-drug conjugate was purified by common procedure E (ε Ｄ，２８０ =7642 、 ε Ｄ，３７０ Using a standard antibody assay (using 24111), the following characteristic values ​​were obtained: antibody concentration: 2.36 mg / mL, antibody yield: 35.4 mg (86%), average number of drugs bound per antibody molecule (n) measured by standard procedure E: 5.1, and average number of drugs bound per antibody molecule (n) measured by standard procedure F: 7.7.

[0266] 8)-2 Preparation of antibody-drug conjugate cMAb1_hIgG1LALA-ADC2

[0267] Antibody reduction: cMAb1_hIgG1LALA prepared in Example 6 was reduced to 1.60 mL mg as an extinction coefficient at 280 nm using the common procedure B described in Production Method 1. －１ cm －１ Using 10.3 mg / mL of TCEP (Tokyo Chemical Industry Co., Ltd.) and C, a 10 mM TCEP solution (0.169 mL; 6.0 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate solution (Nacalai Tesque, Inc.; 0.0600 mL) were added to this solution (4.0 mL). After confirming that the pH of this solution was within 7.0±0.1, the solution was incubated at 37°C for 2 hours to reduce the intra-chain disulfide bond between the antibody and the drug linker. Conjugation of the antibody and the drug linker: The above solution was incubated at 15°C for 10 minutes. Next, a 10 mM dimethyl sulfoxide solution (0.282 mL; 10 equivalents per antibody molecule) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide (WO2015 / 115091) was added, and the mixture was incubated at 15°C for 1 hour to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0282 mL; 10 equivalents per antibody molecule) was added, and the mixture was stirred at room temperature for a further 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 15.0 mL of a solution containing the title antibody-drug conjugate "cMAb1_hIgG1LALA-ADC2". Characterization: The antibody-drug conjugate "cMAb1_hIgG1LALA-ADC2" was purified by common procedure E (ε Ｄ，２８０ =5440、 ε Ｄ，３７０ Using a standard antibody assay (using HPLC with a HPLC spectrophotometer), the following characteristic values ​​were obtained: antibody concentration: 1.74 mg / mL, antibody yield: 26.2 mg (63%), average number of drugs bound per antibody molecule (n) measured by standard procedure E: 6.2, average number of drugs bound per antibody molecule (n) measured by standard procedure F: 7.9.

[0268] 8)-3 Preparation of antibody-drug conjugate cMAb2_hIgG1LALA-ADC

[0269] Antibody reduction: cMAb2_hIgG1LALA prepared in Example 6 was reduced to 1.45 mL mg as an extinction coefficient at 280 nm using the common procedure B described in Production Method 1. －１ cm －１Using 10.1 mg / mL of TCEP (Tokyo Chemical Industry Co., Ltd.) and C, a 10 mM TCEP solution (0.0419 mL; 6.0 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate solution (Nacalai Tesque, Inc.; 0.0150 mL) were added to this solution (1.0 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the interchain disulfide bond between the antibody. Conjugation of antibody and drug linker: The above solution was incubated at 15°C for 10 minutes. Next, a 10 mM dimethyl sulfoxide solution (0.0698 mL; 10 equivalents per antibody molecule) of N-{3-[2-(2-{[3-(2,5-dioxo2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide was added, and the mixture was incubated at 15°C for 1 hour to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0070 mL; 10 equivalents per antibody molecule) was added, and the mixture was stirred at room temperature for an additional 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 6.0 mL of a solution containing the title antibody-drug conjugate "cMAb2_hIgG1LALA-ADC". Characterization: The antibody-drug conjugate "cMAb2_hIgG1LALA-ADC" was purified by common procedure E (ε Ｄ，２８０ =7642 、 ε Ｄ，３７０ Using a standard antibody assay (using 24111), the following characteristic values ​​were obtained: antibody concentration: 1.38 mg / mL, antibody yield: 8.30 mg (82%), average number of drugs bound per antibody molecule (n) measured by standard procedure E: 5.5, and average number of drugs bound per antibody molecule (n) measured by standard procedure F: 7.9.

[0270] 8)-4 Preparation of antibody-drug conjugate cMAb2_hIgG1LALA-ADC2

[0271] Antibody reduction: cMAb2_hIgG1LALA prepared in Example 6 was reduced to 1.45 mL mg as an extinction coefficient at 280 nm using the common procedure B described in Production Method 1. －１ cm －１ Using 10.1 mg / mL of TCEP (Tokyo Chemical Industry Co., Ltd.) and C, a 10.1 mg / mL solution was prepared in PBS 6.0 / EDTA. To this solution (4.0 mL) was added a 10 mM aqueous solution of TCEP (Tokyo Chemical Industry Co., Ltd.) (0.168 mL; 6.0 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.0600 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 2 hours to reduce the intra-chain disulfide bond of the antibody. Conjugation of antibody and drug linker: The above solution was incubated at 15°C for 10 minutes. Next, a 10 mM dimethyl sulfoxide solution (0.278 mL; 10 equivalents per antibody molecule) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide was added, and the mixture was incubated at 15°C for 1 hour to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0278 mL; 10 equivalents per antibody molecule) was added, and the mixture was stirred at room temperature for an additional 20 minutes to terminate the drug linker reaction. Purification: The above solution was purified by common procedure D described in Production Method 1 to obtain 17.5 mL of a solution containing the title antibody-drug conjugate "cMAb2_hIgG1LALA-ADC2". Characterization: The antibody-drug conjugate "cMAb2_hIgG1LALA-ADC2" was purified by common procedure E (ε Ｄ，２８０ =5440 、 ε Ｄ，３７０Using a standard antibody assay (using HPLC with HPLC = 21240), the following characteristic values ​​were obtained: antibody concentration: 2.06 mg / mL, antibody yield: 36.1 mg (90%), average number of drugs bound per antibody molecule (n) measured by standard procedure E: 6.0, average number of drugs bound per antibody molecule (n) measured by standard procedure F: 7.9.

[0272] Example 9 Properties of cMAb1_hIgG1LALA-ADC 9)-1 Cytostatic Activity of cMAb1_hIgG1LALA-ADC 9)-1-1 Cell Culture The CD25-positive human tumor cell line Karpas-299 was passaged in RPMI 1640 (Thermo Fisher Scientific) medium containing 2 mM glutamine (Life Technologies) and 20% FBS at 37°C and 5% CO. The CD25-negative human tumor cell line Daudi was passaged in RPMI 1640 medium containing 10% FBS at 37°C and 5% CO.

[0273] 9)-1-2 Cell proliferation inhibition assay The cultured Karpas-299 cells were cultured in RPMI 1640 medium containing 2 mM glutamine and 20% FBS at a concentration of 1 × 10 3Cells were seeded onto a 96-well clear U-bottom microplate (Corning) at 50 μL / well. cMAb1_hIgG1LALA was added at concentrations ranging from 0.015 μg / mL to 15 μg / mL, control hIgG1LALA-ADC, and cMAb1_hIgG1LALA-ADC at concentrations ranging from 0.0015 μg / mL to 1.5 μg / mL. The cells were cultured at 37°C in 5% CO for 6 days. The cultured plate was left at room temperature for 30 minutes, and then 100 μL / well of CellTiter-Glo™ Luminescent Cell Viability Assay (Promega) reagent solution was added. The mixture was then mixed for 10 minutes at room temperature in the dark using a plate mixer (Tytec). 100 μL of the solution from each well was added to a 96-well white flat-bottom microplate (Corning). The luminescence intensity (relative light unit, RLU) was measured using a plate reader (Ensight, PerkinElmer) and used as an index of cell number. In addition, the cell growth inhibitory activity of the CD25-negative human tumor cell line DauDi was measured in the same manner using RPMI 1640 medium (Thermo Fisher Scientific) containing 10% FBS (GE Healthcare). 4 The cells were seeded at 50 μL / well.

[0274] As a result, cMAb1_hIgG1LALA-ADC exhibited cell growth inhibitory activity at lower concentrations than human IgG1LALA-ADC only in the case of the CD25-positive human tumor cell line, Karpas-299 (Figure 12). Furthermore, addition of cMAb1_hIgG1LALA did not exhibit cell growth inhibitory activity. These results demonstrate that cMAb1_hIgG1LALA-ADC has the ability to inhibit the proliferation of CD25-positive cells. cMAb1_hIgG1LALA-ADC2 also exhibited a similar effect. It can be inferred that the inhibition of CD25-positive cell proliferation by these agents is due to the internalization of cMAb1_hIgG1LALA-ADC and cMAb1_hIgG1LALA-ADC2 into CD25-positive cells and the induction of cell death.

[0275] 9)-2 In vivo immunostimulatory effect of cMAb1_hIgG1LALA-ADC 9)-2-1 Tumor tissue collection and cell suspension preparation 5 x 10 human breast adenocarcinoma-derived MDA-MB-231 cells (American Type Culture Collection) subcultured in 10% FBS-containing Leibovitz's L-15 medium. 6 The cells were suspended in Matrigel and subcutaneously implanted into female humanized NOG mice (huNOG mice) transfected with human CD34 cells. The mice were divided into groups based on tumor volume, and 7 days after implantation, 1 mg / kg of control hIgG1LALA-ADC or 0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg of cMAb1_hIgG1LALA-ADC was intravenously administered (5 mice per group). 28 days after ADC administration, tumors were harvested. The harvested tumor masses were treated with gentleMACS (Miltenyi Biotec) and the cells were collected. The cells were washed and resuspended in PBS, and the cell suspension was used as a sample for flow cytometry. The following amounts of PBS were added during resuspension, depending on the tumor weight: The volume was 0.6 mL for tumor weights up to 50 mg, 0.9 mL for tumor weights of 50-100 mg, 1.2 mL for tumor weights of 100-150 mg, 1.8 mL for tumor weights of 150-200 mg, and 2.4 mL for tumor weights of 200-300 mg.

[0276] 9)-2-2 Preparation of staining solution for flow cytometry analysis Zombie NIR dye solution was prepared by diluting the Zombie NIR dye solution included in the Zombie NIR Fixable Viability Kit (BioLegend) 1000-fold with PBS. The Fc Block solution was prepared by mixing Mouse BD Fc Block (BD Biosciences) solution, Human BD Fc Block solution, and 0.5% BSA-containing autoMACS Rinse Solution (Miltenyi Biotec) (hereinafter referred to as "MACS buffer") in a ratio of 1:1:48. Foxp3 Fixation / Permeability buffer was prepared by mixing 3.5 mL of Concentrate and 10.5 mL of Diluent from Foxp3 Fixation / Permeability Concentrate and Diluent (eBioscience). Perm wash buffer was prepared by diluting Intracellular Staining Perm Wash Buffer (BioLegend) 10-fold with HO. The cell surface marker antibody cocktail solution consisted of 2 μL of FITC anti-human CD25 antibody (BioLegend), 1 μL of APC anti-human CD8 antibody (BioLegend), 3 μL of Brilliant Violet 421 anti-human CD4 antibody (BioLegend), 2.5 μL of PE / Cyanine7 anti-human CD45RA antibody (BioLegend), 2 μL of Alexa Fluor 700 anti-mouse CD45 antibody, and 2 μL of Brilliant Violet 605 anti-human CD45 antibody (BD Biosciences) and Brilliant Violet 650 anti-human CD3 antibody were added, and the total volume was adjusted to 50 μL with MACS buffer.The intracellular marker antibody cocktail solution was prepared by adding 3 μL of PE Mouse anti-Human FoxP3 (BD ​​Biosciences), 3 μL of PerCP / Cyanine5.5 anti-human / mouse Granzyme B Recombinant Antibody (BioLegend), 1 μL of Mouse BD Fc Block (BD Biosciences), and 1 μL of Human BD Fc Block, and then adding Perm wash buffer to make up the total volume to 100 μL.

[0277] 9)-2-3 Antibody staining of samples for flow cytometry analysis To 0.2 mL of the flow cytometry sample prepared in 9)-2-1, 0.1 mL of Zombie NIR dye diluted solution was added and incubated at 4°C for 30 minutes. 0.1 mL of MACS buffer was added and centrifuged, and the supernatant was aspirated. 0.1 mL of Fc Block solution was added to the remaining cells and incubated at 4°C for 5 minutes. 0.1 mL of the cell surface marker antibody cocktail solution prepared in 9)-2-2 was added to the cells and incubated at 4°C for 30 minutes. Next, 0.1 mL of MACS buffer was added and centrifuged. The supernatant was aspirated. 0.2 mL of MACS buffer was added to the remaining cells, and the cells were further centrifuged to remove the supernatant. This procedure was repeated two more times to wash the cells. 0.2 mL of Foxp3 Fixation / Permeabilization buffer was added and incubated at 4°C for 1 hour. 0.2 mL of intracellular marker antibody cocktail solution was added to the washed cells and incubated at 4°C for 1 hour. After washing twice with Perm wash buffer, 4% paraformaldehyde solution was added to the cells and incubated at 4°C for 15 minutes. After washing twice with MACS buffer, 0.18 mL of MACS buffer was added to the cells. The sample was incubated overnight at 4°C and then analyzed by flow cytometry.

[0278] 9)-2-4 Flow cytometry analysis The samples prepared in 9)-2-3 were used to measure the number of human CD45-positive cells, human FoxP3-positive CD4-positive CD3-positive CD45-positive cells (human Treg cells), human CD8-positive CD3-positive CD45-positive cells, CD8-positive T cells (human CD8-positive T cells), and human granzyme...

Claims

1. (1) to (2) below: (1) CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 1, CDRL2 consisting of the amino acid sequence (KAS) described in SEQ ID NO: 2, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO:

3. (2) CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence (FVS) described in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO:

6. CDRL1, CDRL2, and CDRL3 described in any one selected from the group consisting of, (3) to (4) below: (3) CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 8, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO:

9. (4) CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 10, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 11, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO:

12. CDRH1, CDRH2, and CDRH3 are included in any one selected from the group consisting of the following: An antibody or an antigen-binding fragment of said antibody.

2. (1) to (2) below: (1) CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 1, CDRL2 consisting of the amino acid sequence (KAS) described in SEQ ID NO: 2, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 3, and CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 8, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 9, (2) CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence (FVS) described in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 6, and CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 10, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 11, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO:

12. CDRL1, CDRL2 and CDRL3 selected from the group consisting of, and CDRH1, CDRH2 and CDRH3, The antibody according to claim 1, or an antigen-binding fragment of the antibody.

3. The antibody according to claim 1 or an antigen-binding fragment of the antibody, characterized by having the following properties. (1) It does not have IL-2 blocking capability. (2) When conjugated with cytotoxic compounds, it exhibits the ability to eliminate human regulatory T cells and / or promote the proliferation of human granzyme-positive CD8-positive cells.

4. The antibody according to claim 1, or an antigen-binding fragment of the antibody, which has the activity of being internalized in CD25-expressing cells by binding to CD25.

5. A humanized antibody according to claim 1, or an antigen-binding fragment of the antibody.

6. The following (1) to (4): (1) The light chain variable region consisting of amino acid sequences 21 to 127 of SEQ ID NO: 13, (2) Light chain variable region consisting of amino acid sequences 21 to 127 of SEQ ID NO: 14, (3) Light chain variable regions consisting of amino acid sequences having at least 95% sequence identity with respect to the sequences of framework regions other than each CDR sequence in the amino acid sequences of (1) to (2), and (4) Light chain variable regions consisting of amino acid sequences in which one or more amino acids are deleted, substituted, or added in the amino acid sequences of (1) to (2) in the framework regions other than each CDR sequence. A light chain variable region selected from any one of the group consisting of, (5) to (8) below: (5) Heavy chain variable region consisting of amino acid sequences 20 to 146 of SEQ ID NO: 15, (6) Heavy chain variable region consisting of amino acid sequences 20 to 146 of sequence number 16, (7) A heavy chain variable region consisting of amino acid sequences having at least 95% sequence identity with respect to the sequences of framework regions other than each CDR sequence in the amino acid sequences of (5) to (6), and (8) Heavy chain variable region consisting of amino acid sequences in which one or more amino acids are deleted, substituted, or added in the amino acid sequences of (5) to (6) in the framework region other than each CDR sequence. A heavy chain variable region described in any one selected from the group consisting of the following, The antibody according to claim 1, or an antigen-binding fragment of the antibody, comprising the above.

7. (1) to (2) below: (1) The light chain variable region consisting of amino acid sequences 21 to 127 of SEQ ID NO: 13 and the heavy chain variable region consisting of amino acid sequences 20 to 146 of SEQ ID NO: 15 (2) Light chain variable region consisting of amino acid sequences 21 to 127 of SEQ ID NO: 14 and heavy chain variable region consisting of amino acid sequences 20 to 146 of SEQ ID NO: 16 Either of the light chain variable region and the heavy chain variable region, The antibody according to claim 1, or an antigen-binding fragment of the antibody, comprising the above.

8. (1) to (2) below: (1) A light chain consisting of amino acid sequences 21 to 233 of SEQ ID NO: 13, and a heavy chain consisting of amino acid sequences 20 to 476 of SEQ ID NO: 15, or a heavy chain in which one or two amino acids are deleted at the carboxyl terminus. (2) A light chain consisting of the amino acid sequence from position 21 to 233 of SEQ ID NO: 14, and a heavy chain consisting of the amino acid sequence from position 20 to 476 of SEQ ID NO: 16, or a heavy chain in which one or two amino acids are deleted at the carboxyl terminus. The antibody according to claim 1, or an antigen-binding fragment of the antibody, comprising any of the above.

9. The antibody or antigen-binding fragment of the antibody according to claim 8, comprising a light chain consisting of the amino acid sequence from 21 to 233 of SEQ ID NO: 13, and a heavy chain consisting of the amino acid sequence from 20 to 476 of SEQ ID NO: 15, or a heavy chain having one or two amino acids deleted at its carboxyl terminus.

10. The antibody or antigen-binding fragment of the antibody according to claim 8, comprising a light chain consisting of the amino acid sequence from 21 to 233 of SEQ ID NO: 14, and a heavy chain consisting of the amino acid sequence from 20 to 476 of SEQ ID NO: 16, or a heavy chain having one or two amino acids deleted at its carboxyl terminus.

11. The antigen-binding fragment of an antibody according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', and Fv.

12. The antigen-binding fragment of the antibody according to claim 1, wherein the antigen-binding fragment is scFv.

13. A humanized antibody containing a light chain variable region consisting of amino acid sequences 21-127 of SEQ ID NO: 13 and a heavy chain variable region consisting of amino acid sequences 20-146 of SEQ ID NO:

15.

14. A humanized antibody comprising a light chain consisting of amino acid sequences 21-233 of SEQ ID NO: 13, and a heavy chain consisting of amino acid sequences 20-476 of SEQ ID NO: 15, or a heavy chain having one or two amino acids deleted at its carboxyl terminus.

15. A humanized antibody containing a light chain variable region consisting of amino acid sequences 21-127 of SEQ ID NO: 14 and a heavy chain variable region consisting of amino acid sequences 20-146 of SEQ ID NO:

16.

16. A humanized antibody comprising a light chain consisting of amino acid sequences 21-233 of SEQ ID NO: 14, and a heavy chain consisting of amino acid sequences 20-476 of SEQ ID NO: 16, or a heavy chain having one or two amino acids deleted at its carboxyl terminus.

17. A polynucleotide encoding an antibody according to any one of claims 1 to 16, or an antigen-binding fragment of said antibody.

18. (1) to (2) below: (1) A polynucleotide encoding a light chain variable region including CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 1, CDRL2 consisting of the amino acid sequence (KAS) described in SEQ ID NO: 2, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 3, and a polynucleotide encoding a heavy chain variable region including CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 8, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO:

9. (2) A polynucleotide encoding a light chain variable region including CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 4, CDRL2 consisting of the amino acid sequence (FVS) described in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 6, and a polynucleotide encoding a heavy chain variable region including CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 10, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 11, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO:

12. The polynucleotide according to claim 17, comprising any one selected from the group consisting of the following.

19. The polynucleotide according to claim 17, comprising a polynucleotide encoding a light chain consisting of the amino acid sequence from 21 to 233 of SEQ ID NO: 13, and a polynucleotide encoding a heavy chain consisting of the amino acid sequence from 20 to 476 of SEQ ID NO: 15 or a heavy chain having one or two amino acids deleted at its carboxyl terminus.

20. The polynucleotide according to claim 17, comprising a polynucleotide encoding a light chain consisting of the amino acid sequence from 21 to 233 of SEQ ID NO: 14, and a polynucleotide encoding a heavy chain consisting of the amino acid sequence from 20 to 476 of SEQ ID NO: 16 or a heavy chain having one or two amino acids deleted at its carboxyl terminus.

21. An expression vector containing the polynucleotide described in claim 17.

22. A host cell transformed with the expression vector described in claim 21.

23. The host cell according to claim 22, wherein the host cell is a eukaryotic cell.

24. A method for producing an antibody or an antigen-binding fragment of an antibody, comprising the steps of culturing host cells as described in claim 22, and collecting a target antibody or an antigen-binding fragment of the antibody from the culture obtained in the said step.

25. An antibody or antigen-binding fragment of the antibody according to any one of claims 1 to 16, wherein the heavy chain or light chain has one or more modifications selected from the group consisting of addition of N-linked glycans, addition of O-linked glycans, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of a proline residue, pyroglutamine oxidation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus.

26. The antibody according to claim 25, wherein one amino acid is deleted at the carboxyl terminus of both heavy chains.

27. A humanized antibody according to any one of claims 13 to 16, wherein one amino acid is deleted at the carboxyl terminus of both heavy chains.

28. The humanized antibody according to claim 27, wherein the missing amino acid is lysine.

29. The antibody according to claim 25, wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.

30. An antibody or antigen-binding fragment of the antibody according to any one of claims 1 to 16, having glycosylation to enhance antibody-dependent cytotoxic activity.

31. An antibody-drug conjugate in which a drug is bound to an antibody or an antigen-binding fragment of the antibody according to any one of claims 1 to 16.

32. The antibody-drug conjugate according to claim 31, wherein the drug is one or more selected from the group consisting of substances having cytotoxic activity, cytotoxic compounds, substances having antitumor activity, antitumor compounds, chemotherapeutic agents, molecular targeted drugs, immunoactivators, immunosuppressants, toxins, photosensitive substances, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, vitamins, hormones, substances having an effect on blood disorders, substances having an effect on autoimmune diseases, anti-inflammatory substances, antibacterial substances, antifungal substances, antiparasitic substances, antiviral substances, and antianesthetic substances.

33. The antibody-drug conjugate according to claim 32, wherein the drug is a cytotoxic compound.

34. The cytotoxic compound is given by the following formula: 【Chemistry 1】 The antibody-drug conjugate according to claim 33, wherein the cytotoxic compound is represented by [formula].

35. Antibodies and drugs are expressed by the following equations (a) to (f): (a)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-、 (b)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-、 (c)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -O-CH 2 -C(=O)-、 (d)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 -O-CH 2 -C(=O)-、 (e)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-, and (f)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-、 The antibody-drug conjugate according to claim 31, which is bound via a linker of any structure selected from the group consisting of the above. (Here, the antibody is bound at the -(Succinimid-3-yl-N) terminus. The drug is bound to the carbonyl group at the other terminus. In the above formula, GGFG represents an amino acid sequence linked by a peptide bond consisting of glycine-glycine-phenylalanine-glycine.) -(Succinimid-3-yl-N)- is the following equation: 【Chemistry 2】 This is the structure shown, where the antibody is bound at position 3, and the nitrogen atom at position 1 is bound to a methylene group within the linker structure containing it.

36. The antibody-drug conjugate according to claim 31, wherein the linker is represented by any formula selected from the group consisting of (c), (d), and (e). (c)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -O-CH 2 -C(=O)-、 (d)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 -O-CH 2 -C(=O)-、 (e)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-。

37. The antibody-drug conjugate according to claim 31, wherein the linker is represented by the following formula (c) or (e). (c)-(Succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 -O-CH 2 -C(=O)-、 (e)-(Succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CH 2 -C(=O)-GGFG-NH-CH 2 CH 2 CH 2 -C(=O)-。

38. The following formula: 【Transformation 3】 The antibody-drug conjugate according to claim 31, having the structure shown. (Here, AB represents an antibody or an antigen-binding fragment of the antibody. n represents the average number of bindings per antibody or antigen-binding fragment of the drug-linker structure bound to the antibody or the antigen-binding fragment of the antibody. The antibody or the antigen-binding fragment and the linker are bound via sulfhydryl groups derived from the antibody.)

39. The following formula: 【Chemistry 4】 The antibody-drug conjugate according to claim 31, having the structure shown. (Here, AB represents the antibody or the antigen-binding fragment of the antibody. n represents the average number of bindings per antibody or antigen-binding fragment of the drug-linker structure bound to the antibody or the antigen-binding fragment of the antibody. The antibody or the antigen-binding fragment and the linker are bound via sulfhydryl groups derived from the antibody or the antigen-binding fragment of the antibody.)

40. The antibodies are as follows (1) to (2): (1) The light chain variable region consisting of amino acid sequences 21 to 127 of SEQ ID NO: 13 and the heavy chain variable region consisting of amino acid sequences 20 to 146 of SEQ ID NO: 15 (2) Light chain variable region consisting of amino acid sequences 21 to 127 of SEQ ID NO: 14 and heavy chain variable region consisting of amino acid sequences 20 to 146 of SEQ ID NO: 16 The antibody-drug conjugate according to claim 31, comprising an antibody or an antigen-binding fragment of said antibody, which includes a light chain variable region and a heavy chain variable region selected from the group consisting of said.

41. The antibody-drug conjugate according to claim 40, wherein the antibody is an antibody comprising a light chain variable region consisting of amino acid sequences 21 to 127 of SEQ ID NO: 13 and a heavy chain variable region consisting of amino acid sequences 20 to 146 of SEQ ID NO: 15, or an antigen-binding fragment of said antibody.

42. The antibody-drug conjugate according to claim 40, wherein the antibody is an antibody comprising a light chain variable region consisting of amino acid sequences 21 to 127 of SEQ ID NO: 14 and a heavy chain variable region consisting of amino acid sequences 20 to 146 of SEQ ID NO: 16, or an antigen-binding fragment of said antibody.

43. The antibodies are as follows (1) to (2): (1) A light chain consisting of amino acid sequences 21 to 233 of SEQ ID NO: 13, and a heavy chain consisting of amino acid sequences 20 to 476 of SEQ ID NO: 15, or a heavy chain in which one or two amino acids are deleted at the carboxyl terminus. (2) A light chain consisting of the amino acid sequence from position 21 to 233 of SEQ ID NO: 14, and a heavy chain consisting of the amino acid sequence from position 20 to 476 of SEQ ID NO: 16, or a heavy chain in which one or two amino acids are deleted at the carboxyl terminus. The antibody-drug conjugate according to claim 31, which is an antibody comprising a light chain and a heavy chain selected from any one of the group consisting of the above, or an antigen-binding fragment of said antibody.

44. The antibody-drug conjugate according to claim 43, wherein the antibody is an antibody comprising a light chain consisting of amino acid sequences 21 to 233 of SEQ ID NO: 13 and a heavy chain consisting of amino acid sequences 20 to 476 of SEQ ID NO: 15 or a heavy chain having one or two amino acids deleted at its carboxyl terminus, or an antigen-binding fragment of said antibody.

45. The antibody-drug conjugate according to claim 43, wherein the antibody is an antibody comprising a light chain consisting of amino acid sequences 21 to 233 of SEQ ID NO: 14 and a heavy chain consisting of amino acid sequences 20 to 476 of SEQ ID NO: 16 or a heavy chain having one or two amino acids deleted at its carboxyl terminus, or an antigen-binding fragment of said antibody.

46. The antibody-drug conjugate according to claim 31, wherein the heavy chain or light chain has one or more modifications selected from the group consisting of addition of N-linked glycans, addition of O-linked glycans, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of a proline residue, pyroglutamine oxidation of N-terminal glutamine or N-terminal glutamic acid, and deletion of one or two amino acids at the carboxyl terminus.

47. The antibody-drug conjugate according to claim 46, wherein one amino acid is deleted at the carboxyl terminus of both heavy chains.

48. The antibody-drug conjugate according to claim 46, wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.

49. The antibody-drug conjugate according to claim 31, having glycosylation to enhance antibody-dependent cytotoxic activity.

50. The antibody-drug conjugate according to claim 31, wherein the average number of drug-linker structures bound per antibody is in the range of 1 to 10.

51. The antibody-drug conjugate according to claim 50, wherein the average number of drug-linker structures bound per antibody is in the range of 2 to 8.

52. The antibody-drug conjugate according to claim 51, wherein the average number of drug-linker structures bound per antibody is in the range of 5 to 8.

53. The antibody-drug conjugate according to claim 52, wherein the average number of drug-linker structures bound per antibody is 7 to 8.

54. The following formula: 【Transformation 5】 An antibody-drug conjugate having the structure shown. (Here, AB represents a humanized antibody containing a light chain variable region consisting of amino acid sequences 21-127 of SEQ ID NO: 13 and a heavy chain variable region consisting of amino acid sequences 20-146 of SEQ ID NO:

15. n indicates the average number of drug-linker structures bound to the antibody per antibody, ranging from 7 to 8. The antibody and linker are bound via sulfhydryl groups derived from the antibody.)

55. The following formula: 【Transformation 6】 An antibody-drug conjugate having the structure shown. (Here, AB represents a humanized antibody containing a light chain consisting of amino acid sequences 21-233 of SEQ ID NO: 13, and a heavy chain consisting of amino acid sequences 20-476 of SEQ ID NO: 15, or a heavy chain with one or two amino acids deleted at its carboxyl terminus. n indicates the average number of drug-linker structures bound to the antibody per antibody, ranging from 7 to 8. The antibody and linker are bound via sulfhydryl groups derived from the antibody.)

56. The following formula: 【Transformation 7】 An antibody-drug conjugate having the structure shown. (Here, AB represents a humanized antibody containing a light chain variable region consisting of amino acid sequences 21-127 of SEQ ID NO: 14 and a heavy chain variable region consisting of amino acid sequences 20-146 of SEQ ID NO:

16. n represents the average number of drug-linker structures bound to the antibody per antibody, ranging from 7 to 8. The antibody and linker are bound via sulfhydryl groups derived from the antibody.)

57. The following formula: 【Transformation 8】 An antibody-drug conjugate having the structure shown. (Here, AB represents a humanized antibody containing a light chain consisting of amino acid sequences 21-233 of SEQ ID NO: 14, and a heavy chain consisting of amino acid sequences 20-476 of SEQ ID NO: 16, or a heavy chain with one or two amino acids deleted at its carboxyl terminus. n represents the average number of drug-linker structures bound to the antibody per antibody, ranging from 7 to 8. The antibody and linker are bound via sulfhydryl groups derived from the antibody.)

58. The following formula: 【Chemistry 9】 An antibody-drug conjugate having the structure shown. (Here, AB represents an antibody containing a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:

20. n represents the average number of drug-linker structures bound to the antibody per antibody, ranging from 7 to 8. The antibody and linker are bound via sulfhydryl groups derived from the antibody.)

59. The following formula: 【Chemistry 10】 An antibody-drug conjugate having the structure shown. (Here, AB represents an antibody containing a light chain consisting of amino acid sequences 21-232 of SEQ ID NO: 26, and a heavy chain consisting of amino acid sequences 20-476 of SEQ ID NO: 27, or a heavy chain with one or two amino acids deleted at its carboxyl terminus. n represents the average number of drug-linker structures bound to the antibody per antibody, ranging from 7 to 8. The antibody and linker are bound via sulfhydryl groups derived from the antibody.)

60. The antibody-drug conjugate according to any one of claims 54 to 59, wherein one amino acid is deleted at the carboxyl terminus of both heavy chains of the antibody.

61. The antibody-drug conjugate according to claim 60, wherein the missing amino acid is lysine.

62. A pharmaceutical composition characterized by comprising an antibody according to any one of claims 1 to 16 or an antigen-binding fragment of said antibody, or an antibody-drug conjugate according to any one of claims 54 to 59, a salt thereof, or a hydrate thereof.

63. The pharmaceutical composition according to claim 62, characterized in that it is an antitumor drug.

64. The pharmaceutical composition according to claim 63, characterized in that tumor cells express CD25, or tumor immunity is suppressed by regulatory T cells.

65. The pharmaceutical composition according to claim 64, characterized in that regulatory T cells are present in the tumor environment and / or in the lymph nodes.

66. The pharmaceutical composition according to claim 62, characterized in that the tumor is a blood cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, brain tumor, glioma, eye tumor, esophageal cancer, gastric cancer, colorectal cancer, colon cancer, liver cancer, pancreatic cancer, kidney cancer, renal cancer, bladder cancer, adrenocortical cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, melanoma, or sarcoma.

67. A pharmaceutical composition characterized by comprising the antibody-drug conjugate described in Claim 31, a salt thereof, or a hydrate thereof.

68. The pharmaceutical composition according to claim 67, characterized in that it is an antitumor drug.

69. The pharmaceutical composition according to claim 68, characterized in that tumor cells express CD25, or tumor immunity is suppressed by regulatory T cells.

70. The pharmaceutical composition according to claim 69, characterized in that regulatory T cells are present in the tumor environment and / or in the lymph nodes.

71. The pharmaceutical composition according to claim 67, characterized in that the tumor is a hematological cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, brain tumor, glioma, eye tumor, esophageal cancer, gastric cancer, colorectal cancer, colon cancer, liver cancer, pancreatic cancer, kidney cancer, renal cancer, bladder cancer, adrenocortical cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, melanoma, or sarcoma.

72. A method for producing an antibody-drug conjugate, comprising the steps of: culturing host cells as described in claim 22; collecting a target antibody or an antigen-binding fragment of the antibody from the culture obtained in the step; and reacting the antibody or antigen-binding fragment of the antibody obtained in the step with a drug-linker intermediate compound.

73. A pharmaceutical composition comprising the antibody-drug conjugate and immune checkpoint inhibitor described in claim 31, administered in combination.

74. The pharmaceutical composition according to claim 73, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are each contained as active ingredients in separate formulations and administered simultaneously or at different times.

75. The pharmaceutical composition according to claim 73, wherein the antibody-drug conjugate and the immune checkpoint inhibitor are contained as active ingredients in the same formulation.

76. The pharmaceutical composition according to claim 73, wherein the immune checkpoint inhibitor is one or more selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

77. The pharmaceutical composition according to claim 76, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

78. The pharmaceutical composition according to claim 77, wherein the anti-PD-1 antibody is one or more selected from the group consisting of nivolumab, pembrolizumab, cintilimab, spartalizumab, dostallimab, serpullimab, tislerizumab, penprimab, tripalimab, zimmerelimab, camrelizumab, retifanlimab, semiprimab, prolgolimab, geptanolimab, enlonstobart, QL-1604, pucotenlimab, and finotonlimab.

79. The pharmaceutical composition according to claim 77, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

80. The pharmaceutical composition according to claim 76, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

81. The pharmaceutical composition according to claim 80, wherein the anti-PD-L1 antibody is one or more selected from the group consisting of atezolizumab, durvalumab, cosibelimab, adebrelimab, sugemalimab, avelumab, socazolimab, KL-A167, and envafolimab.

82. The pharmaceutical composition according to claim 80, wherein the anti-PD-L1 antibody is one or more selected from the group consisting of atezolizumab, durvalumab, and avelumab.

83. The pharmaceutical composition according to claim 76, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

84. The pharmaceutical composition according to claim 83, wherein the anti-CTLA-4 antibody is one or more selected from the group consisting of ipilimumab, botensilimab, and tremelimumab.

85. The pharmaceutical composition according to claim 83, wherein the anti-CTLA-4 antibody is ipilimumab.

86. A pharmaceutical composition according to claim 73 for the treatment of cancer.

87. The pharmaceutical composition according to claim 73 for the treatment of at least one selected from the group consisting of blood cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, brain tumor, glioma, eye tumor, esophageal cancer, gastric cancer, colorectal cancer, colon cancer, liver cancer, pancreatic cancer, kidney cancer, renal cancer, bladder cancer, adrenocortical cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, melanoma, and sarcoma.

88. The pharmaceutical composition according to claim 73 for the treatment of cancer that is resistant to immune checkpoint inhibitors.

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