Cytotoxicity-inducing therapeutic agent

Abstract

To provide novel multispecific antigen-binding molecules that exhibit strong cytotoxicity against cells expressing DLL3.SOLUTION: Provided are multispecific antigen-binding molecules comprising (1) a first domain comprising a first antigen-binding domain that binds to human DLL3 and (2) a second domain comprising a second antigen-binding domain that binds to a T cell receptor complex, where the first antigen-binding domain of (1) binds to an epitope within a region defined by a specific amino acid sequence in human DLL3.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to multispecific antigen-binding molecules comprising a first domain comprising a first antigen-binding domain that binds to human DLL3 and a second domain comprising a second antigen-binding domain that binds to a T cell receptor complex, uses thereof, etc. The present invention also relates to novel monospecific antigen-binding molecules comprising an antigen-binding domain that binds to human DLL3, and uses thereof, etc. [Background technology]

[0002] Cancer is one of the leading causes of death worldwide. With the exception of certain carcinomas, tumors are often inoperable when they are discovered. Traditional cancer treatments include radiation therapy, chemotherapy, and immunotherapy. These treatments are often not sufficiently effective, and ultimately, cancer recurrence or metastasis occurs after treatment. Lack of tumor specificity is one of the factors limiting maximum efficacy; therefore, more tumor-specific molecular targeted therapy has become an additional viable option in cancer treatment.

[0003] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Among the various therapeutic antibodies, some types of antibodies require effector cells to exert antitumor responses. Antibody-dependent cellular cytotoxicity (ADCC) is a cytotoxicity mechanism in which effector cells exert cytotoxicity against antibody-bound cells through the binding of the Fc region of an antibody to Fc receptors present on NK cells and macrophages. To date, several therapeutic antibodies capable of inducing ADCC to exert antitumor effects have been developed as pharmaceuticals for treating cancer (Non-Patent Document 1). Therapies targeting tumor-specific antigens using conventional therapeutic antibodies have shown excellent antitumor activity, but administration of such antibodies has not always produced satisfactory results.

[0004] In addition to antibodies that employ ADCC by recruiting NK cells or macrophages as effector cells, T cell-recruiting antibodies (TR antibodies) that employ cytotoxicity by recruiting T cells as effector cells have been known since the 1980s (Non-Patent Documents 2-4). TR antibodies are bispecific antibodies that recognize and bind to one of the subunits forming the T cell receptor complex on T cells, particularly the CD3 epsilon chain, and an antigen on cancer cells. Several TR antibodies are currently under development. Catumaxomab, a TR antibody against EpCAM, has been approved in the EU for the treatment of malignant ascites. Furthermore, a type of TR antibody called a "bispecific T cell engager (BiTE)" has recently been found to exhibit potent antitumor activity (Non-Patent Documents 5 and 6). Blinatumomab, a BiTE molecule against CD19, was the first to receive FDA approval in 2014. Compared with rituximab, blinatumomab has been shown to exhibit much stronger cytotoxic activity against CD19 / CD20-positive cancer cells in vitro, and has been shown to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Non-Patent Document 7).

[0005] However, it is known that trifunctional antibodies simultaneously bind to both T cells and cells such as NK cells or macrophages in a cancer antigen-independent manner, resulting in cross-linking of receptors expressed on these cells and induction of the expression of various cytokines in a cancer antigen-independent manner. Systemic administration of trifunctional antibodies is thought to cause side effects such as cytokine storm as a result of the induction of such cytokine expression. In fact, in a phase I clinical trial, a very low dose of 5 micrograms (μg) / systemic administration was shown to be effective in treating non-small cell lung cancer. This is the maximum tolerated dose for systemic administration of catumaxomab to cancer patients, and administration of higher doses has been reported to cause various serious side effects (Non-Patent Document 8). When administered at such low doses, catumaxomab never reaches an effective blood level. In other words, administering catumaxomab at such low doses will not achieve the expected antitumor effect.

[0006] On the other hand, unlike catumaxomab, BiTEs lack Fc gamma receptor binding sites and therefore do not crosslink T cells with receptors expressed on cells such as NK cells and macrophages in a cancer antigen-independent manner. Thus, it has been demonstrated that BiTEs do not cause the cancer antigen-independent cytokine induction observed when catumaxomab is administered. However, because BiTEs are low-molecular-weight engineered antibody molecules lacking an Fc region, their blood half-life after administration to patients is significantly shorter than that of IgG antibodies traditionally used as therapeutic antibodies. In fact, the blood half-life of BiTEs administered in vivo has been reported to be approximately several hours (Non-Patent Documents 9 and 10). In clinical trials of blinatumomab, it was administered by continuous intravenous infusion using a minipump. This administration method is not only extremely inconvenient for patients but also carries the potential risk of medical accidents due to device failure. Thus, such an administration method is not desirable.

[0007] Delta-like protein 3 (DLL3) is a type I membrane protein belonging to the Notch ligand family. DLL3 is required for normal somitogenesis and patterning. Mutations in DLL3 cause rib defects or spondylolysis in patients with autosomal recessive spondylocostal dysostosis (Non-Patent Documents 11 and 12). Previous studies have reported chromosomal amplification of the DLL3 gene and increased expression of this gene in pancreatic cancer cell lines (Non-Patent Document 13) and increased DLL3 expression in some glioma cases (Non-Patent Document 14). Furthermore, DLL3 has previously been suggested in methods for diagnosing and treating glioma, in addition to SCLC, using ADCC-enhancing antibodies, antibody-drug conjugates (ADCs), and T-cell-engaging bispecific molecules using the BiTE-Fc format (Patent Documents 1, 2, and 3). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2011 / 093097 [Patent Document 2] WO2013 / 126746 [Patent Document 3] WO2017 / 021349 [Non-patent literature]

[0009] [Non-Patent Document 1] Clin Cancer Res.2010 Jan 1;16(1):11~20 pages [Non-patent document 2] Nature.1985 Apr 18~24;314(6012):628~31 pages [Non-patent document 3] Int J Cancer.1988 Apr 15;41(4):609~15 pages [Non-patent document 4] Proc Natl Acad Sci USA.1986 Mar;83(5):1453~7 pages [Non-Patent Document 5] Proc Natl Acad Sci USA.1995 Jul 18;92(15):7021~5 pages [Non-patent document 6] Drug Discov Today.2005 Sep 15;10(18):1237~44 pages [Non-Patent Document 7] Int J Cancer.2002 Aug 20;100(6):690~7 pages [Non-patent document 8] Cancer Immunol Immunother (2007) 56(10), pp. 1637-44 [Non-Patent Document 9] Cancer Immunol Immunother.(2006)55(5), pp.503-14 [Non-Patent Document 10] Cancer Immunol Immunother.(2009)58(1), pp.95-109 [Non-Patent Document 11] Bulman, MP et al. (2000) Nat Genet 24, 438-441 [Non-Patent Document 12] Turnpenny, PD et al. (2003) J Med Genet 40, 333-339 [Non-Patent Document 13] Phillips, HS (2006) Cancer Cell 9, pp. 157-173 [Non-Patent Document 14] Mulledndore, ME (2009) Clin Cancer Res 15, pp. 2291-2301 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a multispecific antigen-binding molecule that enables cancer treatment by bringing T cells into proximity with DLL3-expressing cells and utilizing the cytotoxicity of T cells against DLL3-expressing cancer cells, a method for producing the multispecific antigen-binding molecule, and a therapeutic agent comprising such a multispecific antigen-binding molecule as an active ingredient for inducing cancer cell cytotoxicity. Another object of the present invention is to provide a pharmaceutical composition for use in the treatment or prevention of various cancers, comprising one of the above-mentioned antigen-binding molecules as an active ingredient, and a therapeutic method using the pharmaceutical composition. Another object of the present invention is to provide a novel monospecific antigen-binding molecule having human DLL3-binding activity, a therapeutic agent comprising such a monospecific antigen-binding molecule as an active ingredient, and a therapeutic method using such a therapeutic agent. [Means for solving the problem]

[0011] The present inventors have found that a multispecific antigen-binding molecule comprising a first domain containing a first antigen-binding domain that binds to human DLL3 and a second domain containing a second antigen-binding domain that binds to the T cell receptor complex can damage cells expressing DLL3 and exhibit excellent cytotoxicity / anti-tumor activity. The present invention provides multispecific antigen-binding molecules and pharmaceutical compositions that contain the antigen-binding molecules as active ingredients and can treat various cancers, particularly cancers associated with DLL3, such as DLL3-positive tumors. The present invention also provides novel monospecific antigen-binding molecules containing an antigen-binding domain that binds to human DLL3, and pharmaceutical compositions containing such antigen-binding molecules.

[0012] More specifically, the present invention provides: [1] The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, A multispecific antigen-binding molecule in which the first antigen-binding domain of (1) binds to an epitope within the region defined by SEQ ID NO: 7 in human DLL3. [2] The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, The first antigen-binding domain of (1) is selected from the following (a1) to (a12): (a1) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 28, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (a2) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 33, an HVR-H2 sequence of SEQ ID NO: 34, an HVR-H3 sequence of SEQ ID NO: 35, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (a3) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 39, an HVR-H2 sequence of SEQ ID NO: 40, an HVR-H3 sequence of SEQ ID NO: 41, an HVR-L1 sequence of SEQ ID NO: 42, an HVR-L2 sequence of SEQ ID NO: 43, and an HVR-L3 sequence of SEQ ID NO: 44; (a4) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 45, an HVR-H2 sequence of SEQ ID NO: 46, an HVR-H3 sequence of SEQ ID NO: 47, an HVR-L1 sequence of SEQ ID NO: 48, an HVR-L2 sequence of SEQ ID NO: 49, and an HVR-L3 sequence of SEQ ID NO: 50; (a5) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 51, an HVR-H2 sequence of SEQ ID NO: 52, an HVR-H3 sequence of SEQ ID NO: 53, an HVR-L1 sequence of SEQ ID NO: 54, an HVR-L2 sequence of SEQ ID NO: 55, and an HVR-L3 sequence of SEQ ID NO: 56; (a6) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 75, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (a7) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 76, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (a8) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 79, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (a9) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 80, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (a10) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the HVR-H3 sequence of SEQ ID NO: 80, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L2 sequence of SEQ ID NO: 37, and the HVR-L3 sequence of SEQ ID NO: 81; (a11) An antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (a1) to (a10); (a12) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a10). A multispecific antigen-binding molecule comprising any one of: [3] The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, The first antigen-binding domain of (1) is selected from the following (b1) to (b21): (b1) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 15, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 15, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 15, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 16, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 16, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 16; (b2) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 25, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 25, and an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 25 an antibody variable fragment comprising an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 26, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 26, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 26; (b3) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 19, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 19, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 19, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 20, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 20, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 20; (b4) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 23, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 23, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 23, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 24, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 24, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 24; (b5) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 11, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 11, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 11, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 12, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 12, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 12; (b6) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 13, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 13, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 13, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 14, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 14, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 14; (b7) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 17, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 17, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 17, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 18, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 18, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 18; (b8) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 21, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 21, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 21, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 22, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 22, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 22; (b9) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 85, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 85, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 85, and an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 93; an antibody variable fragment comprising an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 93, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 93; (b10) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (b11) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (b12) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (b13) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 66, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b14) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b15) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 74; (b16) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, and an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73; and an antibody variable fragment comprising an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73; (b17) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 69, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 69, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b18) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 70, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b19) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 71, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b20) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (b1) to (b19); (b21) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (b1) to (b19). A multispecific antigen-binding molecule comprising any one of: [4] The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, The first antigen-binding domain of (1) is selected from the following (c1) to (c22): (c1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 16; (c2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; (c3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 20; (c4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 24; (c5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12; (c6) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 14; (c7) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 18; (c8) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22; (c9) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 85 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 93 a light chain variable region having the sequence: (c10) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c11) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 64 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c12) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c13) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 66 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c14) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c15) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 74; (c16) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 68 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c17) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c18) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 70 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c19) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 71 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c20) a heavy chain variable region having greater than 80% identity to any one of the heavy chain variable regions of (c1) to (c19), and a light chain variable region having greater than 80% identity to any one of the light chain variable regions of (c1) to (c19); (c21) a heavy chain variable region having greater than 90% identity to any one of the heavy chain variable regions of (c1) to (c19), and a light chain variable region having greater than 90% identity to any one of the light chain variable regions of (c1) to (c19); (c22) A heavy chain variable region having an identity of more than 95% to any one of the heavy chain variable regions of (c1) to (c19), and a light chain variable region having an identity of more than 95% to any one of the light chain variable regions of (c1) to (c19). A multispecific antigen-binding molecule comprising any one of the combinations of heavy chain variable regions and light chain variable regions selected from: [5] The multispecific antigen-binding molecule according to any one of [1] to [4], which has cytotoxic activity. [6] The multispecific antigen-binding molecule according to [5], wherein the cytotoxic activity is T cell-dependent cytotoxicity. [7] The multispecific antigen-binding molecule according to any one of [1] to [6], wherein the second antigen-binding domain in (2) binds to the CD3 epsilon chain. [8] The multispecific antigen-binding molecule according to any one of [1] to [6], wherein the second antigen-binding domain in (2) binds to a T cell receptor. [9] The second antigen-binding domain in (2) is selected from the following (d1) to (d12): (d1) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 57, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 58; (d2) HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions contained in SEQ ID NO: 98, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions contained in SEQ ID NO: 103, respectively. antibody variable fragments; (d3) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 99, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d4) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 100, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d5) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 101, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d6) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 102, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d7) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 298, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 299; (d8) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 300, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 301; (d9) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 302, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 303; (d10) HVR-H1, HVR-H2 and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2 and HVR-H3 regions included in any one of SEQ ID NOs: 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388 and 390; and an antibody variable fragment comprising HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2 and HVR-L3 regions contained in any one of SEQ ID NOs: 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389 and 391; (d11) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (d1) to (d10); (d12) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (d1) to (d10). The multispecific antigen-binding molecule according to any one of [1] to [8], comprising any one of:

[10] (2) wherein the second antigen-binding domain is one of the following (e1) to (e12): (e1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 57 and a light chain variable region having the amino acid sequence of SEQ ID NO: 58; (e2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 98 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 99 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 100 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 101 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e6) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e7) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 300 and a light chain variable region having the amino acid sequence of SEQ ID NO: 301; (e8) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 302 and a light chain variable region having the amino acid sequence of SEQ ID NO: 303; (e9) a heavy chain variable region and a light chain variable region having any one of the amino acid sequence combinations in Table 2A; (e10) a heavy chain variable region having greater than 80% identity to any one of the heavy chain variable regions of (e1) to (e9), and a light chain variable region having greater than 80% identity to any one of the light chain variable regions of (e1) to (e9); (e11) a heavy chain variable region having greater than 90% identity to any one of the heavy chain variable regions of (e1) to (e9), and a light chain variable region having greater than 90% identity to any one of the light chain variable regions of (e1) to (e9); (e12) A heavy chain variable region having an identity of more than 95% to any one of the heavy chain variable regions of (e1) to (e9), and a light chain variable region having an identity of more than 95% to any one of the light chain variable regions of (e1) to (e9). The multispecific antigen-binding molecule according to any one of [1] to [8], comprising any one of:

[11] (2) wherein the second antigen-binding domain is one of the following (j1) to (j5): (j1) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 136, an HVR-H2 sequence of SEQ ID NO: 137, an HVR-H3 sequence of SEQ ID NO: 138, an HVR-L1 sequence of SEQ ID NO: 139, an HVR-L2 sequence of SEQ ID NO: 140, and an HVR-L3 sequence of SEQ ID NO: 141; (j2) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 142, the HVR-H2 sequence of SEQ ID NO: 143, the HVR-H3 sequence of SEQ ID NO: 144, the HVR-L1 sequence of SEQ ID NO: 145, the HVR-L2 sequence of SEQ ID NO: 146, and the HVR-L3 sequence of SEQ ID NO: 147; (j3) an antibody variable fragment comprising HVR sequences selected from any of the combinations in Table 2B; (j4) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (j1) to (j3); (j5) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (j1) to (j3). The multispecific antigen-binding molecule according to any one of [1] to [8], comprising any one of:

[12] The multispecific antigen-binding molecule of any one of [1] to

[11] , wherein the first antigen-binding domain or the second antigen-binding domain is an antibody variable fragment, or wherein both the first and second antigen-binding domains are antibody variable fragments.

[13] The multispecific antigen-binding molecule according to

[12] , wherein the antibody variable fragment is Fab.

[14] (3) The multispecific antigen-binding molecule according to any one of [1] to

[13] , further comprising a third domain comprising an Fc region with reduced binding activity to an Fc gamma receptor.

[15] The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; and (3) a third domain containing an Fc region with reduced binding activity to Fc gamma receptors; A multispecific antigen-binding molecule comprising:

[16] The multispecific antigen-binding molecule of

[15] , wherein the first antigen-binding domain or the second antigen-binding domain is an antibody variable fragment, or both the first and second antigen-binding domains are antibody variable fragments.

[17] The multispecific antigen-binding molecule according to

[16] , wherein the antibody variable fragment is Fab.

[18] The multispecific antigen-binding molecule according to any one of

[14] to

[17] , wherein the Fc region has an amino acid mutation in any of the amino acids constituting the Fc region of SEQ ID NOs: 112 to 115 (IgG1 to IgG4).

[19] The Fc region comprises the following amino acid positions, as identified by EU numbering: 220th, 226th, 229th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 264th, 265th, 266th, 2 67th, 269th, 270th, 295th, 296th, 297th, 298th, 299th, 300th, 325th, 327th, 328th, 329th, 330th, 331st, and 332nd The multispecific antigen-binding molecule according to

[18] , wherein the Fc region has a mutation of at least one amino acid selected from the group consisting of:

[20] The multispecific antigen-binding molecule according to any one of [1] to

[19] , which is a bispecific antibody.

[21] The bispecific antibody according to

[20] , wherein the antibody is a monoclonal antibody.

[22] A pharmaceutical composition comprising the multispecific antigen-binding molecule according to any one of [1] to

[19] or the bispecific antibody according to

[20] or

[21] , and a pharmaceutically acceptable carrier.

[23] The pharmaceutical composition according to

[22] , which induces T cell-dependent cytotoxicity.

[24] A pharmaceutical composition for use in the treatment or prevention of cancer, comprising the multispecific antigen-binding molecule according to any one of [1] to

[19] or the bispecific antibody according to

[20] or

[21] .

[25] A method for treating or preventing cancer, comprising administering to a patient in need thereof an antigen-binding molecule according to any one of [1] to

[19] or a bispecific antibody according to

[20] or

[21] .

[26] Use of an antigen-binding molecule according to any one of [1] to

[19] or a bispecific antibody according to

[20] or

[21] in the manufacture of a pharmaceutical composition for treating or preventing cancer.

[27] Use of an antigen-binding molecule according to any one of [1] to

[19] or a bispecific antibody according to

[20] or

[21] for treating or preventing cancer.

[28] The method according to

[25] , wherein the cancer is lung cancer (including small cell lung cancer), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, adrenal cancer, kidney cancer, bladder cancer, uterine cancer, esophageal cancer, urothelial cancer, brain cancer, lymphoma, carcinoma, or sarcoma. .

[29] The use according to

[26] or

[27] , wherein the cancer is lung cancer (including small cell lung cancer), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, adrenal cancer, kidney cancer, bladder cancer, uterine cancer, esophageal cancer, urothelial cancer, brain cancer, lymphoma, carcinoma or sarcoma.

[30] The pharmaceutical composition according to

[24] , wherein the cancer is lung cancer (including small cell lung cancer), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, adrenal cancer, kidney cancer, bladder cancer, uterine cancer, esophageal cancer, urothelial cancer, brain cancer, lymphoma, carcinoma or sarcoma.

[31] An antigen-binding molecule that binds to an epitope within the region defined by SEQ ID NO: 7 in human DLL3.

[32] (f1)~(f11) below: (f1) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 28, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (f2) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 33, an HVR-H2 sequence of SEQ ID NO: 34, an HVR-H3 sequence of SEQ ID NO: 35, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (f3) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 39, an HVR-H2 sequence of SEQ ID NO: 40, an HVR-H3 sequence of SEQ ID NO: 41, an HVR-L1 sequence of SEQ ID NO: 42, an HVR-L2 sequence of SEQ ID NO: 43, and an HVR-L3 sequence of SEQ ID NO: 44; (f4) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 45, an HVR-H2 sequence of SEQ ID NO: 46, an HVR-H3 sequence of SEQ ID NO: 47, an HVR-L1 sequence of SEQ ID NO: 48, an HVR-L2 sequence of SEQ ID NO: 49, and an HVR-L3 sequence of SEQ ID NO: 50; (f5) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 75, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (f6) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 76, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (f7) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 79, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (f8) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 80, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (f9) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 80, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 81; (f10) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (f1) to (f9); (f11) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (f1) to (f9). An antigen-binding molecule comprising an antigen-binding domain comprising any one of:

[33] (g1)~(g20) below: (g1) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 15, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 15, and an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 15 an antibody variable fragment comprising an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 16, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 16, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 16; (g2) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 25, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 25, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 25, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 26, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 26, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 26; (g3) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 19, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 19, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 19, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 20, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 20, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 20; (g4) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 23, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 23, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 23, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 24, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 24, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 24; (g5) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 11, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 11, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 11, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 12, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 12, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 12; (g6) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 13, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 13, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 13, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 14, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 14, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 14; (g7) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 17, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 17, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 17, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 18, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 18, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 18; (g8) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 21, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 21, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 21, and an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 22; an antibody variable fragment comprising an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 22, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 22; (g9) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (g10) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (g11) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (g12) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 66, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g13) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g14) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 74; (g15) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, and an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73; and an antibody variable fragment comprising an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73; (g16) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 69, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 69, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g17) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 70, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g18) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 71, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g19) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (g1) to (g18); (g20) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (g1) to (g18). An antigen-binding molecule comprising an antigen-binding domain comprising any one of:

[34] (h1)~(h21) below: (h1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 16; (h2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; (h3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 20; (h4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 24; (h5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12; (h6) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 14; (h7) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 18; (h8) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22; (h9) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (h10) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 64 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (h11) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 72 a light chain variable region having the sequence (h12) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 66 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h13) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h14) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 74; (h15) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 68 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (h16) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h17) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 70 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h18) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 71 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h19) a heavy chain variable region having an identity of more than 80% to any one of the heavy chain variable regions of (h1) to (h18), and a light chain variable region having an identity of more than 80% to any one of the light chain variable regions of (h1) to (h18); (h20) a heavy chain variable region having an identity of more than 90% to any one of the heavy chain variable regions of (h1) to (h18), and a light chain variable region having an identity of more than 90% to any one of the light chain variable regions of (h1) to (h18); (h21) A heavy chain variable region having an identity of more than 95% to any one of the heavy chain variable regions of (h1) to (h18), and a light chain variable region having an identity of more than 95% to any one of the light chain variable regions of (h1) to (h18). An antigen-binding molecule comprising an antigen-binding domain comprising any one of:

[35] The antigen-binding molecule according to any one of

[31] to

[34] , which has cytotoxic activity.

[36] The antigen-binding molecule according to

[35] , wherein the cytotoxic activity is antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity.

[37] The antigen-binding molecule according to any one of

[31] to

[36] , which has internalization activity.

[38] The antigen-binding molecule according to any one of

[31] to

[37] , which is conjugated to a toxic compound.

[39] The antigen-binding molecule according to any one of

[31] to

[38] , which is an antibody variable fragment.

[40] The antigen-binding molecule according to

[39] , wherein the antibody variable fragment is Fab.

[41] The antigen-binding molecule according to any one of

[31] to

[40] , which is an antibody.

[42] The antigen-binding molecule according to

[41] , which is a monoclonal antibody.

[43] An antibody-drug conjugate compound comprising the antibody according to

[42] .

[44] A pharmaceutical composition comprising the antigen-binding molecule according to any one of

[31] to

[40] or the antibody according to

[41] or

[42] , and a pharmaceutically acceptable carrier.

[45] A pharmaceutical composition for use in the treatment or prevention of cancer, comprising the antigen-binding molecule according to any one of

[31] to

[40] or the antibody according to

[41] or

[42] .

[46] A method for treating or preventing cancer, comprising administering to a patient in need thereof an antigen-binding molecule according to any one of

[31] to

[40] or an antibody according to

[41] or

[42] .

[47] Use of an antigen-binding molecule according to any one of

[31] to

[40] or an antibody according to

[41] or

[42] in the manufacture of a pharmaceutical composition for treating or preventing cancer.

[48] ​​Use of an antigen-binding molecule according to any one of

[31] to

[40] or an antibody according to

[41] or

[42] for treating or preventing cancer.

[49] The method according to

[46] , wherein the cancer is lung cancer (including small cell lung cancer), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, adrenal cancer, kidney cancer, bladder cancer, uterine cancer, esophageal cancer, urothelial cancer, brain cancer, lymphoma, carcinoma or sarcoma.

[50] The use according to

[47] or

[48] , wherein the cancer is lung cancer (including small cell lung cancer), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, adrenal cancer, kidney cancer, bladder cancer, uterine cancer, esophageal cancer, urothelial cancer, brain cancer, lymphoma, carcinoma or sarcoma.

[51] The pharmaceutical composition according to

[45] , wherein the cancer is lung cancer (including small cell lung cancer), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, adrenal cancer, kidney cancer, bladder cancer, uterine cancer, esophageal cancer, urothelial cancer, brain cancer, lymphoma, carcinoma or sarcoma.

[52] A kit comprising the antigen-binding molecule according to any one of [1] to

[19] or the bispecific antibody according to

[20] or

[21] , and instructions for use.

[53] A kit comprising the antigen-binding molecule according to any one of

[31] to

[40] or the antibody according to

[41] or

[42] , and instructions for use. [Effects of the Invention]

[0013] The present invention provides multispecific antigen-binding molecules that enable cancer treatment by bringing T cells into contact with DLL3-expressing cells and using T cell cytotoxicity against DLL3-expressing cancer cells, methods for producing the multispecific antigen-binding molecules, and therapeutic agents containing such multispecific antigen-binding molecules as an active ingredient for inducing cytotoxicity, as a new approach to cancer treatment. The multispecific antigen-binding molecules of the present invention have potent antitumor activity that induces cytotoxicity and can target and damage DLL3-expressing cells, thereby enabling the treatment and prevention of various cancers. The present invention also provides novel monospecific antigen-binding molecules with human DLL3-binding activity, therapeutic agents containing such monospecific antigen-binding molecules as an active ingredient, and therapeutic methods using such therapeutic agents. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a schematic diagram showing the structures of full-length human DLL3 and the human DLL3 ECD fragment protein prepared in Example 1. The epitopes recognized by each of the anti-DLL3 antibodies selected in Example 3 are also shown. The EGF domain has six regions, EGF1 to EGF6, from the N-terminus to the C-terminus. [Figure 2] Figure 2 shows antibody binding to human DLL3 ECD fragment protein. The names of the anti-DLL3 antibodies tested are listed above each graph. [Figure 3] Figure 3 shows the expression of DLL3 in the SK-MEL-30 (A), NCI-H1436 (B), and NCI-H2227 (C) cancer cell lines. Black dotted line: secondary antibody (Beckman Coulter) only; gray solid line: control rabbit IgG (Cell Lab); black solid line: DLL3 antibody (bivalent DLA0316). [Figure 4] Figure 4 shows the T cell-dependent cytotoxicity (TDCC) of anti-DLL3 / CD3 bispecific antibodies (DL312 / No. 12, DLA0316 / No. 12, DLA0580 / No. 12, DLA0814 / No. 12, and DLA0769 / No. 12) against the SK-MEL-30 cell line. [Figure 5] FIG. 5 shows the TDCC of the anti-DLL3 / CD3 bispecific antibody (DLA0316 / No. 12) assessed by calcein release assay against NCI-H1436 (A) and NCI-H2227 (B) SCLC cell lines. [Figure 6] Figure 6 shows the in vivo antitumor efficacy of anti-DLL3 / CD3 bispecific antibodies (DLA0316 / No. 12, DLA0580 / No. 12, and DLA0841 / No. 12) in an SK-MEL-30 tumor-bearing T cell injection model. The antibodies and doses used are also shown. [Figure 7] Figure 7 shows the in vivo antitumor efficacy of the anti-DLL3 / CD3 bispecific antibody (DLA0316 / No. 12) in an SCLC tumor-bearing T cell infusion model. The cancer cell lines used were NCI-H1436 (A) and NCI-H2227 (B). [Figure 8] FIG. 8 shows the competition ratio of antibody B to antibody A. [Figure 9]Figure 9 shows the TDCC of humanized anti-DLL3 / CD3 bispecific antibodies against the SK-MEL-30 cell line. The anti-DLL3 arms of the bispecific antibodies tested were derived from DLA0136 or its humanized variant D30316AE03 (A), and DLA0841 or its humanized variants D30841AE08 and D30841AE11 (B), respectively. [Figure 10] Figure 10 shows the TDCC of humanized anti-DLL3 / CD3 bispecific antibodies against the SK-MEL-30 cell line. The graph shows SK-MEL-30 cytotoxicity when co-cultured with PBMCs at an E:T ratio of 5 in the presence of bispecific humanized DLL3 antibodies with various anti-CD3 arms at 0.001 nM to 10 nM. DETAILED DESCRIPTION OF THE INVENTION

[0015] The techniques and procedures described or referenced herein will generally be understood by those skilled in the art as described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M.A.usubel et al. (eds.), (2003)); Methods in Enzymology Series (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames, and G.R. Taylor, eds., 1995), Harlow and Lane, eds., (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed., (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-98) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immuno biology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty (ed.), IRL Press, 1988-1989); Monoclonal Antibodies: A Practical These are well understood and commonly used methods using conventional methods, such as the widely used methods described in *Antibodies: A Laboratory Manual* (P. Shepherd and C. Dean (eds.), Oxford University Press, 2000); *Using Antibodies: A Laboratory Manual* (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); *The Antibodies* (M. Zanetti and JD Capra (eds.), Harwood Academic Publishers, 1995); and *Cancer: Principles and Practice of Oncology* (V.T. DeVita et al. (eds.), J.B. Lippincott Company, 1993). The following definitions and detailed description are provided to facilitate understanding of the invention described herein.

[0016] "Amino acid sequence identity percentage (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, without considering any conservative substitution as part of the sequence identity.Alignment for determining amino acid sequence identity percentage can be achieved by various means within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.).Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the sequences to be compared.

[0017] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and its source code, along with user documentation, is on file with the U.S. Copyright Office, Washington, DC 20559, and is registered under U.S. Copyright Office Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a certain percent amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues in an alignment of A and B by the sequence alignment program ALIGN-2 that are scored as identical matches by the program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are based on the ALIGN-2 program. This is obtained using a computer program as described in the immediately preceding paragraph.

[0018] amino acid As used herein, amino acids are designated by one-letter or three-letter codes or both, e.g., Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V.

[0019] Amino acid changes To change the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, pp. 488-492)) and overlap extension PCR can be appropriately used. Furthermore, several known methods can be used as amino acid alteration methods for substituting unnatural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, pp. 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100(11), pp. 6353-6357). For example, it is preferable to use a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA having an unnatural amino acid bound to a complementary amber suppressor tRNA of one of the stop codons, the UAG codon (amber codon).

[0020] As used herein, the term "and / or" when describing the site of an amino acid change includes all suitable combinations of "and" and "or." Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes the following amino acid change variations: (a) 33, (b) 55, (c) 96, (d) 33 and 55, (e) 33 and 96, (f) 55 and 96, and (g) 33, 55, and 96.

[0021] Furthermore, herein, expressions indicating amino acid changes and expressions indicating positions before and after a number indicating a specific position may appropriately use the one-letter or three-letter codes of the amino acids before and after the change. For example, the change N100bL or Asn100bLeu, used when substituting an amino acid in an antibody variable region, indicates a substitution of Asn at position 100b (according to Kabat numbering) with Leu. That is, the number indicates the amino acid position according to Kabat numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution. Similarly, the change P238D or Pro238Asp, used when substituting an amino acid in an Fc region in an antibody constant region, indicates a substitution of Pro at position 238 (according to EU numbering) with Asp. That is, the number indicates the amino acid position according to EU numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0022] antigen binding molecule As used herein, the term "antigen-binding molecule" refers to any molecule containing an antigen-binding domain, and may further refer to molecules such as peptides or proteins having a length of about 5 amino acids or more. Peptides and proteins are not limited to those derived from organisms; for example, they may be polypeptides produced from artificially designed sequences. They may also be naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, etc.

[0023] A preferred example of the antigen-binding molecule of the present invention is an antigen-binding molecule comprising multiple antigen-binding domains. In certain embodiments, the antigen-binding molecule of the present invention comprises two antigen-binding domains with different antigen-binding specificities. In certain embodiments, the antigen-binding molecule of the present invention comprises two antigen-binding domains with different antigen-binding specificities and an FcRn-binding domain contained in the antibody Fc region. A well-known method for extending the blood half-life of a protein administered to a living body is to add an FcRn-binding domain of an antibody to the protein of interest and utilize the FcRn-mediated recycling function.

[0024] Another preferred example of the antigen-binding molecule of the present invention is an antigen-binding molecule that contains only one type of antigen-binding domain.In certain embodiments, the antigen-binding molecule of the present invention is an antigen-binding molecule that contains two antigen-binding domains with the same antigen-binding specificity.In certain embodiments, the antigen-binding molecule of the present invention is an antigen-binding molecule that contains two antigen-binding domains with the same antigen-binding specificity and an Fc region.

[0025] antigen-binding domain As used herein, the term "antigen-binding domain" refers to an antibody portion comprising a region that specifically binds to and is complementary to all or a portion of an antigen. When the antigen has a large molecular weight, the antibody can bind only to a specific portion of the antigen. This specific portion is called an "epitope." An antigen-binding domain can be provided by one or more antibody variable domains. Preferably, the antigen-binding domain contains both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen-binding domains include, for example, "single-chain Fv (scFv)," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," and "F(ab')2."

[0026] The antigen-binding domains of the antigen-binding molecules of the present invention may bind to the same epitope. The epitopes may be present in a protein comprising the amino acid sequence of SEQ ID NO: 9 or 111. Alternatively, the antigen-binding domains of the multispecific antigen-binding molecules of the present invention may individually bind to different epitopes. The epitopes may be present in a protein comprising the amino acid sequence of SEQ ID NO: 9 or 111.

[0027] The antigen-binding domain of the antigen-binding molecule of the present invention "binds to DLL3 or the T cell receptor complex." That is, DLL3 and the T cell receptor complex are preferred antigens of interest. As used herein, the phrase "binds to an antigen" refers to the binding activity of an antigen-binding domain, antibody, antigen-binding molecule, antibody variable fragment, etc. (hereinafter referred to as "antigen-binding domain, etc.") that binds to an antigen of interest at a level of specific binding that is higher than the level of non-specific binding or background binding. In other words, such an antigen-binding domain, etc., "specifically / significantly binds to" an antigen of interest. Specificity can be measured by any method for detecting affinity or binding activity described herein or known in the art. The above level of specific binding may be sufficiently high to be recognized as significant by those skilled in the art. For example, if a person skilled in the art can detect or observe a significant or relatively strong signal or value of binding between an antigen-binding domain, etc., and an antigen of interest in a suitable binding assay, the antigen-binding domain, etc., can be said to "specifically / significantly bind to" an antigen of interest. The phrase "binds to an antigen" can have substantially the same meaning in the art as the phrase "specifically / significantly binds to an antigen."

[0028] DLL3 As used herein, the term "DLL3" refers to any mammal, including, unless otherwise indicated, mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term "DLL3" refers to any native DLL3 (Delta-like protein 3) of vertebrate origin. The term encompasses "full-length," unprocessed DLL3 as well as any form of DLL3 that results from processing in the cell. The term also encompasses naturally occurring variants of DLL3, such as splice variants or allelic variants. The amino acid sequence of an exemplary human DLL3 is known as NCBI Reference Sequence (RefSeq) NM_016941.3, the amino acid sequence of an exemplary cynomolgus monkey DLL3 is known as NCBI Reference Sequence XP_005589253.1, and the amino acid sequence of an exemplary mouse DLL3 is known as NCBI Reference Sequence XP_005589253.1. It is known as Reference Sequence NM_007866.2. The amino acid sequence of cynomolgus monkey DLL3 used in the examples is shown in SEQ ID NO:8.

[0029] The human DLL3 protein contains a transmembrane (TM) region and an intracellular domain at the C-terminus, and a DSL (Notch) domain at the N-terminus (see, for example, Figure 1). Furthermore, DLL3 has six regions, EGF domains including EGF1 to EGF6, from the N-terminus to the C-terminus. In some embodiments, antigen-binding molecules or antibodies of the present invention bind to an epitope within the extracellular domain (ECD), i.e., the domain from the N-terminus to just before the TM region, rather than the TM region or the C-terminal intracellular domain. The molecules / antibodies of the present invention may bind to an epitope within any of the above domains / regions within the ECD. In a preferred embodiment, the molecules / antibodies of the present invention bind to an epitope within the region from EGF6 to just before the TM region. More specifically, the molecules / antibodies of the present invention can bind to an epitope within the region defined by SEQ ID NO: 7 in human DLL3. In some embodiments, the molecules / antibodies of the invention bind to the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region, or an epitope within the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region.

[0030] In human DLL3, the above domains / regions have the following amino acid residues (see, for example, www.uniprot.org / uniprot / Q9NYJ7 or WO2013 / 126746): Extracellular domain (ECD): amino acid residues 1 to 492; DSL domain: amino acid residues 176–215; EGF domain: amino acid residues 216 to 465; EGF1 region: amino acid residues 216 to 249; EGF2 region: amino acid residues 274 to 310; EGF3 region: amino acid residues 312 to 351; EGF4 region: amino acid residues 353 to 389; EGF5 region: amino acid residues 391 to 427; EGF6 region: amino acid residues 429 to 465; The region from EGF6 to just before the TM region: amino acid residues 429 to 492; TM region: amino acid residues 493 to 513; and C-terminal intracellular domain: amino acid residues at positions 516 to 618 (or positions 516 to 587 in some isoforms). The above amino acid positions also refer to the amino acid positions in the amino acid sequence set forth in SEQ ID NO: 9. Thus, the antigen-binding molecules or antibodies of the present invention can bind to the above region / domain having the amino acid residues at the above positions in human DLL3. That is, the antigen-binding molecules or antibodies of the present invention can bind to an epitope within the above region / domain having the amino acid residues at the above positions in human DLL3.

[0031] In some embodiments, due to their specificity, the antigen-binding molecules / antibodies of the present invention do not specifically bind to the above regions / domains of human DLL3 or epitopes within the above regions / domains of human DLL3. In some embodiments, the molecules / antibodies of the present invention do not specifically bind to an epitope within the region / domain having an amino acid residue at the position in human DLL3. In this context, "specifically" may be replaced with "substantially."

[0032] As used herein, the phrase "specifically binds to" refers to the activity of an antigen-binding molecule / antibody that binds to an antigen / region / domain / epitope of interest at a binding level that includes specific binding. As used herein, the phrase "specifically binds to" refers to the activity of an antigen-binding molecule / antibody that binds to an antigen / region / domain / epitope other than that of interest at a binding level that includes nonspecific binding or background binding, but does not include specific binding. Specificity can be measured by any method described herein or known in the art, for example, epitope mapping or competition assays described herein. The level of nonspecific binding or background binding may be zero, or may be close to zero but not zero, or may be so low that it is technically negligible by those skilled in the art. For example, if a person skilled in the art cannot detect or observe a significant or relatively strong signal for the binding of the molecule / antibody to the antigen / region / domain / epitope other than that of interest in a suitable binding assay, the molecule / antibody can be said to "not specifically bind to" the antigen / region / domain / epitope other than that of interest. The phrase "does not specifically bind to" may have substantially the same meaning in the art as the phrase "does not bind to."

[0033] The DLL3 used in the present invention may be a DLL3 protein having the above sequence, or may be a modified protein having a sequence derived from the above sequence by modifying one or more amino acids. Examples of modified proteins having a sequence derived from the above sequence by modifying one or more amino acids may include polypeptides having 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more identity to the above amino acid sequence. Alternatively, partial peptides of these DLL3 proteins can be used.

[0034] The origin of the DLL3 protein used in the present invention is not limited, and it is preferably a human or cynomolgus monkey DLL3 protein.

[0035] In some embodiments, a DLL3 ECD fragment protein (or ECD variant) can be used for the DLL3 protein. Depending on the truncation site, the fragment / variant may include, from the N-terminus to the C-terminus, the DSL domain to EGF6, EGF1 to EGF6, EGF2 to EGF6, EGF3 to EGF6, EGF4 to EGF6, EGF5 and EGF6, or EGF6. The fragment / variant may further include a region extending from immediately after the EGF6 region to immediately before the TM region. A Flag tag can be attached to the C-terminus of the fragment / variant using techniques well known in the art.

[0036] affinity "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule or antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific demonstrative and exemplary embodiments for measuring binding affinity are described below.

[0037] Methods for determining affinity In certain embodiments, the antigen-binding domain of an antigen-binding molecule or antibody provided herein has a specificity for its antigen of 1 μM or less, 120 nM or less, 100 nM or less, 80 nM or less, 70 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, 10 -8 M~10 -13 M, 10 -9 M~10 -13In certain embodiments, the Kd value of the first antigen-binding domain of the antibody / antigen-binding molecule for DLL3 is within the range of 1 to 40, 1 to 50, 1 to 70, 1 to 80, 30 to 50, 30 to 70, 30 to 80, 40 to 70, 40 to 80, or 60 to 80 nM.

[0038] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by binding the Fab to the antigen at a minimum concentration of ( 125 I) After equilibration with labeled antigen, the bound antigen is measured using a plate coated with an anti-Fab antibody to capture the antigen (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation may be continued for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., for 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20™) in PBS. When the plate has dried, 150 μL / well of scintillant (MICROSCINT-20™; Packard) is added, and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that give less than or equal to 20% of the maximum binding are selected for use in competitive binding assays.

[0039] According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using an immobilized antigen CM5 chip of approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. To achieve approximately 10 response units (RU) of coupled protein, the antigen is diluted to 5 μg / ml (approximately 0.2 μM) using 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 5 μL / min. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C. The binding rate (K on ) and dissociation rate (k off ) is calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated by the ratio K off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The surface plasmon resonance assay described above yields an on-rate of 10 6 M -1 s -1 If the concentration exceeds 100 ppm, a stopped-flow equipped spectrophotometer (Aviv) with a stirred cuvette is used. Binding rates can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nM, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of high concentrations of antigen, as measured in a spectrophotometer, such as a Thermo Instruments or 8000 series SLM-AMINCO™ spectrophotometer (Thermo Spectronic).

[0040] Methods for measuring the affinity of an antigen-binding domain of an antibody are described above, and one skilled in the art can perform affinity measurements for other antigen-binding domains.

[0041] antibody The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0042] In one aspect, the present invention provides multispecific antigen-binding molecules or antibodies. In some embodiments, the multispecific antigen binding molecule comprises: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, The first antigen-binding domain of (1) binds to an epitope within the region of human DLL3 defined by SEQ ID NO:7. In some embodiments, the first antigen-binding domain (1) in the multispecific antigen-binding molecule comprises one of the following (a1) to (a12): (a1) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 28, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (a2) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 33, an HVR-H2 sequence of SEQ ID NO: 34, an HVR-H3 sequence of SEQ ID NO: 35, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (a3) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 39, an HVR-H2 sequence of SEQ ID NO: 40, an HVR-H3 sequence of SEQ ID NO: 41, an HVR-L1 sequence of SEQ ID NO: 42, an HVR-L2 sequence of SEQ ID NO: 43, and an HVR-L3 sequence of SEQ ID NO: 44; (a4) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 45, an HVR-H2 sequence of SEQ ID NO: 46, an HVR-H3 sequence of SEQ ID NO: 47, an HVR-L1 sequence of SEQ ID NO: 48, an HVR-L2 sequence of SEQ ID NO: 49, and an HVR-L3 sequence of SEQ ID NO: 50; (a5) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 51, an HVR-H2 sequence of SEQ ID NO: 52, an HVR-H3 sequence of SEQ ID NO: 53, an HVR-L1 sequence of SEQ ID NO: 54, an HVR-L2 sequence of SEQ ID NO: 55, and an HVR-L3 sequence of SEQ ID NO: 56; (a6) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 75, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (a7) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 76, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (a8) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 79, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (a9) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 80, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (a10) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the HVR-H3 sequence of SEQ ID NO: 80, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L2 sequence of SEQ ID NO: 37, and the HVR-L3 sequence of SEQ ID NO: 81; (a11) An antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (a1) to (a10); (a12) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a10). It is one of the following. In some embodiments, the first antigen-binding domain of (1) in the multispecific antigen-binding molecule comprises one of the following (b1) to (b21): (b1) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 15, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 15, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 15, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 16, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 16, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 16; (b2) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 25, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 25, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 25, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 26, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 26, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 26; (b3) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 19, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 19, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 19, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 20, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 20, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 20; (b4) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 23, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 23, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 23, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 24, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 24, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 24; (b5) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 11, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 11, and an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 11 an antibody variable fragment comprising an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 12, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 12, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 12; (b6) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 13, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 13, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 13, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 14, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 14, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 14; (b7) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 17, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 17, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 17, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 18, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 18, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 18; (b8) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 21, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 21, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 21, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 22, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 22, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 22; (b9) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 85, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 85, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 85, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 93, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 93, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 93; (b10) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (b11) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (b12) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 65, and an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72 an antibody variable fragment comprising an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 72; (b13) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 66, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b14) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b15) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 74; (b16) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b17) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 69, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 69, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b18) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 70, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b19) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 71, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, and an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73; and an antibody variable fragment comprising an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73; (b20) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (b1) to (b19); (b21) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (b1) to (b19). It is one of the following. In some embodiments, the first antigen-binding domain (1) in the multispecific antigen-binding molecule comprises one of the following (c1) to (c22): (c1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 16; (c2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; (c3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 20; (c4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 24; (c5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12; (c6) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 14; (c7) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 18; (c8) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22; (c9) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 85 and a light chain variable region having the amino acid sequence of SEQ ID NO: 93; (c10) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c11) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 64 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c12) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c13) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 66 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c14) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c15) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 74; (c16) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 68 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c17) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c18) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 70 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c19) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 71 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c20) a heavy chain variable region having greater than 80% identity to any one of the heavy chain variable regions of (c1) to (c19), and a light chain variable region having greater than 80% identity to any one of the light chain variable regions of (c1) to (c19); (c21) a heavy chain variable region having greater than 90% identity to any one of the heavy chain variable regions of (c1) to (c19), and a light chain variable region having greater than 90% identity to any one of the light chain variable regions of (c1) to (c19); (c22) A heavy chain variable region having an identity of more than 95% to any one of the heavy chain variable regions of (c1) to (c19), and a light chain variable region having an identity of more than 95% to any one of the light chain variable regions of (c1) to (c19). The variable region comprises any one of the combinations of heavy chain and light chain variable regions selected from: In some embodiments, the multispecific antigen-binding molecule has cytotoxic activity. More specifically, the cytotoxic activity is T-cell dependent cytotoxicity (T-cell dependent cytotoxicity (TDCC)). In some embodiments, the second antigen-binding domain in (2) in the multispecific antigen-binding molecule binds to CD3. More specifically, in some embodiments, the second antigen-binding domain in (2) in the multispecific antigen-binding molecule binds to the CD3 epsilon chain. In some embodiments, the second antigen-binding domain in (2) in the multispecific antigen-binding molecule binds to a T cell receptor. In some embodiments, the second antigen-binding domain in (2) in the multispecific antigen-binding molecule comprises one of the following (d1) to (d12): (d1) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 57, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 58; (d2) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 98, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d3) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 99, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d4) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 100, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d5) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 101, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d6) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 102, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 103; (d7) HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions contained in SEQ ID NO: 298, respectively and HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2 and HVR-L3 regions, respectively, contained in SEQ ID NO: 299; (d8) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 300, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 301; (d9) an antibody variable fragment comprising HVR-H1, HVR-H2, and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2, and HVR-H3 regions, respectively, contained in SEQ ID NO: 302, and HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions, respectively, contained in SEQ ID NO: 303; (d10) HVR-H1, HVR-H2 and HVR-H3 sequences identical to the amino acid sequences of the HVR-H1, HVR-H2 and HVR-H3 regions included in any one of SEQ ID NOs: 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388 and 390; and an antibody variable fragment comprising HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2 and HVR-L3 regions contained in any one of SEQ ID NOs: 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389 and 391; (d11) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (d1) to (d10); (d12) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (d1) to (d10). It is one of the following. In some embodiments, the second antigen-binding domain in (2) in the multispecific antigen-binding molecule is selected from the following (e1) to (e12): (e1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 57 and a light chain variable region having the amino acid sequence of SEQ ID NO: 58; (e2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 98 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 99 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 100 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 101 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e6) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102 and a light chain variable region having the amino acid sequence of SEQ ID NO: 103; (e7) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 300 and a light chain variable region having the amino acid sequence of SEQ ID NO: 301; (e8) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 302 and a light chain variable region having the amino acid sequence of SEQ ID NO: 303; (e9) a heavy chain variable region and a light chain variable region having any one of the amino acid sequence combinations in Table 2A; (e10) a heavy chain variable region having greater than 80% identity to any one of the heavy chain variable regions of (e1) to (e9), and a light chain variable region having greater than 80% identity to any one of the light chain variable regions of (e1) to (e9); (e11) a heavy chain variable region having greater than 90% identity to any one of the heavy chain variable regions of (e1) to (e9), and a light chain variable region having greater than 90% identity to any one of the light chain variable regions of (e1) to (e9); (e12) A heavy chain variable region having an identity of more than 95% to any one of the heavy chain variable regions of (e1) to (e9), and a light chain variable region having an identity of more than 95% to any one of the light chain variable regions of (e1) to (e9). It is one of the following. In some embodiments, the second antigen-binding domain in (2) in the multispecific antigen-binding molecule is selected from the following (j1) to (j5): (j1) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 136, an HVR-H2 sequence of SEQ ID NO: 137, an HVR-H3 sequence of SEQ ID NO: 138, an HVR-L1 sequence of SEQ ID NO: 139, an HVR-L2 sequence of SEQ ID NO: 140, and an HVR-L3 sequence of SEQ ID NO: 141; (j2) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 142, the HVR-H2 sequence of SEQ ID NO: 143, the HVR-H3 sequence of SEQ ID NO: 144, the HVR-L1 sequence of SEQ ID NO: 145, the HVR-L2 sequence of SEQ ID NO: 146, and the HVR-L3 sequence of SEQ ID NO: 147; (j3) an antibody variable fragment comprising HVR sequences selected from any of the combinations in Table 2B; (j4) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (j1) to (j3); (j5) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (j1) to (j3). It is one of the following. In some embodiments, the multispecific antigen binding molecule comprises: (3) a third domain containing an Fc region with reduced binding activity to Fc gamma receptors; Further includes: In some embodiments, the present invention provides (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; and (3) a third domain containing an Fc region with reduced binding activity to Fc gamma receptors; The present invention provides a multispecific antigen-binding molecule comprising: In some embodiments, the Fc region in the multispecific antigen-binding molecule is an Fc region having an amino acid mutation in any of the amino acids constituting the Fc region of SEQ ID NOs: 112 to 115 (IgG1 to IgG4). In some embodiments, in the multispecific antigen binding molecule, the Fc region comprises the following amino acid positions identified by EU numbering: 220th, 226th, 229th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 264th, 265th, 266th, 2 67th, 269th, 270th, 295th, 296th, 297th, 298th, 299th, 300th, 325th, 327th, 328th, 329th, 330th, 331st, and 332nd The Fc region has at least one amino acid mutation selected from the group consisting of: In some embodiments, the multispecific antigen-binding molecule is a bispecific antibody. In some embodiments, the bispecific antibody is a monoclonal antibody. In a preferred embodiment, the multispecific antigen-binding molecule / bispecific antibody / monoclonal antibody has T cell-dependent cytotoxicity (TDCC) activity against cells expressing DLL3. Has.

[0043] In another aspect, the present invention provides an antigen-binding molecule that binds to an epitope within the region defined in SEQ ID NO:7 in human DLL3. In some embodiments, the antigen-binding molecule comprises one of the following (f1) to (f11): (f1) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 28, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (f2) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 33, an HVR-H2 sequence of SEQ ID NO: 34, an HVR-H3 sequence of SEQ ID NO: 35, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (f3) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 39, an HVR-H2 sequence of SEQ ID NO: 40, an HVR-H3 sequence of SEQ ID NO: 41, an HVR-L1 sequence of SEQ ID NO: 42, an HVR-L2 sequence of SEQ ID NO: 43, and an HVR-L3 sequence of SEQ ID NO: 44; (f4) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 45, an HVR-H2 sequence of SEQ ID NO: 46, an HVR-H3 sequence of SEQ ID NO: 47, an HVR-L1 sequence of SEQ ID NO: 48, an HVR-L2 sequence of SEQ ID NO: 49, and an HVR-L3 sequence of SEQ ID NO: 50; (f5) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 75, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (f6) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 27, an HVR-H2 sequence of SEQ ID NO: 76, an HVR-H3 sequence of SEQ ID NO: 29, an HVR-L1 sequence of SEQ ID NO: 30, an HVR-L2 sequence of SEQ ID NO: 31, and an HVR-L3 sequence of SEQ ID NO: 32; (f7) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 79, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (f8) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 80, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 38; (f9) an antibody variable fragment comprising an HVR-H1 sequence of SEQ ID NO: 77, an HVR-H2 sequence of SEQ ID NO: 78, an HVR-H3 sequence of SEQ ID NO: 80, an HVR-L1 sequence of SEQ ID NO: 36, an HVR-L2 sequence of SEQ ID NO: 37, and an HVR-L3 sequence of SEQ ID NO: 81; (f10) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (f1) to (f9); (f11) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (f1) to (f9). The antigen-binding domain comprises any one of: In some embodiments, the antigen-binding molecule comprises one of the following (g1) to (g20): (g1) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 15, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 15, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 15, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 16, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 16, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 16; (g2) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 25, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 25, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 25, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 26, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 26, and and an antibody variable fragment comprising an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 26; (g3) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 19, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 19, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 19, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 20, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 20, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 20; (g4) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 23, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 23, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 23, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 24, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 24, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 24; (g5) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 11, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 11, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 11, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 12, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 12, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 12; (g6) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 13, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 13, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 13, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 14, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 14, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 14; (g7) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 17, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 17, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 17, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 18, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 18, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 18; (g8) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 21, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 21, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 21, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 22, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 22, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 22; (g9) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72 an antibody variable fragment comprising: (g10) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (g11) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (g12) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 66, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g13) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g14) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 74; (g15) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g16) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 69, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 69, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g17) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 70, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g18) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 71, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (g19) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (g1) to (g18); (g20) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (g1) to (g18). The antigen-binding domain comprises any one of: In some embodiments, the antigen-binding molecule has the following structure (h1) to (h21): (h1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 16; (h2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; (h3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 20; (h4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 24; (h5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12; (h6) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 14; (h7) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 18; (h8) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22; (h9) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (h10) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 64 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (h11) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (h12) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 66 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h13) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h14) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 74; (h15) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 68 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (h16) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h17) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 70 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h18) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 71 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (h19) a heavy chain variable region having an identity of more than 80% to any one of the heavy chain variable regions of (h1) to (h18), and a light chain variable region having an identity of more than 80% to any one of the light chain variable regions of (h1) to (h18); (h20) a heavy chain variable region having an identity of more than 90% to any one of the heavy chain variable regions of (h1) to (h18), and a light chain variable region having an identity of more than 90% to any one of the light chain variable regions of (h1) to (h18); (h21) A heavy chain variable region having an identity of more than 95% to any one of the heavy chain variable regions of (h1) to (h18), and a light chain variable region having an identity of more than 95% to any one of the light chain variable regions of (h1) to (h18). The antigen-binding domain comprises any one of: In some embodiments, the antigen binding molecule has cytotoxic activity. In some embodiments, the cytotoxic activity of the antigen-binding molecule is antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. In some embodiments, the antigen binding molecule has internalization activity. In some embodiments, the antigen binding molecule is conjugated to a toxic compound. In some embodiments, the antigen-binding molecule is an antibody. In some embodiments, the antigen-binding molecule is a monoclonal antibody. In some embodiments, the present invention provides an antibody-drug conjugate compound comprising an antibody.

[0044] Antibody classes The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. In a preferred embodiment, the antibody of the present invention is an IgG type antibody.

[0045] Framework "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0046] Human Consensus Framework A "human consensus framework" is a framework that represents the amino acid residues most commonly occurring in selected human immunoglobulin VL or VH framework sequences. Typically, the selected human immunoglobulin VL or VH sequences are derived from a subgroup of variable domain sequences. Typically, the subgroup of sequences is selected from the set of sequences listed in Kabat et al., Sequences of Proteins of Immunological In subgroups such as those described in Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3 In one embodiment, for the VL, the subgroup is subgroup kappa I as described in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as described in Kabat et al., supra.

[0047] HVR As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Generally, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein are: (a) hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda MD (1991)); (c) antigenic contacts present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) a combination of (a), (b), and / or (c) comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Includes. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0048] Variable region The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using a VH or VL domain derived from an antibody that binds to that antigen, and a library of complementary VL or VH domains, respectively, can be screened. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0049] Chimeric antibodies The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy and / or light chain variable region is derived from a particular source or species, while the remaining portions of the heavy and / or light chain variable region are derived from a different source or species.

[0050] humanized antibodies A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may also comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0051] Human antibodies A "human antibody" is one having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequence. This definition of human antibody specifically excludes humanized antibodies, which comprise non-human antigen-binding residues. A "human antibody variable region" refers to the variable region of a human antibody.

[0052] Method for producing antibodies with desired binding activity Methods for producing antibodies with the desired binding activity are known to those skilled in the art. The following is an example describing a method for producing the above-mentioned antibody that binds to DLL3 (anti-DLL3 antibody). Antibodies that bind to T cell receptor complexes, etc., can also be produced as described below.

[0053] Anti-DLL3 antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. The preferred anti-DLL3 antibodies are monoclonal antibodies derived from mammals. Such mammalian monoclonal antibodies include antibodies produced by hybridomas or host cells transformed with an expression vector carrying an antibody gene by genetic engineering techniques.

[0054] Monoclonal antibody-producing hybridomas can be produced using known techniques, for example, as described below. Specifically, a mammal is immunized using a conventional immunization method with a DLL3 protein as a sensitizing antigen. The resulting immune cells are fused with known parent cells using a conventional cell fusion method. Hybridomas producing anti-DLL3 antibodies can then be selected by screening the monoclonal antibody-producing cells using conventional screening methods.

[0055] Specifically, monoclonal antibodies are prepared as follows. First, the DLL3 gene, whose nucleotide sequence is disclosed in NCBI Reference Sequence NM_016941.3 or XP005589253.1, can be expressed to produce a DLL3 protein used as a sensitizing antigen for antibody preparation. Alternatively, a polynucleotide encoding the extracellular domain (ECD) of DLL3 can be expressed to produce a DLL3 ECD-containing protein. That is, a gene sequence encoding full-length DLL3 or DLL3 ECD is inserted into a known expression vector, and an appropriate host cell is transformed with this vector. The desired full-length DLL3 or DLL3 ECD protein is purified from the host cell or its culture supernatant by known methods. Alternatively, purified natural DLL3 protein can be used as a sensitizing antigen. Purified full-length DLL3 or DLL3 ECD protein was used for immunization of mammals. The DLL3 peptide can be used as a sensitizing antigen for use in the treatment of HIV-1 infection. A partial peptide of the full-length DLL3 or the DLL3 ECD can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the DLL3 amino acid sequence. Furthermore, it can also be obtained by incorporating a portion of the DLL3 gene into an expression vector and expressing it. It can also be obtained by degrading the DLL3 protein using a protease. However, the region and size of the DLL3 peptide used as a partial peptide are not particularly limited in certain embodiments. Any sequence derived from the amino acid sequence can be selected as a preferred region. The number of amino acids constituting the peptide used as a sensitizing antigen is at least 5 or more, or preferably, for example, 6 or more, or 7 or more. More specifically, a peptide consisting of 8 to 50 residues, or preferably, 10 to 30 residues, can be used as a sensitizing antigen.

[0056] Alternatively, a fusion protein prepared by fusing a desired partial polypeptide or peptide of the full-length DLL3 or DLL3 ECD protein with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment and a peptide tag are preferably used to produce a fusion protein used as a sensitizing antigen. A vector for expressing such a fusion protein can be constructed by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into the above-mentioned expression vector. The method for producing a fusion protein is described in Molecular Cloning, 2nd Edition (Sambrook, J. et al., Molecular Cloning, 2nd Edition, 9.47-9.58 (1989) Cold Spring, NY). Methods for preparing DLL3 for use as a sensitizing antigen, and immunization methods using DLL3, are also described later in the Examples herein.

[0057] There is no particular limitation on the mammal to be immunized with the sensitizing antigen. However, it is preferable to select a mammal by considering compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

[0058] The animal is immunized with the sensitizing antigen by known methods. Common immunization methods include intraperitoneal or subcutaneous injection of the sensitizing antigen into a mammal. Specifically, the sensitizing antigen is appropriately diluted with phosphate-buffered saline (PBS), saline, or the like. If necessary, a conventional adjuvant, such as complete Freund's adjuvant, is mixed with the antigen, and the mixture is emulsified. The sensitizing antigen is then administered to the mammal several times at intervals of 4 to 21 days. An appropriate carrier can be used for immunization with the sensitizing antigen. Particularly when using a low-molecular-weight partial peptide as the sensitizing antigen, it may be desirable to couple the sensitizing antigen peptide to a carrier protein, such as albumin or keyhole limpet hemocyanin, for immunization.

[0059] Alternatively, hybridomas producing the desired antibodies can be prepared using DNA immunization, as described below. DNA immunization is an immunization method that provides immune stimulation by expressing a sensitizing antigen in an immunized animal as a result of administering a vector DNA that is constructed to enable the expression of a gene encoding an antigen protein in the animal. Compared to conventional immunization methods in which a protein antigen is administered to an animal to be immunized, DNA immunization has the following advantages: - can provide immune stimulation while preserving the structure of membrane proteins such as DLL3; and - No need to purify the antigen for immunization; It is expected to be superior in these respects.

[0060] To prepare the monoclonal antibodies of the present invention using DNA immunization, DNA expressing the DLL3 protein is first administered to the animal to be immunized. DNA encoding DLL3 can be synthesized by known methods such as PCR. The resulting DNA is inserted into an appropriate expression vector, which is then administered to the animal to be immunized. Preferred expression vectors include commercially available expression vectors such as pcDNA3.1. The vector can be administered to an organism using conventional methods. For example, DNA immunization is carried out by using a gene gun to introduce gold particles coated with the expression vector into cells in the body of the animal to be immunized. Antibodies that recognize DLL3 can also be produced by the method described in WO2011 / 093097.

[0061] After immunizing the above-mentioned mammal, confirm the increase in the titer of DLL3 binding antibody in serum.Then, collect immune cells from the mammal and then carry out cell fusion.In particular, splenocytes are preferably used as immune cells.

[0062] Mammalian myeloma cells are used as the cells to be fused with the immune cells. Myeloma cells preferably contain a suitable selection marker for screening. The selection marker confers a characteristic to the cells regarding their survival (or death) under specific culture conditions. Hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency) are known as selection markers. Cells with HGPRT or TK deficiency are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT selection medium and are thus killed. However, when cells are fused with normal cells, they can continue DNA synthesis using the salvage pathway of normal cells and therefore can grow even in HAT selection medium.

[0063] HGPRT-deficient and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells incorporate these pyrimidine analogs into their DNA and are killed. On the other hand, cells deficient in these enzymes cannot incorporate these pyrimidine analogs and can therefore survive in selective media. Furthermore, a selectable marker called G418 resistance, provided by the neomycin resistance gene, confers resistance to the antibiotic 2-deoxystrepamine (a gentamicin analog). Various types of myeloma cells suitable for cell fusion are known.

[0064] For example, myeloma cells including the following cells can be preferably used: P3(P3x63Ag8.653)(J.Immunol.(1979)123(4), pp. 1548-1550); P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, pp. 1-7); NS-1 (C.Eur.J.Immunol.(1976)6(7), pp. 511-519); MPC-11 (Cell (1976) 8(3), pp. 405-415); SP2 / 0 (Nature (1978) 276 (5685), pp. 269-270); FO (J. Immunol. Methods (1980) 35(1-2), pp. 1-21); S194 / 5.XXO.BU.1 (J.Exp.Med.(1978)148(1), pp. 313-323); R210 (Nature (1979) 277 (5692), pp. 131-133), etc.

[0065] The cell fusion between the immune cells and the myeloma cells is carried out using essentially known methods, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73:3-46). More specifically, cell fusion can be carried out in a conventional culture medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) and Sendai virus (HVJ). If necessary, auxiliary substances such as dimethyl sulfoxide can be added to improve fusion efficiency. The ratio of immune cells to myeloma cells may be determined at the discretion of those skilled in the art; for example, one myeloma cell for every 1 to 10 immune cells is preferred. Culture media used for cell fusion include media suitable for the growth of myeloma cell lines, such as RPMI1640 medium and MEM medium, as well as other conventional culture media used for this type of cell culture. Furthermore, it is preferable to add a serum additive, such as fetal calf serum (FCS), to the culture medium.

[0066] For cell fusion, a predetermined amount of the immune cells and myeloma cells is thoroughly mixed in the culture medium. Next, a PEG solution (e.g., with an average molecular weight of approximately 1,000-6,000) pre-warmed to approximately 37°C is added, typically at a concentration of 30%-60% (w / v). The mixture is gently mixed to produce the desired fused cells (hybridomas). The appropriate culture medium is then gradually added to the cells, and the mixture is repeatedly centrifuged to remove the supernatant. This process allows for the removal of cell fusion agents and other substances that are undesirable for hybridoma growth.

[0067] The hybridomas thus obtained can be selected by culturing them in a conventional selective medium, such as HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Cells other than the desired hybridoma (unfused cells) can be killed by continuing to culture them in the above-mentioned HAT medium for a sufficient period of time. Typically, this period is several days to several weeks. Hybridomas that produce the desired antibody are then screened and single-cloned by conventional limiting dilution.

[0068] The hybridomas thus obtained can be selected using a selective medium based on the selection marker possessed by the myeloma used for cell fusion. For example, HGPRT- or TK-deficient cells can be selected by culturing in HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Specifically, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can be selectively grown in HAT medium. Cells other than the desired hybridoma (unfused cells) can be killed by continuing to culture in the above-mentioned HAT medium for a sufficient period of time. Specifically, the desired hybridoma can be selected by culturing for a period of time generally ranging from several days to several weeks. Hybridomas producing the desired antibody are then screened and single cloned by conventional limiting dilution methods.

[0069] Preferably, the desired antibody can be selected by a screening method based on known antigen / antibody reaction and then single-cloned. For example, a DLL3-binding monoclonal antibody can bind to DLL3 expressed on the cell surface. Such a monoclonal antibody can be screened by fluorescence-activated cell sorting (FACS). FACS is a system that uses laser light to analyze cells contacted with fluorescent antibodies and measures the fluorescence emitted from individual cells to evaluate the binding of antibodies to cell surfaces.

[0070] To screen for hybridomas producing the monoclonal antibodies of the present invention by FACS, DLL3-expressing cells are first prepared. The cells preferably used for screening are mammalian cells in which DLL3 is forcibly expressed. As a control, the activity of an antibody that binds to cell surface DLL3 can be selectively detected using non-transformed mammalian cells as host cells. Specifically, hybridomas producing anti-DLL3 monoclonal antibodies can be isolated by selecting hybridomas that produce antibodies that bind to cells forcibly expressing DLL3 but do not bind to host cells.

[0071] Alternatively, the activity of antibodies binding to immobilized DLL3-expressing cells can be evaluated based on the principle of ELISA. For example, DLL3-expressing cells are immobilized in the wells of an ELISA plate. The hybridoma culture supernatant is contacted with the immobilized cells in the wells, and antibodies binding to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected using an anti-mouse immunoglobulin antibody. Hybridomas producing the desired antibody with antigen-binding ability can be selected by the above screening and then cloned by limiting dilution or other methods.

[0072] The monoclonal antibody-producing hybridomas thus prepared can be passaged in conventional culture media and stored in liquid nitrogen for long periods of time.

[0073] The hybridomas can be cultured by conventional methods, and the desired monoclonal antibodies can be prepared from the culture supernatant. Alternatively, the hybridomas can be administered to a suitable mammal, where they are allowed to grow, and the monoclonal antibodies can be prepared as ascites. The former method is suitable for preparing highly pure antibodies.

[0074] Preferably, antibodies encoded by antibody genes cloned from antibody-producing cells such as the above-mentioned hybridomas can also be used. The cloned antibody genes are inserted into an appropriate vector, which is then introduced into a host to express the antibody encoded by the genes. Methods for isolating antibody genes, inserting the genes into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192(3), pp. 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0075] Preferably, the present invention provides nucleic acids encoding the multispecific or monospecific antigen-binding molecules of the present invention. The present invention also provides vectors into which nucleic acids encoding the multispecific or monospecific antigen-binding molecules are introduced, i.e., vectors containing the nucleic acids. Furthermore, the present invention provides cells containing the nucleic acids or vectors. The present invention also provides methods for producing multispecific or monospecific antigen-binding molecules by culturing cells. The present invention further provides multispecific or monospecific antigen-binding molecules produced by the methods.

[0076] For example, cDNA encoding the variable region (V region) of an anti-DLL3 antibody is prepared from hybridoma cells expressing the anti-DLL3 antibody. To this end, total RNA is first extracted from the hybridoma. Methods used to extract mRNA from cells include, for example, - guanidine ultracentrifugation (Biochemistry (1979) 18(24), pp. 5294-5299), and - AGPC method (Anal.Biochem.(1987) 162(1), pp. 156-159) Examples include:

[0077] The extracted mRNA can be purified using an mRNA purification kit (GE Healthcare Bioscience) or similar. Alternatively, kits for extracting total mRNA directly from cells, such as the QuickPrep mRNA purification kit (GE Healthcare Bioscience), are commercially available. mRNA can be prepared from hybridomas using such kits. cDNA encoding the V region of an antibody can be synthesized from the prepared mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Co.) or similar. Furthermore, cDNA can be synthesized and amplified using the SMART RACE cDNA Amplification Kit (Clontech) and the PCR-based 5'-RACE method (Proc. Natl. Acad. Sci. USA (1988) 85(23), pp. 8998-9002; Nucleic Acids Res. (1989) 17(8), pp. 2919-2932). In such a cDNA synthesis process, suitable restriction enzyme sites, as described below, can be introduced at both ends of the cDNA.

[0078] The desired cDNA fragment is purified from the resulting PCR product and then ligated into vector DNA. Thus, a recombinant vector is constructed and introduced into E. coli or other bacteria. After colony selection, the desired recombinant vector can be prepared from the colony-forming E. coli. The recombinant vector is then tested for the presence of the desired cDNA nucleotide sequence by known methods, such as the dideoxynucleotide chain termination method.

[0079] The 5'-RACE method, which uses primers to amplify variable region genes, is conveniently used to isolate genes encoding variable regions.First, the 5'-RACE cDNA library is constructed by cDNA synthesis using RNA extracted from hybridoma cells as a template.The 5'-RACE cDNA library is synthesized by appropriately using a commercially available kit such as SMART RACE cDNA amplification kit.

[0080] The antibody genes are amplified by PCR using the prepared 5'-RACE cDNA library as a template. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. The nucleotide sequences of the primers vary depending on the immunoglobulin subclass. Therefore, it is preferable to determine the subclass in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0081] Specifically, the gene encoding mouse IgG is isolated using primers that allow amplification of genes encoding gamma 1, gamma 2a, gamma 2b, and gamma 3 heavy chains and kappa and lambda light chains. Generally, a primer annealing to the constant region site close to the variable region is used as the 3' primer to amplify the IgG variable region gene. Meanwhile, a primer attached to the 5' RACE cDNA library construction kit is used as the 5' primer.

[0082] The PCR product thus amplified is used to reconstitute immunoglobulins composed of a combination of heavy and light chains. The DL3-binding activity of the reconstituted immunoglobulins can be used as an index to select desired antibodies. For example, if the purpose is to isolate antibodies against DLL3, it is more preferable that the antibody binds specifically to DLL3. DLL3-binding antibodies can be isolated, for example, by the following steps: (1) contacting a DLL3-expressing cell with an antibody comprising a V region encoded by a cDNA isolated from a hybridoma; (2) detecting binding of the antibody to the DLL3-expressing cells; and (3) Screening can be carried out by selecting an antibody that binds to DLL3-expressing cells.

[0083] Methods for detecting the binding of antibodies to DLL3-expressing cells are known. Specifically, the binding of antibodies to DLL3-expressing cells can be detected by the above-mentioned techniques such as FACS. The binding activity of antibodies is evaluated by appropriately using a sample of DLL3-expressing cells immobilized on a solid phase.

[0084] A preferred antibody screening method using binding activity as an index also includes a panning method using a phage vector. Screening methods using a phage vector are advantageous when antibody genes are isolated from heavy and light chain subclass libraries derived from a cell population expressing polyclonal antibodies. Genes encoding heavy and light chain variable regions can be linked with an appropriate linker sequence to form a single-chain Fv (scFv). Phages displaying scFvs on their surface can be generated by inserting a gene encoding the scFv into a phage vector. The phage is contacted with the antigen of interest. DNA encoding the scFv with the desired binding activity can then be isolated by collecting the phage bound to the antigen. This process can be repeated as necessary to enrich for scFvs with the desired binding activity.

[0085] After isolating cDNA encoding the V region of the anti-DLL3 antibody of interest, the cDNA is digested with restriction enzymes that recognize the restriction sites introduced at both ends of the cDNA. Preferred restriction enzymes are those that recognize and cleave nucleotide sequences that occur at low frequencies in the nucleotide sequence of the antibody gene. Furthermore, restriction sites for enzymes that create cohesive ends are preferably introduced into the vector to allow insertion of a single copy of the digested fragment in the correct orientation. The cDNA encoding the V region of the anti-DLL3 antibody is digested as described above and inserted into an appropriate expression vector to construct an antibody expression vector. In this case, a chimeric antibody is obtained when the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in frame. As used herein, the term "chimeric antibody" refers to an antibody in which the origin of the constant region is different from that of the variable region. Thus, in addition to mouse / human heterogeneous chimeric antibodies, human / human homogeneous chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region genes described above into an expression vector that already contains the constant region. Specifically, for example, the recognition sequence for the restriction enzyme that excises the V region gene can be appropriately placed at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusing two genes digested with the same combination of restriction enzymes in frame.

[0086] To produce an anti-DLL3 monoclonal antibody, the antibody gene is inserted into an expression vector so that the gene is expressed under the control of an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. Furthermore, an appropriate signal sequence may be attached to the amino terminus so that the expressed antibody is secreted outside the cell. Alternatively, other appropriate signal sequences may be attached. The expressed polypeptide is cleaved at the carboxyl terminus of the sequence, and the resulting polypeptide is secreted outside the cell as a mature polypeptide. Then, an appropriate host cell is transformed with the expression vector to obtain a recombinant cell expressing DNA encoding the anti-DLL3 antibody.

[0087] The DNA encoding the antibody heavy chain (H chain) and light chain (L chain) are separately inserted into different expression vectors to express the antibody genes. An antibody molecule having H and L chains can be expressed by co-transfecting vectors carrying the H chain gene and the L chain gene into the same host cell. Alternatively, the host cell can be transfected with vectors carrying the H and L chain genes. The encoding DNA can be inserted into a single expression vector and transformed (see WO94 / 11523).

[0088] There are a variety of known host cell / expression vector combinations for preparing antibodies by introducing isolated antibody genes into a suitable host. All of these expression systems are applicable to the isolation of domains containing the antibody variable regions of the present invention. Suitable eukaryotic cells for use as host cells include animal cells, plant cells, and fungal cells. Specifically, animal cells include, for example, the following cells: (1) Mammalian cells: CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero, etc.; (2) amphibian cells, such as Xenopus oocytes; and (3) Insect cells: sf9, sf21, Tn5, etc.

[0089] Furthermore, antibody gene expression systems using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, are known. Callus culture cells can be suitably used to transform plant cells.

[0090] Additionally, the following cells can be used as fungal cells: Yeasts: Saccharomyces, such as Saccharomyces cerevisiae, and Pichia, such as Pichia pastoris; and Filamentous fungi: Aspergillus genus, such as Aspergillus niger.

[0091] Furthermore, antibody gene expression systems using prokaryotic cells are also known. For example, when bacterial cells are used, E. coli cells, Bacillus subtilis cells, and the like can be suitably used in the present invention. An expression vector carrying the antibody gene of interest is introduced into these cells by transfection. The transfected cells are cultured in vitro, and the desired antibody can be prepared from the culture of the transformed cells.

[0092] In addition to the host cells described above, recombinant antibodies can also be produced using transgenic animals. That is, antibodies can be obtained from animals transfected with a gene encoding the desired antibody. For example, an antibody gene can be constructed as a fusion gene by inserting in-frame a gene encoding a protein specifically produced in milk. For example, caprine beta-casein can be used as a protein secreted into milk. A DNA fragment containing the fusion gene inserted together with the antibody gene is injected into a goat embryo, and the embryo is then introduced into a female goat. The desired antibody can be obtained as a fusion protein with a milk protein from the milk produced by the transgenic goat born from the recipient goat (or its offspring). Furthermore, hormones can be administered to the transgenic goat as needed to increase the volume of milk containing the desired antibody produced by the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12(7), pp. 699-702).

[0093] Methods for producing humanized antibodies When the antigen-binding molecules described herein are administered to humans, domains derived from genetically engineered antibodies that have been artificially modified to reduce heterologous antigenicity to humans or the like can be appropriately used as domains of the antigen-binding molecules containing antibody variable regions. Examples of such genetically engineered antibodies include humanized antibodies. These modified antibodies can be prepared using known Furthermore, the binding specificity of one antibody can generally be transferred to another antibody by CDR grafting.

[0094] Specifically, humanized antibodies are known, which are prepared by grafting the CDRs (or "HVRs" as defined herein) of non-human animal antibodies, such as mouse antibodies, onto human antibodies, etc. General genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known as a method for grafting mouse antibody CDRs onto human FRs. In overlap extension PCR, nucleotide sequences encoding the mouse antibody CDRs to be grafted are added to primers for synthesizing human antibody FRs. Primers are prepared for each of the four FRs. When grafting mouse CDRs onto human FRs, it is generally considered advantageous to select human FRs that are highly identical to the mouse FRs in order to maintain CDR function. That is, it is generally preferred to use human FRs that contain amino acid sequences that are highly identical to the amino acid sequences of the FRs adjacent to the mouse CDRs to be grafted.

[0095] The nucleotide sequences to be ligated are designed so that they are connected in frame with each other.Human FRs are synthesized separately using each primer.As a result, a product is obtained in which the DNA encoding mouse CDRs is linked to the DNA encoding each FR.The nucleotide sequences encoding mouse CDRs of each product are designed so that they overlap with each other.Then, complementary strand synthesis reaction is carried out to anneal the overlapping CDR regions of the products synthesized using human antibody genes as templates.Human FRs are ligated through the mouse CDR sequences by this reaction.

[0096] The full-length V region gene, in which three CDRs and four FRs are finally ligated, is amplified using a primer annealing to its 5' or 3' end, which is added with a suitable restriction enzyme recognition sequence. The DNA obtained as described above and DNA encoding the human antibody C region are inserted into an expression vector so that they are ligated in frame, thereby producing an expression vector for a humanized antibody. After transfecting the recombinant vector into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, thereby producing a humanized antibody in cell culture (see European Patent Application Publication No. EP239400 and International Patent Application Publication No. WO1996 / 002576).

[0097] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody produced as described above, it is possible to select suitable human antibody FRs whose CDRs can form a suitable antigen-binding site when ligated via the CDRs. Amino acid residues in the FRs can be substituted as necessary so that the reconstructed CDRs of the human antibody form a suitable antigen-binding site. For example, amino acid sequence mutations can be introduced into the FRs by applying the PCR method used to graft mouse CDRs onto human FRs. More specifically, partial nucleotide sequence mutations can be introduced into primers annealing to the FRs. The nucleotide sequence mutations are introduced into the FRs synthesized using such primers. By measuring and evaluating the antigen-binding activity of antibody mutants with amino acid substitutions using the above method, FR sequence mutants with desired characteristics can be selected (Sato, K. et al., Cancer Res. (1993) 53:851-856).

[0098] Methods for producing human antibodies Alternatively, transgenic animals carrying a full repertoire of human antibody genes can be immunized by DNA immunization to obtain the desired human antibodies (WO1993 / 012227; WO1992 / 003918; WO1994 / 002602; W (See WO1994 / 025585; WO1996 / 034096; WO1996 / 033735).

[0099] Furthermore, techniques for preparing human antibodies by panning using a human antibody library are also known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage by phage display. Phages expressing scFvs that bind to an antigen can be selected. The DNA sequence encoding the V region of a human antibody that binds to the antigen can be determined by analyzing the genes of the selected phage. The DNA sequence of the scFv that binds to the antigen is determined. An expression vector is prepared by fusing the V region sequence in frame with the C region sequence of a desired human antibody and inserting it into an appropriate expression vector. The expression vector is introduced into a cell suitable for expression, such as those described above. Human antibodies can be produced by expressing genes encoding human antibodies in the cells. These methods are already known (see WO1992 / 001047; WO1992 / 020791; WO1993 / 006213; WO1993 / 011236; WO1993 / 019172; WO1995 / 001438; WO1995 / 015388).

[0100] vector As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0101] host cell The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom, regardless of the number of transfers. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are also included herein.

[0102] epitope "Epitope" refers to an antigenic determinant in an antigen and refers to the antigenic site to which the antigen-binding domain of an antigen-binding molecule or antibody disclosed herein binds. Thus, for example, an epitope can be defined according to its structure. Alternatively, an epitope can be defined according to the antigen-binding activity of an antigen-binding molecule or antibody that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that form the epitope. Alternatively, when the epitope is a carbohydrate chain, the epitope can be specified by its specific carbohydrate structure.

[0103] A linear epitope is one that contains a recognized primary amino acid sequence, typically at least three, most commonly at least five, e.g., about 8-10 or 6-20 amino acids in that particular sequence.

[0104] In contrast to a linear epitope, a "conformational epitope" is an epitope in which the primary amino acid sequence comprising the epitope is not the sole determinant of the recognized epitope (e.g., the primary amino acid sequence of a conformational epitope is not necessarily recognized by the antibody that defines the epitope). Conformational epitopes contain a greater number of amino acids than linear epitopes. The antigen-binding domain that recognizes a conformational epitope may recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds and forms a three-dimensional structure, the amino acids and / or polypeptide backbone that form the conformational epitope are aligned, and the epitope can be recognized by the antigen-binding domain. Methods for determining epitope conformation include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-directed spin labeling, and electron paramagnetic resonance. For example, see Epitope Mapping Protocols in See Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0105] An example of a method for assessing epitope binding by a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain is described below. According to the following example, a method for assessing epitope binding by a test antigen-binding molecule or antibody containing an antigen-binding domain for an antigen other than DLL3 can also be performed appropriately.

[0106] For example, whether a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain recognizes a linear epitope in a DLL molecule can be confirmed, for example, as described below. A linear peptide containing the amino acid sequence forming the extracellular domain of DLL3 is synthesized for the above purpose. The peptide can be chemically synthesized or obtained by genetic engineering using a region in DLL3 cDNA encoding the amino acid sequence corresponding to the extracellular domain. The test antigen-binding molecule or antibody containing the anti-DLL3 antigen-binding domain is then evaluated for its binding activity to the linear peptide containing the amino acid sequence forming the extracellular domain. For example, the immobilized linear peptide can be used as an antigen in ELISA to evaluate the binding activity of the polypeptide complex to the peptide. Alternatively, the binding activity to the linear peptide can be evaluated based on the level at which the linear peptide inhibits the binding of the antigen-binding molecule or antibody to DLL3-expressing cells. These tests can demonstrate the binding activity of the antigen-binding molecule or antibody to the linear peptide.

[0107] Whether a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain recognizes a conformational epitope can be assessed as follows: DLL3-expressing cells are prepared for the above purpose. A test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain can be determined to recognize a conformational epitope if it strongly binds to DLL3-expressing cells upon contact and does not substantially bind to an immobilized linear peptide containing an amino acid sequence forming the extracellular domain of DLL3. As used herein, "does not substantially bind" means that the binding activity is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less, compared to the binding activity to DLL3-expressing cells.

[0108] Methods for assaying the binding activity of a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain to DLL3-expressing cells include, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) pp. 359-420). Specifically, evaluation can be performed based on the principles of ELISA or fluorescence-activated cell sorting (FACS) using DLL3-expressing cells as antigens.

[0109] In the ELISA format, the binding activity of a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain to DLL3-expressing cells can be quantitatively evaluated by comparing the level of the signal generated by the enzyme reaction. The tide complex is added to an ELISA plate on which DLL3-expressing cells are immobilized. The test antigen-binding molecule or antibody bound to the cells is then detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule or antibody. Alternatively, when using FACS, a dilution series of the test antigen-binding molecule or antibody can be prepared, and the antibody binding titer for DLL3-expressing cells can be determined to compare the binding activity of the test antigen-binding molecule or antibody to DLL3-expressing cells.

[0110] The binding of a test antigen-binding molecule or antibody to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices: FACSCanto™ II FACSAria™ FACSArray™ FACSVantage™ SE FACSCalibur™ (all trademarks of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (all trademarks of Beckman Coulter) Examples include:

[0111] A preferred method for assaying the binding activity of a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain to an antigen is, for example, the following method. First, DLL3-expressing cells are reacted with the test antigen-binding molecule or antibody, and then stained with an FITC-labeled secondary antibody that recognizes the antigen-binding molecule or antibody. The test antigen-binding molecule or antibody is appropriately diluted with a suitable buffer to prepare the antigen-binding molecule or antibody at the desired concentration. For example, the antigen-binding molecule or antibody can be used at a concentration ranging from 10 μg / ml to 10 ng / ml. The fluorescence intensity and cell number are then determined using a FACSCalibur (BD). The fluorescence intensity, i.e., the geometric mean value, obtained by analysis using CELL QUEST Software (BD) reflects the amount of antibody bound to the cells. That is, the binding activity of the test antigen-binding molecule or antibody, represented by the amount of bound test antigen-binding molecule or antibody, can be determined by measuring the geometric mean value.

[0112] Whether the test antigen-binding molecule or antibody that contains anti-DLL3 antigen binding domain shares a common epitope with another antigen-binding molecule or antibody can be evaluated based on the competition between two antigen-binding molecules or antibodies for the same epitope.The competition between antigen-binding molecules or antibodies can be detected by cross-blocking assays, etc.For example, competitive ELISA assays are preferred cross-blocking assays.

[0113] Specifically, in a cross-blocking assay, DLL3 protein immobilized on the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competitor antigen-binding molecule or antibody, and then a test antigen-binding molecule or antibody is added. The amount of test antigen-binding molecule or antibody bound to DLL3 protein in the well indirectly correlates with the binding ability of the candidate competitor antigen-binding molecule or antibody that competes for binding to the same epitope. That is, the higher the affinity of the competitor antigen-binding molecule or antibody for the same epitope, the lower the binding activity of the test antigen-binding molecule or antibody to the DLL3 protein-coated wells.

[0114] The amount of test antigen-binding molecule or antibody bound to the well via DLL3 protein can be easily determined by labeling the antigen-binding molecule or antibody in advance.For example, biotin-labeled antigen-binding molecule or antibody is measured using avidin / peroxidase conjugate and an appropriate substrate.In particular, cross-blocking assays using enzyme labels such as peroxidase are called "competitive ELISA assays."Antigen-binding molecules or antibodies can also be labeled with other labeling substances that allow detection or measurement.Specifically, radiolabels, fluorescent labels, etc. are known.

[0115] When a candidate competitor antigen-binding molecule or antibody can block binding by a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity in a control experiment performed in the absence of the competitor antigen-binding molecule or antibody, the test antigen-binding molecule or antibody is determined to substantially bind to the same epitope as the competitor antigen-binding molecule or antibody, or to compete for binding to the same epitope.

[0116] When the structure of the epitope bound by a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain has already been identified, whether the test and control antigen-binding molecules or antibodies share a common epitope can be evaluated by comparing the binding activity of the two antigen-binding molecules or antibodies against a peptide prepared by introducing amino acid mutations into the peptide that forms the epitope.

[0117] The above-mentioned binding activity, for example, the binding activity of test and control antigen-binding molecules or antibodies to the linear peptide into which mutations have been introduced, is measured and compared using the above-mentioned ELISA format. In addition to the ELISA method, the binding activity to the peptide variant bound to the column can be determined by passing the test and control antigen-binding molecules or antibodies through a column and then quantifying the antigen-binding molecules or antibodies eluted in the elution solution. For example, methods for adsorbing peptide variants to a column in the form of GST-fusion peptides are known.

[0118] Alternatively, if the identified epitope is a conformational epitope, the following method can be used to assess whether the test and control antigen-binding molecules or antibodies share a common epitope. First, DLL3-expressing cells and cells expressing DLL3 with an epitope-specific mutation are prepared. The test and control antigen-binding molecules or antibodies are added to a cell suspension prepared by suspending these cells in an appropriate buffer, such as PBS. The cell suspension is then appropriately washed with buffer, and FITC-labeled antibodies that recognize the test and control antigen-binding molecules or antibodies are added. The fluorescence intensity and the number of cells stained with the labeled antibodies are determined using a FACSCalibur (BD). The test and control antigen-binding molecules or antibodies are appropriately diluted using a suitable buffer and used at the desired concentration. For example, they can be used at a concentration ranging from 10 μg / ml to 10 ng / ml. The fluorescence intensity, i.e., the geometric mean value, determined by analysis using CELL QUEST Software (BD) reflects the amount of labeled antibody bound to the cells. That is, the binding activity of the test and control antigen-binding molecules or antibodies, represented by the amount of bound labeled antibody, can be determined by measuring the geometric mean value.

[0119] In the above-mentioned method, whether an antigen-binding molecule or antibody "does not substantially bind to cells expressing a DLL3 mutant (or DLL3 variant)" can be evaluated, for example, by the following method. First, test and control antigen-binding molecules or antibodies bound to cells expressing a DLL3 mutant are stained with a labeled antibody. The fluorescence intensity of the cells is then determined. When a FACSCalibur is used for fluorescence detection by flow cytometry, the determined fluorescence intensity can be analyzed using CELL QUEST Software. From the geometric mean values ​​in the presence and absence of an antigen-binding molecule or antibody, a comparative value (delta geometric mean) can be calculated according to the following formula to determine the ratio of increase in fluorescence intensity as a result of binding by the antigen-binding molecule or antibody.

[0120] Delta geometric mean = geometric mean (in the presence of antigen-binding molecules or antibodies) / geometric mean (in the absence of antigen-binding molecules or antibodies).

[0121] The comparative geometric mean value determined by the above analysis, which reflects the amount of test antigen-binding molecule or antibody bound to cells expressing a DLL3 mutant (delta geometric mean value for DLL3 mutant molecules), is compared with the comparative delta geometric mean value reflecting the amount of test antigen-binding molecule or antibody bound to DLL3-expressing cells. In this case, the concentrations of the test antigen-binding molecule or antibody used to determine the comparative delta geometric mean values ​​for DLL3-expressing cells and DLL3 mutant-expressing cells are particularly preferably adjusted to be equal or substantially equal. An antigen-binding molecule or antibody confirmed to recognize an epitope in DLL3 is used as a control antigen-binding molecule or antibody.

[0122] If the delta geometric mean comparison value of the test antigen-binding molecule or antibody for cells expressing a DLL3 mutant is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% less than the delta geometric mean comparison value of the test antigen-binding molecule or antibody for DLL3-expressing cells, the test antigen-binding molecule or antibody "does not substantially bind to cells expressing a DLL3 mutant (or DLL3 variant)." The formula for determining the geometric mean is described in the CELL QUEST Software User Guide (BD biosciences). If the comparison shows that the comparison values ​​are substantially equivalent, the epitopes of the test and control antigen-binding molecules or antibodies can be determined to be the same.

[0123] Antibodies that bind to the same epitope An antigen-binding molecule or antibody comprising an antigen-binding domain that "binds to the same epitope" as a reference antibody refers to an antigen-binding molecule or antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay; conversely, the reference antibody blocks the binding of the antigen-binding molecule or antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays, such as cross-blocking assays, are provided above.

[0124] specificity "Specific" means that a molecule that specifically binds to one or more binding partners does not exhibit significant binding to molecules other than the partners. Furthermore, "specific" is also used when an antigen-binding domain is specific to a particular epitope among multiple epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to various antigens that have the epitope.

[0125] antibody fragment "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and monospecific or multispecific antibodies formed from antibody fragments.

[0126] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to antibodies that have a structure substantially similar to a native antibody structure, or that are F as defined herein. It refers to an antibody having a heavy chain containing a c region.

[0127] Variable fragment (Fv) As used herein, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain consisting of a pair of antibody light chain variable region (VL) and heavy chain variable region (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be prepared from the periplasmic fraction of Escherichia coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing gene expression in Escherichia coli (Science (1988) 240 (4855), pp. 1038-1041). In Fv prepared from the periplasmic fraction, the VH associates with the VL in a manner that binds to an antigen. As used herein, the term "antibody variable fragment" refers to any fragment comprising at least one light chain variable region (VL) and at least one antibody heavy chain variable region (VH).

[0128] scFv, single chain antibodies, and sc(Fv)2 As used herein, the terms "scFv," "single-chain antibody," and "sc(Fv)2" all refer to antibody fragments of a single polypeptide chain containing variable regions from heavy and light chains but no constant region. Generally, single-chain antibodies also contain a polypeptide linker between the VH and VL domains that enables the formation of the desired structure thought to enable antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994)." See also International Patent Application Publication No. WO 1988 / 001649; U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may be bispecific and / or humanized.

[0129] An scFv is an antigen-binding domain in which the VH and VL that form the Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), pp. 5879-5883). The VH and VL can be held in close proximity by the peptide linker.

[0130] sc(Fv)2 is a single-chain antibody in which four variable regions, two VL and two VH, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231(1-2), pp. 177-189). The two VH and two VL may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes a bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen, as disclosed in, for example, Journal of Immunology (1994) 152(11), pp. 5368-5374. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, sc(Fv)2 can be produced by linking scFvs with a linker such as a peptide linker.

[0131] As used herein, forms of antigen-binding domains that form sc(Fv)2 include antibodies in which two VH units and two VL units are arranged in the order VH, VL, VH, and VL, starting from the N-terminus of the single-chain polypeptide ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). The order of the two VH units and two VL units is not limited to the above form, and they may be arranged in any order. Examples of such forms are listed below. [VL]-linker-[VH]-linker-[VH]-linker-[VL] [VH]-linker-[VL]-linker-[VL]-linker-[VH] [VH]-linker-[VH]-linker-[VL]-linker-[VL] [VL]-linker-[VL]-linker-[VH]-linker-[VH] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0132] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352. Based on these descriptions, a person skilled in the art can appropriately prepare a desired sc(Fv)2 for producing the polypeptide complexes disclosed herein.

[0133] Furthermore, the antigen-binding molecules or antibodies of the present invention can be conjugated to carrier polymers such as PEG or organic compounds such as anticancer drugs. Alternatively, it is preferable to insert a glycosylation sequence into the antigen-binding molecules or antibodies so that the sugar chains exert the desired effect.

[0134] Linkers used to link antibody variable regions include any peptide linker that can be introduced by genetic engineering, synthetic linkers, and linkers such as those disclosed in Protein Engineering, 9(3), pp. 299-305, 1996. However, peptide linkers are preferred in the present invention. The length of the peptide linker is not particularly limited, and those skilled in the art can select an appropriate length depending on the purpose. The length is preferably 5 amino acids or more (although not particularly limited, the upper limit is generally 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When an sc(Fv)2 contains three peptide linkers, their lengths may be the same or different.

[0135] For example, such peptide linkers include: Ser Gly Ser Gly Gly Ser Ser Gly Gly Gly Gly Gly Ser (SEQ ID NO: 116) Ser Gly Gly Gly (SEQ ID NO: 117) Gly Gly Gly Gly Ser (SEQ ID NO: 118) Ser Gly Gly Gly Gly (SEQ ID NO: 119) Gly Gly Gly Gly Gly Ser (SEQ ID NO: 120) Ser Gly Gly Gly Gly Gly (SEQ ID NO: 121) Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 122) Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 123) (Gly Gly Gly Gly Ser (SEQ ID NO: 118))n (Ser Gly Gly Gly Gly (SEQ ID NO: 119)) (wherein n is an integer of 1 or greater). Those skilled in the art can select the length or sequence of the peptide linker depending on the purpose.

[0136] Synthetic linkers (chemical cross-linkers) are routinely used to cross-link peptides, examples of which include: N-hydroxysuccinimide (NHS), Disuccinimidyl suberate (DSS), Bis(sulfosuccinimidyl) suberate (BS3), Dithiobis(succinimidyl propionate) (DSP), Dithiobis(sulfosuccinimidyl propionate) (DTSSP), Ethylene glycol bis(succinimidyl succinate) (EGS), Ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), Disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DS) T), Bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and Bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available.

[0137] Generally, three linkers are required to link four antibody variable regions together. The linkers used may be of the same type or different types.

[0138] Fab, F(ab')2, and Fab' A "Fab" consists of one light chain and the CH1 domain and variable region from one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0139] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, digesting the immunoglobulin (monoclonal antibody) near the disulfide bond between the hinge regions of each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond between the hinge regions of each of the two H chains to produce two homologous antibody fragments in which an L chain containing a VL (light chain variable region) and a CL (light chain constant region) is linked by a disulfide bond in their C-terminal regions to an H chain fragment containing a VH (heavy chain variable region) and a CH gamma 1 (gamma 1 region in the heavy chain constant region). Each of these two homologous antibody fragments is called Fab'.

[0140] "F(ab')2" consists of two light chains and two heavy chains, each containing the constant region of the CH1 domain and a portion of the CH2 domain, with disulfide bonds formed between the two heavy chains. The F(ab')2 disclosed herein can be preferably produced as follows: a whole monoclonal antibody containing the desired antigen-binding domain is partially digested with a protease such as pepsin, and the Fc fragment is removed by adsorption onto a protein A column. The protease is not particularly limited, as long as it can selectively cleave the whole antibody to produce F(ab')2 under appropriately set enzymatic reaction conditions, such as pH. Examples of such proteases include pepsin and ficin.

[0141] Fc area The term "Fc region" or "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and Fc region variants. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 toward the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0142] Native sequence Fc region A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A native sequence human Fc region comprises a native sequence human IgG1 Fc region (non-A and and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0143] Variant Fc region A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region herein will preferably have at least about 80% sequence identity with a native-sequence Fc region and / or the Fc region of a parent polypeptide, most preferably at least about 90% sequence identity, and more preferably at least about 95% sequence identity.

[0144] Fc receptors The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds to IgG antibodies (gamma receptors), including receptors of the FcgammaRI, FcgammaRII, and FcgammaRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcgammaRII receptors include FcgammaRIIA (an "activating receptor") and FcgammaRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor, FcgammaRIIA, contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor, Fc gamma RIIB, contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0145] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587 (1976); and Kim et al., J. Immunol., 24:249 (1994)) and for regulating immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today, 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem., 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0146] In vivo binding to human FcRn and plasma half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides having variant Fc regions are administered. WO2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See, for example, Shields et al., J. Biol. Chem. 9(2):6591-66. See also page 04 (2001).

[0147] Fc gamma receptor Fc gamma receptor refers to a receptor capable of binding to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies, and includes all members of a family of proteins substantially encoded by Fc gamma receptor genes. In humans, this family includes Fc gamma RI (CD64), which includes isoforms Fc gamma RIa, Fc gamma RIb and Fc gamma RIc; Fc gamma RII (CD32), which includes isoforms Fc gamma RIIa (including allotypes H131 and R131), Fc gamma RIIb (including Fc gamma RIIb-1 and Fc gamma RIIb-2) and Fc gamma RIIc; and Fc gamma RIII (CD16), which includes isoforms Fc gamma RIIIa (including allotypes V158 and F158) and Fc gamma RIIIb (including allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2); and all unidentified human Fc gamma receptors, Fc gamma receptor isoforms, and their allotypes.However, Fc gamma receptors are not limited to these examples. Fc gamma receptors include, but are not limited to, those derived from humans, mice, rats, rabbits, and monkeys. Fc gamma receptors can be derived from any organism. Mouse Fc gamma receptors include, but are not limited to, Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD16), and Fc gamma RIII-2 (CD16-2), as well as all unidentified mouse Fc gamma receptors, Fc gamma receptor isoforms, and allotypes thereof. Such preferred Fc gamma receptors include, for example, human Fc gamma RI (CD64), Fc gamma RIIA (CD32), Fc gamma RIIB (CD32), Fc gamma RIIIA (CD16), and / or Fc gamma RIIIB (CD16).The polynucleotide and amino acid sequences of Fc gamma RI are set forth in NCBI Reference Sequence NM_000566.3 (SEQ ID NO: 124) and NP_000557.1 (SEQ ID NO: 125), respectively; the polynucleotide and amino acid sequences of Fc gamma RIIA are set forth in BC020823.1 (SEQ ID NO: 126) and AAH20823.1 (SEQ ID NO: 127), respectively; and the polynucleotide and amino acid sequences of Fc gamma RIIB are set forth in BC146678.1 (SEQ ID NO: 128) and AAI46679, respectively. The polynucleotide and amino acid sequences of Fc gamma RIIIA are set forth in BC033678.1 (SEQ ID NO: 130) and AAH33678.1 (SEQ ID NO: 131), respectively; and the polynucleotide and amino acid sequences of Fc gamma RIIIB are set forth in BC128562.1 (SEQ ID NO: 132) and AAI28563.1 (SEQ ID NO: 133), respectively (RefSeq accession numbers). In addition to the above-mentioned FACS and ELISA formats, whether an Fc gamma receptor has binding activity to the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be evaluated by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method based on surface plasmon resonance (SPR), and others (Proc. Natl. Acad. Sci. USA (2006) 103(11), pp. 4005-4010).

[0148] In contrast, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, that binds to an antibody Fc domain and forms an Fc / Fc ligand complex. The molecule may be derived from any organism. Binding of the Fc ligand to the Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, Fc gamma receptors, Fc alpha receptors, Fc beta receptors, FcRn, C1q, and C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, staphylococcal protein G, and Examples of Fc ligands include viral Fc gamma receptors. Fc ligands also include Fc receptor homologs (FcRHs), a family of Fc receptors homologous to Fc gamma receptors (Davis et al. (2002), Immunological Reviews 190, 123-136). Fc ligands also include unidentified molecules that bind to Fc.

[0149] Fc gamma receptor binding activity The reduced binding activity of the Fc domain to any of the Fc gamma receptors Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and / or Fc gamma RIIIB can be assessed using the above-mentioned FACS and ELISA formats, as well as ALPHA screens (Amplified Luminescent Proximity Homogeneous Assays) and surface plasmon resonance (SPR)-based BIACORE methods (Proc. Natl. Acad. Sci. USA (2006) 103 (11), pp. 4005-4010). In some embodiments, the antigen-binding molecules or antibodies of the present invention comprise a domain containing an Fc region with reduced binding activity to Fc gamma receptors.

[0150] The ALPHA screen is performed using two types of beads: donor beads and acceptor beads, using ALPHA technology based on the principles described below. A luminescent signal is detected only when a molecule linked to a donor bead biologically interacts with a molecule linked to an acceptor bead and when the two beads are located in close proximity. When excited by a laser beam, a photosensitizer in the donor bead converts oxygen around the bead into excited singlet oxygen. When the singlet oxygen diffuses around the donor bead and reaches an acceptor bead located in close proximity, a chemiluminescent reaction is induced in the acceptor bead. This reaction ultimately results in light emission. If the molecule linked to the donor bead does not interact with the molecule linked to the acceptor bead, the singlet oxygen generated by the donor bead does not reach the acceptor bead, and no chemiluminescent reaction occurs.

[0151] For example, a biotin-labeled antigen-binding molecule or antibody is immobilized on donor beads, and a glutathione S-transferase (GST)-tagged Fc gamma receptor is immobilized on acceptor beads. In the absence of an antigen-binding molecule or antibody containing a competitive mutant Fc domain, the Fc gamma receptor interacts with an antigen-binding molecule or antibody containing a wild-type Fc domain, resulting in a signal at 520-620 nm. An antigen-binding molecule or antibody containing an untagged mutant Fc domain competes with an antigen-binding molecule or antibody containing a wild-type Fc domain for interaction with the Fc gamma receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from competition. Methods for biotinylating antigen-binding molecules or antibodies, such as antibodies using sulfo-NHS-biotin, are known. Suitable method for adding GST tag to Fc gamma receptor includes: Fc gamma receptor and GST coding polypeptide are fused in frame, the vector carrying fusion gene is used to transfect the cell, express the gene, and then use glutathione column to purify.Preferably, the signal induced can be analyzed by fitting to one-site competition model based on nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad; San Diego).

[0152] One of the substances for observing their interaction is immobilized as a ligand on the thin gold layer of the sensor chip. When light is irradiated onto the backside of the sensor chip so that total reflection occurs at the interface between the thin gold layer and the glass, the intensity of the reflected light is partially reduced at a specific site (SPR signal). Another substance for observing their interaction is injected onto the surface of the sensor chip as an analyte. When the analyte binds to the ligand, it binds to the immobilized ligand. The mass of the molecule increases, which changes the refractive index of the solvent on the surface of the sensor chip. The change in refractive index causes a position shift of the SPR signal (conversely, dissociation shifts the signal back to its original position). In the Biacore system, the amount of this shift (i.e., the change in mass on the sensor chip surface) is plotted on the vertical axis, thus showing the change in mass over time as measurement data (sensorgram). The reaction rate parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the sensorgram curve, and the affinity (KD) is determined from the ratio between these two constants. Inhibition assays are preferably used in the BIACORE method. An example of such an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), pp. 4005-4010.

[0153] Functional Fc region A "functional Fc region" has the "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors; BCRs); and the like. Such effector functions generally require the combination of an Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays, for example, as disclosed in the definitions herein. In some embodiments, the antigen-binding molecules or antibodies of the invention comprise a domain comprising a functional Fc region having an effector function.

[0154] "Effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0155] Human effector cells "Human effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least Fc gamma RIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood.

[0156] Fc region with reduced Fc gamma receptor binding activity As used herein, "reduced Fc gamma receptor binding activity" means, for example, that the competitive activity of a test antigen-binding molecule or antibody is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, and particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the competitive activity of a control antigen-binding molecule or antibody, based on the above-mentioned analytical method.

[0157] Antigen-binding molecules or antibodies containing the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be suitably used as control antigen-binding molecules or antibodies. The Fc domain structures are shown in SEQ ID NO: 112 (RefSeq Accession No. AAC82527.1 with an A added to the N-terminus), SEQ ID NO: 113 (RefSeq Accession No. AAB59393.1 with an A added to the N-terminus), SEQ ID NO: 114 (RefSeq Accession No. CAA27268.1 with an A added to the N-terminus), and SEQ ID NO: 115 (RefSeq Accession No. AAB59394.1 with an A added to the N-terminus). Furthermore, when antigen-binding molecules or antibodies containing Fc domain variants of antibodies of a specific isotype are used as test substances, the effect of the mutations on the Fc gamma receptor binding activity can be assessed using the same antibody. An antigen-binding molecule or antibody containing a variant Fc domain of the same type is used as a control for evaluation. As described above, an antigen-binding molecule or antibody containing an Fc domain variant determined to have reduced Fc gamma receptor binding activity is appropriately prepared.

[0158] Such known mutants include, for example, a mutant having a deletion of amino acids 231A to 238S (EU numbering) (WO2009 / 011941), as well as mutants C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54); C226S, C229S, E233P, L234V, and L235A (Blood (2007) 109, 1185-1192).

[0159] Specifically, among the amino acids that form the Fc domain of an antibody of a particular isotype, preferred antigen-binding molecules or antibodies include those that contain an Fc domain with a mutation (e.g., substitution) of at least one amino acid selected from the following amino acid positions: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering). The antibody isotype from which the Fc domain originates is not particularly limited, and an appropriate Fc domain derived from a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be used. It is preferable to use an Fc domain derived from an IgG1 antibody.

[0160] Preferred antigen-binding molecules or antibodies include, for example, the following substitutions among the amino acids forming the Fc domain of an IgG1 antibody, the positions of which are identified according to EU numbering (each number represents the position of an amino acid residue in EU numbering, the one-letter amino acid code before the number represents the amino acid residue before substitution, and the one-letter amino acid code after the number represents the amino acid residue after substitution): (a) L234F, L235E, P331S; (b) C226S, C229S, P238S; (c) C226S, C229S; or (d)C226S, C229S, E233P, L234V, L235A and those having an Fc domain with a deletion in the amino acid sequence at positions 231 to 238.

[0161] Furthermore, preferred antigen-binding molecules or antibodies include those containing the following substitutions in the amino acids forming the Fc domain of an IgG2 antibody, the positions of which are identified according to EU numbering: (e) H268Q, V309L, A330S, and P331S; (f)V234A; (g)G237A; (h) V234A and G237A; (i) A235E and G237A; or (j) V234A, A235E, and G237A Each number represents the position of an amino acid residue according to the EU numbering system, with the single-letter amino acid code preceding the number representing the amino acid residue before substitution and the single-letter amino acid code following the number representing the amino acid residue after substitution.

[0162] Furthermore, preferred antigen-binding molecules or antibodies include those that form the Fc domain of an IgG3 antibody. The substitutions shown below, in which the positions are identified according to EU numbering, are included in the amino acids that make up the sequence: (k)F241A; (l) D265A; or (m)V264A Each number represents the position of an amino acid residue according to the EU numbering system, with the single-letter amino acid code preceding the number representing the amino acid residue before substitution and the single-letter amino acid code following the number representing the amino acid residue after substitution.

[0163] Furthermore, preferred antigen-binding molecules or antibodies include those containing the following substitutions in the amino acids forming the Fc domain of an IgG4 antibody, the positions of which are identified according to EU numbering: (n) L235A, G237A, and E318A; (o) L235E; or (p)F234A and L235A Each number represents the position of an amino acid residue according to the EU numbering system, with the single-letter amino acid code preceding the number representing the amino acid residue before substitution and the single-letter amino acid code following the number representing the amino acid residue after substitution.

[0164] Other preferred antigen-binding molecules or antibodies include, for example, those comprising an Fc domain in which any amino acid at positions 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody has been substituted with an amino acid at the corresponding position (EU numbering) in the corresponding IgG2 or IgG4.

[0165] Preferred antigen-binding molecules or antibodies also include, for example, those comprising an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 (EU numbering) in the amino acid sequence that forms the Fc domain of an IgG1 antibody have been substituted with other amino acids. The type of amino acid after substitution is not particularly limited, but particularly preferred are antigen-binding molecules or antibodies comprising an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 have been substituted with alanine.

[0166] Preferred antigen-binding molecules or antibodies also include, for example, those comprising an Fc domain in which the amino acids at positions 1 and 265 (EU numbering) of the amino acids forming the Fc domain of an IgG1 antibody have been substituted with another amino acid. The type of amino acid after substitution is not particularly limited, but antigen-binding molecules or antibodies comprising an Fc domain in which the amino acid at position 265 has been substituted with alanine are particularly preferred.

[0167] Antigen-binding domain that binds to DLL3 As used herein, the phrase "antigen-binding domain that binds to DLL3" or "anti-DLL3 antigen-binding domain" refers to an antigen-binding domain that specifically binds to the above-mentioned DLL3 protein, or all or a portion of a partial peptide of the DLL3 protein.

[0168] In certain embodiments, the antigen-binding domain that binds to DLL3 is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," "F(ab')2," and the like. In certain embodiments, the antigen-binding domain that binds to DLL3 is an antibody variable fragment. The domain comprising an antibody variable fragment can be provided from the variable domain of one or more antibodies.

[0169] In certain embodiments, the antigen-binding domain that binds to DLL3 comprises the heavy chain variable region and the light chain variable region of an anti-DLL3 antibody. In certain embodiments, the antigen-binding domain that binds to DLL3 is a domain that comprises a Fab structure.

[0170] Preferably, the anti-DLL3 antigen-binding domain comprises a heavy chain variable region of any one of SEQ ID NO: 15, SEQ ID NO: 25, and SEQ ID NOs: 63 to 71, and a light chain variable region of any one of SEQ ID NO: 16, SEQ ID NO: 26, and SEQ ID NOs: 72 to 74.

[0171] In some embodiments, the anti-DLL3 antigen binding domain specifically binds to the extracellular domain of DLL3. In some embodiments, the anti-DLL3 antigen binding domain specifically binds to an epitope within the extracellular domain of DLL3. In some embodiments, the anti-DLL3 antigen binding domain binds to a DLL3 protein expressed on the surface of a eukaryotic cell. In some embodiments, the anti-DLL3 antigen binding domain binds to a DLL3 protein expressed on the surface of a cancer cell.

[0172] In certain embodiments, the antigen binding domain that binds to DLL3 comprises any one of the antibody variable / constant region sequences shown in Table 1A and Table 1B below.

[0173] Table 1A shows the sequence numbers of the anti-DLL3 antibodies generated.

[0174] [Table 1A]

[0175] Table 1B shows the SEQ ID NOs for the HVR (CDR) sequences of the generated anti-DLL3 antibodies.

[0176] [Table 1B]

[0177] In certain embodiments, an antigen-binding domain that binds to DLL3 is a domain comprising an antibody variable fragment that competes for binding to DLL3 with any one of the antibody variable regions set forth in Table 1A, or that competes for binding to DLL3 with any antibody variable fragment that comprises HVR sequences identical to the HVR regions of the antibody variable regions set forth in Table 1A, or that competes for binding to DLL3 with any antibody variable fragment that comprises HVR sequences identical to those set forth in Table 1B. In certain embodiments, an antigen-binding domain that binds to DLL3 is a domain comprising an antibody variable fragment that binds to the same epitope in DLL3 as any one of the antibody variable regions set forth in Table 1A, or that binds to the same epitope in DLL3 as any antibody variable fragment that comprises HVR sequences identical to the HVR regions of the antibody variable regions set forth in Table 1A, or that binds to the same epitope in DLL3 as any antibody variable fragment that comprises HVR sequences identical to those set forth in Table 1B.

[0178] Alternatively, the antigen-binding domain that binds to DLL3 comprises an antibody variable fragment that competes with any one of the above-mentioned antibody variable fragments for binding to DLL3. Alternatively, the antigen-binding domain that binds to DLL3 comprises an antibody variable fragment that binds to the same epitope on DLL3 as any one of the above-mentioned antibody variable fragments.

[0179] Antigen-binding domain that binds to the T cell receptor complex As used herein, the phrase "antigen-binding domain that binds to a T cell receptor complex" or "anti-T cell receptor complex antigen-binding domain" refers to an antigen-binding domain that specifically binds to all or part of a partial peptide of a T cell receptor complex. The T cell receptor complex may be the T cell receptor itself or an adaptor molecule that constitutes the T cell receptor complex together with the T cell receptor. CD3 is preferred as the adaptor molecule.

[0180] In certain embodiments, the antigen-binding domain that binds to the T cell receptor complex is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," "F(ab')2," and the like. In certain embodiments, the antigen-binding domain that binds to the T cell receptor complex is a domain comprising an antibody variable fragment. The domain comprising an antibody variable fragment can be provided from the variable domains of one or more antibodies.

[0181] In certain embodiments, the antigen-binding domain that binds to the T cell receptor complex comprises the heavy chain variable region and the light chain variable region of an anti-T cell receptor complex antibody. The antigen-binding domain that binds to the T cell receptor complex binding activity is the domain that comprises the Fab structure.

[0182] Antigen-binding domain that binds to the T cell receptor As used herein, the phrase "antigen-binding domain that binds to a T cell receptor" or "anti-T cell receptor antigen-binding domain" refers to an antigen-binding domain that specifically binds to all or a portion of a partial peptide of a T cell receptor. The portion of the T cell receptor to which the antigen-binding domain binds may be the variable region of the T cell receptor or the constant region of the T cell receptor, with epitopes present in the constant region being preferred. Examples of constant region sequences include the T cell receptor alpha chain of RefSeq Accession No. CAA26636.1 (SEQ ID NO: 104), the T cell receptor beta chain of RefSeq Accession No. C25777 (SEQ ID NO: 105), the T cell receptor gamma 1 chain of RefSeq Accession No. A26659 (SEQ ID NO: 106), the T cell receptor gamma 2 chain of RefSeq Accession No. AAB63312.1 (SEQ ID NO: 107), and the T cell receptor delta chain of RefSeq Accession No. AAA61033.1 (SEQ ID NO: 108).

[0183] In certain embodiments, the antigen-binding domain that binds to a T cell receptor is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," "F(ab')2," etc. In certain embodiments, the antigen-binding domain that binds to a T cell receptor is a domain comprising an antibody variable fragment. The domain comprising an antibody variable fragment can be provided from the variable domains of one or more antibodies.

[0184] In certain embodiments, the antigen-binding domain that binds to a T cell receptor comprises the heavy chain variable region and the light chain variable region of an anti-T cell receptor antibody. In certain embodiments, the antigen-binding domain that binds to a T cell receptor is a domain that comprises a Fab structure.

[0185] Antigen-binding domain that binds to CD3 As used herein, the phrase "antigen-binding domain that binds to CD3" or "anti-CD3 antigen-binding domain" refers to an antigen-binding domain that specifically binds to all or a portion of a partial peptide of CD3. The antigen-binding domain that binds to CD3 may be any epitope-binding domain, as long as the epitope is present in the gamma, delta, or epsilon chain sequence that constitutes human CD3. The polynucleotide sequences of the gamma, delta, or epsilon chain structures that constitute CD3 are disclosed in RefSeq Accession Nos. NM_000073.2, NM_000732.4, and NM_000733.3, and the polypeptide sequences are shown in NP_000064.1 (SEQ ID NO: 109), NP_000723.1 (SEQ ID NO: 110), and NP_000724.1 (SEQ ID NO: 111) (RefSeq Accession Nos.). In some embodiments, the antigen binding molecules or antibodies of the invention comprise a domain comprising an antigen variable region that binds to the CD3 epsilon chain.

[0186] In certain embodiments, the antigen-binding domain that binds to CD3 is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," "F(ab')2," and the like. In certain embodiments, the antigen-binding domain that binds to CD3 is a domain comprising an antibody variable fragment. The domain comprising an antibody variable fragment can be provided from the variable domains of one or more antibodies.

[0187] In certain embodiments, the antigen-binding domain that binds to CD3 is a heavy chain of an anti-CD3 antibody. In certain embodiments, the antigen-binding domain that binds to CD3 is a domain comprising a Fab structure.

[0188] The anti-CD3 antigen-binding domain of the present invention may bind to any epitope, as long as the epitope is located within the gamma, delta, or epsilon chain sequence that forms human CD3. In the present invention, preferred anti-CD3 antigen-binding domains include those comprising a CD3 antibody light chain variable region (VL) and a CD3 antibody heavy chain variable region (VH) that bind to an epitope in the extracellular domain of the epsilon chain of the human CD3 complex. Such preferred anti-CD3 antigen-binding domains include those comprising the CD3 antibody light chain variable region (VL) and CD3 antibody heavy chain variable region (VH) of various known CD3 antibodies (Int. J. Cancer Suppl. 7, pp. 45-50 (1992)), such as the OKT3 antibody (Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4914-4917) or an antibody having the light chain variable region (VL) of NCBI accession number AAB24132 and the heavy chain variable region (VH) of NCBI accession number AAB24133. Furthermore, such suitable anti-CD3 antigen-binding domains include those derived from CD3 antibodies with desired characteristics obtained by immunizing a desired animal with the gamma, delta, or epsilon chains that form human CD3 by the above-described methods. Suitable anti-CD3 antibodies from which the anti-CD3 antigen-binding domain is derived include human antibodies and antibodies that have been appropriately humanized as described above.

[0189] Preferably, the anti-CD3 antigen-binding domain comprises a heavy chain variable region of any one of the heavy chain variable regions shown in Table 2B and a light chain variable region of any one of the light chain variable regions shown in Table 2B.

[0190] In some embodiments, the anti-CD3 antigen binding domain specifically binds to the CD3 epsilon chain. In some embodiments, the anti-CD3 antigen binding domain specifically binds to an epitope within the CD3 epsilon chain. In some embodiments, the anti-CD3 antigen binding domain binds to a CD3 epsilon chain expressed on the surface of a eukaryotic cell. In some embodiments, the anti-CD3 antigen binding domain binds to a CD3 epsilon chain expressed on the surface of a T cell.

[0191] In certain embodiments, the antigen-binding domain that binds to CD3 comprises any one of the antibody variable region sequences shown in Table 2A below. In certain embodiments, the antigen-binding domain that binds to CD3 comprises any one of the combinations of heavy chain variable region and light chain variable region shown in Table 2A. In certain embodiments, the antigen-binding domain that binds to CD3 comprises an HVR sequence contained in an antibody variable region shown in Table 2A.

[0192] Table 2A shows the SEQ ID NOs of the variable regions of the anti-CD3 antigen-binding domain.

[0193] [Table 2A]

[0194] In certain embodiments, the antigen-binding domain that binds to CD3 is shown in Table 2B below. It contains any one of the combinations of HVR sequences.

[0195] Table 2B shows the SEQ ID NOs of the HVR (CDR) sequences of the anti-CD3 antigen-binding domain.

[0196] [Table 2B]

[0197] In certain embodiments, the antigen-binding domain that binds to CD3 is a domain comprising an antibody variable fragment that competes for binding to CD3 with any one of the antibody variable regions set forth in Table 2A, or that competes for binding to CD3 with any antibody variable fragment that comprises HVR sequences identical to the HVR regions of the antibody variable regions set forth in Table 2A, or that competes for binding to CD3 with any antibody variable fragment that comprises HVR sequences identical to those set forth in Table 2B. In certain embodiments, the antigen-binding domain that binds to CD3 binds to the same epitope within CD3 as any one of the antibody variable regions set forth in Table 2A, or that competes for binding to CD3 with any antibody variable fragment that comprises HVR sequences identical to those set forth in Table 2B. A domain comprising an antibody variable fragment that binds to the same epitope in CD3 as any antibody variable fragment containing HVR sequences identical to the HVR regions of the variable region, or that binds to the same epitope in CD3 as any antibody variable fragment containing HVR sequences identical to those shown in Table 2B.

[0198] Alternatively, the antigen-binding domain that binds to CD3 comprises an antibody variable fragment that competes with any one of the above antibody variable fragments / antibody variable regions for binding to CD3. Alternatively, the antigen-binding domain that binds to CD3 comprises an antibody variable fragment that binds to the same epitope on CD3 as any one of the above antibody variable fragments / antibody variable regions binds to.

[0199] Multispecific antigen binding molecules A "multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to more than one antigen. In a preferred embodiment, a multispecific antigen-binding molecule of the present invention comprises two or more antigen-binding domains, and different antigen-binding domains specifically bind to different antigens.

[0200] The multispecific antigen-binding molecules of the present invention comprise a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to the T cell receptor complex. A combination of an antigen-binding domain that binds to DLL3 selected from those described above in "Antigen-binding domains that bind to DLL3" and an antigen-binding domain that binds to the T cell receptor complex selected from those described above in "Antigen-binding domains that bind to the T cell receptor complex" with the above-mentioned "Antigen-binding domain that binds to CD3" can be used.

[0201] For example, the first antigen-binding domain is a domain comprising antibody heavy and light chain variable regions, and / or the second antigen-binding domain is a domain comprising antibody heavy and light chain variable regions. Alternatively, the first antigen-binding domain is a domain comprising an antibody variable fragment, and / or the second antigen-binding domain is a domain comprising an antibody variable fragment. Alternatively, the first antigen-binding domain is a domain comprising an Fab structure, and / or the second antigen-binding domain is a domain comprising an Fab structure.

[0202] In certain embodiments, the present invention provides a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to the T cell receptor complex. In certain embodiments, the present invention provides a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to DLL3, a second antigen-binding domain that binds to the T cell receptor complex, and a domain comprising an Fc region with reduced Fc gamma receptor binding activity. The Fc region may have reduced Fc gamma receptor binding activity compared to the Fc domain of an IgG1, IgG2, IgG3, or IgG4 antibody. In embodiments, the Fc region has an amino acid mutation in any of the amino acids constituting the Fc regions of SEQ ID NOs: 112 to 115 (IgG1 to IgG4).

[0203] In certain embodiments, the present invention provides bispecific antibodies comprising a first antibody variable fragment that binds to DLL3 and a second antibody variable fragment that binds to CD3. In certain embodiments, the present invention provides bispecific antibodies comprising a first antibody variable fragment that binds to DLL3, a second antibody variable fragment that binds to CD3, and an Fc region with reduced Fc gamma receptor binding activity. In certain embodiments, the present invention provides bispecific antibodies comprising a first antibody variable fragment that binds to DLL3, a second antibody variable fragment that binds to the CD3 epsilon chain, and an Fc region with reduced Fc gamma receptor binding activity compared to a naturally occurring IgG Fc region.

[0204] A preferred embodiment of the "multispecific antigen-binding molecule" of the present invention is, for example, a multispecific antibody. When an Fc region with reduced Fc gamma receptor binding activity is used as the multispecific antibody F region, the Fc region derived from the multispecific antibody can be appropriately used. Bispecific antibodies are particularly preferred as multispecific antibodies of the present invention. In this case, the bispecific antibody is an antibody having two different specificities. IgG-type bispecific antibodies can be secreted from hybrid hybridomas (quadromas) generated by fusing two types of hybridomas that produce IgG antibodies (Milstein et al., Nature (1983) 305, 537-540).

[0205] Furthermore, IgG-type bispecific antibodies can be secreted by introducing into cells the genes for the L and H chains that constitute two types of desired IgG, i.e., a total of four genes, and simultaneously expressing them. However, these methods theoretically allow for the production of 10 combinations of IgG H and L chains. Therefore, it is difficult to purify IgG containing the desired combination of H and L chains from the 10 types of IgG. Furthermore, theoretically, the secreted amount of IgG containing the desired combination would be significantly reduced, thus necessitating large-scale culture, which would further increase production costs.

[0206] Therefore, techniques for promoting binding between H chains and between L and H chains having desired combinations can be applied to the multispecific antigen-binding molecules of the present invention. For example, techniques for suppressing undesired heavy chain binding by introducing electrostatic repulsion into the interface of the second or third constant region (CH2 or CH3) of the antibody heavy chain can be applied to multispecific antibody binding (WO2006 / 106905).

[0207] In the technique for suppressing unintended H-chain binding by introducing electrostatic repulsion into the CH2 or CH3 interface, examples of amino acid residues that contact the interface of other constant regions of the H-chain include regions corresponding to residues at positions 356, 439, 357, 370, 399, and 409 (EU numbering) in the CH3 region.

[0208] More specifically, examples include antibodies comprising two types of H chain CH3 regions, in which one to three pairs of amino acid residues in the first H chain CH3 region carry the same type of charge, selected from the pairs of amino acid residues shown in (1) to (3) below: (1) amino acid residues in the H chain CH3 region at positions 356 and 439, EU numbering; (2) amino acid residues in the H chain CH3 region at positions 357 and 370, EU numbering; and (3) amino acid residues in the H chain CH3 region at positions 399 and 409, EU numbering.

[0209] Furthermore, the antibody may be an antibody in which pairs of amino acid residues in a second H chain CH3 region different from the first H chain CH3 region are selected from the pairs of amino acid residues (1) to (3) above, and one to three pairs of amino acid residues corresponding to the pairs of amino acid residues (1) to (3) in the first H chain CH3 region that carry the same type of charge carry the opposite charge derived from the corresponding amino acid residues in the first H chain CH3 region.

[0210] The amino acid residues shown in (1) to (3) above are located close to each other during binding. Those skilled in the art can find the positions corresponding to the amino acid residues in (1) to (3) above in the desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can appropriately modify the amino acid residues at these positions.

[0211] In the above-described antibodies, the "charged amino acid residues" are preferably selected from, for example, the following groups: (a) glutamic acid (E) and aspartic acid (D); and (b) lysine (K), arginine (R), and histidine (H) The amino acid residues are selected from those included in either one of:

[0212] In the above-mentioned antibodies, the phrase "carrying the same charge" means, for example, that two or more amino acid residues are all selected from amino acid residues included in either group (a) or (b) above. The phrase "carrying the opposite charge" means, for example, that when at least one amino acid residue among two or more amino acid residues is selected from amino acid residues included in either group (a) or (b) above, the remaining amino acid residue is selected from amino acid residues included in the other group.

[0213] In a preferred embodiment, the antibody may have its first heavy chain CH3 region and second heavy chain CH3 region cross-linked by a disulfide bond.

[0214] In the present invention, the amino acid residues to be modified are not limited to the above-mentioned amino acid residues in antibody variable region or antibody constant region.Those skilled in the art can identify the amino acid residues that form the interface in polypeptide variants or heteromultimers by homology modeling using commercially available software; then, the amino acid residues at these positions can be modified to adjust binding.

[0215] Other known techniques can also be used for conjugating the multispecific antibodies of the present invention. Fc region-containing polypeptides containing different amino acids can be efficiently conjugated to each other by replacing an amino acid side chain present in one of the antibody heavy chain Fc regions with a longer side chain (knob) and replacing the amino acid side chain present in the corresponding Fc region of the other heavy chain with a smaller side chain (hole), allowing the knob to be positioned within the hole (WO 1996 / 027011; Ridgway JB et al., Protein Engineering (1996), 9, pp. 617-621; Merchant AM et al., Nature Biotechnology (1998), 16, pp. 677-681; and US 20130336973).

[0216] Furthermore, other known techniques can also be used to form the multispecific antibodies of the present invention. By altering a portion of one of the antibody H chain CH3s to a corresponding IgA-derived sequence and introducing the corresponding IgA-derived sequence into the complementary portion of the other H chain CH3, a chain-exchange-engineered CH3 domain can be produced, allowing efficient binding of polypeptides with different sequences through complementary CH3 binding (Protein Engineering Design & Selection, 23; pp. 195-202, 2010). This known technique can also be used to efficiently form the desired multispecific antibodies.

[0217] Further, techniques for producing antibodies using antibody CH1-CL binding and VH-VL binding described in WO2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb;32(2):191-8; techniques for producing bispecific antibodies using a combination of separately prepared monoclonal antibodies (Fab arm exchange) described in WO2008 / 119353 and WO2011 / 131746; techniques for modulating the binding between antibody heavy chain CH3s described in WO2012 / 058768 and WO2013 / 063702; techniques for producing bispecific antibodies composed of two types of light chains and one type of heavy chain described in WO2012 / 023053; For the formation of multispecific antibodies, a technique for producing bispecific antibodies using two bacterial cell lines that individually express one of the chains of an antibody comprising a single H chain and a single L chain, such as that described by I. M., et al., J. Immunol., 1999, 10, 1449-1452 (2013), can be used.

[0218] Alternatively, even if the desired multispecific antibody cannot be efficiently formed, the multispecific antibody of the present invention can be obtained by separating the desired multispecific antibody from the produced antibodies and These antibodies can be obtained by purification. For example, a method has been reported for purifying two types of homomeric and heteromeric antibodies of interest by ion exchange chromatography by introducing amino acid substitutions into the variable regions of the two types of H chains to impart different isoelectric points (WO2007114325). Previously, methods for purifying heteromeric antibodies have been reported in which Protein A is used to purify heterodimeric antibodies comprising a mouse IgG2a H chain that binds to Protein A and a rat IgG2b H chain that does not (WO98050431 and WO95033844). Furthermore, by using H chains containing substitutions of amino acid residues at positions 435 and 436 (EU numbering), which are the IgG-Protein A binding site, with amino acids such as Tyr and His that confer different Protein A affinities, or by using H chains with different Protein A affinities, the interaction between each H chain and Protein A can be altered, and then the heterodimeric antibody can be efficiently purified directly using a Protein A column.

[0219] Alternatively, a common L chain capable of providing binding ability to multiple different H chains can be obtained and used as the common L chain of a multispecific antibody. Efficient expression of multispecific IgG can be achieved by introducing genes for such a common L chain and multiple different H chains into cells and expressing IgG (Nature Biotechnology (1998) 16, pp. 677-681). When selecting a common H chain, a method for selecting a common L chain that exhibits strong binding ability to any of the different H chains can also be used (WO2004 / 065611).

[0220] Furthermore, Fc regions with improved C-terminal heterogeneity can be suitably used as the Fc regions of the present invention. More specifically, the present invention provides Fc regions produced by deleting glycine at position 446 and lysine at position 447 (EU numbering) from the amino acid sequences of the two polypeptides that constitute the Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0221] These techniques can be used in combination, for example, two or more of them. Furthermore, these techniques can be applied separately to the two H chains to be combined. Furthermore, these techniques can be used in combination with the above-mentioned Fc region with reduced binding activity to Fc gamma receptors. Furthermore, the antigen-binding molecules of the present invention may be separately produced molecules that have the same amino acid sequence as the antigen-binding molecules that have been modified as described above.

[0222] Preferably, the antigen-binding molecule of the present invention may comprise a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to the T cell receptor complex. In one embodiment, the second antigen-binding domain binds to the T cell receptor. In another embodiment, the second antigen-binding domain binds to the CD3 epsilon chain. In another embodiment, the first antigen-binding domain binds to human DLL3. In a further embodiment, the first antigen-binding domain binds to DLL3 on the surface of a eukaryotic cell. In another embodiment, the first antigen-binding domain binds to human DLL3 on the surface of a eukaryotic cell, preferably a cancer cell.

[0223] As used herein, the phrase "anti-DLL3 arm" refers to the antibody heavy chain and antibody light chain that bind to DLL3 in a bispecific antibody. As used herein, the phrase "anti-CD3 arm" refers to the antibody heavy chain and antibody light chain that bind to CD3 in a bispecific antibody.

[0224] Preferably, the antigen-binding molecule of the present invention may have cellular cytotoxicity (also referred to as "cytotoxicity"). In an embodiment, the cellular cytotoxicity is T-cell dependent cytotoxicity (TDCC). In another embodiment, the cytotoxicity is directed against cells expressing DLL3 on their surface. The DLL3-expressing cells may be cancer cells.

[0225] In a preferred embodiment, the antibody (or antigen-binding molecule) of the present invention has cytotoxicity (or cellular cytotoxicity), or preferably T-cell dependent cytotoxicity (TDCC), against DLL3-expressing cells, such as cancer cells. DLL3 can be expressed on the surface of such cells. The (cellular) cytotoxicity or TDCC of the antibody (or antigen-binding molecule) of the present invention can be evaluated by any suitable method known in the art. For example, the methods described in some Examples can be used to measure TDCC. In this case, cytotoxic activity is evaluated by the rate of cell growth inhibition by the antibody (or antigen-binding molecule) of the present invention. Cell growth is measured using a suitable analyzer, such as the xCELLigence Real-Time Cell Analyzer. Cancer cells are used as target cells, and they are cultured at a suitable cell concentration (e.g., about 10 4The cells are seeded onto a multi-well plate at 1000 x 1000 cells / well. The next day, a test antibody prepared at an appropriate concentration (e.g., 0.001-10 nM) is added to the plate. After 15 minutes of reaction, a solution containing T cells (e.g., PBMCs) is added to it at an appropriate effector (PBMC) / target (cancer cell) ratio, such as a ratio of 10. The reaction is carried out using carbon dioxide gas. After the addition of T cells, the cell growth inhibition (CGI) rate (%) is determined using the formula: CGI rate (%) = (A-B) x 100 / (A-1), where A represents the average cell index value of wells containing no antibody (or antigen-binding molecule), i.e., containing only target cells and T cells, and B represents the average cell index value of wells containing antibody (or antigen-binding molecule). The cell index value used in the calculation is a normalized value; i.e., the cell index value immediately before antibody addition is defined as 1. When the CGI rate of an antibody (or antigen-binding molecule) is high, i.e., has a significantly positive value, the antibody (or antigen-binding molecule) can be said to have TDCC activity and to be more preferable in the present invention.

[0226] Alternatively, cytotoxic activity can be assessed by a calcein-acetoxymethyl release assay. Cancer cells are used as target cells. The target cells are labeled with calcein-acetoxymethyl and then washed. Test antibodies (e.g., 0.001–10 nM) are pipetted onto the plate, and the calcein-labeled target cell suspension is added. After the plate is left at room temperature, an effector cell (e.g., PBMC) suspension is added. The plate is agitated, centrifuged, and incubated in a CO2 incubator. After thorough agitation and centrifugation, the culture medium from each well is transferred to another plate. The absorbance (495 nm, reference 515 nm) is measured. For maximum release, cells may be lysed with 0.5% NP-40. The background fluorescence value of the culture medium is subtracted from the experimental release (A), spontaneous target cell release (B), and maximum target cell release (C). Cytotoxicity is calculated using the following formula: Cytotoxicity (%) = (AB) / (CB) × 100. When this value for an antibody (or antigen-binding molecule) is high, i.e., when it has a significantly positive value, it can be said that the antibody (or antigen-binding molecule) has TDCC activity and is more preferable in the present invention.

[0227] Monospecific antigen-binding molecules The term "monospecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to only one type of antigen. A preferred example of a monospecific antigen-binding molecule is an antigen-binding molecule that contains a single type of antigen-binding domain. A monospecific antigen-binding molecule may contain a single antigen-binding domain or multiple antigen-binding domains of the same type. A preferred example of a monospecific antigen-binding molecule is a monospecific antibody. When a monospecific antigen-binding molecule is an IgG-type monospecific antibody, the monospecific antibody contains two antibody variable fragments with the same antigen-binding specificity.

[0228] The monospecific antigen-binding molecules of the present invention contain an antigen-binding domain that binds to DLL3. The antigen-binding domain that binds to LL3 may be any one of those described above under "Antigen-binding domain that binds to DLL3."

[0229] The term "monospecific antigen-binding molecule that binds to DLL3" refers to a monospecific antigen-binding molecule that can bind to DLL3 with sufficient affinity so as to be useful as a diagnostic and / or therapeutic agent when the antibody targets DLL3. In one embodiment, the extent of binding of a monospecific antigen-binding molecule that binds to DLL3 to an unrelated non-DLL3 protein is less than about 10% of the binding to DLL3, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, a monospecific antigen-binding molecule that binds to DLL3 has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0230] In some embodiments, the monospecific antigen binding molecules of the present invention that bind to DLL3 comprise a functional Fc region that has effector functions such as C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; or downregulation of cell surface receptors (e.g., B cell receptors; BCR).

[0231] Antibody-dependent cytotoxicity "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a type of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and then kill them using cytotoxins. NK cells, the primary cells for mediating ADCC, express only Fc gamma RIII, whereas monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as that described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., PNAS (USA), 95:652-656 (1998).

[0232] Immunoconjugates The present invention also provides immunoconjugates comprising the antigen-binding molecules herein, e.g., monospecific antigen-binding molecules that bind to DLL3, conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxic compounds, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes. In some embodiments, the present invention provides antigen-binding molecules or antibodies conjugated to toxic compounds. In other words, the present invention provides antibody-drug conjugate compounds comprising the antigen-binding molecules or antibodies.

[0233] In one embodiment, the immunoconjugate is an immunoconjugate in which the antibody is coupled to a steroid or steroid-resistant steroid, including, but not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. EP 0425235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358~362 (2006);Torgov et al., Bioconj.Chem.16:717~721 (2005);Nagy et al., Proc.Natl.Acad.Sci.USA 97:829~834 (2000);Dubowchik et al., Bioorg.&Med.Chem.Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and antibody-drug conjugates (ADCs) conjugated to one or more drugs including CC1065.

[0234] In another embodiment, the immunoconjugate comprises an antigen binding molecule described herein (e.g., a monospecific antigen binding molecule that binds DLL3) conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0235] In another embodiment, the immunoconjugate comprises an antigen-binding molecule described herein (such as a monospecific antigen-binding molecule that binds to DLL3) conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioactive isotopes of Pb and Lu are included. When a radioactive conjugate is used for detection, it is used as a radioactive atom for scintigraphic examination, such as Tc-99m or 123 I, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as, again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0236] Conjugates of antigen-binding molecules (e.g., monospecific antigen-binding molecules that bind to DLL3) with cytotoxic agents can be prepared using N-succinimidyl-3-(2-pyridyldithio)propionic acid (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-diazoniumbenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-azidobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6 1,5-Difluoro-2,4-dinitrobenzene)。 For example, ricin immunotoxins can be made using a variety of bifunctional protein coupling agents, such as bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026. The linker may also be a "cleavable linker" that facilitates the release of the cytotoxic drug in cells. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) can be used.

[0237] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared using cross-linking reagents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0238] cancer The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0239] tumor The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor," as referred to herein, are not mutually exclusive.

[0240] In preferred embodiments, the cancer is a cancer (including cancer tissues or cells) that expresses DLL3. In some embodiments, the cancer is pancreatic cancer, glioma, small cell lung cancer (SCLC), or melanoma.

[0241] Pharmaceutical preparations The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0242] Pharmaceutically acceptable carrier A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to: Buffers, excipients, stabilizers, or preservatives may be included.

[0243] treatment As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease appearance or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0244] In one aspect, the present invention is based in part on a multispecific antigen-binding molecule comprising a first antigen-binding domain that binds to DLL3 and a second antigen-binding domain that binds to the T cell receptor complex, and its use. The antigen-binding molecules and antibodies of the present invention are useful, for example, for the diagnosis or treatment of tumors, particularly colorectal and gastric tumors.

[0245] Pharmaceutical Composition The pharmaceutical compositions of the present invention, therapeutic agents for inducing cellular cytotoxicity, cell growth inhibitors, or anticancer agents of the present invention can be formulated with various types of antigen-binding molecules, as needed. For example, the cytotoxic effect on antigen-expressing cells can be enhanced by a cocktail of multiple antigen-binding molecules of the present invention.

[0246] If necessary, the antigen-binding molecules of the present invention can be encapsulated in microcapsules (microcapsules made from hydroxymethylcellulose, gelatin, poly(methyl methacrylate), etc.) and prepared as components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remington's Pharmaceutical Science, 16th Edition," edited by Oslo (1980)). Furthermore, methods for preparing drugs as sustained-release agents are known and can be applied to the antigen-binding molecules of the present invention (J. Biomed. Mater. Res. (1981) 15, pp. 267-277; Chemtech. (1982) 12, pp. 98-105; U.S. Patent No. 3,773,719; European Patent Applications EP 58481 and EP 133988; Biopolymers (1983) 22, pp. 547-556).

[0247] The pharmaceutical composition, cytostatic agent, or anticancer agent of the present invention can be administered to a patient orally or parenterally. Parenteral administration is preferred. Specific administration methods include injection, intranasal administration, pulmonary administration, and transdermal administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition, therapeutic agent, cytostatic agent, or anticancer agent for inducing cellular cytotoxicity of the present invention can be administered locally or systemically by injection. Furthermore, an appropriate administration method can be selected according to the patient's age and symptoms. The administered dose can be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dose can be selected, for example, from the range of 0.001 mg to 100,000 mg per patient's body weight. However, the dose of the pharmaceutical composition of the present invention is not limited to these doses.

[0248] The pharmaceutical compositions of the present invention can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Sciences, latest edition, Mark Publishing Company, Easton, USA) and are well known in the art. The composition may contain a commercially acceptable carrier and additive. Examples include, but are not limited to, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, glidants, and flavoring agents, and other commonly used carriers can also be used. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc.

[0249] Preferably, the pharmaceutical composition of the present invention comprises an antigen-binding molecule of the present invention. In one embodiment, the composition is a pharmaceutical composition for use in inducing cellular cytotoxicity. In another embodiment, the composition is a pharmaceutical composition for use in treating or preventing cancer. Preferably, the cancer is one of the cancers described above. The pharmaceutical composition of the present invention can be used to treat or prevent cancer. Thus, the present invention provides a method for treating or preventing cancer, comprising administering an antigen-binding molecule of the present invention to a patient in need thereof.

[0250] Furthermore, the present invention provides use of the antigen-binding molecule or antibody described above in the manufacture of a pharmaceutical composition for treating or preventing cancer. The present invention also provides use of the antigen-binding molecule / antibody / pharmaceutical composition for treating or preventing cancer.

[0251] The present invention also provides a method for damaging or inhibiting cell proliferation of DLL3-expressing cells by contacting the cells with an antigen-binding molecule of the present invention that binds to DLL3. Monoclonal antibodies that bind to DLL3 are described above as antigen-binding molecules of the present invention that are included in therapeutic agents, cell growth inhibitors, and anticancer agents for inducing cellular cytotoxicity. Cells to which the antigen-binding molecules of the present invention bind are not particularly limited, as long as they express DLL3. Specifically, preferred cancer antigen-expressing cells in the present invention include pancreatic cancer cells, glioma cells, melanoma cells, and small cell lung cancer (SCLC) cells.

[0252] In the present invention, "contacting" can be performed, for example, by adding an antigen-binding molecule of the present invention to the culture medium of in vitro cultured cells expressing DLL3. In this case, the antigen-binding molecule to be added can be in an appropriate form, such as a solution or a solid prepared by lyophilization. When the antigen-binding molecule of the present invention is added as an aqueous solution, the solution may be a pure aqueous solution containing only the antigen-binding molecule, or a solution containing, for example, the above-mentioned surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, glidants, and flavoring agents. The concentration of the added molecule is not particularly limited, but the final concentration in the culture medium is preferably in the range of 1 pg / ml to 1 g / ml, more preferably 1 ng / ml to 1 mg / ml, and even more preferably 1 μg / ml to 1 mg / ml.

[0253] In another embodiment of the present invention, "contacting" can also be performed by administration to a non-human animal transplanted with DLL3-expressing cells in vivo or to an animal having cancer cells that endogenously express DLL3. The administration method may be oral or parenteral. Parenteral administration is particularly preferred. Specifically, parenteral administration methods include injection, intranasal administration, pulmonary administration, and transdermal administration. Examples of injection include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition, therapeutic agent, cytostatic agent, or anticancer agent for inducing cellular cytotoxicity of the present invention can be administered locally or systemically by injection. Furthermore, an appropriate administration method can be selected depending on the age and symptoms of the animal subject. When the antigen-binding molecule is administered as an aqueous solution, the solution contains only the antigen-binding molecule. The antigen-binding molecule of the present invention may be a pure aqueous solution containing the antigen-binding molecule of the present invention, or a solution containing, for example, the above-mentioned surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, glidants, and flavoring agents. The administered dose can be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight for each administration. Alternatively, the dose can be selected, for example, from the range of 0.001 mg to 100,000 mg per patient's body weight. However, the dose of the antigen-binding molecule of the present invention is not limited to these examples.

[0254] The method described below is preferably used as a method for evaluating or determining cellular cytotoxicity caused by contacting an antigen-binding molecule of the present invention with a DLL3-expressing cell to which the antigen-binding domain forming the antigen-binding molecule of the present invention binds. In vitro methods for evaluating or determining cytotoxic activity include methods for determining the activity of cytotoxic T cells, etc. Whether an antigen-binding molecule of the present invention has the activity of inducing T cell-mediated cellular cytotoxicity can be determined by known methods (see, for example, Current Protocols in Immunology, Chapter 7, Immunologic Studies in Human, John E., Coligan et al. (eds.), John Wiley & Sons, Inc. (1993)). In cytotoxicity assays, an antigen-binding molecule whose antigen-binding domain binds to an antigen other than DLL3 and is not expressed in cells is used as a control antigen-binding molecule. The control antigen-binding molecule is assayed in the same manner. The activity of the antigen-binding molecule of the present invention is then evaluated by testing whether it exhibits stronger cytotoxic activity than that of the control antigen-binding molecule.

[0255] Meanwhile, in vivo cytotoxic activity is evaluated or determined, for example, by the following procedure. Cells expressing an antigen to which the antigen-binding domain forming the antigen-binding molecule of the present invention binds are implanted intradermally or subcutaneously into a non-human animal subject. Then, starting on or after the day of implantation, a test antigen-binding molecule is administered intravenously or intraperitoneally daily or at intervals of several days. Tumor size is measured over time. The difference in change in tumor size can be defined as cytotoxic activity. As in in vitro assays, a control antigen-binding molecule is administered. The antigen-binding molecule of the present invention can be determined to have cytotoxic activity if the tumor size is smaller in the group administered with the antigen-binding molecule of the present invention than in the group administered with the control antigen-binding molecule.

[0256] Preferably, the effect of contact with the antigen-binding molecule of the present invention is evaluated or determined using the MTT method and measurement of cellular incorporation of isotope-labeled thymidine to inhibit the proliferation of cells expressing an antigen to which the antigen-binding domain forming the antigen-binding molecule binds. On the other hand, preferably, the activity of inhibiting cell proliferation in vivo can be evaluated or determined using the same methods described above for evaluating or determining in vivo cytotoxic activity.

[0257] The present invention also provides kits for use in the methods of the present invention, which contain the antigen-binding molecules of the present invention or antigen-binding molecules produced by the methods of the present invention. The kits can be packaged together with additional pharmaceutically acceptable carriers or vehicles, or instructions describing how to use the kit.

[0258] Furthermore, the present invention relates to the antigen-binding molecules of the present invention or antigen-binding molecules produced by the methods of the present invention, for use in the methods of the present invention.

[0259] In another embodiment, an internalizing antibody is provided. In other words, the present invention provides an antibody or antigen-binding molecule with internalizing activity. Such an antibody increases the delivery of the antibody into cells. The antibody may have certain characteristics that enhance its internalization or can be modified to have such characteristics. Techniques for achieving this are known in the art. For example, cationization of an antibody is known to facilitate its uptake into cells (see, e.g., U.S. Pat. No. 6,703,019). Antibodies can also be delivered into cells using lipofection or liposomes. When using antibody fragments, the smallest inhibitory fragment that specifically binds to the target protein can be used. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA 90:7889-7893 (1993). The internalization activity of an antibody (or antigen-binding molecule) can be determined, for example, by the method described in Reference Example 18 of European Patent Application Publication No. 3015115 (WO 2014 / 208482).

[0260] Expression and purification of DLL3 extracellular domain (ECD) fragment protein A DLL3 extracellular domain (ECD) fragment protein (or a truncated variant thereof), which may have a Flag tag at the C-terminus, can be transiently expressed using an appropriate vector and cells. The culture supernatant containing the protein is loaded onto a column packed with affinity resin and eluted. The fractions containing the protein are collected and then loaded onto a filtration column equilibrated with a buffer solution. The fractions containing the protein are then pooled and stored at -80°C until use. The ECD fragment protein can be used for epitope mapping or competitive assays by known methods or the methods described herein.

[0261] Establishment of a cell line expressing DLL3 To establish a cell line expressing DLL3, DLL3 cDNA is inserted into an expression vector, which is then introduced into cells, for example, by electroporation. After introduction, a selective agent (such as geneticin) is added, and the cells are cultured to obtain a cell line that is resistant to the agent. The transfected cell line can be plated and grown by limiting dilution. The established cell line can be used to evaluate the TDCC activity of the antigen-binding molecule or antibody of the present invention against cells expressing DLL3.

[0262] Generation and screening of anti-DLL3 antibodies (monospecific) Anti-DLL3 antibodies can be prepared, selected, and assayed as described below. Animals, such as rabbits, are immunized with DLL3 or a fragment thereof. After the final immunization, spleens and blood are collected from the immunized animals. Antigen-specific B cells are stained and sorted using a cell sorter, e.g., EL4 cells (European Collection of The cells are seeded into plates at a density of 1 cell per well with DLL3-expressing cells (B cell cultures) and cultured. After culture, the B cell culture supernatant can be collected for further analysis, and the pellet can be cryopreserved. ELISA screening can be performed to test the specificity of the antibody in the B cell culture supernatant. DLL3-expressing cells are immobilized on plates pre-coated with BSA and a biocompatible anchor for the cell membrane. The immobilized cells are incubated with the B cell culture supernatant. The cells are washed, and a goat anti-rabbit IgG polyclonal antibody HRP conjugate, for example, is added. The cells are further incubated on ice, substrate is added, and the optical density is suitably analyzed. B cell clones are screened for binding to DLL3-expressing cells, and clones are selected as DLL3-specific binders. The selected clones are purified from the cryopreserved cell pellet. The DNA of the antibody heavy chain variable region is amplified by reverse transcription PCR and ligated with DNA encoding the human IgG1 heavy chain constant region to form the corresponding heavy chain. The DNA encoding the antibody light chain variable region was amplified by reverse transcription PCR and ligated to the DNA encoding the light chain constant region. The cloned antibodies are expressed in cells and purified from the culture supernatant for functional evaluation. The monospecific antibodies may be used to produce anti-DLL3 / anti-CD3 bispecific antibodies by known methods.

[0263] Epitope mapping of selected anti-DLL3 antibodies (monospecific) The structures of DLL3 and DLL3 ECD fragment proteins are illustrated in Figure 1. These DLL3 ECD fragment proteins can be used, for example, for epitope mapping of anti-DLL3 antibodies, as described below. A plate is coated with the fragment protein and blocked with buffer. The blocking buffer is removed, and an anti-DLL3 antibody is incubated with the immobilized protein and washed with buffer. Antibody binding can be assessed using any suitable detection system, including an anti-Flag antibody. For example, monoclonal ANTI-FLAG M2-peroxidase, clone M2 (Sigma-Aldrich) is added, incubated, and washed. Substrate is then added, and the optical density (e.g., OD405) is determined. The OD405 value represents the reactivity of the test antibody against the fragment protein. Antibody epitopes can be inferred by testing which domain deletions (see Figure 1) abolish antibody binding. That is, if an antibody cannot bind to a particular deletion construct, for example, among a panel of sequential deletion constructs truncated from the N-terminus (Figure 1), then this construct does not contain the antibody's epitope, and a longer deletion construct to which the antibody can bind, or more specifically the N-terminal region of the shortest one among such longer deletion constructs, can be said to contain the antibody's epitope.

[0264] Functional evaluation of anti-DLL3 / CD3 bispecific antibodies Anti-DLL3 / CD3 bispecific antibodies can be evaluated for their in vivo anti-tumor efficacy in a xenograft model, as described below. Cancer cell lines are transplanted into NOD scid mice, and NOD scid mice with confirmed tumor formation are transplanted with T cells expanded by in vitro culture of human PBMCs. The mice (referred to as the T cell injection model) are treated by administering the bispecific antibody. For example, in testing the anti-tumor efficacy of bispecific antibodies using the T cell injection model, the following can be performed: T cells are expanded using PBMCs and a suitable medium, such as a T cell activation / expansion kit / human (MACS Miltenyi biotec). Human cancer cell lines are mixed with a suitable support material, such as Matrigel™ Basement Membrane Matrix (BD), and transplanted into the area of ​​NOD scid mice. The day before transplantation (day -1, if the day of tr...

Claims

1. The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, A multispecific antigen-binding molecule (1) in which the first antigen-binding domain binds to an epitope within the region defined by SEQ ID NO: 7 in human DLL3.

2. The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, The first antigen-binding domain of (1) is selected from the following (a1) to (a12): (a1) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 27, the HVR-H2 sequence of SEQ ID NO: 28, the HVR-H3 sequence of SEQ ID NO: 29, the HVR-L1 sequence of SEQ ID NO: 30, the HVR-L2 sequence of SEQ ID NO: 31, and the HVR-L3 sequence of SEQ ID NO: 32; (a2) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 33, the HVR-H2 sequence of SEQ ID NO: 34, the HVR-H3 sequence of SEQ ID NO: 35, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L2 sequence of SEQ ID NO: 37, and the HVR-L3 sequence of SEQ ID NO: 38; (a3) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 39, the HVR-H2 sequence of SEQ ID NO: 40, the HVR-H3 sequence of SEQ ID NO: 41, the HVR-L1 sequence of SEQ ID NO: 42, the HVR-L2 sequence of SEQ ID NO: 43, and the HVR-L3 sequence of SEQ ID NO: 44; (a4) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 45, the HVR-H2 sequence of SEQ ID NO: 46, the HVR-H3 sequence of SEQ ID NO: 47, the HVR-L1 sequence of SEQ ID NO: 48, the HVR-L2 sequence of SEQ ID NO: 49, and the HVR-L3 sequence of SEQ ID NO: 50; (a5) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 51, the HVR-H2 sequence of SEQ ID NO: 52, the HVR-H3 sequence of SEQ ID NO: 53, the HVR-L1 sequence of SEQ ID NO: 54, the HVR-L2 sequence of SEQ ID NO: 55, and the HVR-L3 sequence of SEQ ID NO: 56; (a6) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 27, the HVR-H2 sequence of SEQ ID NO: 75, the HVR-H3 sequence of SEQ ID NO: 29, the HVR-L1 sequence of SEQ ID NO: 30, the HVR-L2 sequence of SEQ ID NO: 31, and the HVR-L3 sequence of SEQ ID NO: 32; (a7) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 27, the HVR-H2 sequence of SEQ ID NO: 76, the HVR-H3 sequence of SEQ ID NO: 29, the HVR-L1 sequence of SEQ ID NO: 30, the HVR-L2 sequence of SEQ ID NO: 31, and the HVR-L3 sequence of SEQ ID NO: 32; (a8) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the HVR-H3 sequence of SEQ ID NO: 79, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L2 sequence of SEQ ID NO: 37, and the HVR-L3 sequence of SEQ ID NO: 38; (a9) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the HVR-H3 sequence of SEQ ID NO: 80, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L2 sequence of SEQ ID NO: 37, and the HVR-L3 sequence of SEQ ID NO: 38; (a10) an antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the HVR-H3 sequence of SEQ ID NO: 80, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L2 sequence of SEQ ID NO: 37, and the HVR-L3 sequence of SEQ ID NO: 81; (a11) an antibody variable fragment that binds to the same epitope as any of the antibody variable fragments selected from (a1) to (a10); (a12) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a10). A multispecific antigen-binding molecule comprising any one of:

3. The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, The first antigen-binding domain of (1) is selected from the following (b1) to (b21): (b1) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 15, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 15, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 15, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 16, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 16, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 16; (b2) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 25, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 25, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 25, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 26, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 26, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 26; (b3) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 19, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 19, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 19, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 20, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 20, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 20; (b4) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 23, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 23, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 23, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 24, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 24, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 24; (b5) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 11, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 11, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 11, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 12, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 12, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 12; (b6) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 13, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 13, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 13, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 14, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 14, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 14; (b7) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 17, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 17, and an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 17 an antibody variable fragment comprising an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 18, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 18, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 18; (b8) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 21, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 21, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 21, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 22, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 22, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 22; (b9) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 85, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 85, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 85, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 93, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 93, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 93; (b10) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (b11) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (b12) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 72, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 72, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 72; (b13) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 66, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b14) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, and an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73 an antibody variable fragment comprising an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73; (b15) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 74; (b16) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b17) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 69, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 69, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b18) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 70, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b19) an antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region included in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region included in SEQ ID NO: 71, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region included in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region included in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region included in SEQ ID NO: 73, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region included in SEQ ID NO: 73; (b20) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (b1) to (b19); (b21) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (b1) to (b19). A multispecific antigen-binding molecule comprising any one of:

4. The following domains: (1) a first domain comprising a first antigen-binding domain that binds to human DLL3; and (2) a second domain comprising a second antigen-binding domain that binds to the T cell receptor complex; Including, The first antigen-binding domain of (1) is selected from the following (c1) to (c22): (c1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16 a light chain variable region having the sequence: (c2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and a light chain variable region having the amino acid sequence of SEQ ID NO: 26; (c3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and a light chain variable region having the amino acid sequence of SEQ ID NO: 20; (c4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and a light chain variable region having the amino acid sequence of SEQ ID NO: 24; (c5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 12; (c6) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and a light chain variable region having the amino acid sequence of SEQ ID NO: 14; (c7) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 18; (c8) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22; (c9) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 85 and a light chain variable region having the amino acid sequence of SEQ ID NO: 93; (c10) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c11) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 64 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c12) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c13) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 66 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c14) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c15) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and a light chain variable region having the amino acid sequence of SEQ ID NO: 74; (c16) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 68 and a light chain variable region having the amino acid sequence of SEQ ID NO: 72; (c17) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c18) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 70 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c19) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 71 and a light chain variable region having the amino acid sequence of SEQ ID NO: 73; (c20) a heavy chain variable region having greater than 80% identity to any one of the heavy chain variable regions of (c1) to (c19) and a light chain variable region having greater than 80% identity to any one of the light chain variable regions of (c1) to (c19); (c21) A heavy chain variable region having greater than 90% identity to any one of the heavy chain variable regions of (c1) to (c19) and a light chain variable region having greater than 90% identity to any one of the light chain variable regions of (c1) to (c19); (c22) A heavy chain variable region having more than 95% identity to any one of the heavy chain variable regions of (c1) to (c19), and a light chain variable region having more than 95% identity to any one of the light chain variable regions of (c1) to (c19). A multispecific antigen-binding molecule comprising any one of the combinations of heavy chain variable regions and light chain variable regions selected from:

5. The multispecific antigen-binding molecule of any one of claims 1 to 4, which has cytotoxic activity. child.

6. The multispecific antigen-binding molecule of claim 5 , wherein the cytotoxic activity is T-cell dependent cytotoxicity.

7. The multispecific antigen-binding molecule of any one of claims 1 to 6, wherein the second antigen-binding domain in (2) binds to the CD3 epsilon chain.

8. The multispecific antigen-binding molecule of any one of claims 1 to 6, wherein the second antigen-binding domain in (2) binds to a T cell receptor.

9. The multispecific antigen-binding molecule of any one of claims 1 to 8, wherein the first antigen-binding domain or the second antigen-binding domain is an antibody variable fragment, or both the first and second antigen-binding domains are antibody variable fragments.

10. (3) A third domain comprising an Fc region with reduced binding activity to an Fc gamma receptor.

10. The multispecific antigen-binding molecule of claim 1, further comprising:

11. The multispecific antigen-binding molecule of claim 10, wherein the Fc region has an amino acid mutation in any of the amino acids constituting the Fc region of SEQ ID NOs: 112 to 115 (IgG1 to IgG4).

12. The Fc region comprises the following amino acid positions identified by EU numbering: 220th, 226th, 229th, 231st, 232nd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 264th, 265th, 266th, 2 67th, 269th, 270th, 295th, 296th, 297th, 298th, 299th, 300th, 325th, 327th, 328th, 329th, 330th, 331st, and 332nd The multispecific antigen-binding molecule of claim 11, wherein the Fc region has at least one amino acid mutation selected from the group consisting of:

13. The multispecific antigen-binding molecule of claim 1 , which is a bispecific antibody.

14. A pharmaceutical composition comprising the multispecific antigen-binding molecule of any one of claims 1 to 12 or the bispecific antibody of claim 13 and a pharmaceutically acceptable carrier.

15. 14. A pharmaceutical composition for use in the treatment or prevention of cancer, comprising a multispecific antigen-binding molecule according to any one of claims 1 to 12 or a bispecific antibody according to claim 13.

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