Cytotoxic Inducing Therapeutic Agent

Abstract

To provide a novel pharmaceutical composition that comprises a molecule or an antibody to show strong cellular cytotoxicity against cells expressing DLL3 and to treat or prevent various cancers associated with DLL3.SOLUTION: The present invention provides a novel multispecific antigen-binding molecule which comprise a first domain comprising a first antigen variable region which binds to an epitope in a specific region of DLL3, and a second domain comprising a second antigen variable region which binds to T cell receptor complex. The multispecific antigen-binding molecule shows excellent T cell-dependent cell cytotoxicity (TDCC). There is also provided a pharmaceutical composition comprising the same.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to 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, and uses thereof. The present invention also relates to a novel monospecific antigen-binding molecule containing an antigen-binding domain that binds to human DLL3, and uses thereof. and the like. The present invention also relates to a novel monospecific antigen-binding molecule containing an antigen-binding domain that binds to human DLL3, and uses thereof. and the like.

Background Art

[0002] Cancer is one of the leading causes of death worldwide. Except for certain carcinomas, tumors are often inoperable when they are discovered. Conventional cancer treatments include radiotherapy, chemotherapy, and immunotherapy. These treatments are often not sufficiently effective and ultimately result in cancer recurrence or metastasis after treatment. The lack of tumor specificity is one of the factors limiting maximum efficacy; therefore, more tumor-specific molecular target therapies are becoming additional viable options in cancer treatment. and the like. and the like. and ultimately result in cancer recurrence or metastasis after treatment. The lack of tumor specificity is one of the factors limiting maximum efficacy; therefore, more tumor-specific molecular target therapies are becoming additional viable options in cancer treatment.

[0003] Antibodies are attracting attention as pharmaceuticals because of their high stability in plasma and low side effects. Among multiple therapeutic antibodies, some types of antibodies require effector cells to exert an antitumor response. Antibody-dependent cell-mediated cytotoxicity (ADCC) is the cytotoxicity shown by effector cells against cells bound by an antibody due to the binding of the Fc region of the antibody to Fc receptors present on NK cells and macrophages. To date, multiple therapeutic antibodies capable of inducing ADCC to exert antitumor efficacy have been used as pharmaceuticals for treating cancer. and the like. and the like. and the like. and the like. To date, multiple therapeutic antibodies capable of inducing ADCC to exert antitumor efficacy have been used as pharmaceuticals for treating cancer. ​​​and developed (Non-Patent Document 1). Therapies targeting antigens specifically expressed in tumors using conventional therapeutic antibodies exhibit excellent antitumor activity, but the administration of such antibodies has not necessarily led to satisfactory results. In addition to antibodies that employ ADC by mobilizing NK cells or macrophages as effector cells, T cell-recruiting (TR) antibodies that employ cytotoxicity by mobilizing T cells as effector cells have also been known since the 1980s (Non-Patent Documents 2 to 4). TR antibodies are bispecific antibodies that recognize and bind to any one of the subunits that form the T cell receptor complex on T cells, particularly the CD3 epsilon chain, and an antigen on target cells. Some TR antibodies are currently under development. Catumaxomab, a TR antibody against EpCAM, is approved in the EU for the treatment of malignant ascites. Furthermore, a type of TR antibody called "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, received FDA approval for the first time in 2014. Blinatumomab has been shown to have much stronger cytotoxic activity against CD19 / CD20-positive cancer cells in vitro compared to Rituximab, and has been proven to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Non-Patent Document 7).

[0004] NK cells or macrophages as effector cells to employ ADC In addition to antibodies that employ ADC by mobilizing NK cells or macrophages as effector cells, cytotoxicity is employed by mobilizing T cells as effector cells T cell-recruiting (TR) antibodies have also been known since the 1980s (Non-Patent Documents 2 to 4). TR antibodies are bispecific antibodies that recognize and bind to any one of the subunits that form the T cell receptor complex on T cells, particularly the CD3 epsilon chain, and an antigen on target cells. Some TR antibodies are currently under development. Catumaxomab, a TR antibody against EpCAM, is approved in the EU for the treatment of malignant ascites. Furthermore, a type of TR antibody called "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, received FDA approval for the first time in 2014. Blinatumomab has been shown to have much stronger cytotoxic activity against CD19 / CD20-positive cancer cells in vitro compared to Rituximab, and has been proven to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Non-Patent Document 7). FDA approval. Blinatumomab has been shown to have much stronger cytotoxic activity against CD19 / CD20-positive cancer cells in vitro compared to Rituximab, and has been proven to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) compared to Rituximab, and has been proven to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Non-Patent Document 7).

[0005] ​​​However, trifunctional antibodies bind simultaneously to both T cells and cells such as NK cells or macrophages in a cancer antigen-independent manner, resulting in cross-linking of the 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 storms as a result of such induction of cytokine expression. In fact, in a phase I clinical trial, a very low dose of 5 micrograms (μg) / systemic was the maximum tolerated dose for the systemic administration of catumaxomab to patients with non-small cell lung cancer, and administration of higher doses has been reported to cause various severe side effects (Non-Patent Document 8). When administered at such low doses, catumaxomab never reaches effective blood levels. That is, by administering catumaxomab at such low doses, the expected anti-tumor effect cannot be achieved. As a result, the receptors expressed on these cells are cross-linked, and it is known that the expression of various cytokines is induced in a cancer antigen-independent manner. Systemic administration of trifunctional antibodies is thought to cause side effects such as cytokine storms as a result of such induction of cytokine expression. In fact, in a phase I clinical trial, a very low dose of 5 micrograms (μg) / systemic was the maximum tolerated dose for the systemic administration of catumaxomab to patients with non-small cell lung cancer, and administration of higher doses has been reported to cause various severe side effects (Non-Patent Document 8). When administered at such low doses, catumaxomab never reaches effective blood levels. That is, by administering catumaxomab at such low doses, the expected anti-tumor effect cannot be achieved. (Non-Patent Document 8).

[0006] On the other hand, unlike catumaxomab, BiTE does not have an Fc gamma receptor binding site, and thus does not cross-link the receptors expressed on cells such as T cells, NK cells, and macrophages in a cancer antigen-independent manner. Thus, it has been demonstrated that BiTE does not cause cancer antigen-independent cytokine induction observed when catumaxomab is administered. However, since BiTE is a low molecular weight modified antibody molecule lacking an Fc region, the problem is that its blood half-life after administration to patients is significantly shorter than that of IgG-type antibodies conventionally used as therapeutic antibodies. In fact, the blood ​​​​​​​​​The half-life is reported to be about several hours (Non-Patent Document 9 and Non-Patent Document 10). In the clinical trial of blinatumomab, it is administered by continuous intravenous infusion using a minipump This administration method is not only extremely inconvenient for patients, but also has the potential risk of medical accidents due to device malfunctions and the like. Thus, such an administration method cannot be said to be desirable .

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

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Literature

[0009]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Non-Patent Literature 6

Non-Patent Literature 7

Non-Patent Literature 8

Non-Patent Literature 9

Non-Patent Literature 10

Non-Patent Literature 11

Non-Patent Literature 12

Non - Patent Document 13

Non - Patent Document 14

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to enable cancer treatment by using the cytotoxicity of T cells against cancer cells that express DLL3 by bringing T cells into proximity with DLL3 - expressing cells, a multispecific antigen - binding molecule, a method for producing the multispecific antigen - binding molecule, and a therapeutic agent containing 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, which contains one of the above - mentioned antigen - binding molecules as an active ingredient, and a treatment method using the pharmaceutical composition. Another object of the present invention is to provide a novel single - specificity antigen - binding molecule having human DLL3 - binding activity, a therapeutic agent containing such a single - specificity antigen - binding molecule as an active ingredient, and a treatment method using such a therapeutic agent.

Means for Solving the Problems

Means for Solving the Problems

[0011] The inventors of the present invention have found that a multispecific antigen - binding molecule containing a first domain having a first antigen - binding domain that binds to human DLL3 and a second domain having a second antigen - binding domain that binds to the T - cell receptor complex damages cells expressing DLL3 and has excellent cytotoxic / anti - tumor activity. activity. The present invention has been found to be effective in treating cancer by using an antigen-binding molecule as an active ingredient. By including The present invention provides a multispecific antigen-binding molecule and a pharmaceutical composition capable of treating The present invention also provides a novel monospecific antigen-binding molecule comprising an antigen-binding domain that binds to human DLL3. and pharmaceutical compositions comprising 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, The first antigen-binding domain of (1) is a region defined by SEQ ID NO: 7 in human DLL3. A multispecific antigen-binding molecule that binds to an epitope. [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) the HVR-H1 sequence of SEQ ID NO: 27, the HVR-H2 sequence of SEQ ID NO: 28, HVR-H3 sequence of SEQ ID NO: 29, HVR-L1 sequence of SEQ ID NO: 30, HVR-L sequence of SEQ ID NO: 31 an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 32; (a2) the HVR-H1 sequence of SEQ ID NO: 33, the HVR-H2 sequence of SEQ ID NO: 34, and the HVR-H3 sequence of SEQ ID NO: HVR-H3 sequence of SEQ ID NO: 35, HVR-L1 sequence of SEQ ID NO: 36, HVR-L1 sequence of SEQ ID NO: 37 An antibody variable fragment comprising a 2-array and an HVR-L3 array of SEQ ID NO: 38; (a3) The HVR-H1 array of SEQ ID NO: 39, the HVR-H2 array of SEQ ID NO: 40, the SEQ ID NO: 41 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 42, the HVR-L 2-array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 44; (a4) The HVR-H1 array of SEQ ID NO: 45, the HVR-H2 array of SEQ ID NO: 46, the SEQ ID NO: 47 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 48, the HVR-L of SEQ ID NO: 49 2-array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 50; (a5) The HVR-H1 array of SEQ ID NO: 51, the HVR-H2 array of SEQ ID NO: 52, the SEQ ID NO: 53 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 54, the HVR-L of SEQ ID NO: 55 2-array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 56; (a6) The HVR-H1 array of SEQ ID NO: 27, the HVR-H2 array of SEQ ID NO: 75, the SEQ ID NO: 29 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 30, the HVR-L of SEQ ID NO: 31 2-array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 32; (a7) The HVR-H1 array of SEQ ID NO: 27, the HVR-H2 array of SEQ ID NO: 76, the SEQ ID NO: 29 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 30, the HVR-L of SEQ ID NO: 31 2-array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 32; (a8) The HVR-H1 array of SEQ ID NO: 77, the HVR-H2 array of SEQ ID NO: 78, the SEQ ID NO: 79 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 36, the HVR-L of SEQ ID NO: 37 2-array, and an antibody variable fragment comprising the HVR-L3 array 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-L 2 sequence, 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 SEQ ID NO<, : 80 HVR-H3 sequence, the HVR-L1 sequence of SEQ ID NO: 36, the HVR- L2 sequence, and the HVR-L3 sequence of SEQ ID NO: 81; (a11) An antibody variable fragment that binds to the same epitope of any of the antibody variable fragments selected from (a1) to (a10); (a12) An antibody variable fragment that competes with the binding of any of the antibody variable fragments selected from (a1) to (a10) Any one of A multispecific antigen-binding molecule comprising. [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 comprising, (1) The first antigen-binding domain of is as follows (b1) to (b21): (b1) 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 sequence, 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 sequence, 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, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 16 An antibody variable fragment comprising; (b2) An HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 25 sequence, an HVR-H2 sequence having the same amino acid sequence as the HVR-H2 region contained in SEQ ID NO: 25, an HVR-H3 sequence having the same amino acid sequence as the HVR-H3 region contained in SEQ ID NO: 25, an HVR-L1 sequence having the same amino acid sequence as the HVR-L1 region contained in SEQ ID NO: 26, an HVR-L2 sequence having the same amino acid sequence as the HVR-L2 region contained in SEQ ID NO: 26, and an HVR-L3 sequence having the same amino acid sequence as the HVR-L3 region contained in SEQ ID NO: 26 An antibody variable fragment comprising; (b3) An HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 19 sequence, an HVR-H2 sequence having the same amino acid sequence as the HVR-H2 region contained in SEQ ID NO: 19, an HVR-H3 sequence having the same amino acid sequence as the HVR-H3 region contained in SEQ ID NO: 19 an HVR-L1 sequence having the same amino acid sequence as the HVR-L1 region contained in SEQ ID NO: 20, an HVR-L2 sequence having the same amino acid sequence as the HVR-L2 region contained in SEQ ID NO: 20, and an HVR-L3 sequence having the same amino acid sequence as the HVR-L3 region contained in SEQ ID NO: 20 An antibody variable fragment comprising; (b4) An HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 23 sequence, an HVR-H2 sequence having the same amino acid sequence as the HVR-H2 region contained in SEQ ID NO: 23, an HVR-H3 sequence having the same amino acid sequence as the HVR-H3 region contained in SEQ ID NO: 23 an HVR-L1 sequence having the same amino acid sequence as the HVR-L1 region contained in SEQ ID NO: 24, an HVR-L2 sequence having the same amino acid sequence as 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 An antibody variable fragment comprising; (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, 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 An antibody variable fragment comprising; (b6) 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 HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 17, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 17, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 17, 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 An antibody variable fragment comprising; (b8) 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 (b7) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 17, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 17, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 17, 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 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: 18, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 18 An antibody variable fragment comprising; (b8) 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 contained in SEQ ID NO: 85, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 85, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 85, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 93, 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 HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 An HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72 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; an antibody variable fragment comprising the sequence; (b11) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72 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; an antibody variable fragment comprising the sequence; (b12) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72 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; an antibody variable fragment comprising the sequence; (b13) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 66, an HVR-H3(b13) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 66, an HVR-H3 An antibody variable fragment comprising an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74; 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: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74; 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: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 75, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 75, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 75; (b14) 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: 67, an HVR-H 1 sequence, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H 2 sequence, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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 contained in SEQ ID NO: 67, an HVR-H 1 sequence, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H 2 sequence, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained 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 contained in SEQ ID NO: 68, an HVR-H 1 An HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 68 An HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 68 An HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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 Antibody variable fragment comprising the sequence; (b17) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 69 An HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 69 An HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 69 An HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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 Antibody variable fragment comprising the sequence; (b18) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 70 An HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 70 An HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 70 An HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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 Antibody variable fragment comprising the sequence; (b19) An HVR-H sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 71, an HVR-H 2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 71, an HVR-H3 sequence, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 , 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 comprising an antibody variable fragment; (b20) An antibody variable fragment that binds to the same epitope of 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) and binds to any one of them, a multispecific antigen-binding molecule. [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 comprising, wherein the first antigen-binding domain of (1) is any 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 A light chain variable region having an array; (c5) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and the amino acid A light chain variable region having an array; (c6) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and the amino acid A light chain variable region having an array; (c7) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and the amino acid A light chain variable region having an array; (c8) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and the amino acid A light chain variable region having an array; (c9) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 85 and the amino acid A light chain variable region having an array; (c10) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 heavy chain variable region having the amino acid sequence of SEQ ID NO: 73 a light chain variable region having the sequence (c18) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 70 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 73 a light chain variable region having the sequence (c19) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 71 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 73 a light chain variable region having the sequence (c20) More than 80% of any one of the heavy chain variable regions (c1) to (c19) a heavy chain variable region having identity with any one of the light chain variable regions (c1) to (c19) a light chain variable region with greater than 80% identity to (c21) More than 90% of any one of the heavy chain variable regions (c1) to (c19) a heavy chain variable region having identity with any one of the light chain variable regions (c1) to (c19) a light chain variable region with greater than 90% identity to (c22) More than 95% of any one of the heavy chain variable regions (c1) to (c19) a heavy chain variable region having identity with any one of the light chain variable regions (c1) to (c19) Light chain variable region with greater than 95% identity to A multispecific antibody comprising any one of the combinations of heavy chain variable regions and light chain variable regions selected from sexual antigen-binding molecule. [5] A multispecific antibody according to any one of [1] to [4], which has cytotoxic activity. Original binding molecule. [6] The multispecific antibody according to [5], wherein the cytotoxic activity is T cell-dependent cytotoxic activity. Original binding molecule. [7] The second antigen-binding domain in (2) binds to the CD3 epsilon chain, [1 [6] The multispecific antigen-binding molecule according to any one of [1] to [5]. The second antigen-binding domain in [8](2) binds to the T cell receptor, from [1] to The multispecific antigen-binding molecule according to any one of [6]. The second antigen-binding domain in [9](2) is the following (d1) to (d12): (d1) respectively, HVR-H1, HVR-H2 and HVR- contained in SEQ ID NO: 57 The HVR-H1, HVR-H2 and HVR-H3 sequences identical to the amino acid sequences of the H3 regions, and respectively, HVR-L1, HVR-L2 and HVR-L3 regions contained in SEQ ID NO: 58 The HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the regions, and containing Antibody variable fragment; (d2) respectively, HVR-H1, HVR-H2 and HVR- contained in SEQ ID NO: 98 The HVR-H1, HVR-H2 and HVR-H3 sequences identical to the amino acid sequences of the H3 regions, and respectively, HVR-L1, HVR-L2 and HVR-L3 contained in SEQ ID NO: 103 The HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the regions, and containing Antibody variable fragment; (d3) respectively, HVR-H1, HVR-H2 and HVR- contained in SEQ ID NO: 99 The HVR-H1, HVR-H2 and HVR-H3 sequences identical to the amino acid sequences of the H3 regions, and respectively, HVR-L1, HVR-L2 and HVR-L3 contained in SEQ ID NO: 103 The HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the regions, and containing Antibody variable fragment; (d4) respectively, HVR-H1, HVR-H2 and HVR contained in SEQ ID NO: 100 The HVR-H1, HVR-H2 and HVR-H3 sequences identical to the amino acid sequences of the -H3 regions and, respectively, HVR-L1, HVR-L2 and HVR-L contained in SEQ ID NO: 103 An antibody variable fragment comprising HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the 3 regions; ; (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 contained in SEQ ID NO: 101, 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 contained in SEQ ID NO: 103, respectively; ; ; An antibody variable fragment comprising HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the 3 regions; ; (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 contained in SEQ ID NO: 102, 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 contained in SEQ ID NO: 103, respectively; ; ; An antibody variable fragment comprising HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the 3 regions; ; (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 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 contained in SEQ ID NO: 299, respectively; ; ; An antibody variable fragment comprising HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the 3 regions; ; (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 contained in SEQ ID NO: 300, 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 contained in SEQ ID NO: 301, respectively; ; ; An antibody variable fragment comprising HVR-L1, HVR-L2 and HVR-L3 sequences identical to the amino acid sequences of the 3 regions; ; (d9) respectively, HVR-H1, HVR-H2 and HVR contained in SEQ ID NO: 302 -H3 region amino acid sequences identical to the HVR-H1, HVR-H2 and HVR-H3 sequences and, respectively, HVR-L1, HVR-L2 and HVR-L contained in SEQ ID NO: 303 3 region amino acid sequences identical to the HVR-L1, HVR-L2 and HVR-L3 sequences containing antibody variable fragments; (d10) 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 any one selected from 390 contained in the amino acid sequences of the HVR-H1, HVR-H2 and HVR-H3 regions identical HVR-H1, HVR-H2 and HVR-H3 sequences, and 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 any one selected from 391 contained in the HVR-L1, HVR-L 2 and amino acid sequences of the HVR-L3 region identical to the HVR-L1, HVR-L2 and H VR-L3 sequences containing antibody variable fragments; (d11) Any one of the antibody variable fragments selected from (d1) to (d10) binds to the same epitope Antibody variable fragment; (d12) Any one of the antibody variable fragments selected from (d1) to (d10) competes with the binding Antibody variable fragment Any one of [1] to [8] containing any one of the above, the multispecific antigen-binding molecule .

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

[11] (2) The second antigen-binding domain in is as follows (j1) to (j5): (j1) An antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 136, the HVR-H2 sequence of SEQ ID NO: 137, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 139, the HVR-L2 sequence of SEQ ID NO: 140, and the 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 an HVR sequence selected from any of the combinations in Table 2B; (j4) An antibody variable fragment that binds to the same epitope of any of the antibody variable fragments selected from (j1) to (j3); (j5) An antibody variable fragment having at least 80% sequence identity to any of the antibody variable fragments selected from (j1) to (j4). A multispecific antigen-binding molecule according to any one of [1] to [8], comprising any one of (j1) to (j5).

[11] (2) The second antigen-binding domain in is any one of the following (j1) to (j5): (j1) An antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 136, the HVR-H2 sequence of SEQ ID NO: 137, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 139, the HVR-L2 sequence of SEQ ID NO: 140, and the 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-H? 2 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 an HVR sequence selected from any of the combinations in Table 2B; (j4) An antibody variable fragment that binds to the same epitope of any of the antibody variable fragments selected from (j1) to (j3); (j5) An antibody variable fragment having at least 80% sequence identity to any of the antibody variable fragments selected from (j1) to (j4). (j3) An antibody variable fragment comprising an HVR sequence selected from any of the combinations in Table 2B; (j4) An antibody variable fragment that binds to the same epitope of any of the antibody variable fragments selected from (j1) to (j3); (j5) An antibody variable fragment having at least 80% sequence identity to any of the antibody variable fragments selected from (j1) to (j4). An antibody that competes with the binding of any of the antibody variable fragments selected from (j5), (j1) to (j3) variable fragment A multispecific antigen-binding molecule according to any one of [1] to [8], comprising any one of them.

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

[0011] , 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.

[13] The multispecific antigen-binding molecule according to

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

[14] Further comprising a third domain comprising an Fc region with reduced binding activity to the Fc gamma receptor, a multispecific antigen-binding molecule according to any one of [1] to

[13] . them.

[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 and (3) A third domain comprising an Fc region with reduced binding activity to the Fc gamma receptor A multispecific antigen-binding molecule comprising.

[16] A multispecific antigen-binding molecule according to

[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

[0017] , wherein the Fc region is an Fc region having an amino acid mutation in any of the amino acids constituting the Fc regions of SEQ ID NOs: 112 to 115 (IgG1 to IgG4).

[19] The Fc region has the following amino acid positions specified by EU numbering: Position 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, and 33 Position 2 and has a mutation of at least one amino acid selected from the Fc region described in

[18] . The multispecific antigen-binding molecule described in

[20] A bispecific antibody, which is the multispecific antigen-binding molecule described in any one of [1] to

[19] .

[21] The bispecific antibody described in

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

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

[19] or the bispecific antibody described in

[20] or

[21] , and a pharmaceutically acceptable carrier.

[23] The pharmaceutical composition described in

[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 described in any one of [1] to

[19] or the bispecific antibody described in

[20] or

[21] .

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

[19] or the bispecific antibody described in

[20] or

[21] .

[26] Use of the antigen-binding molecule described in any one of [1] to

[19] or the bispecific antibody described in

[20] or

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

[27] The 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

[0027] , 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] The following (f1) to (f11): (f1) 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-L An antibody variable fragment comprising a 2-array and an HVR-L3 array of SEQ ID NO: 32; (f2) The HVR-H1 array of SEQ ID NO: 33, the HVR-H2 array of SEQ ID NO: 34, the SEQ ID NO: 35 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 36, the HVR-L 2 array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 38; (f3) The HVR-H1 array of SEQ ID NO: 39, the HVR-H2 array of SEQ ID NO: 40, the SEQ ID NO: 41 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 42, the HVR-L 2 array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 44; (f4) The HVR-H1 array of SEQ ID NO: 45, the HVR-H2 array of SEQ ID NO: 46, the SEQ ID NO: 47 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 48, the HVR-L 2 array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 50; (f5) The HVR-H1 array of SEQ ID NO: 27, the HVR-H2 array of SEQ ID NO: 75, the SEQ ID NO: 29 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 30, the HVR-L 2 array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 32; (f6) The HVR-H1 array of SEQ ID NO: 27, the HVR-H2 array of SEQ ID NO: 76, the SEQ ID NO: 29 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 30, the HVR-L 2 array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 32; (f7) The HVR-H1 array of SEQ ID NO: 77, the HVR-H2 array of SEQ ID NO: 78, the SEQ ID NO: 79 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 36, the HVR-L 2 array, and an antibody variable fragment comprising the HVR-L3 array of SEQ ID NO: 38; (f8) The HVR-H1 array of SEQ ID NO: 77, the HVR-H2 array of SEQ ID NO: 78, the SEQ ID NO: An antibody variable fragment comprising the HVR-H3 sequence of 80, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L 2 sequence, and the HVR-L3 sequence of SEQ ID NO: 38; (f9) The HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the SEQ ID NO: 80 HVR-H3 sequence, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L of SEQ ID NO: 37 2 sequence, and an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 81; (f10) An antibody variable fragment that binds to the same epitope as any of the antibody variable fragments selected from (f1) to (f9) ; (f11) Competes with the binding of any of the antibody variable fragments selected from (f1) to (f9) Antibody variable fragment An antigen-binding molecule comprising an antigen-binding domain comprising any one of

[33] The following (g1) to (g20): (g1) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 15, an HVR-H1 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 15, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 15, an HVR-H3 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 16, an HVR-L1 sequence, 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 Antibody variable fragment; (g2) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 25, an HVR-H1 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 25, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 25, an HVR-H3 sequence 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 and contained in an antibody variable fragment; (g3) 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 and contained in an antibody variable fragment; (g4) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 23,[[ID=[]] 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 and contained in an antibody variable fragment; (g5) 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 and an antibody variable fragment comprising the same; (g6) 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 and an antibody variable fragment comprising the same; (g7) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 17, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 17, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 17, 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 and an antibody variable fragment comprising the same; (g8) An HVR-H1 An antibody variable fragment comprising 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; An antibody variable fragment comprising 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; An antibody variable fragment comprising 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; An antibody variable fragment comprising 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; An antibody variable fragment comprising 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; An antibody variable fragment comprising 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 antibody variable fragment comprising an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; 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: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; 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: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; 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: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; 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: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; 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: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; 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: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; (g10) 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: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; An antibody variable fragment comprising an HVR-H, sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; An antibody variable fragment comprising an HVR-H, sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; An antibody variable fragment comprising an HVR-H, sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; An antibody variable fragment comprising an HVR-H, sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; An antibody variable fragment comprising an HVR-H, sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; An antibody variable fragment comprising an HVR-H, sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; (g11) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72 , 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; (g12) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 66, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 , 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; (g13) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 , 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; ​​​​​Antibody variable fragments comprising columns; (g14) An HVR-H identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 67 1 sequence, an HVR-H2 identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67 sequence, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74 an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74 Antibody variable fragments; and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74 Antibody variable fragments comprising columns; (g15) An HVR-H identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 68 1 sequence, an HVR-H2 identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 68 sequence, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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 Antibody variable fragments; and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73 Antibody variable fragments comprising columns; (g16) An HVR-H identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 69 1 sequence, an HVR-H2 identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 69 sequence, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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 Antibody variable fragments; 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; a variable antibody fragment containing the sequence; (g17) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 70, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 73, and an antibody variable fragment containing an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73; a variable antibody fragment containing the sequence; a variable antibody fragment containing the sequence; an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 73, and an antibody variable fragment containing an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73; a variable antibody fragment containing the sequence; (g18) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 71, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 73, and an antibody variable fragment containing an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73; a variable antibody fragment containing the sequence; a variable antibody fragment containing the sequence; an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: c73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 73, and an antibody variable fragment containing an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 73; a variable antibody fragment containing the sequence; (g19) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (g1) to (g18); a variable antibody 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); a variable antibody 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 containing any one of the above.

[34] The following (h1) to (h21): (h1) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence of the light chain variable region; (h2) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and an amino acid sequence of the light chain variable region; (h3) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and an amino acid sequence of the light chain variable region; (h4) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and an amino acid sequence of the light chain variable region; (h5) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and an amino acid sequence of the light chain variable region; (h6) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and an amino acid sequence of the light chain variable region; (h7) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence of the light chain variable region; (h8) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and an amino acid sequence of the light chain variable region; (h9) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and an amino acid sequence of the light chain variable region; (h10) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 64 and an amino acid sequence of the light chain variable region; (h11) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and an amino acid sequence of the light chain variable region; (h12) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 66 and an amino acid sequence of the light chain variable region; (h13) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and an amino A light chain variable region having an acid sequence; (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; A light chain variable region having an acid sequence; (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; A light chain variable region having an acid sequence; (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; A light chain variable region having an acid sequence; (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; A light chain variable region having an acid sequence; (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; A light chain variable region having an acid sequence; (h19) A heavy chain variable region having more than 80% identity to any one of the heavy chain variable regions of (h1) to (h18) and a light chain variable region having more than 80% identity to any one of the light chain variable regions of (h1) to (h18); A heavy chain variable region having more than 80% identity to any one of the heavy chain variable regions of (h) to (h18) and a light chain variable region having more than 80% identity to any one of the light chain variable regions of (h1) to (h18); A light chain variable region having more than 80% identity to any one of the light chain variable regions of (h1) to (h18); (h20) A heavy chain variable region having more than 90% identity to any one of the heavy chain variable regions of (h1) to (h18) and a light chain variable region having more than 90% identity to any one of the light chain variable regions of (h1) to (h18); A heavy chain variable region having more than 90% identity to any one of the heavy chain variable regions of (h1) to (h18) and a light chain variable region having more than 90% identity to any one of the light chain variable regions of (h1) to (h18); A light chain variable region having more than 90% identity to any one of the light chain variable regions of (h1) to (h18); (h21) A heavy chain variable region having more than 95% identity to any one of the heavy chain variable regions of (h1) to (h18) and a light chain variable region having more than 95% identity to any one of the light chain variable regions of (h1) to (h18); A heavy chain variable region having more than 95% identity to any one of the heavy chain variable regions of (h1) to (h18) and a light chain variable region having more than 95% identity to any one of the light chain variable regions of (h1) to (h18); A light chain variable region having more than 95% identity to any one of the light chain variable regions of (h1) to (h18); An antigen-binding molecule comprising an antigen-binding domain containing any one of the above.

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

[31] to

[34] , having cytotoxic activity. An antigen-binding molecule.

[36] The antigen-binding molecule according to

[0035] , wherein the cytotoxic activity is antibody-dependent cell cytotoxicity or complement-dependent cell 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 [the antibody described in

[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 the antigen-binding molecule according to any one of

[31] to

[40] or the antibody according to

[41] or

[42] .

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

[31] to

[0040] or the antibody according to

[41] or

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

[48] For treating or preventing cancer, according to any one of

[31] to

[40] . Use of the antigen-binding molecule described above or the antibody described in

[41] or

[42] .

[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

[0048] , 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 described in any one of [1] to

[19] or the bispecific antibody described in

[20] or

[21] , and instructions for use.

[53] A kit comprising the antigen-binding molecule described in any one of

[31] to

[40] or the antibody described in

[41] or

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

[0013] The present invention enables cancer treatment by bringing T cells into proximity to DLL3-expressing cells and using the cytotoxicity of T cells against DLL3-expressing cancer cells. The invention provides a multispecific antigen-binding molecule, a method for producing the multispecific antigen-binding molecule, and a therapeutic agent containing such a multispecific antigen-binding molecule as an active ingredient for inducing cytotoxicity, as a new approach to cancer treatment. The multispecific antigen-binding molecule of the present invention has a strong antitumor activity for inducing cytotoxicity, targets DLL3-expressing cells, and can damage them, thus enabling the treatment and prevention of various cancers. The present invention also provides a novel monospecific antigen-binding molecule having human DLL3-binding activity, a therapeutic agent containing such a monospecific antigen-binding molecule as an active ingredient, and a treatment method using such a therapeutic agent. By using the cytotoxicity of T cells against DLL3-expressing cancer cells, a multispecific antigen-binding molecule that enables cancer treatment is provided. The multispecific antigen-binding molecule, a method for producing the multispecific antigen-binding molecule, and a therapeutic agent containing such a multispecific antigen-binding molecule as an active ingredient for inducing cytotoxicity, as a new approach to cancer treatment. The invention provides a multispecific antigen-binding molecule having a strong antitumor activity for inducing cytotoxicity, targeting DLL3-expressing cells, and capable of damaging them, thus enabling the treatment and prevention of various cancers. The multispecific antigen-binding molecule of the present invention has a strong antitumor activity for inducing cytotoxicity, targets DLL3-expressing cells, and can damage them, thus enabling the treatment and prevention of various cancers. The multispecific antigen-binding molecule of the present invention has a strong antitumor activity for inducing cytotoxicity, targets DLL3-expressing cells, and can damage them, thus enabling the treatment and prevention of various cancers. The invention also provides a novel monospecific antigen-binding molecule having human DLL3-binding activity, a therapeutic agent containing such a monospecific antigen-binding molecule as an active ingredient, and a treatment method using such a therapeutic agent. The invention provides a novel monospecific antigen-binding molecule having human DLL3-binding activity, a therapeutic agent containing such a monospecific antigen-binding molecule as an active ingredient, and a treatment method using such a therapeutic agent. The invention provides a novel monospecific antigen-binding molecule having human DLL3-binding activity, a therapeutic agent containing such a monospecific antigen-binding molecule as an active ingredient, and a treatment method using such a therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Best Mode for Carrying Out the Invention

[0015] The techniques and procedures described or referenced in this specification are generally known to those skilled in the art, for example, Sambrook et al., Molecular Cloning: A La boratory Manual, 3rd Edition (2001) Cold Spring Ha rbor Laboratory Press, Cold Spring Harbor , N.Y.; Current Protocols in Molecular Bio logy (F.M. Ausubel 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 Laborator y Manual, and Animal Cell Culture (R.I.F reshney (eds.), (1987)); Oligonucleotide Synth esis (M.J. Gait (ed.), 1984); Methods in Molecu lar Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis (ed.), 1998) Academic Press; Animal Cell Culture (R.I.F reshney) (eds.), 1987); Introduction to Cell a nd Molecular Biology (J. M. Walker (ed.), 1987); Principles of Protein nd Tissue Culture (J.P.Mather and P.E.Robe rts, 1998) Plenum Press; Cell and Tissue Cu lture: Laboratory Procedures (A.Doyle, J.B. Griffiths, and D.G.Newell (eds.), 1993~8) J.Wil ey and Sons; Handbook of Experimental Imm unology (D.M.Weir and C.C.Blackwell (eds)); Gen e Transfer Vectors for Mammalian Cells (J .M.Miller and M.P.Calos (eds.), 1987); PCR: The P olymerase Chain Reaction (Mullis et al. (eds), 1994 ); Current Protocols in Immunology (J.E.Co ligan et al. (eds.), 1991); Short Protocols in Molec ular Biology (Wiley and Sons, 1999); Immuno biology (C.A.Janeway and P.Travers, 1997); An tibodies (P.Finch, 1997); Antibodies: A Prac tical Approach (D.Catty (ed), IRL Press, 1988 ~1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C.Dean (eds), Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E.Harlow and D.Lane (Co Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra (eds.), Harwood Academic Publishers, 1995); and Can cer: Principles and Practice of Oncology( V. T. DeVita et al. (eds.), J. B. Lippincott Company, 19 93), are well understood and commonly used using conventional methods such as those described in widely used methods such as the above. The following definitions and detailed descriptions are provided to facilitate understanding of the present invention described herein for.

[0016] The "percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is determined by aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity without considering any conservative substitutions as part of the sequence identity. After that, it is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. Alignments for determining percent amino acid sequence identity can be achieved by various means within the scope of techniques in the art, such as BLAST, BLAST-2, ALIGN ment, Megalign (DNASTAR) software, or publicly available computer soft ware such as GENETYX® (Genetyx Co., Ltd.). One of ordinary skill in the art can use any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared, including the alignment algorithms included in these software. the alignment algorithms included in these software. ware such as BLAST, BLAST-2, ALIGN ment, Megalign (DNASTAR) software, or publicly available computer soft ware such as GENETYX® (Genetyx Co., Ltd.). One of ordinary skill in the art can use any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared, including the alignment algorithms included in these software. the alignment algorithms included in these software. Appropriate parameters for aligning columns can be determined.

[0017] The ALIGN-2 array comparison computer program was authored by Genentech, Inc. and its source code is filed with the U.S. Copyright Office, Washington, D.C., 20559 together with user documentation and is registered under U.S. Copyright Office Registration No. 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 a UNIX (trademark) operating system including Digital UNIX ( trademark) V4.0D. All array comparison parameters are set by the ALIGN-2 program and do not change. 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 against a given amino acid sequence B (or can be expressed as a given amino acid sequence A having or containing a particular percent amino acid sequence identity to, with, or against 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 scored as identical matches by the program in the alignment of the alignment program ALIGN-2 of A and B, and Y is the total number of amino acid residues in B). If the length of amino acid sequence A is amino acid ... ... ... ... ... ... 100 times the fraction X / Y (where X is the number of amino acid residues scored as identical matches by the program in the alignment of the alignment program ALIGN-2 of A and B, and Y is the total number of amino acid residues in B). ... ... If it is not equal to the length of array B, it will be understood that the amino acid sequence identity percentage of A with respect to B is not equal to the amino acid sequence identity percentage of B with respect to A. Unless specifically stated otherwise, all values of amino acid sequence identity percentages used in this specification are obtained as described in the previous paragraph using the ALIGN-2 computer program. It will be understood that. Unless specifically stated otherwise, all values of amino acid sequence identity percentages used in this specification are obtained as described in the previous paragraph using the ALIGN-2 computer program.

[0018] Amino acid In this specification, amino acids are described by one-letter or three-letter codes or both, for example, 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 change Due to amino acid changes in the amino acid sequence of the antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlapping extension PCR can be appropriately used. Furthermore, some known methods can also be used as amino acid change methods for substitution with non-natural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. U.S.A. (2003) 100(11), 6353-6357). For example, one of the stop codons, the complementary amber suppressor tRNA of the UAG codon (amber codon) ​​​​​​​​​It is preferable to use a cell-free translation system (Clove r Direct(Protein Express)) containing tRNA having a non-natural amino acid bound to RNA.

[0020] In this specification, the meaning of the term "and / or" when describing the site of amino acid change includes all combinations in which "and" and "or" are preferably combined. Specifically, for example , "the amino acid at position 33, 55 and / or 96 is substituted" includes the following amino acid change variations: (a) position 33, (b) position 55, (c) position 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96, and (g) positions 33, 55 and 96.

[0021] Furthermore, in this specification, as expressions indicating before and after an amino acid change and expressions indicating before and after a number indicating a specific position , the one-letter or three-letter codes of the amino acids before and after the change can be appropriately used respectively. For example, when substituting an amino acid contained in the antibody variable region , the change N100bL or Asn100bLeu used indicates the substitution of Asn at position 100b (by Kabat's numbering) with Leu. That is, the number indicates the amino acid position by Kab at's 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 the Fc region of the antibody constant region indicates the substitution of Pro at position 238 (by EU numbering) with Asp. That is, The numbers indicate the amino acid positions according to EU numbering, and 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 further, it can refer to a molecule such as a peptide or protein 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 any of natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. For example, they may be polypeptides produced from artificially designed sequences. They may also be any of natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. They may also be any of natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc.

[0023] Preferred examples of the antigen-binding molecules of the present invention are antigen-binding molecules containing a plurality of antigen-binding domains. In certain embodiments, the antigen-binding molecules of the present invention are antigen-binding molecules containing two antigen-binding domains having different antigen-binding specificities. In certain embodiments, the antigen-binding molecules of the present invention are antigen-binding molecules containing two antigen-binding domains having different antigen-binding specificities and an FcRn-binding domain contained in the Fc region of an antibody. As a method for extending the blood half-life of a protein administered to a living body, a method of adding an FcRn-binding domain of an antibody to the target protein and utilizing the recycling function via FcRn is well-known. In certain embodiments, the antigen-binding molecules of the present invention are antigen-binding molecules containing two antigen-binding domains having different antigen-binding specificities. In certain embodiments, the antigen-binding molecules of the present invention are antigen-binding molecules containing two antigen-binding domains having different antigen-binding specificities. In certain embodiments, the antigen-binding molecules of the present invention are antigen-binding molecules containing two antigen-binding domains having different antigen-binding specificities and an FcRn-binding domain contained in the Fc region of an antibody. As a method for extending the blood half-life of a protein administered to a living body, a method of adding an FcRn-binding domain of an antibody to the target protein and utilizing the recycling function via FcRn is well-known. As a method for extending the blood half-life of a protein administered to a living body, a method of adding an FcRn-binding domain of an antibody to the target protein and utilizing the recycling function via FcRn is well-known. As a method for extending the blood half-life of a protein administered to a living body, a method of adding an FcRn-binding domain of an antibody to the target protein and utilizing the recycling function via FcRn is well-known.

[0024] Another preferred example of the antigen-binding molecules of the present invention is an antibody containing only one type of antigen-binding domain. ​​​It is the original binding molecule. In certain embodiments, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising two antigen-binding domains having the same antigen-binding specificity. In certain embodiments, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising two antigen-binding domains having the same antigen-binding specificity. In certain embodiments, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising two antigen-binding domains having 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 that specifically binds to all or part of an antigen and includes a region that is complementary thereto. When the molecular weight of the antigen is large, the antibody can bind only to a specific portion of the antigen. That specific portion is called an "epitope". The antigen-binding domain can be provided from 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 epitope 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 epitope may be present in a protein comprising the amino acid sequence of SEQ ID NO: 9 or 111.

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

[0028] DLL3 As used herein, the term "DLL3", unless otherwise indicated, refers to any native DLL3 (delta-like protein 3) of vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term refers to unprocessed "full-length" DLL3 as well as those resulting from processing in cells. ​​​​​​​​​​​​​​​includes any form of DLL3. This term also encompasses naturally occurring variants of DLL3, such as splice variants or allelic variants. Exemplary amino acid sequences of human DLL3 are known as NCBI Reference Sequence (R efSeq) NM_016941.3, and an exemplary amino acid sequence of cynomolgus monkey DLL3 is known as NCBI Reference Sequence XP_0055 89253.1, and an exemplary amino acid sequence of mouse DLL3 is known as NCBI 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 on the C-terminal side and a DSL (Notch) domain on the N-terminal side (see, for example, FIG. 1). Further, DLL3 has an EGF domain containing six regions, EGF1 to EGF6, from the N-terminal side to the C-terminal side. In some embodiments, the antigen-binding molecule or antibody of the present invention binds to an epitope within a domain that is not the transmembrane (TM) region or the C-terminal intracellular domain, but rather within the domain from the N-terminal to immediately before the TM region. The molecule / antibody of the present invention may bind to an epitope within any of the above domains / regions within the ECD. In preferred embodiments, the molecule / antibody of the present invention binds to an epitope within the region from EGF6 to immediately before the TM region. More specifically, the molecule / antibody 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 present invention bind to EGF1, EGF2, EGF3, EGF4, EGFR, and EGFR of human DLL3. F5, or the EGF6 region, or the region from EGF6 to just before the TM region, or human EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 domain of DLL3 Or, it binds to an epitope within the region from EGF6 to just before the TM region.

[0030] In human DLL3, the above domains / regions are separated by the following amino acid residues (e.g., ww w.uniprot.org / uniprot / Q9NYJ7 or WO2013 / 12 6746): 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: positions 516-618 (or 516 in some isoforms) The amino acid positions are also the amino acid residues shown in SEQ ID NO: 9. The amino acid sequence also refers to the amino acid position in the amino acid sequence. Thus, the antigen-binding molecules or antibodies of the present invention are DLL3, which can bind to the above region / domain having the amino acid residue at the above position That is, the antigen-binding molecule or antibody of the present invention can bind to an epitope within the above region / domain having the amino acid residue at the above position in human DLL3. acid residue within the above region / domain having the amino acid residue at the above position in human DLL3.

[0031] In some embodiments, due to its specificity, the antigen-binding molecule / antibody of the present invention does not specifically bind to the above region / domain of human DLL 3, or to an epitope within the above region / domain of human DLL3. In some embodiments, the molecule / antibody of the present invention does not specifically bind to the above region / domain having the amino acid residue at the above position in human DLL3. In some embodiments, the molecule / antibody of the present invention does not specifically bind to an epitope within the above region / domain having the amino acid residue at the above position in human DLL3. In some embodiments, the molecule / antibody of the present invention does not specifically bind to an epitope within the above region / domain having the amino acid residue at the above position in human DLL3. In this context, "specifically" may be equivalently expressed as "substantially". "Specifically binds to" as used herein refers to the activity of an antigen-binding molecule / antibody that binds to the antigen / region / domain / epitope of interest at a binding level that includes specific binding.

[0032] "Specifically binds to" as used herein refers to the activity of an antigen-binding molecule / antibody that binds to an antigen / region / domain / epitope that is not the target at a binding level that includes non-specific binding or background binding but does not include specific binding. "Specifically binds to" as used herein refers to the activity of an antigen-binding molecule / antibody that binds to an antigen / region / domain / epitope that is not the target at a binding level that includes non-specific binding or background binding but does not include specific binding. Specificity can be measured by any method described herein or known in the art, such as the epitope mapping or competition assay described herein. The above level of non-specific binding or background binding may be 0, or may not be 0 but close to 0, or may be very low enough to be technically ignored by those skilled in the art. The above level of non-specific binding or background binding may be 0, or may not be 0 but close to 0, or may be very low enough to be technically ignored by those skilled in the art. Specificity can be measured by any method described herein or known in the art, such as the epitope mapping or competition assay described herein. Specificity can be measured by any method described herein or known in the art, such as the epitope mapping or competition assay described herein. The above level of non-specific binding or background binding may be 0, or may not be 0 but close to 0, or may be very low enough to be technically ignored by those skilled in the art. The above level of non-specific binding or background binding may be 0, or may not be 0 but close to 0, or may be very low enough to be technically ignored by those skilled in the art. It may be low. For example, if a person skilled in the art cannot detect or observe a significant or relatively strong signal regarding the binding of the molecule / antibody to an antigen / region / domain / epitope that is not the target in a suitable binding assay, then it can be said that the molecule / antibody "does not specifically bind" to the antigen / region / domain / epitope that is not the target. In the art, the phrase "does not specifically bind" may sometimes have substantially the same meaning as the phrase "does not bind". In the case where a person skilled in the art cannot detect or observe a significant or relatively strong signal regarding the binding of the molecule / antibody to an antigen / region / domain / epitope that is not the target in a suitable binding assay, then it can be said that the molecule / antibody "does not specifically bind" to the antigen / region / domain / epitope that is not the target. In the art, the phrase "does not specifically bind" may sometimes have substantially the same meaning as the phrase "does not bind". In the case where a person skilled in the art cannot detect or observe a significant or relatively strong signal regarding the binding of the molecule / antibody to an antigen / region / domain / epitope that is not the target in a suitable binding assay, then it can be said that the molecule / antibody "does not specifically bind" to the antigen / region / domain / epitope that is not the target. In the art, the phrase "does not specifically bind" may sometimes have substantially the same meaning as the phrase "does not bind". In the case where a person skilled in the art cannot detect or observe a significant or relatively strong signal regarding the binding of the molecule / antibody to an antigen / region / domain / epitope that is not the target in a suitable binding assay, then it can be said that the molecule / antibody "does not specifically bind" to the antigen / region / domain / epitope that is not the target. In the art, the phrase "does not specifically bind" may sometimes have substantially the same meaning as the phrase "does not bind". In the case where a person skilled in the art cannot detect or observe a significant or relatively strong signal regarding the binding of the molecule / antibody to an antigen / region / domain / epitope that is not the target in a suitable binding assay, then it can be said that the molecule / antibody "does not specifically bind" to the antigen / region / domain / epitope that is not the target. In the art, the phrase "does not specifically bind" may sometimes have substantially the same meaning as the phrase "does not bind". In the case where a person skilled in the art cannot detect or observe a significant or relatively strong signal regarding the binding of the molecule / antibody to an antigen / region / domain / epitope that is not the target in a suitable binding assay, then it can be said that the molecule / antibody "does not specifically bind" to the antigen / region / domain / epitope that is not the target. In the art, the phrase "does not specifically bind" may sometimes have substantially the same meaning as the phrase "does not bind".

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

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

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

[0036] Affinity "Affinity" refers to the ability of a molecule (e.g., an antigen-binding molecule or antibody) to bind to a single binding site. It refers to the strength of the sum of non-covalent interactions with its binding partner (e.g., antigen). Unless otherwise specified, "binding affinity" as used herein refers to the affinity of a binding pair (e.g., antigen binding). Specific binding reflects a one-to-one interaction between members of a molecule (molecule and antigen, or antibody and antigen) The affinity of a molecule X for its partner Y is generally expressed as the dissociation constant (Kd) Affinity can be expressed by any of the methods known in the art, including those described herein. Specific experimental and quantitative methods for measuring binding affinity can be used. and exemplary embodiments are described below.

[0037] Method for determining affinity In certain embodiments, the antigen-binding domain of an antigen-binding molecule or antibody provided herein The main targets are 1 μM or less, 120 nM or less, 100 nM or less, and 80 nM or less for the antigen. Bottom, 70nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10n M or less, 2nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.00 1 nM or less (e.g., 10 -8 M or less, 10 -8M~10 -13 M, 10 -9 M~10 - 13 has a dissociation constant (Kd) for M). In certain embodiments, the antibody / the Kd value of the first antigen-binding domain of the antigen-binding molecule is 1-40, 1-50, 1-70, 1-80, 30-50, 30-70, 30-80, 40-70, 40-80, or 60 ~80 nM.

[0038] In one embodiment, the Kd is measured by a radiolabeled antigen-binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined in the presence of a titration series of unlabeled antigen by equilibrating the Fab with a minimum concentration of ([[]] 125 I)-labeled antigen and then capturing the bound antigen using a plate coated with an anti-Fa b antibody (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (19 99)). To establish the conditions for the assay, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of the 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 (about 23 °C). In a non-adsorbing plate (Nunc #269620) ), 100 pM or 26 pM of [[[[]]] I]-antigen is mixed with serial dilutions of the Fab of interest 125 (see, for example, Presta et al., Cancer Res. 57:4593-4599 ). (Consistent with the evaluation of anti-VEGF antibody, Fab-12 in (1997)). Then, the desired Fab is incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., for 1 hour). Then, the solution is removed and the plate is washed 8 times with 0.1% polysorbate 2 0 (TWEEN-20 (trademark)) in PBS. When the plate is dry, 1 50 μL / well of scintillant (MICROSCINT-20 (trademark); Packar d) is added and the plate is counted on a TOPCOUNT (trademark) gamma counter (Packard) for 10 minutes. The concentration of each Fab that gives a maximum binding of less than or equal to 20% is selected for use in the competitive binding assay.

[0039] According to another embodiment, Kd is measured using a BIACORE (registered trademark) surface plasmon resonance assay. For example, an assay using a BIACORE (registered trademark)-2000 or BI ACORE (registered trademark)-3000 (BIAcore, Inc., Piscataway , NJ) is performed at 25° C. using a solid-phase antigen CM5 chip of about 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. About 10 response units To achieve the coupled protein in (RU), the antigen was dissolved in 10 mM sodium acetate, pH 4.8, and diluted to 5 μg / ml (about 0.2 μM ) at a flow rate of 5 μL / min before injection. After injection of the antigen, 1 M ethanolamine was injected to block the unreacted groups . For kinetic measurements, serial two-fold dilutions of Fab (0.78 nM - 500 nM ) were injected at a flow rate of approximately 25 μL / min at 25 °C into PBS (PBST) containing 0.05% polysorbate 20 (Tween -20 (trademark)) surfactant. The association rate (K ) and dissociation rate (k on ) were calculated using the BIAcore (registered off ) Evaluation Software version 3.2 by fitting the association and dissociation sensorgrams simultaneously to a simple 1:1 Langmuir binding model . The equilibrium dissociation constant (Kd) was calculated as the ratio K / k . See, for example, Chen et al., J. Mol. Biol. 293:865 - 881 (1999). In the surface plasmon resonance assay described above, when the on-rate is greater than 10 off M on s , for cases measured in a spectrophotometer, such as a stopped-flow equipped spectrophotometer with a stirred cuvette (Aviv Instruments) or an 8000 series SLM-AMINCO (trademark) spectrophotometer (Thermo Spectronic), in the presence of a high concentration of antigen, the fluorescence emission intensity at 25 °C of 20 nM anti-antigen antibody (Fab type 6 M -1 s -1 ) in PBS, pH 7.2 (excitation = 295 nm; emission = 340 nM, 16 nm bandwidth . Instruments) or an 8000 series SLM-AMINCO (trademark) spectrophotometer (Thermo Spectronic), in the presence of a high concentration of antigen, the fluorescence emission intensity at 25 °C of 20 nM anti-antigen antibody (Fab type ) in PBS, pH 7.2 (excitation = 295 nm; emission = 340 nM, 16 nm bandwidth . ) in PBS, pH 7.2 (excitation = 295 nm; emission = 340 nM, 16 nm bandwidth By using fluorescence quenching techniques that measure increases or decreases in (a certain parameter), the binding rate can be determined. The rate can be determined.

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

[0041] Antibody As used herein, the term "antibody" is used in the broadest sense and is not limited, but includes various antibody structures such as monoclonal antibodies, polyclonal antibodies, single-specificity antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity. but includes various antibody structures such as monoclonal antibodies, polyclonal antibodies, single-specificity antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity. but includes various antibody structures such as monoclonal antibodies, polyclonal antibodies, single-specificity antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity. but includes various antibody structures such as monoclonal antibodies, polyclonal antibodies, single-specificity antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired antigen-binding activity.

[0042] In one aspect, the present invention provides a multispecific antigen-binding molecule or antibody. In some embodiments, the multispecific antigen-binding molecule (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 and the first antigen-binding domain of (1) binds to an epitope within the region defined by SEQ ID NO: 7 in human DLL3. the first antigen-binding domain of (1) binds to an epitope within the region defined by SEQ ID NO: 7 in human DLL3. In some embodiments, the first antigen-binding domain of (1) in the multispecific antigen-binding molecule is the following (a1)-(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-L 2 sequence of SEQ ID NO: 31, and the HVR-L3 sequence of SEQ ID NO: 32; (a2) the HVR-H1 sequence of SEQ ID NO: 33, the HVR-H2 sequence of SEQ ID NO: 34, and the HVR-H3 sequence of SEQ ID NO: HVR-H3 sequence of SEQ ID NO: 35, HVR-L1 sequence of SEQ ID NO: 36, HVR-L1 sequence of SEQ ID NO: 37 an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 38; (a3) the HVR-H1 sequence of SEQ ID NO: 39, the HVR-H2 sequence of SEQ ID NO: 40, HVR-H3 sequence of SEQ ID NO: 41, HVR-L1 sequence of SEQ ID NO: 42, HVR-L1 sequence of SEQ ID NO: 43 an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 44; (a4) the HVR-H1 sequence of SEQ ID NO: 45, the HVR-H2 sequence of SEQ ID NO: 46, HVR-H3 sequence of SEQ ID NO: 47, HVR-L1 sequence of SEQ ID NO: 48, HVR-L sequence of SEQ ID NO: 49 an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 50; (a5) the HVR-H1 sequence of SEQ ID NO: 51, the HVR-H2 sequence of SEQ ID NO: 52, HVR-H3 sequence of SEQ ID NO: 53, HVR-L1 sequence of SEQ ID NO: 54, HVR-L1 sequence of SEQ ID NO: 55 an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 56; (a6) the HVR-H1 sequence of SEQ ID NO: 27, the HVR-H2 sequence of SEQ ID NO: 75, and the HVR-H3 sequence of SEQ ID NO: HVR-H3 sequence of SEQ ID NO: 29, HVR-L1 sequence of SEQ ID NO: 30, HVR-L sequence of SEQ ID NO: 31 an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 32; (a7) the HVR-H1 sequence of SEQ ID NO: 27, the HVR-H2 sequence of SEQ ID NO: 76, and the HVR-H3 sequence of SEQ ID NO: HVR-H3 sequence of SEQ ID NO: 29, HVR-L1 sequence of SEQ ID NO: 30, HVR-L sequence of SEQ ID NO: 31 an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 32; (a8) the HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, HVR-H3 sequence of SEQ ID NO: 79, HVR-L1 sequence of SEQ ID NO: 36, HVR-L1 sequence of SEQ ID NO: 37 An antibody variable fragment comprising a 2-array and an HVR-L3 array of SEQ ID NO: 38; (a9) The HVR-H1 array of SEQ ID NO: 77, the HVR-H2 array of SEQ ID NO: 78, the SEQ ID NO: 80 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 36, the HVR-L of SEQ ID NO: 37 An antibody variable fragment comprising a 2-array and an HVR-L3 array of SEQ ID NO: 38; (a10) The HVR-H1 array of SEQ ID NO: 77, the HVR-H2 array of SEQ ID NO: 78, the SEQ ID NO: 80 HVR-H3 array, the HVR-L1 array of SEQ ID NO: 36, the HVR-L of SEQ ID NO: 37 An antibody variable fragment comprising an L2 array and an HVR-L3 array of SEQ ID NO: 81; (a11) An antibody variable fragment that binds to the same epitope of any of the antibody variable fragments selected from (a1) to (a10); An antibody variable fragment; (a12) An antibody variable fragment that competes with the binding of any of the antibody variable fragments selected from (a1) to (a10); One of the antibody variable fragments is any one of them. In some embodiments, the first antigen-binding domain in the multispecific antigen-binding molecule is (1): as follows (b1) to (b21): (b1) An HVR-H1 array identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 15, an HVR-H2 array identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 15, an HVR-H3 array identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 15, an HVR-L1 array identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 16, an HVR-L2 array identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 16, and an HVR-L3 array identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 16 An antibody variable fragment comprising; (b2) An HVR-H1 array identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 25, an HVR-H2 array identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 25, an HVR-H3 array identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 25, an HVR-L1 array identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 26, an HVR-L2 array identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 26, and an HVR-L3 array identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 26 An antibody variable fragment comprising; and an HVR-L3 array identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 26 (b2) An HVR-H1 array identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 25, an HVR-H2 array identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 25, an HVR-H3 array identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 25, an HVR-L1 array identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 26, An antibody variable fragment comprising 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; ; ; ; 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; ; ; ; ; 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; ; ; ; ; 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 identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 11 sequence, an HVR-H2 identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 11 sequence, an HVR-H3 identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 11 , an HVR-L1 identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 12, an HVR-L2 identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 12, and an HVR-L3 identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 12 antibody variable fragment; (b6) An HVR-H1 identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 13 sequence, an HVR-H2 identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 13 sequence, an HVR-H3 identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 13 , an HVR-L1 identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 14, an HVR-L2 identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 14, and an HVR-L3 identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 14 antibody variable fragment; (b7) An HVR-H1 identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 17 sequence, an HVR-H2 identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 17 sequence, an HVR-H3 identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 17 , an HVR-L1 identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 18, an HVR-L2 identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 18, and an HVR-L3 identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 18 An antibody variable fragment comprising; (b8) An HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 21 sequence, an HVR-H2 sequence having the same amino acid sequence as the HVR-H2 region contained in SEQ ID NO: 21, an HVR-H3 sequence having the same amino acid sequence as the HVR-H3 region contained in SEQ ID NO: 21, an HVR-L1 sequence having the same amino acid sequence as the HVR-L1 region contained in SEQ ID NO: 22, an HVR-L2 sequence having the same amino acid sequence as the HVR-L2 region contained in SEQ ID NO: 22, and an HVR-L3 sequence having the same amino acid sequence as the HVR-L3 region contained in SEQ ID NO: 22 An antibody variable fragment comprising; (b9) An HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 85 sequence, an HVR-H2 sequence having the same amino acid sequence as the HVR-H2 region contained in SEQ ID NO: 85, an HVR-H3 sequence having the same amino acid sequence as the HVR-H3 region contained in SEQ ID NO: 85, an HVR-L1 sequence having the same amino acid sequence as the HVR-L1 region contained in SEQ ID NO: 93, an HVR-L2 sequence having the same amino acid sequence as the HVR-L2 region contained in SEQ ID NO: 93, and an HVR-L3 sequence having the same amino acid sequence as the HVR-L3 region contained in SEQ ID NO: 93 An antibody variable fragment comprising; (b10) An HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 63<{ 1 sequence, an HVR-H2 sequence having the same amino acid sequence as the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence having the same amino acid sequence as the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence having the same amino acid sequence as the HVR-L1 region contained in SEQ ID NO: 72, an HVR-L2 sequence having the same amino acid sequence as the HVR-L2 region contained in SEQ ID NO: 72, 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: 72; a sequence; (b11) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 64, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72 , 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; an antibody variable fragment comprising a sequence; a sequence; (b12) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 65, an HVR-H 2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72 , 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; an antibody variable fragment comprising a sequence; a sequence; (b13) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 66, an HVR-H 1 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 , an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 73, An HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained 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; (b14) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained 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; (b15) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, 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: 74; (b16) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 68, ​​​​​​​​An HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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; an antibody variable fragment comprising the sequence; (b17) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 69 an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 69, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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; an antibody variable fragment comprising the sequence; (b18) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 70 an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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; an antibody variable fragment comprising the sequence; (b19) an HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 71 an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 71, An antibody variable fragment comprising an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; An antibody variable fragment identical to any one of the antibody variable fragments selected from (b20)(b1) to (b19) that binds to the same epitope; An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (b21)(b1) to (b19); Any one of the above. In some embodiments, the first antigen-binding domain in the multispecific antigen-binding molecule of (1) is as follows (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; In some embodiments, the first antigen-binding domain in the multispecific antigen-binding molecule of (1) is as follows (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: 27 and a light chain variable region having the amino acid sequence of SEQ ID NO: 28; (c8) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 31 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32; (c9) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 35 and a light chain variable region having the amino acid sequence of SEQ ID NO: 36; (c10) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 39 and a light chain variable region having the amino acid sequence of SEQ ID NO: 40; (c11) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 43 and a light chain variable region having the amino acid sequence of SEQ ID NO: 44; (c12) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48; (c13) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; (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 an amino acid of SEQ ID NO: 22 sequence of the light chain variable region; (c9) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 85 and an amino acid of SEQ ID NO: 93 sequence of the light chain variable region; (c10) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and an amino acid of SEQ ID NO: 72 sequence of the light chain variable region; (c11) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 64 and an amino acid of SEQ ID NO: 72 sequence of the light chain variable region; (c12) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 65 and an amino acid of SEQ ID NO: 72 sequence of the light chain variable region; (c13) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 66 and an amino acid of SEQ ID NO: 73 sequence of the light chain variable region; (c14) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and an amino acid of SEQ ID NO: 73 sequence of the light chain variable region; (c15) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 and an amino acid of SEQ ID NO: 74 sequence of the light chain variable region; (c16) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 68 and an amino acid of SEQ ID NO: 72 sequence of the light chain variable region; (c17) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 69 and an amino acid of SEQ ID NO: 73 sequence of the light chain variable region; (c18) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 70 and an amino acid of SEQ ID NO: 73 sequence of the light chain variable region; (c19) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 71 and an amino acid of SEQ ID NO: 73 A light chain variable region having an amino acid sequence; (c20) A heavy chain variable region having more than 80% identity to any one of (c1) to (c19), and a light chain variable region having more than 80% identity to any one of the light chain variable regions of (c1) to (c19); (c21) A heavy chain variable region having more than 90% identity to any one of (c1) to (c19), and a light chain variable region having more 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 (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); Comprising any one of the combinations of a heavy chain variable region and a light chain variable region selected from the above. In some embodiments, the multispecific antigen-binding molecule has cytotoxic activity. More specifically, the cytotoxic activity is T cell-dependent cytotoxic activity (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 the T cell receptor. In some embodiments, the second antigen-binding domain in (2) in the multispecific antigen-binding molecule is any one of the following (d1) to (d12): ​​​(d1) An antibody variable fragment comprising, respectively, 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: 57, and, respectively, 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: 58; (d2) An antibody variable fragment comprising, respectively, 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, respectively, 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; (d3) An antibody variable fragment comprising, respectively, 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: 99, and, respectively, 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; (d4) An antibody variable fragment comprising, respectively, 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: 100, and, respectively, 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; (d5) An antibody variable fragment comprising, respectively, HVR-H1, HVR-H2 and HVR-H3 sequences contained in SEQ ID NO: 101; (d6) An antibody variable fragment comprising, respectively, HVR-H1, HVR-H2 and HVR-H3 sequences contained in SEQ ID NO: 102; (d7) An antibody variable fragment comprising, respectively, HVR-H1, HVR-H2 and HVR-H3 sequences contained in SEQ ID NO: 104; (d8) An antibody variable fragment comprising, respectively, HVR-H1, HVR-H2 and HVR-H3 sequences contained in SEQ ID NO: 105; (d9) An antibody variable fragment comprising, respectively, HVR-H1, HVR-H2 and HVR-H3 sequences contained in SEQ ID NO: 106; An HVR-H1, HVR-H2, and HVR-H3 sequence identical to the amino acid sequence of the -H3 region and, respectively, an HVR-L1, HVR-L2, and HVR-L 3 sequence identical to the amino acid sequence of the HVR-L1, HVR-L2, and HVR-L3 regions contained in SEQ ID NO: 103 An antibody variable fragment comprising; (d6) An HVR-H1, HVR-H2, and HVR -H3 sequence identical to the amino acid sequence of the HVR-H1, HVR-H2, and HVR-H3 regions contained in SEQ ID NO: 102 and, respectively, an HVR-L1, HVR-L2, and HVR-L 3 sequence identical to the amino acid sequence of the HVR-L1, HVR-L2, and HVR-L3 regions contained in SEQ ID NO: 103 An antibody variable fragment comprising; (d7) An HVR-H1, HVR-H2, and HVR -H3 sequence identical to the amino acid sequence of the HVR-H1, HVR-H2, and HVR-H3 regions contained in SEQ ID NO: 298 and, respectively, an HVR-L1, HVR-L2, and HVR-L 3 sequence identical to the amino acid sequence of the HVR-L1, HVR-L2, and HVR-L3 regions contained in SEQ ID NO: 299 An antibody variable fragment comprising; (d8) An HVR-H1, HVR-H2, and HVR -H3 sequence identical to the amino acid sequence of the HVR-H1, HVR-H2, and HVR-H3 regions contained in SEQ ID NO: 300 and, respectively, an HVR-L1, HVR-L2, and HVR-L 3 sequence identical to the amino acid sequence of the HVR-L1, HVR-L2, and HVR-L3 regions contained in SEQ ID NO: 301 An antibody variable fragment comprising; (d9) An HVR-H1, HVR-H2, and HVR -H3 sequence identical to the amino acid sequence of the HVR-H1, HVR-H2, and HVR-H3 regions contained in SEQ ID NO: 302 and, respectively, HVR-L1, HVR-L2, and HVR-L included in SEQ ID NO: 303, antibody variable fragments 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, respectively; (d10) any one selected from 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 having 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 them, 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 included in any one selected from 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, and having HVR-L1, HVR-L2, and HVR-L3 sequences identical to the amino acid sequences of the HVR-L1, HVR-L2, and HVR-L3 regions included in any one of them, respectively; antibody variable fragments 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 included in any one selected from 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, (d11) an antibody variable fragment that binds to the same epitope as any one of the antibody variable fragments selected from (d1) to (d10); and (d12) an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (d1) to (d10). ​ is any one of the following. In some embodiments, the second antigen-binding domain in the multispecific antigen-binding molecule mainly includes 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 combinations of amino acid sequences in Table 2A; (e10) a heavy chain variable region having more than 80% identity to any one of the heavy chain variable regions of (e1) to (e9) and a light chain variable region having more than 80% identity to any one of the light chain variable regions of (e1) to (e9); (e11)A heavy chain variable region having more than 90% identity to any one of the heavy chain variable regions of (e1) to (e9), and a light chain variable region having more than 90% identity to any one of the light chain variable regions of (e1) to (e9); and a light chain variable region having more than 90% identity to any one of the light chain variable regions of (e1) to (e9); (e12)A heavy chain variable region having more than 95% identity to any one of the heavy chain variable regions of (e1) to (e9), and a light chain variable region having more than 95% identity to any one of the light chain variable regions of (e1) to (e9); or a light chain variable region having more than 95% identity to any one of the light chain variable regions of (e1) to (e9). and a light chain variable region having more than 95% identity to any one of the light chain variable regions of (e1) to (e9). (e12)A heavy chain variable region having more than 95% identity to any one of the heavy chain variable regions of (e1) to (e9), and is any one of the following: In some embodiments, the second antigen binding domain in the multispecific antigen binding molecule of (2) is one of the following (j1) to (j5): (j1)An antibody variable fragment comprising the HVR-H1 sequence of SEQ ID NO: 136, the HVR-H2 sequence of SEQ ID NO: 137, the HVR-H3 sequence of SEQ ID NO: 138, the HVR-L1 sequence of SEQ ID NO: 139, the HVR-L2 sequence of SEQ ID NO: 140, and the 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 of the antibody variable fragments selected from (j1) to (j3); (j5)An antibody variable fragment that competes with the binding of any of the antibody variable fragments selected from (j1) to (j3). (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 of the antibody variable fragments selected from (j1) to (j3); (j5)An antibody variable fragment that competes with the binding of any of the antibody variable fragments selected from (j1) to (j3). and is any one of the following: (j4)An antibody variable fragment that binds to the same epitope as any of the antibody variable fragments selected from (j1) to (j3); (j5)An antibody variable fragment that competes with the binding of any of the antibody variable fragments selected from (j1) to (j3). (j5)An antibody variable fragment that competes with the binding of any of the antibody variable fragments selected from (j1) to (j3). and is any one of the following: In some embodiments, the multispecific antigen binding molecule comprises (3)A third domain comprising an Fc region with reduced binding activity to the Fc gamma receptor Further comprises. In some embodiments, the present invention (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 and (3) a third domain comprising an Fc region with reduced binding activity to the Fc gamma receptor to provide a multispecific antigen-binding molecule. In some embodiments, in the multispecific antigen-binding molecule, the Fc region has an amino acid mutation in any of the amino acids constituting the Fc regions of SEQ ID NOs: 112-1 15 (IgG1-IgG4). It is an Fc region having the mutation. In some embodiments, in the multispecific antigen-binding molecule, the Fc region has the following amino acid positions specified by EU numbering: The following amino acid positions: Position 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, and 33 2 It is an Fc region having a mutation in at least one amino acid 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 above 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 by SEQ ID NO: 7 in human DLL3. In some embodiments, the antigen-binding molecule is one of the following (f1)-(f11): (f1) 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-L 2 sequence of SEQ ID NO: 31, and the HVR-L3 sequence of SEQ ID NO: 32; (f2) 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-L 2 sequence of SEQ ID NO: 37, and the HVR-L3 sequence of SEQ ID NO: 38; (f3) 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-L 2 sequence of SEQ ID NO: 43, and the HVR-L3 sequence of SEQ ID NO: 44; (f4) 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-L 2 sequence of SEQ ID NO: 49, and the HVR-L3 sequence of SEQ ID NO: 50; (f5) 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-L 2 sequence of SEQ ID NO: 31, and the HVR-L3 sequence of SEQ ID NO: 32; (f6) 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-L ​An antibody variable fragment comprising a 2-array and an HVR-L3 array of SEQ ID NO: 32; (f7) The HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the SEQ ID NO: 79 HVR-H3 sequence, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L of SEQ ID NO: 37 2 array, and an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 38; (f8) The HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the SEQ ID NO: 80 HVR-H3 sequence, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L of SEQ ID NO: 37 2 array, and an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 38; (f9) The HVR-H1 sequence of SEQ ID NO: 77, the HVR-H2 sequence of SEQ ID NO: 78, the SEQ ID NO: 80 HVR-H3 sequence, the HVR-L1 sequence of SEQ ID NO: 36, the HVR-L of SEQ ID NO: 37 2 array, and an antibody variable fragment comprising the HVR-L3 sequence of SEQ ID NO: 81; (f10) An antibody variable fragment that binds to the same epitope of any of the antibody variable fragments selected from (f1) to (f9) An antibody variable fragment that binds; (f11) Competes with the binding of any of the antibody variable fragments selected from (f1) to (f9) An antibody variable fragment And comprises an antigen-binding domain comprising any one of them. In some embodiments, the antigen-binding molecule is the following (g1) to (g20): (g1) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 15 Sequence, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 15 Column, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 15 Sequence, 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 An antibody variable fragment comprising; (g2) 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 An antibody variable fragment comprising; (g3) 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 (g3) An antibody variable fragment comprising; (g4) 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 An antibody variable fragment comprising; (g4) An antibody variable fragment comprising; An antibody variable fragment comprising; (g4) 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 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: 24; and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 24. an antibody variable fragment comprising: (g5) HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 11 The HVR-H2 sequence is identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 11. 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 and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 12. an antibody variable fragment comprising: (g6) HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 13 The HVR-H2 sequence is identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 13. 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. an antibody variable fragment comprising: (g7) HVR-H1 having the same amino acid sequence as the HVR-H1 region contained in SEQ ID NO: 17 The HVR-H2 sequence is identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 17. 17, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 17 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 comprising an antibody variable fragment; (g8) 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 contained in SEQ ID NO: 63, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 63, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 63, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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 (g10) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 64, an HVR-H2 An antibody variable fragment comprising an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 64, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; 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: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; 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: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 66, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 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: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 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: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74; (g11) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 65, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 65, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 65, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 72, 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; An antibody variable fragment; (g12) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 66, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 66, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 66, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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-L]3 region contained in SEQ ID NO: 73; An antibody variable fragment; (g13) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: e67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; An antibody variable fragment; (g14) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 68, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 68, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 68, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74; An antibody variable fragment; (g15) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 69, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 69, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 75, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 75, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 75; An antibody variable fragment; (g16) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 70, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 76, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 76, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 76; An antibody variable fragment; (g17) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 71, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 77, an HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 77, and an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 77; An antibody variable fragment; (g13) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 67, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73, 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; 1 An HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67 An HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67 An HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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 Antibody variable fragment comprising the sequence; (g14) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 67 An HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 67 An HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 67 An HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 74 An HVR-L2 sequence identical to the amino acid sequence of the HVR-L2 region contained in SEQ ID NO: 74 And an HVR-L3 sequence identical to the amino acid sequence of the HVR-L3 region contained in SEQ ID NO: 74 Antibody variable fragment comprising the sequence; (g15) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 68 An HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 68 An HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 68 An HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 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 Antibody variable fragment comprising the sequence; (g16) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 69, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 69, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 69, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 , 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; An antibody variable fragment comprising; (g17) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 70, an HVR-H 1 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 70, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 70, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 70, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 , 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; An antibody variable fragment comprising; (g18) An HVR-H1 sequence identical to the amino acid sequence of the HVR-H1 region contained in SEQ ID NO: 71, an HVR-H 1 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 71, an HVR-H2 sequence identical to the amino acid sequence of the HVR-H2 region contained in SEQ ID NO: 71, an HVR-H3 sequence identical to the amino acid sequence of the HVR-H3 region contained in SEQ ID NO: 71, an HVR-L1 sequence identical to the amino acid sequence of the HVR-L1 region contained in SEQ ID NO: 73 , 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; An antibody variable fragment comprising; An antibody variable fragment comprising a column; (g19) Any one of the antibody variable fragments selected from (g1) to (g18) binds to the same epi top of the top; an antibody variable fragment that binds to the top; (g20) Any one of the antibody variable fragments selected from (g1) to (g18) competes with the binding of the antibody variable fragment; It contains an antigen-binding domain containing any one of them. In some embodiments, the antigen-binding molecule is the following (h1) to (h21): (h1) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 15 and an amino acid of SEQ ID NO: 16 A light chain variable region having the sequence; (h2) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 25 and an amino acid of SEQ ID NO: 26 A light chain variable region having the sequence; (h3) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 and an amino acid of SEQ ID NO: 20 A light chain variable region having the sequence; (h4) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 23 and an amino acid of SEQ ID NO: 24 A light chain variable region having the sequence; (h5) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 11 and an amino acid of SEQ ID NO: 12 A light chain variable region having the sequence; (h6) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 13 and an amino acid of SEQ ID NO: 14 A light chain variable region having the sequence; (h7) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and an amino acid of SEQ ID NO: 18 A light chain variable region having the sequence; (h8) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and an amino acid of SEQ ID NO: 22 A light chain variable region having the sequence; (h9) A heavy chain variable region having the amino acid sequence of SEQ ID NO: 63 and an amino acid of SEQ ID NO: 72 A light chain variable region having the sequence; (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; acid sequence; (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; acid 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; acid sequence; (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; acid sequence; (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; acid sequence; (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; acid sequence; (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; acid sequence; (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; acid sequence; (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; acid sequence; (h19)(h1) to (h18) A heavy chain variable region having more than 80% identity to any one of the heavy chain variable regions and (h1) to (h18) A light chain variable region having more than 80% identity to any one of the light chain variable regions; identity, and (h1) to (h18) A light chain variable region having more than 80% identity to any one of the light chain variable regions; acid sequence; (h20)(h1) to (h18) A heavy chain variable region having more than 90% identity to any one of the heavy chain variable regions and (h1) to (h18) A light chain variable region having more than 90% identity to any one of the light chain variable regions; identity, and (h1) to (h18) A light chain variable region having more than 90% identity to any one of the light chain variable regions; acid sequence; A heavy chain variable region having more than 95% identity to any one of the heavy chain variable regions of (h21)(h1) to (h18) and a light chain variable region having more than 95% identity to any one of the light chain variable regions of (h1) to (h18) A light chain variable region having more than 95% identity to any one of the light chain variable regions of (h1) to (h18) contains an antigen-binding domain containing any one of the above. In some embodiments, the antigen-binding molecule has cytotoxic activity. In some embodiments, the cytotoxic activity in the antigen-binding molecule is antibody-dependent cell 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 containing an antibody .

[0044] Antibody class The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. Five major antibody classes exist: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as 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 the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain generally consists of four FR domains: FR1, FR 2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3 -H3(L3)-FR4.

[0046] Human consensus framework "Human consensus framework" refers to a framework that represents the amino acid residues that are most commonly present in the selected human immunoglobulin VL or V H framework sequences. Generally, those selected human immunoglobulin VL or VH sequences are derived from subgroups of the variable domain main sequences. Generally, the subgroups of sequences are as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Be thesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I as described by Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as described by Kabat et al., supra.

[0047] HVR As used herein, the terms "hypervariable region" or "HVR" refer to regions of the sequence that are hypervariable (the "complementary determining regions" or "CDRs") and / or are structurally defined forms the loops that have been made ("ultra-variable loops") and / or contains antigen contact residues ("antigen contact"), and refers to each region of the antibody variable domain. Generally, an antibody contains six HVRS, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRS herein are: (a) the ultra-variable loops present 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) the 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) the antigen 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) including 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) of (a), (b), and (a) the ultra-variable loops present 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) the 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) the antigen 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) including 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) of (a), (b), and (a) the ultra-variable loops present 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) the 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) the antigen 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) including 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) of (a), (b), and (a) the ultra-variable loops present 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) the 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) the antigen 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) including 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) of (a), (b), and (a) the ultra-variable loops present 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)); [[ID=***]] (b) the 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) the antigen 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) including 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) of (a), (b), and a combination of (b) and / or (c) is included. Unless otherwise indicated, HVR residues and other residues in the variable domains (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 chain or light chain that is involved in binding of the antibody to antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively ) generally have similar structures, and each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, an antibody that binds a particular antigen can be isolated using a VH or VL domain derived from an antibody that binds that antigen, and that library of complementary VL or VH domains, respectively, can be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 ( 1993); Clarkson et al., Nature 352:624-628 (1991 ). )

[0049] Chimeric antibody The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chains is derived from a particular source or species, while the remainder of the heavy and / or light chains is derived from a different source or species. Similarly, the term “chimeric antibody variable domain” refers to a variable domain of a heavy and / or light chain that​​ A portion of the variable region is derived from a particular source or species, but the heavy and / or light chain variable regions refers to an antibody variable region in which the remaining portions are derived from different sources or species.

[0050] Humanized antibody A "humanized" antibody refers to a chimeric antibody that contains amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody will correspond to an antibody 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, and will typically include substantially all of at least one, typically two variable domains. A humanized antibody may include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., as opposed to a non-human antibody, refers to an antibody that has been humanized. A "humanized antibody variable region" refers to the variable region of a humanized antibody. (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, and will typically include substantially all of at least one, typically two variable domains. A humanized antibody may include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., as opposed to a non-human antibody, refers to an antibody that has been humanized. A "humanized antibody variable region" refers to the variable region of a humanized antibody. will typically include substantially all of at least one, typically two variable domains. A humanized antibody may include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., as opposed to a non-human antibody, refers to an antibody that has been humanized. A "humanized antibody variable region" refers to the variable region of a humanized antibody. will typically include substantially all of at least one, typically two variable domains. A humanized antibody may include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., as opposed to a non-human antibody, refers to an antibody that has been humanized. A "humanized antibody variable region" refers to the variable region of a humanized antibody. A "humanized" antibody refers to a chimeric antibody that contains amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody will correspond to an antibody 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, and will typically include substantially all of at least one, typically two variable domains. A humanized antibody may include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., as opposed to a non-human antibody, refers to an antibody that has been humanized. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

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

[0052] Method for producing an antibody having a desired binding activity Methods for producing antibodies having a desired binding activity are known to those of skill in the art. The following is an example of a method for producing an antibody that binds to DLL3 (anti-DLL3 antibody). T cells Antibodies that bind to receptor complexes and the like can also be produced as described below.

[0053] Anti-DLL3 antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. The preferably produced anti-DLL3 antibodies are monoclonal antibodies derived from mammals. Such mammalian-derived monoclonal antibodies include antibodies produced by hybridomas or host cells transformed with an expression vector carrying the antibody gene by genetic engineering techniques. Monoclonal antibody-producing hybridomas can be generated using known techniques such as those described below. Specifically, a mammal is immunized by a conventional immunization method using DLL3 protein as the sensitizing antigen. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Then, hybridomas producing anti-DLL3 antibodies can be selected by screening of monoclonal antibody-producing cells using conventional screening methods. 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 the DLL3 protein used as the 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, full-length DLL3

[0054] [[ID=?]]

[0055] Alternatively, insert the gene sequence encoding DLL3 ECD into a known expression vector and transform an appropriate host cell 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 it is possible to use the native DLL3 protein purified as the sensitizing antigen. The purified full-length DLL3 or DLL3 ECD protein can be used as a sensitizing antigen for use in immunizing mammals. Partial peptides of full-length DLL3 or DLL3 ECD can also be used as a sensitizing antigen. In this case, the partial peptide can also be obtained by chemical synthesis from the DLL3 amino acid sequence. Furthermore, they can also be obtained by incorporating a part of the DLL3 gene into an expression vector and expressing it thereby. Furthermore, they can also be obtained by degrading the DLL3 protein using a protease, but the region and size of the DLL3 peptide used as the partial peptide are not particularly limited to specific embodiments. As a preferred region any sequence derived from the amino acid sequence can be selected. The number of amino acids constituting the peptide used as the 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 the sensitizing antigen.

[0056] Alternatively, as the sensitizing antigen, a desired partial polypeptide or peptide of the full-length DLL3 or DLL3 ECD protein is fused with a different polypeptide to prepare The fusion protein to be used can be used. For example, an antibody Fc fragment and a peptide ta ng are preferably used to produce a fusion protein used as a sensitizing antigen. A vector for the expression of such a fusion protein is prepared by fusing genes encoding two or more desired polypeptide fra gments in-frame and inserting the fusion gene into the above-described expression vector. A method for producing a fusion protein is described in Mol ecular Cloning, 2nd Edition (Sambrook, J. et al., Molecula r Cloning, 2nd Edition, 9.47-9.58 (1989) Cold Spring Harbor Lab.Press). A method for preparing DLL3 used as a sensitizing antigen and an immunization method using DLL3 are also described in the examples of this specification later. There is no particular limitation on the mammal immunized with the sensitizing antigen. However, it is preferable to select a mammal in consideration of its compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and guinea pigs are preferably used. The above animals are immunized with the sensitizing antigen by a known method. Generally, examples of the immunization method include intraperitoneal or subcutaneous injection of the sensitizing antigen into the mammal. Specifically, the sensitizing antigen is appropriately diluted with PBS (phosphate-buffered saline), physiological saline, etc. If necessary, a conventional adjuvant such as complete Freund's adjuvant is mixed with the antigen and the mixture is emulsified. Then, the sensitizing antigen is administered to the mammal several times at intervals of 4 to 21 days.

[0057] There is no particular limitation on the mammal immunized with the sensitizing antigen. However, it is preferable to select a mammal in consideration of its compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and guinea pigs are preferably used. The above animals are immunized with the sensitizing antigen by a known method. Generally, examples of the immunization method include intraperitoneal or subcutaneous injection of the sensitizing antigen into the mammal.

[0058] Specifically, the sensitizing antigen is appropriately diluted with PBS (phosphate-buffered saline), physiological saline, etc. If necessary, a conventional adjuvant such as complete Freund's adjuvant is mixed with the antigen and the mixture is emulsified. Then, the sensitizing antigen is administered to the mammal several times at intervals of 4 to 21 days. ​​​A suitable carrier can be used in immunization with the sensitizing antigen. When a low molecular weight partial peptide is used as an immunizing antigen, the sensitizing antigen peptide is The antibody is coupled to a carrier protein such as albumin or keyhole limpet hemocyanin. It may be desirable to pull it.

[0059] Alternatively, hybridomas producing the desired antibodies can be isolated by DNA immunization as described below. DNA immunization can be carried out by using a vector encoding an antigen protein in an animal. Immunization results from the administration of vector DNA constructed to allow expression of a gene that Immunization methods that provide immune stimulation by expressing sensitizing antigens in immunized animals Compared to conventional immunization methods in which protein antigens are administered to the animal to be immunized, DNA immunization is - Able to 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, first, DNA DNA expressing the DLL3 protein is administered to the animal to be immunized. The DNA to be used can be synthesized by known methods such as PCR. A is inserted into an appropriate expression vector, which is then administered to the animal to be immunized. The expression vector preferably used is, for example, a commercially available expression vector such as pcDNA3.1. The vectors can be administered to an organism using conventional methods. For example, DNA immunization is carried out by using a gene gun for introducing gold particles encoded by an expression vector into cells in the body of an animal to be immunized. An antibody recognizing DLL3 can also be produced by the method described in WO2011 / 093097. After immunizing the above-mentioned mammal, an increase in the titer of the DLL3-binding antibody is confirmed in the serum. Then, after collecting immune cells from the mammal, they are subjected to cell fusion. In particular, splenocytes are preferably used as immune cells. Mammalian myeloma cells are used as cells to be fused with the above-mentioned immune cells. Myeloma cells preferably contain a suitable selection marker for screening. The selection marker imparts characteristics regarding its survival (or death) under specific culture conditions to the cells. Hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency) are known as selection markers. Cells having HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). 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 by using the salvage pathway of normal cells and can therefore proliferate even in HAT selection medium.

[0061]

[0062]

[0063] HGPRT-deficient and TK-deficient cells are respectively 6-thioguanine, 8-azaguanine ​​​​​​​​​​​​​​​medium containing 8AG (hereafter abbreviated as 8AG) or 5'-bromodeoxyuridine Normal cells incorporate these pyrimidine analogs into their DNA. Cells lacking these enzymes, on the other hand, are killed because they incorporate these pyrimidines. They are unable to incorporate the analogue and are therefore able to survive in selective media. The selection marker called G418 resistance provided by the neomycin resistance gene is -confers resistance to deoxystrepamine antibiotics (gentamicin analogs) Various types of myeloma cells are known that are suitable for cell fusion.

[0064] For example, myeloma cells including the following cells can be preferably used: P3(P3x63Ag8.653)(J.Immunol.(1979)123(4), pages 1548~1550); P3x63Ag8U.1(Current Topics in Microbiolo gy 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), 3 pages 13-323); R210 (Nature (1979) 277 (5692), pp. 131-133), etc.

[0065] Cell fusion between immune cells and myeloma cells can be carried out by essentially known methods, for example, Kohl er and Milstein et al. (Methods Enzymol. (1981) 73: 3-46). More specifically, cell fusion can be carried out, for example, in a conventional culture medium in the presence of a cell fusion promoter . Examples of the fusion promoter include polyethylene glycol (PEG ) and Sendai virus (HVJ). If necessary, auxiliary substances such as dimethyl sulfoxide are also added to improve the fusion efficiency . The ratio of immune cells to myeloma cells can be determined at the discretion of those skilled in the art. For example , one myeloma cell per 1-10 immune cells is preferred. Examples of the culture medium used for cell fusion include media suitable for the growth of myeloma cell lines such as RPMI1640 medium and MEM medium, and other conventional culture media used for this type of cell culture . Furthermore, it is preferable to add serum additives such as fetal calf serum (FCS) to the culture medium . For cell fusion, a predetermined amount of the above-mentioned immune cells and myeloma cells is well mixed in the above-mentioned culture medium . Then, a PEG solution pre-warmed to about 37°C (for example, with an average molecular weight of about

[0066] 1,000-6,000) is generally added thereto at a concentration of 30%-60% (w / v). This is gently mixed to generate the desired fused cells (hybridomas) . Then, the above-mentioned appropriate culture medium is gradually added to the cells, and this is repeatedly centrifuged to remove the supernatant . Thus, cell fusion agents and the like that are not favorable for the growth of hybridomas can be removed . . . .

[0067] The hybridomas thus obtained can be selected by culturing using a conventional selection medium, for example, HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Cells other than the desired hybridomas (non-fused cells) can be killed by continuing the culture in the above-mentioned HAT medium for a sufficient period of time. Typically, the period is from several days to several weeks. Then, the hybridomas producing the desired antibody are screened and cloned singly by the conventional limiting dilution method.

[0068] The hybridomas thus obtained can be selected using a selection 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 using 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 selectively proliferate in HAT medium. Cells other than the desired hybridomas (non-fused cells) can be killed by continuing the culture in the above-mentioned HAT medium for a sufficient period of time. Specifically, the desired hybridomas can generally be selected by culturing for a period of several days to several weeks. Then, the hybridomas producing the desired antibody are screened and cloned singly by the conventional limiting dilution method.

[0069] Preferably, the desired antibody is screened by a screening method based on a known antigen / antibody reaction. Select and can be cloned singly. 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 uses a laser beam to analyze cells contacted with a fluorescent antibody and measure the fluorescence emitted from individual cells, thereby evaluating the binding of the antibody to the cell surface.

[0070] To screen for hybridomas producing the monoclonal antibody of the present invention by FACS, DLL3-expressing cells are first prepared. Cells preferably used for screening are mammalian cells in which DLL3 is forcedly 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 an anti-DLL3 monoclonal antibody can be isolated by selecting hybridomas

[0071] that produce an antibody that binds to cells expressing DLL3 but does not bind to host cells. Alternatively, the activity of an antibody that binds to solid-phase 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 culture supernatant of the hybridoma is It is possible. Hybridomas that produce the desired antibody having antigen-binding ability are selected by the above screening, and they can be cloned by methods such as the limiting dilution method. The monoclonal antibody-producing hybridomas thus prepared can be passaged in a conventional culture medium and stored in liquid nitrogen for a long period of time.

[0072] The above-prepared monoclonal antibody-producing hybridomas can be cultured by conventional methods, and the desired monoclonal antibody can be prepared from the culture supernatant. Alternatively, the hybridomas are administered to a compatible mammal, proliferated therein, and the monoclonal antibody is prepared in ascites. The former method is suitable for preparing high-purity antibodies.

[0073] The above hybridomas are cultured by conventional methods, and the desired monoclonal antibody can be prepared from the culture supernatant. Alternatively, the hybridomas are administered to a compatible mammal, proliferated therein, and the monoclonal antibody is prepared in ascites. The former method is suitable for preparing high-purity antibodies. Preferably, antibodies encoded by antibody genes cloned from antibody-producing cells such as the above hybridomas can also be used. Methods for isolating the cloned antibody gene, inserting the gene into a vector, 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.

[0074] Preferably, the present invention provides a nucleic acid encoding the multispecific antigen-binding molecule or monospecific antigen-binding molecule of the present invention. The present invention also provides a vector into which a nucleic acid encoding a multispecific antigen-binding molecule or monospecific antigen-binding molecule is introduced, that is, a vector containing the nucleic acid. For example, by Vandamme et al. (Eur. J. Biochem. (199 0) 192(3), pp. 767-775). Methods for producing recombinant antibodies are also known as described below.

[0075] Preferably, the present invention provides a nucleic acid encoding the multispecific antigen-binding molecule or monospecific antigen-binding molecule of the present invention. The present invention also provides a vector into which a nucleic acid encoding a multispecific antigen-binding molecule or monospecific antigen-binding molecule is introduced, that is, a vector containing the nucleic acid. ​​​​​​​​​​ also provides. Furthermore, the present invention provides a cell containing a nucleic acid or a vector. The present invention also provides a method for producing a multispecific antigen-binding molecule or a monospecific antigen-binding molecule by culturing cells . The present invention further provides a multispecific antigen-binding molecule or a monospecific antigen-binding molecule produced by the above method .

[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. For this purpose, total RNA is first extracted from the hybridoma . Methods used for extracting mRNA from cells include, for example - the guanidine ultracentrifugation method (Biochemistry (1979) 18(24), 5 294-5299), and - the AGPC method (Anal. Biochem. (1987) 162(1), 156-15 9) are mentioned . The extracted mRNA can be purified using an mRNA purification kit (GE Healthcare Bioscience) or the like. Alternatively, kits for directly extracting total mRNA from cells, such as the QuickPrep

[0077] mRNA purification kit (GE Healthcare Bioscience), are also commercially available. mRNA can be prepared from hybridoma cells using such a kit. cDNA encoding the V region of the antibody can be synthesized from the prepared mRNA using reverse transcriptase . The cDNA can be synthesized from the prepared mRNA using reverse transcriptase . cDNA can be synthesized from the prepared mRNA using reverse transcriptase . cDNA encoding the V region of the antibody can be synthesized from the prepared mRNA using reverse transcriptase . cDNA encoding the V region of the antibody can be synthesized from the prepared mRNA using reverse transcriptase . cDNA can be synthesized from the prepared mRNA using AMV Reverse Transcriptase First- strand cDNA Synthesis Kit (Seikagaku Co.) etc. can be used for synthesis. Furthermore, SMART RACE cDNA amplification kit (Clontech) and 5'-RACE method based on PCR (Proc. Natl . Acad. Sci. USA (1988) 85(23), pp. 8998 - 9002; Nuc leic Acids Res. (1989) 17(8), pp. 2919 - 2932) can be suitably used to synthesize and amplify cDNA. In such cDNA synthesis procedures, the appropriate restriction enzyme sites described below can be introduced to both ends of the cDNA.

[0078] After purifying the target cDNA fragment from the obtained PCR product, this is ligated to vector DNA. Thus, a recombinant vector is constructed and introduced into, for example, Escherichia coli (E. coli). After colony selection, the desired recombinant vector can be prepared from the colony-forming E. coli. Then, whether the recombinant vector has the target cDNA nucleotide

[0079] sequence is tested by a known method such as the dideoxynucleotide chain termination method. The 5'-RACE method using primers for amplifying variable region genes is conveniently used to isolate the gene encoding the variable region. First, a 5'-RACE cDNA library is constructed by cDNA synthesis using RNA extracted from hybridoma cells as a template. A commercially available

[0080] Using the prepared 5’-RACE cDNA library as a template, the antibody gene is amplified by PCR. Based on the known antibody gene sequence, primers for amplifying the mouse antibody gene can be designed. The nucleotide sequence of the primers varies according to the immunoglobulin subclass. Therefore, it is preferable to determine the subclass in advance using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics). Specifically, for example, primers that enable amplification of genes encoding gamma 1, gamma 2a, gamma 2b, and gamma 3 heavy chains as well as kappa and lambda light chains are used to isolate the gene encoding mouse IgG. Generally, a primer that anneals to a constant region site close to the variable region is used as the 3’-side primer for amplifying the IgG variable region gene. On the other hand, a primer bound to the 5’RACE cDNA library construction kit is used as the 5’-side primer. Using the PCR product amplified in this way, an immunoglobulin composed of a combination of heavy and light chains is reconstituted. Using the DL3 binding activity of the reconstituted immunoglobulin as an index, the desired antibody can be selected. For example, when the aim is to isolate an antibody against DLL3, it is more preferable that the binding of the antibody to DLL3 is specific. The DLL3 binding antibody can be obtained, for example, by the following steps: (1) A step of contacting DLL3-expressing cells with an antibody containing the V region encoded by cDNA isolated from a hybridoma;

[0081]

[0082] ​​​​​​​​​​​​​​​(2) detecting the binding of the antibody to DLL3-expressing cells; and (3) screening by selecting an antibody that binds to DLL3-expressing cells can be performed.

[0083] Methods for detecting the binding of an antibody to DLL3-expressing cells are known. Specifically, the binding of the antibody to DLL3-expressing cells can be detected by the above-described techniques such as FACS. An immobilized sample of DLL3-expressing cells is appropriately used to evaluate the binding activity of the antibody.

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

[0085] After isolating the cDNA encoding the V region of the anti-DLL3 antibody of interest, the cDNA is digested with a restriction enzyme that recognizes the restriction sites introduced at both ends of the cDNA. Preferred restriction enzymes are 、recognizes and cleaves nucleotide sequences that are present at low frequencies in the nucleotide sequence of the antibody gene It is. Furthermore, preferably, a restriction site of an enzyme that provides an attachment end is introduced into the vector to insert a single-copy 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 anti body expression vector. In this case, when the gene encoding the antibody constant region (C region) and the gene encoding the above V region are fused in-frame, a chimeric antibody is obtained . As used herein, a "chimeric antibody" means that the origin of the constant region is different from that of the variable region . Thus, in addition to mouse / human heterologous chimeric antibodies, human / human homologous chimeric antibodies are also included in the chimeric antibodies of the present invention. By inserting the above V region gene into an expression vector that already has a constant region, a chimeric antibody expression vector can be constructed . Specifically, for example, the recognition sequence of the restriction enzyme that excises the above V region gene is appropriately placed on the 5' side 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 . Specifically, for example, the recognition sequence of the restriction enzyme that excises the above V region gene is appropriately placed on the 5' side 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 . A chimeric antibody expression vector can be 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 such that the gene is expressed under the control of an expression regulatory region . The expression regulatory region for antibody expression includes, for example, an enhancer and a promoter. Furthermore, an appropriate signal sequence may be attached to the amino terminus so that the expressed antibody is secreted outside the cell . . On the one hand, other suitable signal sequences can be ligated. The expressed polypeptide is cleaved at the carboxyl terminus of the above-mentioned sequence, and the resulting polypeptide is the mature polypeptide and is secreted outside the cell. Then, a suitable host cell is transformed with the expression vector, to obtain recombinant cells expressing DNA encoding the anti-DLL3 antibody.

[0087] DNA encoding the antibody heavy chain (H chain) and light chain (L chain) is inserted separately into different expression vectors to express the antibody gene. Antibody molecules having H and L chains are cotransfected into the same host cell with vectors into which the H chain gene and the L chain gene have been inserted respectively. [[ID=!6]]Alternatively, the host cell can be transformed with a single expression vector into which DNA encoding the H and L chains has been inserted (see WO94 / 11523).

[0088] There are various known combinations of host cells / expression vectors for antibody preparation by introducing the isolated antibody gene into a suitable host. All of these expression systems are applicable to the isolation of domains containing the antibody variable region of the present invention. Suitable eukaryotic cells used 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: Xenopus oocytes, etc.; and (3) Insect cells: sf9, sf21, Tn5, etc.

[0089] Furthermore, as plant cells, tobacco such as Nicotiana tabacum Antibody gene expression systems using cells derived from the genus Coffea are known. Callus cultured cells can be appropriately used to transform plant cells.

[0090] Furthermore, the following cells can be used as fungal cells: Yeast: Saccharomyces cerevisia e) and other Saccharomyces genera, and Pichia pastoris is) and other Pichia genera; and Filamentous fungi: Aspergillus niger and other Asper gillus genera.

[0091] Furthermore, antibody gene expression systems using prokaryotic cells are also known. For example, when using bacterial cells such as Escherichia coli cells and Bacillus subtilis cells 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 product of the transformed cells.

[0092] In addition to the above host cells, transgenic animals can also be used to produce recombinant antibodies. That is, antibodies can be obtained from animals into which the gene encoding the antibody of interest has been introduced. For example, by inserting a gene encoding a protein specifically produced in milk in-frame, an antibody gene can be constructed as a fusion gene. For example, caprine beta-casein and the like can be used as proteins secreted in milk ​​​​can be used. A DNA fragment containing a fusion gene inserted together with an antibody gene is injected into a goat embryo, and then the embryo is introduced into a female goat. From the milk produced by the transgenic goat born from the recipient goat of the embryo (or its offspring), the desired antibody can be obtained as a protein fused with the milk protein. Furthermore, to increase the volume of the milk containing the desired antibody produced by the trans genic goat, hormones may be administered to the transgenic goat as necessary (Ebert , K.M. et al., Bio / Technology (1994) 12(7), 699 - 702 page). ).

[0093] Method for producing a humanized antibody When administering the antigen-binding molecule described herein to a human, a domain derived from a genetically engineered antibody artificially modified to reduce the heterologous antigenicity against humans and the like can be appropriately used as the domain of the antigen-binding molecule including the antibody variable region. Such genetically engineered antibodies include, for example, humanized antibodies. These modified antibodies are appropriately produced by known methods. Furthermore, in general, the binding specificity of a specific antibody can be introduced into another antibody by CD R transplantation. Specifically, humanized antibodies prepared by transplanting the CDR (or "HVR" as defined herein) of non-human animal antibodies such as mouse antibodies into human antibodies and the like are known

[0094] . Specifically, for example, overlap extension PCR is known as a method for transplanting mouse antibody CDR into human FR . General genetic engineering techniques for obtaining humanized antibodies are also known. Specifically, for example , overlap extension PCR is known as a method for transplanting mouse antibody CDR into human FR In overlapping extension PCR, the nucleotide sequence encoding the mouse antibody CDR to be transplanted is added to the primer for synthesizing the human antibody FR. The primers are prepared for each of the four FRs. When transplanting the mouse CDR into the human FR, it is generally considered advantageous to select a human FR having a high identity to the mouse FR in order to maintain the CDR function. That is, it is generally preferred to use a human FR containing an amino acid sequence having a high identity to the amino acid sequence of the FR adjacent to the mouse CDR to be transplanted. The nucleotide sequences to be ligated are designed so that they are connected in-frame with each other. The human FRs are synthesized individually using the respective primers. As a result, a product in which the DNA encoding the mouse CDR is bound to the DNA encoding the individual FRs is obtained. The nucleotide sequences encoding the mouse CDR of each product are designed so that they overlap with each other. Then, a complementary strand synthesis reaction is performed to anneal the overlapping CDR regions of the products synthesized using the human antibody gene as a template. The human FRs are ligated through the mouse CDR sequences by this reaction. The full-length V region gene in which three CDRs and four FRs are finally ligated is amplified using a primer that anneals to its 5' or 3' end, added with a suitable restriction enzyme recognition sequence. The DNA obtained as described above and the DNA encoding the human antibody C region are inserted into an expression vector so that they are ligated in-frame.

[0095]

[0096] ​​​​​​​​​​​​​​​By introducing it, an expression vector for a humanized antibody can be generated. After transfecting the recombinant vector into a host to establish recombinant cells, the recombinant cells are cultured, and DNA encoding the humanized antibody is expressed to produce the humanized antibody in the 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, the CDR can preferably select a human antibody FR that can form a preferable antigen-binding site when ligated via the CDR. In order for the CDR of the re-formed human antibody to form an appropriate antigen-binding site, the amino acid residues in the FR may be substituted as necessary. For example, by applying the PCR method used for transplanting mouse CDR into human FR, amino acid sequence mutations can be introduced into the FR . More specifically, partial nucleotide sequence mutations can be introduced into the primer annealing to the FR. The nucleotide sequence mutations are introduced into the FR synthesized by using such primers . By measuring and evaluating the activity of the amino acid-substituted antibody variant that binds to the antigen by the above method, an FR sequence variant having desirable characteristics can be selected (Sato, K. et al., Cancer R es. (1993) 53: 851-856).

[0098] Method for producing a human antibody Alternatively, a transgenic animal having the entire repertoire of human antibody genes is DN Desired human antibodies can be obtained by immunization through immunization (see WO1 993 / 012227; WO1992 / 003918; WO1994 / 002602; W O1994 / 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 phage surface by the phage display method. Phages expressing scFv that bind to the antigen can be selected. The DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined by analyzing the gene of the selected phage. Determine the DNA sequence of the scFv that binds to the antigen. By fusing the V region sequence in-frame with the C region sequence of the desired human antibody and inserting this into an appropriate expression vector, an expression vector can be prepared. The expression vector is introduced into an appropriate cell for expression, such as the above-mentioned ones. By expressing the gene encoding the human antibody in the cell, human antibodies can be produced. These methods are already known (see WO199 2 / 001047; WO1992 / 020791; WO1993 / 006213; WO1 99 _ 3 / 011236; WO1993 / 019172; WO1995 / 001438; W O1995 / 015388). As used herein, the term "vector" refers to the propagation of another nucleic acid to which it is ligated .

[0100] Vector and ​​​​Refers to a nucleic acid molecule that can effect. This term includes vectors as self-replicating nucleic acid structures — as well as vectors that are integrated into the genome of the host cell into which they are introduced. Certain vectors can direct the expression of nucleic acids to which they are operably 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 a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. A host cell includes "transformants" and "transformed cells" that are "transformed" and progeny derived therefrom, regardless of the number of passages. Progeny may not be exactly identical to the parental cell in nucleic acid content and may contain mutations. Mutant progeny having the same function or biological activity as that originally screened or selected in the originally transformed cell are also included herein.

[0102] Epitope "Epitope" means an antigenic determinant in an antigen and refers to the antigenic site to which the antigen-binding molecule or the antigen-binding domain of an 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 sugar chain, the epitope can be specified by its specific sugar chain structure.

[0103] A linear epitope is one in which the primary amino acid sequence contains the epitope that is recognized. Such linear epitopes typically have at least three sequences in that particular sequence. , most commonly at least 5, e.g., about 8-10 or 6-20 amino acids Contains:

[0104] In contrast to a linear epitope, a "conformational epitope" is a primary antigen containing the epitope. The amino acid sequence is not the only determinant of the recognized epitope (e.g., The primary amino acid sequence of a conformational epitope is not necessarily determined by the antibody that defines the epitope. Conformational epitopes contain a larger number of amino acids than linear epitopes. The antigen-binding domain that recognizes a conformational epitope may contain a peptide or a protein. For example, protein molecules fold and form three-dimensional structures. When forming a conformational epitope, the amino acids and / or polypeptide backbone that form the conformational epitope are The epitope becomes recognizable by the antigen-binding domain. Methods for determining the topographic conformation include, but are not limited to, However, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-directed spin labeling, and electron paramagnetic resonance have been used to characterize the For example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, see Morris (ed.).

[0105] The epitope is identified by a test antigen-binding molecule or antibody containing the anti-DLL3 antigen-binding domain. Examples of methods for assessing peptide binding are described below. According to the following examples, peptides other than DLL3 are A method for evaluating epitope binding by a test antigen-binding molecule or antibody containing an antigen-binding domain for the antigen can also be appropriately performed. For example, whether a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain recognizes a linear epitope in the DLL molecule can be confirmed as described below.

[0106] For example, 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 techniques using the region encoding the amino acid sequence corresponding to the extracellular domain in DLL3 cDNA. Then, the test antigen-binding molecule or antibody containing the anti-DLL3 antigen-binding domain is 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 prove the binding activity of the antigen-binding molecule or antibody to the linear peptide. For example, whether a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain recognizes a linear epitope in the DLL molecule can be confirmed 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 techniques using the region encoding the amino acid sequence corresponding to the extracellular domain in DLL3 cDNA. 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 techniques using the region encoding the amino acid sequence corresponding to the extracellular domain in DLL3 cDNA. Then, the test antigen-binding molecule or antibody containing the anti-DLL3 antigen-binding domain is 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. 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. 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 prove the binding activity of the antigen-binding molecule or antibody to the linear peptide. These tests can prove 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 evaluated as follows. DLL3-expressing cells are prepared for the above purpose. The test antigen-binding molecule containing the anti-DLL3 antigen-binding domain Alternatively, an antibody can be determined to recognize when it does not substantially bind to a solid-phase linear peptide comprising an amino acid sequence forming the extracellular domain of DLL3, which strongly binds to DLL3-expressing cells upon contact with a conformational epitope. 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] As a method for assaying the binding activity of a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain to DLL3-expressing cells, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) pp. 359-4 20) can be mentioned. Specifically, evaluation can be carried out based on the principle of ELISA or fluorescence-activated cell sorting (FACS) using DLL3-expressing cells as an antigen. :A Laboratory Manual(Ed Harlow、David Lan e、Cold Spring Harbor Laboratory(1988)359 ~420 pages) can be used. Specifically, 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 levels of signals generated by an enzymatic reaction. Specifically, a test polypeptide complex is added to an ELISA plate on which DLL3-expressing cells are immobilized. Then, the test antigen-binding molecule or antibody bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule or antibody. Alternatively, when using FACS, the test

[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 levels of signals generated by an enzymatic reaction. Specifically, a test polypeptide complex is added to an ELISA plate on which DLL3-expressing cells are immobilized. Then, the test antigen-binding molecule or antibody bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule or antibody. Alternatively, when using FACS, the test ​​​​​​​​​​​​Prepare a dilution series of the antigen-binding molecule or antibody and determine the antibody-binding titer for DLL3-expressing cells to compare the binding activity of the test antigen-binding molecule or antibody against DLL3-expressing cells This can be done

[0110] The binding of the test antigen-binding molecule or antibody to the antigen expressed on the surface of cells suspended in a buffer or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices: FACSCantoTM II FACSAriaTM FACSArrayTM FACSVantageTM SE FACSCaliburTM (all are trademarks of BD Biosciences) EPICS ALTRA HyPerSort Cytomics FC500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (all are trademarks of Bec kman Coulter) can be mentioned

[0111] Preferred methods for assaying the binding activity of a test antigen-binding molecule or antibody containing an anti-DLL3 antigen-binding domain to an antigen include, for example, the following methods First, after reacting DLL3-expressing cells with the test antigen-binding molecule or antibody, stain them with an FITC-labeled secondary antibody that recognizes the antigen -binding molecule or antibody. Dilute the test antigen-binding molecule or antibody appropriately with a suitable buffer to prepare an antigen-binding molecule or antibody at a desired concentration For example, the antigen-binding molecule or antibody is in the range of 10 μg / ml to 10 ng / ml ​​​It can be used at a concentration. Then, the fluorescence intensity and the number of cells are determined using FACSCalibu r (BD). The fluorescence intensity obtained by the analysis using CELL QUEST Software (BD), that is, the geometric mean value, 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 the bound test antigen-binding molecule or antibody, can be determined by measuring the geometric mean value .

[0112] Whether a test antigen-binding molecule or antibody containing an 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 a cross-blocking assay or the like . For example, a competitive ELISA assay is a preferred cross-blocking assay . Specifically, in a cross-blocking assay, the 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 the test antigen-binding molecule or antibody is added thereto

[0113] . The amount of the test antigen-binding molecule or antibody bound to the DLL3 protein in the well is indirectly correlated 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 for the well coated with the DLL3 protein . ​​​​​​​

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

[0115] If the binding of the candidate competitor antigen-binding molecule or antibody to the test antigen-binding molecule or antibody containing the anti-DLL3 antigen-binding domain is at least 20%, preferably at least 20-50%, more preferably at least 50% blocked compared to the binding activity in a control experiment conducted 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 to which the competitor antigen-binding molecule or antibody binds, or compete for binding to the same epitope.

[0116] If the structure of the epitope to which the test antigen-binding molecule or antibody containing the anti-DLL3 antigen-binding domain binds has already been identified, it can be evaluated by comparing the binding activities of the test and control antigen-binding molecules or antibodies to peptides prepared by introducing amino acid mutations into the peptide forming the epitope to determine whether they share a common epitope. ​​

[0117] The above binding activity, for example, the binding activity of the test and control anti- original binding molecule or antibody to the linear peptide into which the mutation is introduced is compared in the above ELISA format. In addition to the ELISA method, after flowing the test and control antigen-binding molecules or antibodies through a column, the binding activity to the peptide variant bound to the column can be determined by quantifying the antigen-binding molecule or antibody eluted in the elution solution. For example, a method for adsorbing a peptide variant to a column in the form of a GST fusion peptide is known. Alternatively, when the identified epitope is a conformational epitope, it can be evaluated by the following method whether the test and control anti- original binding molecules or antibodies share a common epitope. First, prepare DLL3-expressing cells and cells expressing DLL3 having the mutation introduced into the epitope. Add the test and control antigen-binding molecules or antibodies to the cell suspension prepared by suspending these cells in an appropriate buffer such as PBS. Then, wash the cell suspension appropriately with the buffer and add to it an FITC-labeled antibody that recognizes the test and control antigen-binding

[0118] molecules or antibodies. Determine the fluorescence intensity and the number of cells stained with the labeled antibody using FACSCalibur (BD). Dilute the test and control antigen-binding molecules or antibodies appropriately using a suitable buffer and use them at the desired concentration. For example, they can be used at a concentration in the range of 10 μg / ml to 10 ng / ml. The fluorescence intensity determined by analysis using CELL QUEST Software (BD), that is, the geometric mean value, is the labeled antibody bound to the cells. First, prepare DLL3-expressing cells and cells expressing DLL3 having the mutation introduced into the epitope. Add the test and control antigen-binding molecules or antibodies to the cell suspension prepared by suspending these cells in an appropriate buffer such as PBS. Then, wash the cell suspension appropriately with the buffer and add to it an FITC-labeled antibody that recognizes the test and control antigen-binding molecules or antibodies. Determine the fluorescence intensity and the number of cells stained with the labeled antibody using FACSCalibur (BD). Dilute the test and control antigen-binding molecules or antibodies appropriately using a suitable buffer and use them at the desired concentration. For example, they can be used at a concentration in the range of 10 μg / ml to 10 ng / ml. The fluorescence intensity determined by analysis using CELL QUEST Software (BD), that is, the geometric mean value, is the labeled antibody bound to the cells. Dilute the test and control antigen-binding molecules or antibodies appropriately using a suitable buffer and use them at the desired concentration. For example, they can be used at a concentration in the range of 10 μg / ml to 10 ng / ml. The fluorescence intensity determined by analysis using CELL QUEST Software (BD), that is, the geometric mean value, is the labeled antibody bound to the cells. The fluorescence intensity determined by analysis using CELL QUEST Software (BD), that is, the geometric mean value, is the labeled antibody bound to the cells. reflects the amount. That is, the binding activity of the test and control antigen-binding molecules or antibodies, represented by the amount of labeled antibody bound, can be determined by measuring the geometric mean. It is possible. [[ID=⑥]]

[0119] [[ID=⑦]] In the above method, whether the antigen-binding molecule or antibody "substantially does not bind to cells expressing the DLL3 variant (or DLL3 variant)", for example, can be evaluated by the following method. First, the test and control antigen-binding molecules or antibodies bound to cells expressing the DLL3 variant are stained with a labeled antibody. Then, the fluorescence intensity of the cells is determined. When FACSCalibur is used for fluorescence detection by flow cytometry, the determined fluorescence intensity can be analyzed using CELL QUEST Software. From the geometric means in the presence and absence of the antigen-binding molecule or antibody, a comparison value (delta geometric mean) is calculated according to the following formula to determine the ratio of the increase in fluorescence intensity as a result of binding by the antigen-binding molecule or antibody. When FACSCalibur is used for fluorescence detection by flow cytometry, the determined fluorescence intensity can be analyzed using CELL QUEST Software. From the geometric means in the presence and absence of the antigen-binding molecule or antibody, a comparison value (delta geometric mean) is calculated according to the following formula to determine the ratio of the increase in fluorescence intensity as a result of binding by the antigen-binding molecule or antibody. It is possible to determine the ratio of the 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 the antigen-binding molecule or antibody) / geometric mean (in the absence of the antigen -binding molecule or antibody).

[0121] The geometric mean comparison value (delta geometric mean value for DLL3 variant molecules), which reflects the amount of the test antigen-binding molecule or antibody bound to cells expressing the DLL3 variant, is compared with the delta geometric mean comparison value that reflects the amount of the test antigen-binding molecule or antibody bound to DLL3-expressing cells. In this case, DLL3-expressing cells and DLL3 variants The geometric mean comparison value (delta geometric mean value for DLL3 variant molecules) determined by the above analysis, which reflects the amount of the test antigen-binding molecule or antibody bound to cells expressing the DLL3 variant, is compared with the delta geometric mean comparison value that reflects the amount of the test antigen-binding molecule or antibody bound to DLL3-expressing cells. In this case, DLL3-expressing cells and DLL3 variants The geometric mean comparison value (delta geometric mean value for DLL3 variant molecules), which reflects the amount of the test antigen-binding molecule or antibody bound to cells expressing the DLL3 variant, is compared with the delta geometric mean comparison value that reflects the amount of the test antigen-binding molecule or antibody bound to DLL3-expressing cells. In this case, DLL3-expressing cells and DLL3 variants The geometric mean comparison value (delta geometric mean value for DLL3 variant molecules), which reflects the amount of the test antigen-binding molecule or antibody bound to cells expressing the DLL3 variant, is compared with the delta geometric mean comparison value that reflects the amount of the test antigen-binding molecule or antibody bound to DLL3-expressing cells. In this case, DLL3-expressing cells and DLL3 variants The test antigen-binding molecule or antibody concentration used to determine the delta geometric mean comparison value for the expressing cells is adjusted, particularly preferably, 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. The offspring or antibody concentration is adjusted, particularly preferably, 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. 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 the DLL3 variant is at least 80%, preferably 50%, more preferably 30%, 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 the DLL3 variant (or DLL3 variant)". The formula for determining the geometric mean is described in the user guide of CELL QUEST Software (BD biosciences). When the comparison shows that the comparison values are substantially equivalent, it can be determined that the epitopes of the test and control antigen-binding molecules or antibodies are the same. If the delta geometric mean comparison value of the test antigen-binding molecule or antibody for cells expressing the DLL3 variant is at least 80%, preferably 50%, more preferably 30%, 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 the DLL3 variant (or DLL3 variant)". If the delta geometric mean comparison value of the test antigen-binding molecule or antibody for cells expressing the DLL3 variant is at least 80%, preferably 50%, more preferably 30%, 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 the DLL3 variant (or DLL3 variant)". If the delta geometric mean comparison value of the test antigen-binding molecule or antibody for cells expressing the DLL3 variant is at least 80%, preferably 50%, more preferably 30%, 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 the DLL3 variant (or DLL3 variant)". 3 variant)". The formula for determining the geometric mean is described in the user guide of CELL QUEST Software (BD biosciences). The formula for determining the geometric mean is described in the user guide of CELL QUEST Software (BD biosciences). When the comparison shows that the comparison values are substantially equivalent, it can be determined that the epitopes of the test and control antigen-binding molecules or antibodies are the same. When the comparison shows that the comparison values are substantially equivalent, it can be determined that the epitopes of the test and control antigen-binding molecules or antibodies are the same. When the comparison shows that the comparison values are substantially equivalent, it can be determined that the epitopes of the test and control antigen-binding molecules or antibodies are the same.

[0123] Antibodies that bind to the same epitope An antigen-binding molecule or antibody containing an antigen-binding domain that "binds to the same epitope" as the 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. An antigen-binding molecule or antibody containing an antigen-binding domain that "binds to the same epitope" as the 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. An antigen-binding molecule or antibody containing an antigen-binding domain that "binds to the same epitope" as the 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. An antigen-binding molecule or antibody containing an antigen-binding domain that "binds to the same epitope" as the 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. 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 show significant binding to molecules other than the partners. Further, "specific" is also used in the context where an antigen-binding domain is specific for a particular epitope among a plurality of epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in a plurality of different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope. "Specific" means that a molecule that specifically binds to one or more binding partners does not show significant binding to molecules other than the partners. Further, "specific" is also used in the context where an antigen-binding domain is specific for a particular epitope among a plurality of epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in a plurality of different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope. "Specific" means that a molecule that specifically binds to one or more binding partners does not show significant binding to molecules other than the partners. Further, "specific" is also used in the context where an antigen-binding domain is specific for a particular epitope among a plurality of epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in a plurality of different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope. "Specific" means that a molecule that specifically binds to one or more binding partners does not show significant binding to molecules other than the partners. Further, "specific" is also used in the context where an antigen-binding domain is specific for a particular epitope among a plurality of epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in a plurality of different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope. "Specific" means that a molecule that specifically binds to one or more binding partners does not show significant binding to molecules other than the partners. Further, "specific" is also used in the context where an antigen-binding domain is specific for a particular epitope among a plurality of epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in a plurality of different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope. "Specific" means that a molecule that specifically binds to one or more binding partners does not show significant binding to molecules other than the partners. Further, "specific" is also used in the context where an antigen-binding domain is specific for a particular epitope among a plurality of epitopes contained in an antigen. When the epitope to which the antigen-binding domain binds is contained in a plurality of different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope.

[0125] Antibody fragment "Antibody fragment" refers to a molecule other than an intact antibody that contains a part of the intact antibody that binds to an 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; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and single-specificity or multispecific antibodies formed from antibody fragments. "Antibody fragment" refers to a molecule other than an intact antibody that contains a part of the intact antibody that binds to an 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; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and single-specificity or multispecific antibodies formed from antibody fragments. "Antibody fragment" refers to a molecule other than an intact antibody that contains a part of the intact antibody that binds to an 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; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and single-specificity or multispecific antibodies formed from antibody fragments. "Antibody fragment" refers to a molecule other than an intact antibody that contains a part of the intact antibody that binds to an 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; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and single-specificity or multispecific antibodies formed from antibody fragments. "Antibody fragment" refers to a molecule other than an intact antibody that contains a part of the intact antibody that binds to an 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; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and single-specificity 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 an antibody having a structure substantially similar to the native antibody structure or having a heavy chain containing the Fc region as defined herein. The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to the native antibody structure or having a heavy chain containing the Fc region as defined herein. The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to the native antibody structure or having a heavy chain containing the Fc region as defined herein.

[0127] Variable fragment (Fv) As used herein, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain composed of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun placed an antibody As used herein, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain composed of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun placed an antibody As used herein, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain composed of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun placed an antibody By inserting a gene and inducing the expression of the gene in Escherichia coli, it has been found that a homogeneous and active antibody can be prepared from the periplasm fraction of Escherichia coli (Science ( 1988) 240 (4855), pp. 1038-1041). In the Fv prepared from the periplasm fraction, VH associates with VL in a manner that binds to the antigen. As used herein, the term "antibody variable fragment" refers to any fragment containing at least one light chain variable region (VL) and at least one heavy chain variable region (VH) of an antibody. As used herein, the terms "scFv", "single-chain antibody", and "sc(Fv)2" all refer to a single polypeptide chain antibody fragment that contains variable regions derived from the heavy and light chains but does not contain constant regions. Generally, a single-chain antibody also contains a polypeptide linker between the VH domain and the VL domain that allows for the formation of a desired structure that enables antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monocl onal Antibodies, Vol. 113, Rosenberg and Moor

[0128] scFv, single-chain antibody, and sc(Fv)2 (eds.), Springer-Verlag, New York, pp. 269-315 (1 994)". See also International Patent Application Publication No. WO1988 / 001649; U.S. Pat. Nos. 4,946,778 and 5, 260,203. In certain embodiments, the single-chain antibody may be bispecific and / or may be humanized. An scFv is one in which the VH and VL that form the Fv are linked together by a peptide linker and light chains but does not contain constant regions. Generally, a single-chain antibody also contains a polypeptide linker between the VH domain and the VL domain that allows for the formation of a desired structure that enables antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monocl onal Antibodies, Vol. 113, Rosenberg and Moor (eds.), Springer-Verlag, New York, pp. 269-315 (1 994)". See also International Patent Application Publication No. WO1988 / 001649; U.S. Pat. Nos. 4,946,778 and 5, 260,203. In certain embodiments, the single-chain antibody may be bispecific and / or may be humanized. An scFv is one in which the VH and VL that form the Fv are linked together by a peptide linker and light chains but does not contain constant regions. Generally, a single-chain antibody also contains a polypeptide linker between the VH domain and the VL domain that allows for the formation of a desired structure that enables antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monocl

[0129] and light chains but does not contain constant regions. Generally, a single-chain antibody also contains a polypeptide linker between the VH domain and the VL domain that allows for the formation of a desired structure that enables antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monocl which is the antigen-binding domain (Proc. Natl. Acad. Sci. U.S.A.( 1988) 85(16), pp. 5879-5883). VH and VL can be held very close to each other by a peptide linker -.

[0130] sc(Fv)2 is a single-chain antibody in which four variable regions of two VLs and two VHs are linked by a peptide linker of any linker to form a single chain (J Immunol ol. Methods (1999) 231(1-2), pp. 177-189). The two VHs and the two VLs may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably contains, for example, bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen as disclosed in 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 scFv with a linker such as a peptide linker . .

[0131] In this specification, the form of the antigen-binding domain forming sc(Fv)2 is such that two VH units and...

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, wherein the first antigen-binding domain of (1) is any one of the following (a1), (a2), and (a6) to (a10): (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; (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; A nucleic acid encoding a multispecific antigen-binding molecule, comprising any one of the above.

2. A nucleic acid encoding the multispecific antigen-binding molecule according to claim 1, The first antigen-binding domain of (1) is as follows: (c1), (c2), and (c10) 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; (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: 73; (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 more than 80% identity to any one of the heavy chain variable regions of (c1), (c2), and (c10) to (c19), and a light chain variable region having more than 80% identity to any one of the light chain variable regions of (c1), (c2), and (c10) to (c19); (c21) A heavy chain variable region having more than 90% identity to any one of the heavy chain variable regions of (c1), (c2), and (c10) to (c19), and a light chain variable region having more than 90% identity to any one of the light chain variable regions of (c1), (c2), and (c10) to (c19); A heavy chain variable region having more than 95% identity to any one of the heavy chain variable regions of (c22), (c1), (c2), and (c10)-(c19), and a light chain variable region having more than 95% identity to any one of the light chain variable regions of (c1), (c2), and (c10)-(c19) A nucleic acid encoding a multispecific antigen-binding molecule, comprising any one of the combinations of a heavy chain variable region and a light chain variable region selected from the above. **Claim 3** The nucleic acid according to claim 1 or 2, wherein the multispecific antigen-binding molecule has cytotoxic activity. **Claim 4** The nucleic acid according to claim 3, wherein the cytotoxic activity is T cell-dependent cytotoxic activity. **Claim 5** The nucleic acid according to any one of claims 1 to 4, wherein the second antigen-binding domain in (2) binds to the CD3 epsilon chain. **Claim 6** The nucleic acid according to any one of claims 1 to 4, wherein the second antigen-binding domain in (2) binds to the T cell receptor. **Claim 7** The multispecific antigen-binding molecule further comprises a third domain comprising an Fc region having an amino acid mutation at any one of the amino acids constituting the Fc regions of SEQ ID NOs: 112-115 (IgG1-IgG4), and the Fc region having the amino acid mutation is an Fc region with reduced binding activity to the Fc gamma receptor compared to the Fc region of its isotype. The nucleic acid according to any one of claims 1 to 6. **Claim 8** The Fc region has the following amino acid positions specified by EU numbering: The nucleic acid according to claim 7, which is an Fc region having a mutation in at least one amino acid selected from 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, and 332. **Claim 9** A vector comprising the nucleic acid according to any one of claims 1 to 8. **Claim 10** A cell comprising the nucleic acid according to any one of claims 1 to 8 or the vector according to claim 9. **Claim 11** A method for producing a multispecific antigen-binding molecule by culturing the cell according to claim 10.

Images