DLL3 targeting multispecific antigen-binding molecules and uses thereof

Abstract

The present invention provides multispecific antigen-binding molecules capable of treating cancers by recruiting T cells to the vicinity of DLL3 expressing cells and using the cytotoxicity of T cells against DLL3 expressing cancer cells, and methods for producing the multispecific antigen-binding molecules.SOLUTION: Provided are multispecific antigen-binding molecules comprising a first antigen-binding moiety and a second antigen-binding moiety, each capable of binding to CD3 and CD137, but not to CD3 and CD137 at the same time, and a third antigen-binding moiety capable of binding to DLL3, preferably human DLL3, and methods of production thereof. The multispecific antigen-binding molecules can more efficiently induce T cell-dependent cytotoxic activity and treat cancers associated with DLL3, while avoiding adverse toxicological concerns or side effects that may be associated with other multispecific antigen-binding molecules.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to multispecific antigen-binding molecules comprising a first antigen-binding moiety and a second antigen-binding moiety, each capable of binding to CD3 and CD137 but not simultaneously, and a third antigen-binding moiety capable of binding to DLL3. The present invention also relates to nucleic acids encoding such antigen-binding molecules, methods for preparing such antigen-binding molecules, host cells that express or are capable of expressing such antigen-binding molecules, compositions comprising such antigen-binding molecules, and uses of such antigen-binding molecules or such compositions for therapeutic purposes, particularly in the field of cancer diseases. [Background technology]

[0002] Cancer is one of the leading causes of death worldwide. With the exception of certain carcinomas, tumors are often inoperable when they are discovered. Traditional cancer treatments include radiation therapy, chemotherapy, and immunotherapy. These treatments are often ineffective, and ultimately, cancer recurrence or metastasis occurs after treatment. One of the factors limiting maximum efficacy is the lack of tumor specificity; therefore, molecular targeted therapies with higher tumor specificity have become an increasingly viable option in cancer treatment.

[0003] Antibodies have attracted attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Among the various therapeutic antibodies, some types of antibodies require effector cells to exert antitumor responses. Antibody-dependent cellular cytotoxicity (ADCC) is a cytotoxicity exhibited by effector cells against antibody-bound cells via binding of the antibody Fc region to Fc receptors present on NK cells and macrophages. To date, several therapeutic antibodies capable of inducing ADCC and exerting antitumor effects have been developed as pharmaceuticals for cancer treatment (Non-Patent Document 1). Therapies using conventional therapeutic antibodies targeting tumor-specific antigens exhibit excellent antitumor activity, but administration of such antibodies does not always produce satisfactory results.

[0004] In addition to antibodies that induce ADCC by recruiting NK cells or macrophages as effector cells, T cell-recruiting antibodies (TR antibodies), which incorporate cytotoxicity by recruiting T cells as effector cells, have been known since the 1980s (Non-Patent Documents 2-4). TR antibodies are bispecific antibodies that recognize and bind to one of the subunits forming the T cell receptor complex on T cells, particularly the CD3ε chain, and an antigen on cancer cells. Several TR antibodies are currently under development. Catumaxomab is a TR antibody against EpCAM and has been approved in the EU for the treatment of malignant ascites. Furthermore, a type of TR antibody called a "bispecific T cell-recruiter (BiTE)" has recently been found to exhibit potent antitumor activity (Non-Patent Documents 5 and 6). Blinatumomab is a BiTE molecule against CD19 and was the first to receive FDA approval in 2014. Blinatumomab has been shown to exhibit much stronger cytotoxic activity against CD19 / CD20-positive cancer cells in vitro than rituximab, which induces antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Non-patent Document 7).

[0005] However, it is known that trifunctional antibodies simultaneously bind to both T cells and cells such as NK cells or macrophages in a cancer antigen-independent manner, resulting in cross-linking of receptors expressed on these cells and induction of various cytokines in a cancer antigen-independent manner. Systemic administration of trifunctional antibodies is thought to cause cytokine storm-like side effects as a result of the induction of cytokine expression. In fact, a phase I clinical trial reported that the maximum tolerated dose of systemically administered catumaxomab in patients with non-small cell lung cancer was an ultra-low dose of 5 μg / body, and that higher doses caused various severe side effects (Non-Patent Document 8). When administered at such low doses, catumaxomab cannot reach effective blood levels. In other words, administering catumaxomab at such low doses does not achieve the expected antitumor effect.

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

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

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

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

[0010] An object of the present invention is to provide multispecific antigen-binding molecules that can treat cancer by recruiting T cells to the vicinity of DLL3-expressing cells and utilizing the cytotoxicity of T cells against DLL3-expressing cancer cells; a method for producing the multispecific antigen-binding molecules; and a therapeutic agent containing such a multispecific antigen-binding molecule as an active ingredient for inducing cytotoxic activity. Another object of the present invention is to provide pharmaceutical compositions for use in the treatment or prevention of various cancers, which contain one of the above-mentioned antigen-binding molecules as an active ingredient, and therapeutic methods using the pharmaceutical compositions. [Means for solving the problem]

[0011] The present invention relates to a multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, each capable of binding to CD3 and CD137 but not simultaneously (i.e., capable of binding to CD3 and CD137 but not simultaneously); and a third antigen-binding moiety capable of binding to DLL3, preferably human DLL3, which more efficiently induces T cell-dependent cytotoxicity while avoiding the potentially harmful toxicity or side effects of other multispecific antigen-binding molecules. The present invention provides multispecific antigen-binding molecules and pharmaceutical compositions that contain the antigen-binding molecules as active ingredients and can treat various cancers, particularly cancers associated with DLL3, such as DLL3-positive tumors.

[0012] In one aspect, the multispecific antigen-binding molecules of the present invention have a highly unique structural format that improves or enhances the efficacy of the multispecific antigen-binding molecules. The novel antigen-binding molecules with unique structural formats provide an increased number of antigen-binding domains to generate increased valency and / or specificity for each antigen on effector cells and target cells, while reducing undesirable adverse effects.

[0013] In one particular aspect, the present invention relates to a multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, each capable of binding to CD3 and CD137 but not simultaneously (i.e., capable of binding to CD3 and CD137 but not simultaneously); and a third antigen-binding moiety capable of binding to DLL3, preferably human DLL3, which multispecific antigen-binding molecule efficiently induces T-cell dependent cytotoxicity while avoiding potentially harmful toxicity concerns or side effects of other multispecific antigen-binding molecules. In one such aspect, each of the first and second antigen-binding moieties comprises at least one amino acid mutation, e.g., a cysteine ​​insertion / substitution / mutation, which creates a disulfide bond between the first and second antigen-binding moieties, keeping them close to each other and promoting cis antigen binding to an antigen (CD3 and / or CD137) on the same single effector cell, e.g., as a result of steric hindrance or shorter distance between the two Dual-Fabs, thereby improving the safety profile of the trispecific antibody by preventing undesired cross-linking of two CD3 / CD137-expressing immune cells mediated by the two Dual-Fabs in a DLL3-independent manner. In one specific aspect, each of the first and second antigen-binding moieties is a Fab and comprises at least one cysteine ​​residue (via mutation, substitution, or insertion) in the CH1 region, which is capable of forming at least one disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety. In another specific aspect, each of the first and second antigen-binding moieties comprises a cysteine ​​residue (via mutation, substitution, or insertion) at position 191 (EU numbering) in the CH1 region, which is capable of forming a disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety.

[0014] Antigen-binding molecules having such unique structural formats have surprisingly been found to exhibit superior efficacy compared to other multispecific antibody formats (e.g., BiTEs), while reducing or minimizing off-target side effects due to undesired cross-linking between various cells (e.g., effector cells such as T cells).

[0015] More specifically, the present disclosure provides: [1] a first antigen-binding moiety and a second antigen-binding moiety, each capable of binding to CD3 and CD137, but not simultaneously binding to CD3 and CD137; and a third antigen-binding moiety capable of binding to a third antigen, preferably an antigen expressed on cancer cells / tissues; A multispecific antigen-binding molecule comprising: [1A] a first antigen-binding moiety and a second antigen-binding moiety, each capable of binding to CD3 and CD137, but not simultaneously binding to CD3 and CD137; and a third antigen-binding moiety capable of binding to DLL3, preferably human DLL3; A multispecific antigen-binding molecule comprising: [2] The first antigen-binding portion and the second antigen-binding portion are each any one of the following (a1) to (a17): (a1) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 17, heavy chain CDR 2 of SEQ ID NO: 31, heavy chain CDR 3 of SEQ ID NO: 45, light chain CDR 1 of SEQ ID NO: 64, light chain CDR 2 of SEQ ID NO: 69, and light chain CDR 3 of SEQ ID NO: 74; (a2) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 18, heavy chain CDR 2 of SEQ ID NO: 32, heavy chain CDR 3 of SEQ ID NO: 46, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a3) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 19, heavy chain CDR 2 of SEQ ID NO: 33, heavy chain CDR 3 of SEQ ID NO: 47, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a4) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 19, heavy chain CDR 2 of SEQ ID NO: 33, heavy chain CDR 3 of SEQ ID NO: 47, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75; (a5) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 20, heavy chain CDR 2 of SEQ ID NO: 34, heavy chain CDR 3 of SEQ ID NO: 48, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a6) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 22, heavy chain CDR 2 of SEQ ID NO: 36, heavy chain CDR 3 of SEQ ID NO: 50, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a7) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a8) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a9) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 24, heavy chain CDR 2 of SEQ ID NO: 38, heavy chain CDR 3 of SEQ ID NO: 52, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a10) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 25, heavy chain CDR 2 of SEQ ID NO: 39, heavy chain CDR 3 of SEQ ID NO: 53, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a11) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 26, heavy chain CDR 2 of SEQ ID NO: 40, heavy chain CDR 3 of SEQ ID NO: 54, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a12) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 26, heavy chain CDR 2 of SEQ ID NO: 40, heavy chain CDR 3 of SEQ ID NO: 54, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a13) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 27, heavy chain CDR 2 of SEQ ID NO: 41, heavy chain CDR 3 of SEQ ID NO: 55, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a14) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 28, heavy chain CDR 2 of SEQ ID NO: 42, heavy chain CDR 3 of SEQ ID NO: 56, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a15) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 82, heavy chain CDR 2 of SEQ ID NO: 83, heavy chain CDR 3 of SEQ ID NO: 84, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75; (a16) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a17) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15). The multispecific antigen-binding molecule of any one of [1] to [1A], comprising an antibody variable region comprising: [3] The first antigen-binding portion and the second antigen-binding portion are each any one of the following (a1) to (a17): (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60; (a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a8) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a9) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a10) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a11) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a12) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a13) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a14) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a15) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60; (a16) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a17) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15). The multispecific antigen-binding molecule of any one of [1] to [2], comprising an antibody variable region comprising: [4] The multispecific antigen-binding molecule of any one of [1] to [3], wherein each of the first antigen-binding portion and the second antigen-binding portion is a Fab molecule and comprises at least one disulfide bond formed between the CH1 region of the first antigen-binding portion and the CH1 region of the second antigen-binding portion. [4A] The multispecific antigen-binding molecule of [4], wherein each of the first antigen-binding portion and the second antigen-binding portion is a Fab molecule and comprises one disulfide bond formed between the amino acid residue at position 191 (EU numbering) in the CH1 region of each of the first antigen-binding portion and the second antigen-binding portion. [5] The multispecific antigen-binding molecule of any one of [1] to [4A], wherein the third antigen-binding portion is fused to either the first antigen-binding portion or the second antigen-binding portion. [5A] The multispecific antigen-binding molecule of [5], wherein the third antigen-binding portion is a Fab or scFv. [6] The multispecific antigen-binding molecule of any one of [5] to [5A], wherein each of the first, second, and third antigen-binding moieties is a Fab molecule, and the third antigen-binding moiety is fused at the C-terminus (CH1) of the Fab heavy chain to the N-terminus of the Fab heavy chain of either the first antigen-binding moiety or the second antigen-binding moiety, optionally via a peptide linker. [6A] The multispecific antigen-binding molecule of any one of [5] to [6], wherein the peptide linker is selected from the group consisting of the amino acid sequences of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259. [6B] The multispecific antigen-binding molecule of any one of [1] to [6A], wherein the first antigen-binding portion is identical to the second antigen-binding portion. [7] The multispecific antigen-binding molecule of any one of [1] to [6B], wherein the third antigen-binding portion is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and each of the first and second antigen-binding portions is a conventional Fab molecule. [8] The multispecific antigen-binding molecule of [7], wherein in the constant domain CL of the light chain of each of the first and second antigen-binding moieties, the amino acids at positions 123 and / or 124 are independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the heavy chain of each of the first and second antigen-binding moieties, the amino acids at positions 147 and / or 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU numbering). [9] A multispecific antigen-binding molecule according to [8], wherein the amino acids at positions 123 and 124 in the constant domain CL of the light chain of each of the first and second antigen-binding moieties are arginine (R) and lysine (K), respectively (Kabat numbering), and the amino acids at positions 147 and 213 in the constant domain CH1 of the heavy chain of each of the first and second antigen-binding moieties are glutamic acid (E) (Kabat EU index numbering).

[10] The third antigen-binding moiety capable of binding to DLL3 is any one of the following (a1) to (a5): (a1) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 233, heavy chain CDR 2 of SEQ ID NO: 234, heavy chain CDR 3 of SEQ ID NO: 235, light chain CDR 1 of SEQ ID NO: 237, light chain CDR 2 of SEQ ID NO: 238, and light chain CDR 3 of SEQ ID NO: 239; (a2) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 276, heavy chain CDR 2 of SEQ ID NO: 277, heavy chain CDR 3 of SEQ ID NO: 278, light chain CDR 1 of SEQ ID NO: 279, light chain CDR 2 of SEQ ID NO: 280, and light chain CDR 3 of SEQ ID NO: 281; (a3) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 285, heavy chain CDR 2 of SEQ ID NO: 286, heavy chain CDR 3 of SEQ ID NO: 287, light chain CDR 1 of SEQ ID NO: 288, light chain CDR 2 of SEQ ID NO: 289, and light chain CDR 3 of SEQ ID NO: 290; (a4) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a3); and (a5) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a3). The multispecific antigen-binding molecule of any one of [1] to [9], comprising an antibody variable region comprising:

[11] The third antigen-binding moiety capable of binding to DLL3 is any one of the following (a1) to (a6): (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 232, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 236; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 264, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 265; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 266, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 267; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 268, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 269; (a5) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a4); and (a6) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a4). The multispecific antigen-binding molecule of any one of [1] to

[10] , comprising an antibody variable region comprising:

[12] Any one of the multispecific antigen-binding molecules of [1] to

[11] , further comprising an Fc domain. [12A] A multispecific antigen-binding molecule according to

[12] , wherein the Fc domain is composed of a first and a second Fc region subunit capable of stable association, and the Fc domain exhibits reduced binding affinity to human Fcγ receptors compared to a native human IgG1 Fc domain. [12B] The first Fc region subunit comprises: (a1) an Fc region polypeptide comprising Ala at position 234 and Ala at position 235; (a2) an Fc region polypeptide comprising Ala at position 234, Ala at position 235, and Ala at position 297; (a3) an Fc region polypeptide comprising Ala at position 234, Ala at position 235, Ala at position 297, Cys at position 354, and Trp at position 366; and The second Fc region polypeptide comprises: (a4) an Fc region polypeptide comprising Ala at position 234 and Ala at position 235; (a5) an Fc region polypeptide comprising Ala at position 234, Ala at position 235, and Ala at position 297; (a6) an Fc region polypeptide comprising Ala at position 234, Ala at position 235, Ala at position 297, Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407; selected from the group consisting of The multispecific antigen-binding molecule of [12A], wherein amino acid positions are numbered using EU index numbering. [12C] Any one of the multispecific antigen-binding molecules of

[12] to [12B], wherein the Fc domain exhibits enhanced FcRn-binding activity under acidic pH conditions (e.g., pH 5.8) compared to that of the Fc region of native IgG. [12D] The multispecific antigen-binding molecule of [12C], wherein the Fc domain comprises, according to EU numbering, Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440. [12E] The multispecific antigen-binding molecule of [12D], wherein the Fc domain comprises, according to EU numbering, Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440. [12F] The multispecific antigen-binding molecule of [12E], wherein the Fc domain further comprises Ile or Leu at position 428; and / or Ile, Leu, Val, Thr, or Phe at position 436, according to EU numbering. [12G] The Fc domain is: (a)N434A / Q438R / S440E; (b)N434A / Q438R / S440D; (c)N434A / Q438K / S440E; (d)N434A / Q438K / S440D; (e)N434A / Y436T / Q438R / S440E; (f)N434A / Y436T / Q438R / S440D; (g)N434A / Y436T / Q438K / S440E; (h)N434A / Y436T / Q438K / S440D; (i)N434A / Y436V / Q438R / S440E; (j)N434A / Y436V / Q438R / S440D; (k)N434A / Y436V / Q438K / S440E; (l)N434A / Y436V / Q438K / S440D; (m)N434A / R435H / F436T / Q438R / S440E; (n)N434A / R435H / F436T / Q438R / S440D; (o)N434A / R435H / F436T / Q438K / S440E; (p)N434A / R435H / F436T / Q438K / S440D; (q)N434A / R435H / F436V / Q438R / S440E; (r)N434A / R435H / F436V / Q438R / S440D; (s)N434A / R435H / F436V / Q438K / S440E; (t)N434A / R435H / F436V / Q438K / S440D; (u)M428L / N434A / Q438R / S440E; (v)M428L / N434A / Q438R / S440D; (w)M428L / N434A / Q438K / S440E; (x)M428L / N434A / Q438K / S440D; (y)M428L / N434A / Y436T / Q438R / S440E; (z)M428L / N434A / Y436T / Q438R / S440D; (aa)M428L / N434A / Y436T / Q438K / S440E; (ab)M428L / N434A / Y436T / Q438K / S440D; (ac)M428L / N434A / Y436V / Q438R / S440E; (ad)M428L / N434A / Y436V / Q438R / S440D; (ae)M428L / N434A / Y436V / Q438K / S440E; (af)M428L / N434A / Y436V / Q438K / S440D; (ag)L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (ah)L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E Any one of the multispecific antigen-binding molecules of [12C] to [12F], comprising a combination of amino acid substitutions selected from the group consisting of: [12H] Any one of [12C] to [12G], wherein the Fc domain contains a combination of amino acid substitutions of M428L / N434A / Q438R / S440E. [12I] The multispecific antigen-binding molecule of any one of

[12] to [12H], wherein the Fc domain is an IgG Fc domain, preferably a human IgG Fc domain, more preferably a human IgG1 Fc domain. [12J] The Fc domain is: (a) a first Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 100 and a second Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 111; and (b) a first Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 99 and a second Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 109

[0023] Any one of

[12] to [12I], comprising any one of: [12K] Any one of the multispecific antigen-binding molecules of

[12] to [12J], wherein each of the first and second antigen-binding moieties is a Fab, the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain. [12L] The multispecific antigen-binding molecule of [12K], wherein the third antigen-binding portion is fused at the C-terminus to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion, optionally via a peptide linker.

[13] (a1) to (a15) below: (a1) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 208, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a2) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a3) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a4) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 205, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a5) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 216, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 229, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a6) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 217, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 210, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a7) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 219, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a8) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 220, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a9) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 221, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a10) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 222, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 230, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a11) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 223, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 212, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; (a12) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 225, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; (a13) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 226, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; (a14) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 227, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215. and Preferably, the five polypeptide chains (chain 1 to chain 5) are connected and / or associated with each other according to the orientation shown in Figure 1(a), A multispecific antigen-binding molecule according to any one of [1] to [12L].

[14] An isolated polynucleotide or multiple polynucleotides encoding any one of the multispecific antigen-binding molecules of [1] to

[13] .

[15] A vector encoding the polynucleotide or polynucleotides of

[14] .

[16] A host cell containing the polynucleotide or polynucleotides of

[14] or the vector of

[15] .

[17] A method for producing any one of the multispecific antigen-binding molecules of [1] to

[13] , comprising the steps of: a) culturing the host cell of

[16] under conditions suitable for expression of the antigen-binding molecule; and b) recovering the antigen-binding molecule. [17A] A multispecific antigen-binding molecule produced by the method of

[17] .

[18] A pharmaceutical composition comprising any one of the multispecific antigen-binding molecules of [1] to

[13] and a pharmaceutically acceptable carrier.

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

[13] or a pharmaceutical composition according to

[18] , which induces cytotoxic activity, preferably T cell-dependent cytotoxic activity.

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

[13] or a pharmaceutical composition according to

[18] for use as a pharmaceutical.

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

[13] or a pharmaceutical composition according to

[18] for use in treating or preventing a disease in a subject in need thereof.

[22] The multispecific antigen-binding molecule or pharmaceutical composition for use in the treatment / prevention of the disease of

[21] , wherein the disease is cancer. [22A] A multispecific antigen-binding molecule or pharmaceutical composition for use in the treatment / prevention of the disease of

[22] , wherein the cancer is a DLL3-expressing cancer or a DLL3-positive cancer. [22B] A multispecific antigen-binding molecule or pharmaceutical composition for use in the treatment / prevention of the disease of

[22] or [22A], wherein the cancer is lung cancer (including small cell lung cancer) and melanoma.

[23] Use of any one of the multispecific antigen-binding molecules of [1] to

[13] or the pharmaceutical composition of

[18] for the manufacture of a medicament for treating a disease in an individual in need thereof.

[24] A method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of the multispecific antigen-binding molecule of any one of [1] to

[13] or the pharmaceutical composition of

[18] .

[25] The use of

[23] or the method of

[24] , wherein the disease is cancer, preferably DLL3-positive cancer or DLL3-expressing cancer. [25A] The use or method of

[25] , wherein the cancer is a DLL3-expressing cancer or a DLL3-positive cancer. [25B] The use or method of [25A], wherein the cancer is lung cancer (including small cell lung cancer) or melanoma.

[26] A method for inducing lysis of target cells, comprising contacting the target cells with any one of the multispecific antigen-binding molecules of [1] to

[13] or the pharmaceutical composition of

[18] in the presence of T cells.

[27] A kit comprising the pharmaceutical composition of

[18] and a package insert containing instructions for administering the composition to a subject to treat or delay the progression of cancer, preferably a DLL3-positive cancer or a DLL3-expressing cancer. [27A] The kit of

[27] , in which the cancer is lung cancer (including small cell lung cancer) and melanoma.

[0016] Another aspect of the present invention relates to:

[28] The following: an antigen-binding portion that is capable of binding to CD3 and CD137, but does not simultaneously bind to CD3 and CD137; and Antigen-binding moieties capable of binding to DLL3, preferably human DLL3 A multispecific antigen-binding molecule comprising:

[29] The antigen-binding portion that can bind to CD3 and CD137 but does not simultaneously bind to CD3 and CD137 is any one of the following (a1) to (a17): (a1) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 17, heavy chain CDR 2 of SEQ ID NO: 31, heavy chain CDR 3 of SEQ ID NO: 45, light chain CDR 1 of SEQ ID NO: 64, light chain CDR 2 of SEQ ID NO: 69, and light chain CDR 3 of SEQ ID NO: 74; (a2) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 18, heavy chain CDR 2 of SEQ ID NO: 32, heavy chain CDR 3 of SEQ ID NO: 46, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a3) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 19, heavy chain CDR 2 of SEQ ID NO: 33, heavy chain CDR 3 of SEQ ID NO: 47, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a4) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 19, heavy chain CDR 2 of SEQ ID NO: 33, heavy chain CDR 3 of SEQ ID NO: 47, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75; (a5) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 20, heavy chain CDR 2 of SEQ ID NO: 34, heavy chain CDR 3 of SEQ ID NO: 48, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a6) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 22, heavy chain CDR 2 of SEQ ID NO: 36, heavy chain CDR 3 of SEQ ID NO: 50, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a7) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a8) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a9) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 24, heavy chain CDR 2 of SEQ ID NO: 38, heavy chain CDR 3 of SEQ ID NO: 52, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a10) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 25, heavy chain CDR 2 of SEQ ID NO: 39, heavy chain CDR 3 of SEQ ID NO: 53, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a11) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 26, heavy chain CDR 2 of SEQ ID NO: 40, heavy chain CDR 3 of SEQ ID NO: 54, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a12) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 26, heavy chain CDR 2 of SEQ ID NO: 40, heavy chain CDR 3 of SEQ ID NO: 54, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a13) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 27, heavy chain CDR 2 of SEQ ID NO: 41, heavy chain CDR 3 of SEQ ID NO: 55, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a14) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 28, heavy chain CDR 2 of SEQ ID NO: 42, heavy chain CDR 3 of SEQ ID NO: 56, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a15) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 82, heavy chain CDR 2 of SEQ ID NO: 83, heavy chain CDR 3 of SEQ ID NO: 84, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75; (a16) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a17) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15).

[28] A multispecific antigen-binding molecule comprising an antibody variable region comprising:

[30] The antigen-binding portion that can bind to CD3 and CD137 but does not simultaneously bind to CD3 and CD137 is any one of the following (a1) to (a17): (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60; (a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a8) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a9) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a10) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a11) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a12) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a13) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a14) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a15) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60; (a16) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a17) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15).

[28] -

[29] . The multispecific antigen-binding molecule of any one of

[28] to

[29] , comprising an antibody variable region comprising:

[0017] In yet another aspect of the present invention, there is provided a method for treating a vascular endothelial cell, comprising:

[31] (a1) to (a15) below: (a1) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 208, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a2) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a3) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a4) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 205, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a5) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 216, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 229, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a6) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 217, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 210, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a7) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 219, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a8) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 220, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a9) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 221, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a10) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 222, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 230, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214; (a11) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 223, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 212, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; (a12) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 225, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; (a13) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 226, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; (a14) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 227, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215. wherein the five polypeptide chains (chain 1 to chain 5) are linked and / or associated with each other according to the orientation shown in Figure 1(a).

[0018] Yet another aspect of the present invention relates to:

[32] One of the following (a1) to (a5): (a1) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 233, heavy chain CDR 2 of SEQ ID NO: 234, heavy chain CDR 3 of SEQ ID NO: 235, light chain CDR 1 of SEQ ID NO: 237, light chain CDR 2 of SEQ ID NO: 238, and light chain CDR 3 of SEQ ID NO: 239; (a2) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 276, heavy chain CDR 2 of SEQ ID NO: 277, heavy chain CDR 3 of SEQ ID NO: 278, light chain CDR 1 of SEQ ID NO: 279, light chain CDR 2 of SEQ ID NO: 280, and light chain CDR 3 of SEQ ID NO: 281; (a3) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 285, heavy chain CDR 2 of SEQ ID NO: 286, heavy chain CDR 3 of SEQ ID NO: 287, light chain CDR 1 of SEQ ID NO: 288, light chain CDR 2 of SEQ ID NO: 289, and light chain CDR 3 of SEQ ID NO: 290; (a4) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a3); and (a5) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a3). An antigen-binding molecule capable of binding to DLL3, comprising an antibody variable region comprising:

[33] One of the following (a1) to (a6): (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 232, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 236; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 264, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 265; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 266, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 267; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 268, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 269; (a5) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a4); and (a6) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a4). An antigen-binding molecule capable of binding to DLL3, comprising an antibody variable region comprising: Yet another aspect of the present invention relates to: [2-1] (a) A first antigen-binding moiety and a second antigen-binding moiety, each of which binds to human CD3 and comprises antibody variable regions which may be the same or different, wherein the antibody variable regions are independently selected from the group consisting of: (a1) an antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 17, a heavy chain CDR 2 of SEQ ID NO: 31, a heavy chain CDR 3 of SEQ ID NO: 45, a light chain CDR 1 of SEQ ID NO: 64, a light chain CDR 2 of SEQ ID NO: 69, and a light chain CDR 3 of SEQ ID NO: 74; (a2) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 18, a heavy chain CDR 2 of SEQ ID NO: 32, a heavy chain CDR 3 of SEQ ID NO: 46, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a3) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, a heavy chain CDR 2 of SEQ ID NO: 33, a heavy chain CDR 3 of SEQ ID NO: 47, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a4) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, a heavy chain CDR 2 of SEQ ID NO: 33, a heavy chain CDR 3 of SEQ ID NO: 47, a light chain CDR 1 of SEQ ID NO: 65, a light chain CDR 2 of SEQ ID NO: 70, and a light chain CDR 3 of SEQ ID NO: 75; (a5) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, a heavy chain CDR 2 of SEQ ID NO: 34, a heavy chain CDR 3 of SEQ ID NO: 48, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a6) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 22, a heavy chain CDR 2 of SEQ ID NO: 36, a heavy chain CDR 3 of SEQ ID NO: 50, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a7) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, a heavy chain CDR 2 of SEQ ID NO: 37, a heavy chain CDR 3 of SEQ ID NO: 51, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a8) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, a heavy chain CDR 2 of SEQ ID NO: 37, a heavy chain CDR 3 of SEQ ID NO: 51, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76; (a9) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 24, a heavy chain CDR 2 of SEQ ID NO: 38, a heavy chain CDR 3 of SEQ ID NO: 52, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a10) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 25, a heavy chain CDR 2 of SEQ ID NO: 39, a heavy chain CDR 3 of SEQ ID NO: 53, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76; (a11) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, a heavy chain CDR 2 of SEQ ID NO: 40, a heavy chain CDR 3 of SEQ ID NO: 54, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76; (a12) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, a heavy chain CDR 2 of SEQ ID NO: 40, a heavy chain CDR 3 of SEQ ID NO: 54, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a13) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 27, a heavy chain CDR 2 of SEQ ID NO: 41, a heavy chain CDR 3 of SEQ ID NO: 55, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a14) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, a heavy chain CDR 2 of SEQ ID NO: 42, a heavy chain CDR 3 of SEQ ID NO: 56, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; and (a15) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 82, a heavy chain CDR 2 of SEQ ID NO: 83, a heavy chain CDR 3 of SEQ ID NO: 84, a light chain CDR 1 of SEQ ID NO: 65, a light chain CDR 2 of SEQ ID NO: 70, and a light chain CDR 3 of SEQ ID NO: 75; and (b) a third antigen-binding portion that binds to human delta-like 3 (DLL3) and comprises an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 233, a heavy chain CDR2 comprising SEQ ID NO: 234, a heavy chain CDR3 comprising SEQ ID NO: 235, a light chain CDR1 comprising SEQ ID NO: 237, a light chain CDR2 comprising SEQ ID NO: 238, and a light chain CDR3 comprising SEQ ID NO: 239. A multispecific antigen-binding molecule comprising: [2-2] Each of the first and second antigen-binding moieties comprises an antibody variable region, which may be the same or different, and the antibody variable region is selected from the group consisting of: (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60; (a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a8) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a9) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a10) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a11) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a12) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a13) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a14) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; and (a15) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60 The multispecific antigen-binding molecule of [2-1], further independently selected from the above. [2-2A] The first antigen-binding portion and the second antigen-binding portion each comprise: an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO:20, heavy chain CDR 2 of SEQ ID NO:34, heavy chain CDR 3 of SEQ ID NO:48, light chain CDR 1 of SEQ ID NO:63, light chain CDR 2 of SEQ ID NO:68, and light chain CDR 3 of SEQ ID NO:73 The multispecific antigen-binding molecule of any one of [2-1] to [2-2], comprising: [2-2B] The first antigen-binding portion and the second antigen-binding portion each comprise: An antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58. The multispecific antigen-binding molecule of [2-2A], comprising: [2-2C] a third antigen-binding moiety, An antibody variable region comprising a VH comprising SEQ ID NO: 232 and a VL comprising SEQ ID NO: 236. The multispecific antigen-binding molecule of any one of [2-1] to [2-2B], comprising: [2-3] The multispecific antigen-binding molecule of any one of [2-1] to [2-2C], wherein each of the first and second antigen-binding moieties is a Fab having a cysteine ​​residue at position 191 (EU numbering), and wherein a disulfide bond is present between the two cysteine ​​residues. [2-4] The multispecific antigen-binding molecule of [2-3], wherein each of the first, second, and third antigen-binding moieties is a Fab comprising a heavy chain comprising a VH and CH1 domain and a light chain comprising a VL and a light-chain constant (CL) domain, and the C-terminus of the heavy chain of the third antigen-binding moiety is fused, directly or via a peptide linker, to the N-terminus of the Fab heavy chain of either the first or second antigen-binding moiety. [2-5] The multispecific antigen-binding molecule of [2-4], wherein the C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion via a peptide linker, and the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259. [2-6] The multispecific antigen-binding molecule of [2-5], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding moieties are arginine and lysine, respectively, and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding moieties are glutamic acid. [2-7] The multispecific antigen-binding molecule of [2-6], further comprising an Fc domain. [2-8] the Fc domain comprises a first and a second Fc region subunit; The first Fc region subunit comprises: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 354, and a tryptophan at position 366; and the second Fc region subunit comprising: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 349, a serine at position 366, an alanine at position 368, and a valine at position 407; and selected from the group comprising: All positions are according to EU numbering, [2-7] A multispecific antigen-binding molecule. Yet another aspect of the present invention relates to: [3-1] (a) A first antigen-binding moiety and a second antigen-binding moiety, each of which binds to human CD137 and which comprise antibody variable regions that may be the same or different, wherein the antibody variable regions are independently selected from the group consisting of: (a1) an antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 17, a heavy chain CDR 2 of SEQ ID NO: 31, a heavy chain CDR 3 of SEQ ID NO: 45, a light chain CDR 1 of SEQ ID NO: 64, a light chain CDR 2 of SEQ ID NO: 69, and a light chain CDR 3 of SEQ ID NO: 74; (a2) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 18, a heavy chain CDR 2 of SEQ ID NO: 32, a heavy chain CDR 3 of SEQ ID NO: 46, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a3) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, a heavy chain CDR 2 of SEQ ID NO: 33, a heavy chain CDR 3 of SEQ ID NO: 47, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a4) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, a heavy chain CDR 2 of SEQ ID NO: 33, a heavy chain CDR 3 of SEQ ID NO: 47, a light chain CDR 1 of SEQ ID NO: 65, a light chain CDR 2 of SEQ ID NO: 70, and a light chain CDR 3 of SEQ ID NO: 75; (a5) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, a heavy chain CDR 2 of SEQ ID NO: 34, a heavy chain CDR 3 of SEQ ID NO: 48, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a6) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 22, a heavy chain CDR 2 of SEQ ID NO: 36, a heavy chain CDR 3 of SEQ ID NO: 50, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a7) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, a heavy chain CDR 2 of SEQ ID NO: 37, a heavy chain CDR 3 of SEQ ID NO: 51, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a8) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, a heavy chain CDR 2 of SEQ ID NO: 37, a heavy chain CDR 3 of SEQ ID NO: 51, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76; (a9) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 24, a heavy chain CDR 2 of SEQ ID NO: 38, a heavy chain CDR 3 of SEQ ID NO: 52, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a10) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 25, a heavy chain CDR 2 of SEQ ID NO: 39, a heavy chain CDR 3 of SEQ ID NO: 53, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76; (a11) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, a heavy chain CDR 2 of SEQ ID NO: 40, a heavy chain CDR 3 of SEQ ID NO: 54, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76; (a12) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, a heavy chain CDR 2 of SEQ ID NO: 40, a heavy chain CDR 3 of SEQ ID NO: 54, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a13) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 27, a heavy chain CDR 2 of SEQ ID NO: 41, a heavy chain CDR 3 of SEQ ID NO: 55, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; (a14) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, a heavy chain CDR 2 of SEQ ID NO: 42, a heavy chain CDR 3 of SEQ ID NO: 56, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; and (a15) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 82, a heavy chain CDR 2 of SEQ ID NO: 83, a heavy chain CDR 3 of SEQ ID NO: 84, a light chain CDR 1 of SEQ ID NO: 65, a light chain CDR 2 of SEQ ID NO: 70, and a light chain CDR 3 of SEQ ID NO: 75; and (b) a third antigen-binding portion that binds to human delta-like 3 (DLL3) and comprises an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 233, a heavy chain CDR2 comprising SEQ ID NO: 234, a heavy chain CDR3 comprising SEQ ID NO: 235, a light chain CDR1 comprising SEQ ID NO: 237, a light chain CDR2 comprising SEQ ID NO: 238, and a light chain CDR3 comprising SEQ ID NO: 239. A multispecific antigen-binding molecule comprising: [3-2] Each of the first and second antigen-binding moieties comprises an antibody variable region, which may be the same or different, and the antibody variable region is selected from the group consisting of: (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60; (a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a8) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a9) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a10) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a11) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61; (a12) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a13) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; (a14) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58; and (a15) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60 The multispecific antigen-binding molecule of [3-1], further independently selected from the above. [3-2A] The first antigen-binding portion and the second antigen-binding portion each comprise: an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO:20, heavy chain CDR 2 of SEQ ID NO:34, heavy chain CDR 3 of SEQ ID NO:48, light chain CDR 1 of SEQ ID NO:63, light chain CDR 2 of SEQ ID NO:68, and light chain CDR 3 of SEQ ID NO:73 The multispecific antigen-binding molecule of any one of [3-1] to [3-2], comprising: [3-2B] The first antigen-binding portion and the second antigen-binding portion each comprise: An antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58. The multispecific antigen-binding molecule of [3-2A], comprising: [3-2C] a third antigen-binding moiety, An antibody variable region comprising a VH comprising SEQ ID NO: 232 and a VL comprising SEQ ID NO: 236 The multispecific antigen-binding molecule of any one of [3-1] to [3-2B], comprising: [3-3] The multispecific antigen-binding molecule of any one of [3-1] to [3-2C], wherein each of the first and second antigen-binding moieties is a Fab having a cysteine ​​residue at position 191 (EU numbering), and wherein a disulfide bond is present between the two cysteine ​​residues. [3-4] The multispecific antigen-binding molecule of [3-3], wherein each of the first, second, and third antigen-binding moieties is a Fab comprising a heavy chain comprising a VH and CH1 domain and a light chain comprising a VL and a light-chain constant (CL) domain, and the C-terminus of the heavy chain of the third antigen-binding moiety is fused, directly or via a peptide linker, to the N-terminus of the Fab heavy chain of either the first or second antigen-binding moiety. [3-5] The multispecific antigen-binding molecule of [3-4], wherein the C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion via a peptide linker, and the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259. [3-6] The multispecific antigen-binding molecule of [3-5], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding moieties are arginine and lysine, respectively, and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding moieties are glutamic acid. [3-7] The multispecific antigen-binding molecule of [3-6], further comprising an Fc domain. [3-8] the Fc domain comprises a first and a second Fc region subunit; The first Fc region subunit comprises: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 354, and a tryptophan at position 366; and the second Fc region subunit comprising: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 349, a serine at position 366, an alanine at position 368, and a valine at position 407; and selected from the group comprising: All positions are according to EU numbering, [3-7] A multispecific antigen-binding molecule. Yet another aspect of the present invention relates to: [4-1] (a) A first antigen-binding moiety and a second antigen-binding moiety, each of which binds to human CD3 and comprises an antibody variable region which may be the same or different, wherein the antibody variable region is independently selected from the group consisting of (a1) to (a15) below: (a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 comprising SEQ ID NO: 17, a heavy chain CDR 2 comprising SEQ ID NO: 31, a heavy chain CDR 3 comprising SEQ ID NO: 45, a light chain CDR 1 comprising SEQ ID NO: 64, a light chain CDR 2 comprising SEQ ID NO: 69, and a light chain CDR 3 comprising SEQ ID NO: 74; (a2) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 18, a heavy chain CDR2 comprising SEQ ID NO: 32, a heavy chain CDR3 comprising SEQ ID NO: 46, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; (a3) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 19, a heavy chain CDR2 comprising SEQ ID NO: 33, a heavy chain CDR3 comprising SEQ ID NO: 47, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; (a4) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 19, a heavy chain CDR2 comprising SEQ ID NO: 33, a heavy chain CDR3 comprising SEQ ID NO: 47, a light chain CDR1 comprising SEQ ID NO: 65, a light chain CDR2 comprising SEQ ID NO: 70, and a light chain CDR3 comprising SEQ ID NO: 75; (a5) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 20, a heavy chain CDR2 comprising SEQ ID NO: 34, a heavy chain CDR3 comprising SEQ ID NO: 48, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; (a6) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 22, a heavy chain CDR2 comprising SEQ ID NO: 36, a heavy chain CDR3 comprising SEQ ID NO: 50, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; (a7) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 23, a heavy chain CDR2 comprising SEQ ID NO: 37, a heavy chain CDR3 comprising SEQ ID NO: 51, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; (a8) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 23, a heavy chain CDR2 comprising SEQ ID NO: 37, a heavy chain CDR3 comprising SEQ ID NO: 51, a light chain CDR1 comprising SEQ ID NO: 66, a light chain CDR2 comprising SEQ ID NO: 71, and a light chain CDR3 comprising SEQ ID NO: 76; (a9) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 24, a heavy chain CDR2 comprising SEQ ID NO: 38, a heavy chain CDR3 comprising SEQ ID NO: 52, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; (a10) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 25, a heavy chain CDR2 comprising SEQ ID NO: 39, a heavy chain CDR3 comprising SEQ ID NO: 53, a light chain CDR1 comprising SEQ ID NO: 66, a light chain CDR2 comprising SEQ ID NO: 71, and a light chain CDR3 comprising SEQ ID NO: 76; (a11) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 26, a heavy chain CDR2 comprising SEQ ID NO: 40, a heavy chain CDR3 comprising SEQ ID NO: 54, a light chain CDR1 comprising SEQ ID NO: 66, a light chain CDR2 comprising SEQ ID NO: 71, and a light chain CDR3 comprising SEQ ID NO: 76; (a12) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 26, a heavy chain CDR2 comprising SEQ ID NO: 40, a heavy chain CDR3 comprising SEQ ID NO: 54, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; (a13) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 27, a heavy chain CDR2 comprising SEQ ID NO: 41, a heavy chain CDR3 comprising SEQ ID NO: 55, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; (a14) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 28, a heavy chain CDR2 comprising SEQ ID NO: 42, a heavy chain CDR3 comprising SEQ ID NO: 56, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; and (a15) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 82, a heavy chain CDR2 comprising SEQ ID NO: 83, a heavy chain CDR3 comprising SEQ ID NO: 84, a light chain CDR1 comprising SEQ ID NO: 65, a light chain CDR2 comprising SEQ ID NO: 70, and a light chain CDR3 comprising SEQ ID NO: 75; and (b) a third antigen-binding portion that binds to human delta-like 3 (DLL3) and comprises an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 233, a heavy chain CDR2 comprising SEQ ID NO: 234, a heavy chain CDR3 comprising SEQ ID NO: 235, a light chain CDR1 comprising SEQ ID NO: 237, a light chain CDR2 comprising SEQ ID NO: 238, and a light chain CDR3 comprising SEQ ID NO: 239. A multispecific antigen-binding molecule comprising: [4-2] (a) A first antigen-binding moiety and a second antigen-binding moiety, each of which comprises an antibody variable region, which may be the same or different, and which is independently selected from the group consisting of: (a1) an antibody variable region comprising a heavy chain variable region (VH) comprising SEQ ID NO: 3 and a light chain variable region (VL) comprising SEQ ID NO: 59; (a2) an antibody variable region comprising a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 58; (a3) an antibody variable region comprising a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 58; (a4) an antibody variable region comprising a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 60; (a5) an antibody variable region comprising a VH comprising SEQ ID NO: 6 and a VL comprising SEQ ID NO: 58; (a6) an antibody variable region comprising a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 58; (a7) an antibody variable region comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 58; (a8) an antibody variable region comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 61; (a9) an antibody variable region comprising a VH comprising SEQ ID NO: 10 and a VL comprising SEQ ID NO: 58; (a10) an antibody variable region comprising a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 61; (a11) an antibody variable region comprising a VH comprising SEQ ID NO: 12 and a VL comprising SEQ ID NO: 61; (a12) an antibody variable region comprising a VH comprising SEQ ID NO: 12 and a VL comprising SEQ ID NO: 58; (a13) an antibody variable region comprising a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 58; (a14) an antibody variable region comprising a VH comprising SEQ ID NO: 14 and a VL comprising SEQ ID NO: 58; and (a15) an antibody variable region comprising a VH comprising SEQ ID NO: 81 and a VL comprising SEQ ID NO: 60; and (b) a third antigen-binding portion comprising an antibody variable region comprising a VH comprising SEQ ID NO: 232 and a VL comprising SEQ ID NO: 236. A multispecific antigen-binding molecule comprising: [4-3] The multispecific antigen-binding molecule of [4-1], wherein the antibody variable regions of the first and second antigen-binding moieties are the same. [4-4] The multispecific antigen-binding molecule of [4-2], wherein the antibody variable regions of the first and second antigen-binding moieties are the same. [4-5] The multispecific antigen-binding molecule of [4-3], wherein each of the first and second antigen-binding moieties is a Fab having a cysteine ​​residue at position 191 (EU numbering), and these two cysteine ​​residues are linked by a disulfide bond. [4-6] The multispecific antigen-binding molecule of [4-4], wherein each of the first and second antigen-binding moieties is a Fab having a cysteine ​​residue at position 191 (EU numbering), and these two cysteine ​​residues are linked by a disulfide bond. [4-7] The multispecific antigen-binding molecule of [4-5], wherein each of the first, second, and third antigen-binding moieties is in a Fab format comprising a VH, a VL, a CH1 domain, and a light chain constant (CL) domain, and the C-terminus of the CH1 domain of the third antigen-binding moiety is fused to the N-terminus of the VH of either the first antigen-binding moiety or the second antigen-binding moiety, directly or via a peptide linker. [4-8] The multispecific antigen-binding molecule of [4-6], wherein each of the first, second, and third antigen-binding moieties is in a Fab format comprising a CH1 domain and a light chain constant (CL) domain, and the C-terminus of the CH1 domain of the third antigen-binding moiety is fused directly or via a peptide linker to the N-terminus of the VH of either the first antigen-binding moiety or the second antigen-binding moiety. [4-9] The multispecific antigen-binding molecule of [4-7], wherein the fusion is via a peptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259. [4-10] The multispecific antigen-binding molecule of [4-8], wherein the fusion is via a peptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259. [4-11] The multispecific antigen-binding molecule of [4-9], wherein the third antigen-binding portion is a crossover Fab in which the VH is linked to a CL domain and the VL is linked to a CH1 domain, and each of the first and second antigen-binding portions is a conventional Fab in which the VH is linked to a CH1 domain and the VL is linked to a CL domain. [4-12] The multispecific antigen-binding molecule of [4-10], wherein the third antigen-binding portion is a crossover Fab in which the VH is linked to a CL domain and the VL is linked to a CH1 domain, and each of the first and second antigen-binding portions is a conventional Fab in which the VH is linked to a CH1 domain and the VL is linked to a CL domain. [4-13] The multispecific antigen-binding molecule of [4-11], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding moieties are arginine and lysine, respectively, and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding moieties are glutamic acid. [4-14] The multispecific antigen-binding molecule of [4-12], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding moieties are arginine and lysine, respectively, and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding moieties are glutamic acid. [4-15] The multispecific antigen-binding molecule of [4-13], further comprising an Fc domain. [4-16] The multispecific antigen-binding molecule of [4-14], further comprising an Fc domain. [4-17] the Fc domain comprises a first and a second Fc region subunit; The first Fc region subunit comprises: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 354, and a tryptophan at position 366; and the second Fc region subunit comprising: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 349, a serine at position 366, an alanine at position 368, and a valine at position 407; and selected from the group comprising: All positions are according to EU numbering, [4-15] Multispecific antigen-binding molecules. [4-18] the Fc domain comprises a first and a second Fc region subunit; The first Fc region subunit comprises: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 354, and a tryptophan at position 366; and the second Fc region subunit comprising: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 349, a serine at position 366, an alanine at position 368, and a valine at position 407; and selected from the group comprising: All positions are according to EU numbering, [4-16] Multispecific antigen-binding molecules. [4-19] (A)~(O) below: (A) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 208, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (B) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (C) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (D) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 205, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (E) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 216, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 229, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (F) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 217, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 210, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (G) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 219, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (H) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 220, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (I) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 221, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (J) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 222, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 230, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214; (K) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 223, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 212, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215; (L) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 225, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215; (M) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 226, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215; (N) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 227, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (O) A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215. A multispecific antigen-binding molecule comprising five polypeptide chains in a combination selected from the group consisting of: [4-20] The multispecific antigen-binding molecule of [4-19], comprising a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214. [4-21] The multispecific antigen-binding molecule of [4-19], comprising a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214. [4-22] The multispecific antigen-binding molecule of [4-19], comprising a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 205, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 214. [4-23] (a) a first antigen-binding portion and a second antigen-binding portion, each of which binds to human CD3 and comprises an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 20, a heavy chain CDR2 comprising SEQ ID NO: 34, a heavy chain CDR3 comprising SEQ ID NO: 48, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73; and (b) a third antigen-binding portion that binds to human DLL3 and comprises an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 233, a heavy chain CDR2 comprising SEQ ID NO: 234, a heavy chain CDR3 comprising SEQ ID NO: 235, a light chain CDR1 comprising SEQ ID NO: 237, a light chain CDR2 comprising SEQ ID NO: 238, and a light chain CDR3 comprising SEQ ID NO: 239. A multispecific antigen-binding molecule comprising: [4-24] The multispecific antigen-binding molecule of [4-23], wherein the antibody variable regions of each of the first and second antigen-binding moieties comprise a VH comprising SEQ ID NO: 6 and a VL comprising SEQ ID NO: 58, and the antibody variable region of the third antigen-binding moiety comprises a VH comprising SEQ ID NO: 232 and a VL comprising SEQ ID NO: 236. [4-25] The multispecific antigen-binding molecule of [4-24], wherein each of the first and second antigen-binding moieties is a Fab having a cysteine ​​residue at position 191 (EU numbering), and these two cysteine ​​residues are linked by a disulfide bond. [4-26] The multispecific antigen-binding molecule of [4-25], wherein each of the first, second, and third antigen-binding moieties is in a Fab format comprising a VH, a VL, a CH1 domain, and a CL domain, and the C-terminus of the CH1 domain of the third antigen-binding moiety is fused to the N-terminus of the VH of either the first antigen-binding moiety or the second antigen-binding moiety, directly or via a peptide linker. [4-27] The multispecific antigen-binding molecule of [4-26], wherein the fusion is via a peptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259. [4-28] The multispecific antigen-binding molecule of [4-27], wherein the third antigen-binding portion is a crossover Fab in which the VH is linked to a CL domain and the VL is linked to a CH1 domain, and each of the first and second antigen-binding portions is a conventional Fab in which the VH is linked to a CH1 domain and the VL is linked to a CL domain. [4-29] The multispecific antigen-binding molecule of [4-28], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding moieties are arginine and lysine, respectively, and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding moieties are glutamic acid. [4-30] The multispecific antigen-binding molecule of [4-29], further comprising an Fc domain. [4-31] The Fc domain comprises a first and a second Fc region subunit; The first Fc region subunit comprises: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 354, and a tryptophan at position 366; and the second Fc region subunit comprising: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 349, a serine at position 366, an alanine at position 368, and a valine at position 407; and selected from the group comprising: All positions are according to EU numbering, [4-30] Multispecific antigen-binding molecules. [Brief explanation of the drawings]

[0019] [Figure 1] 1 shows the design and naming rules for a) trivalent antibodies in DUAL / LINC (1+2) format, b) bispecific antibodies with a conventional antibody format, and c) bispecific antibodies with a BiTE format. [Figure 2] Figure 1 shows the TDCC activity of antibodies against the SK-MEL30 cell line. a) Comparison of TDCC between DUAL / LINC and CD3 bispecific formats. b) Effect of linker length on cytotoxic activity. [Figure 3] 1 shows the in vivo efficacy of antibodies against NCI-H1436 xenografts in the huNOG mouse model. Y-axis represents tumor volume (mm3), and X-axis represents days after tumor implantation. [Figure 4] Figure 1 shows the results of analyzing CD8 T cell infiltration. Tumors were harvested at the indicated time points after antibody injection, and T cell infiltration was analyzed using a flow cytometer. [Figure 5] The results of analyzing exhaustion markers on CD8 T cells are shown. Tumors were harvested 7 days after antibody injection, and the expression of exhaustion markers was analyzed using a flow cytometer. [Figure 6]Schematic diagrams showing the structures of full-length human DLL3 and human DLL3 ECD fragment proteins. The epitopes recognized by each anti-DLL3 antibody are also shown. The EGF domain has six regions, EGF1 to EGF6, from the N-terminus to the C-terminus. DETAILED DESCRIPTION OF THE INVENTION

[0020] The techniques and procedures described or referenced herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (RI Freshney), ed., 1987);Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons;Handbook of Experimental Immunology (DM Weir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and those commonly employed by those skilled in the art using conventional methodologies such as those widely used in Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0021] The following definitions and detailed description are provided to facilitate understanding of the disclosure described herein.

[0022] definition amino acid As used herein, 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.

[0023] Amino acid modification For amino acid modification (herein also referred to as "amino acid substitution" or "amino acid mutation") in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. Furthermore, several known methods for amino acid modification to substitute with unnatural amino acids can also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is suitable to use a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which an unnatural amino acid is bound to a complementary amber suppressor tRNA for the UAG codon (amber codon), which is a type of stop codon.

[0024] As used herein, the term "and / or" when describing the site of an amino acid modification includes any combination of "and" and "or." Specifically, for example, "the amino acids at positions 33, 55, and / or 96 are substituted" includes the following variations of amino acid modification: (a) positions 33, (b) positions 55, (c) positions 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96, and (g) positions 33, 55, and 96.

[0025] Furthermore, as used herein, expressions indicating amino acid modifications may be appropriately expressed by indicating the one-letter or three-letter code of the amino acid before and after the modification, respectively, before and after the number indicating a specific position. For example, the modification N100bL or Asn100bLeu used to substitute an amino acid contained in an antibody variable region represents a substitution of Asn at position 100b (according to Kabat numbering) with Leu. That is, the number indicates the amino acid position according to Kabat numbering, the one-letter or three-letter amino acid code written before the number represents the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number represents the amino acid after substitution. Similarly, the modification P238D or Pro238Asp used to substitute an amino acid in the Fc region contained in an antibody constant region represents a substitution of Pro at position 238 (according to EU numbering) with Asp. That is, the numbers indicate the amino acid positions according to EU numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0026] Polypeptides As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but need not necessarily be translated from a designated nucleic acid. It may be generated by any method, including chemical synthesis. The polypeptides described herein may be about 3 or more amino acids, 5 or more amino acids, 10 or more amino acids, 20 or more amino acids, 25 or more amino acids, 50 or more amino acids, 75 or more amino acids, 100 or more amino acids, 200 or more amino acids, 500 or more amino acids, 1,000 or more amino acids, or 2,000 or more amino acids in size. Polypeptides may have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are said to be folded, while polypeptides that do not have a defined three-dimensional structure but can adopt multiple different conformations are said to be unfolded.

[0027] Percent (%) amino acid sequence identity "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0028] Recombination methods and constructs Antibodies and antigen-binding molecules can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell)). In one embodiment, there is provided a method for making a multispecific antigen-binding molecule of the present invention, comprising culturing a host cell comprising nucleic acid encoding the antibody as described above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0029] For recombinant production of the antibodies described herein, nucleic acid encoding the antibody (e.g., such as those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0030] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste or further purified.

[0031] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0032] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0033] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0034] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). Other useful mammalian host cell lines include DHFR cells; MRC5 cells; and FS4 cells. - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0035] Recombinant production of the antigen-binding molecules described herein can be carried out in a similar manner to that described above, by using host cells containing (e.g., transformed with) one or more vectors containing nucleic acids encoding an amino acid sequence comprising the entire antigen-binding molecule or a portion of the antigen-binding molecule.

[0036] Antigen-binding molecules and multispecific antigen-binding molecules As used herein, the term "antigen-binding molecule" refers to any molecule containing an antigen-binding site or any molecule that has binding activity to an antigen, and may further refer to molecules such as peptides or proteins having a length of about 5 amino acids or more. Peptides and proteins are not limited to those derived from living organisms; for example, they may be polypeptides produced from artificially designed sequences. They may be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Scaffold molecules that contain a known stable three-dimensional structure such as an α / β barrel as a scaffold, and a portion of the molecule serves as the antigen-binding site, are also an embodiment of the antigen-binding molecules described herein.

[0037] A "multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to two or more antigens. The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. The term "trispecific" means that an antigen-binding molecule can specifically bind to at least three different antigenic determinants. In a specific embodiment, the multispecific antigen-binding molecule of the present application is a trispecific antigen-binding molecule, i.e., a trispecific antigen-binding molecule that can specifically bind to three different antigens, i.e., can bind to either CD3 or CD137, but not both antigens simultaneously, and can specifically bind to DLL3.

[0038] In a first aspect, the present disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety, each capable of binding to CD3 and CD137 but not simultaneously; and a third antigen-binding moiety capable of binding to a third antigen, preferably an antigen expressed on cancer cells / tissues. In certain embodiments, the third antigen bound by the third antigen-binding moiety is DLL3, preferably human DLL3.

[0039] The first and second antigen-binding moieties may be "dual antigen-binding moieties" that can bind to both CD3 and CD137, but not simultaneously, as described in more detail below. The third antigen-binding moiety may be a "DLL3 antigen-binding moiety," as described in more detail below.

[0040] In some embodiments, each of the first and second antigen-binding moieties is a Fab molecule and comprises at least one disulfide bond formed between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety. The disulfide bond can be formed between the amino acid residue at position 191 (EU numbering) in the CH1 region of each of the first and second antigen-binding moieties.

[0041] In some embodiments, the third antigen-binding portion may be a Fab or scFv and is fused to either the first or second antigen-binding portion. When each of the first, second, and third antigen-binding portions is a Fab molecule, the third antigen-binding portion may be fused at the C-terminus (CH1) of the Fab heavy chain to the N-terminus of the Fab heavy chain of either the first or second antigen-binding portion, optionally via a peptide linker. Exemplary peptide linkers include those consisting of the amino acid sequence of SEQ ID NO:248, SEQ ID NO:249, or SEQ ID NO:259. In certain embodiments, the first antigen-binding portion is identical to the second antigen-binding portion.

[0042] In some embodiments, the third antigen-binding portion is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and each of the first and second antigen-binding portions is a conventional Fab molecule.

[0043] In some embodiments, in the constant domain CL of the light chain of each of the first and second antigen-binding moieties, the amino acid at position 123 and / or 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the heavy chain of each of the first and second antigen-binding moieties, the amino acid at position 147 and / or 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering). In another embodiment, in the constant domain CL of the light chain of each of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 are arginine (R) and lysine (K), respectively (numbering according to Kabat), and in the constant domain CH1 of the heavy chain of each of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 are glutamic acid (E) (numbering according to EU index, Kabat).

[0044] The multispecific antigen-binding molecule may further comprise an Fc domain, as described in detail below. When each of the first and second antigen-binding moieties is a Fab, the first antigen-binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain. In some embodiments, the third antigen-binding moiety is fused at the C-terminus to the N-terminus of the Fab heavy chain of either the first or second antigen-binding moiety, optionally via a peptide linker.

[0045] In another aspect of the present invention, the present disclosure provides a multispecific antigen-binding molecule comprising an antigen-binding portion capable of binding to CD3 and CD137, but not simultaneously; and an antigen-binding portion capable of binding to DLL3, preferably human DLL3. In certain embodiments, the antigen-binding portion capable of binding to CD3 and CD137, but not simultaneously, is a "dual antigen-binding portion" capable of binding to CD3 and CD137, but not simultaneously, as described in detail below.

[0046] The components of the multispecific antigen-binding molecules of the present invention can be fused to each other in a variety of configurations. Exemplary configurations are illustrated in Figure 1a) read in conjunction with Tables 10-1 to 10-3.

[0047] According to any of the above aspects, the components of the multispecific antigen-binding molecule (e.g., antigen-binding portion, Fc domain) may be fused directly or through various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2 to 20 amino acids, described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n, or G4(SG4)n peptide linkers, where n is generally a number between 1 and 10, typically between 2 and 4.

[0048] Pyroglutamylation It is known that when antibody is expressed in cells, antibody is modified after translation.Examples of post-translational modifications include cleavage of lysine at the C-terminus of heavy chain by carboxypeptidase; modification of glutamine or glutamic acid at the N-terminus of heavy chain and light chain to pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation, and such post-translational modifications are known to occur in various antibodies (Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447).

[0049] In some embodiments, the multispecific antigen-binding molecules of the present invention contain post-translational modifications. Examples of post-translational modifications include pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications, such as pyroglutamylation at the N-terminus and deletion of lysine at the C-terminus, have no effect on antibody activity (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0050] antigen binding part As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigen. In one embodiment, an antigen-binding moiety can direct the entity to which it binds to a target site, such as a specific type of tumor cell expressing a cancer antigen (DLL3). In another embodiment, an antigen-binding moiety can activate signaling through its target antigen, such as a T-cell receptor complex antigen (particularly CD3) and / or a costimulatory receptor (CD137). Antigen-binding moieties include antibodies and fragments thereof, as further defined herein. Specific antigen-binding moieties include the antigen-binding domain of an antibody or antibody variable region, including an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding moiety may comprise an antibody constant region, as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ.

[0051] As used herein, the terms "first," "second," and "third" with respect to antigen-binding moieties, etc., are used for the convenience of distinguishing between two or more different types of moieties, etc. The use of these terms is not intended to confer a particular order or orientation of the multispecific antigen-binding molecules, unless otherwise specified.

[0052] In another aspect, the antigen-binding portion of the present invention disclosed herein can be used in a novel chimeric antigen receptor (CAR) comprising one or more of the antigen-binding portions disclosed herein. In certain embodiments, the CAR of the present invention comprises an scFv construct, and in preferred embodiments, comprises heavy and light chain variable regions as disclosed herein. In preferred embodiments, the disclosed chimeric antigen receptor is useful for treating or preventing proliferative diseases and any recurrence or metastasis thereof.

[0053] An antigen-binding moiety that can bind to CD3 and CD137, but not simultaneously The multispecific antigen-binding molecules described herein comprise at least one antigen-binding moiety (also referred to herein as a "Dual antigen-binding moiety" or "first antigen-binding moiety" or "Dual-Fab" or "Dual-Ig") that can bind to CD3 and CD137, but does not bind to CD3 and CD137 simultaneously. In certain embodiments, the multispecific antigen-binding molecule comprises two Dual antigen-binding moieties (a "first antigen-binding moiety" and a "second antigen-binding moiety," each of which may be referred to as a "Dual-Fab"). In some embodiments, each of the two Dual antigen-binding moieties (a "first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") provides monovalent binding to CD3 or CD137, but does not bind to CD3 and CD137 simultaneously. In certain embodiments, the multispecific antigen-binding molecule comprises no more than two Dual antigen-binding moieties ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab").

[0054] In certain embodiments, the Dual antigen-binding moiety ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") is generally a Fab molecule, particularly a conventional Fab molecule. In certain embodiments, the Dual antigen-binding moiety ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)", "single-chain antibody", "Fv", "single-chain Fv2 (scFv2)", "Fab", "F(ab')2", etc.

[0055] In certain embodiments, the Dual antigen-binding moiety ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") specifically binds to all of CD3 or a portion of a partial peptide thereof. In certain embodiments, the CD3 is human CD3 or cynomolgus CD3, particularly human CD3. In certain embodiments, the first antigen-binding moiety is cross-reactive with (i.e., specifically binds to) human and cynomolgus CD3. In some embodiments, the first antigen-binding moiety can specifically bind to the epsilon subunit of CD3, particularly the human CD3 epsilon subunit of CD3 set forth in SEQ ID NO:7 (NP_000724.1) (RefSeq accession number shown in parentheses). In some embodiments, the Dual antigen-binding moiety ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") can specifically bind to the CD3 epsilon chain expressed on the surface of a eukaryotic cell. In some embodiments, the Dual antigen-binding moiety ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") binds to the CD3 epsilon chain expressed on the surface of a T cell.

[0056] In certain embodiments, CD137 is human CD137. In some embodiments, suitable examples of antigen-binding molecules of the present invention include the following: an antibody recognizing a region including the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 21); an antibody recognizing a region containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 35); An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 49), and An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQIC (SEQ ID NO: 105) in the human CD137 protein and a dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") that binds to the same human CD137 epitope as that bound by an antibody selected from the group consisting of:

[0057] In certain embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises any one of the antibody variable region sequences shown in Table 1 below. In certain embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises any one of the combinations of heavy chain variable region and light chain variable region shown in Table 1. Table 1: SEQ ID NOs of the variable regions of the dual antigen-binding moieties ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") TIFF2026031947000002.tif97170

[0058] In one embodiment, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:58. In one embodiment, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:58.

[0059] In one embodiment, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. In one embodiment, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.

[0060] In one embodiment, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. In one embodiment, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.

[0061] In certain embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises any one of the combinations of HVR sequences shown in Table 2 below. Table 2: SEQ ID NOs of HVR (CDR) sequences of dual antigen-binding portions ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") TIFF2026031947000003.tif91170

[0062] In some embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") is each any one of the following (a1) to (a17): (a1) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 17, heavy chain CDR 2 of SEQ ID NO: 31, heavy chain CDR 3 of SEQ ID NO: 45, light chain CDR 1 of SEQ ID NO: 64, light chain CDR 2 of SEQ ID NO: 69, and light chain CDR 3 of SEQ ID NO: 74; (a2) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 18, heavy chain CDR 2 of SEQ ID NO: 32, heavy chain CDR 3 of SEQ ID NO: 46, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a3) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 19, heavy chain CDR 2 of SEQ ID NO: 33, heavy chain CDR 3 of SEQ ID NO: 47, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a4) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 19, heavy chain CDR 2 of SEQ ID NO: 33, heavy chain CDR 3 of SEQ ID NO: 47, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75; (a5) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 20, heavy chain CDR 2 of SEQ ID NO: 34, heavy chain CDR 3 of SEQ ID NO: 48, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a6) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 22, heavy chain CDR 2 of SEQ ID NO: 36, heavy chain CDR 3 of SEQ ID NO: 50, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a7) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a8) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a9) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 24, heavy chain CDR 2 of SEQ ID NO: 38, heavy chain CDR 3 of SEQ ID NO: 52, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a10) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 25, heavy chain CDR 2 of SEQ ID NO: 39, heavy chain CDR 3 of SEQ ID NO: 53, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a11) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 26, heavy chain CDR 2 of SEQ ID NO: 40, heavy chain CDR 3 of SEQ ID NO: 54, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76; (a12) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 26, heavy chain CDR 2 of SEQ ID NO: 40, heavy chain CDR 3 of SEQ ID NO: 54, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a13) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 27, heavy chain CDR 2 of SEQ ID NO: 41, heavy chain CDR 3 of SEQ ID NO: 55, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a14) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 28, heavy chain CDR 2 of SEQ ID NO: 42, heavy chain CDR 3 of SEQ ID NO: 56, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73; (a15) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 82, heavy chain CDR 2 of SEQ ID NO: 83, heavy chain CDR 3 of SEQ ID NO: 84, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75; (a16) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a17) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15). The antibody variable region comprises:

[0063] In some embodiments, the multispecific antigen-binding molecules or Dual antigen-binding portions of the present invention ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") also contain post-translational modifications. Examples of post-translational modifications include pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications as pyroglutamylation at the N-terminus and deletion of lysine at the C-terminus have no effect on antibody activity (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0064] An antigen-binding moiety capable of binding to DLL3 The multispecific antigen-binding molecules described herein comprise an antigen-binding portion capable of binding to Delta-like 3 (DLL3) (referred to herein as a "DLL3 antigen-binding portion" or a "third antigen-binding portion" or "Antigen-binding moiety that binds to DLL3" (also called

[0065] In certain embodiments, a multispecific antigen-binding molecule comprises one antigen-binding portion capable of binding to DLL3. In certain embodiments, a multispecific antigen-binding molecule comprises two antigen-binding portions capable of binding to DLL3 ("DLL3 antigen-binding portions"). In certain such embodiments, each of these antigen-binding portions specifically binds to the same epitope of DLL3. In even more specific embodiments, all of these "DLL3 antigen-binding portions" are identical. In one embodiment, a multispecific antigen-binding molecule comprises an immunoglobulin molecule ("DLL3 antigen-binding portion") capable of specifically binding to DLL3. In one embodiment, a multispecific antigen-binding molecule comprises no more than two antigen-binding portions ("DLL3 antigen-binding portions") capable of binding to DLL3.

[0066] In certain embodiments, the DLL3 antigen-binding portion is a crossover Fab molecule, i.e., a DLL3 molecule in which either the variable or constant regions of the Fab heavy and light chains have been exchanged. In certain embodiments, the DLL3 antigen-binding portion is a crossover Fab molecule in which the variable regions of the Fab light and heavy chains have been exchanged.

[0067] In some embodiments, the DLL3 antigen binding moiety specifically binds to the extracellular domain of DLL3. In some embodiments, the DLL3 antigen binding moiety specifically binds to an epitope within the extracellular domain of DLL3. In some embodiments, the DLL3 antigen binding moiety binds to a DLL3 protein expressed on the surface of a eukaryotic cell. In some embodiments, the DLL3 antigen binding moiety binds to a DLL3 protein expressed on the surface of a cancer cell.

[0068] In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion binds to an epitope within the extracellular domain (ECD), i.e., from the N-terminus to just before the TM region, but not to the TM region or the C-terminal intracellular domain. The multispecific antigen-binding molecule or DLL3 antigen-binding portion may bind to an epitope within any of the above-mentioned domains / regions within the ECD. In preferred embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion binds to an epitope within the region from EGF6 to just before the TM region. More specifically, the multispecific antigen-binding molecule or DLL3 antigen-binding portion may bind to an epitope within the region defined by SEQ ID NO: 89 in human DLL3. In some embodiments, the multispecific antigen binding molecule or DLL3 antigen binding portion binds to an epitope within the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region, or the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region. In some embodiments, the multispecific antigen binding molecule or DLL3 antigen binding portion can be derived from previously reported anti-DLL3 antibodies (e.g., WO2019131988 and WO2011093097) in which the DLL3 epitope that binds has been characterized.

[0069] In certain embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion comprises any one of the antibody variable region sequences shown in Table 1C below. In certain embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion comprises any one of the combinations of heavy chain variable region and light chain variable region shown in Table 3. In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion comprises a domain comprising an antibody variable fragment that competes for binding to DLL3 with any one of the antibody variable regions shown in Table 3. Table 3. SEQ ID NOs of exemplary variable regions of DLL3 antigen binding moieties TIFF2026031947000004.tif139170

[0070] In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 232, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 236. In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 232, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 236.

[0071] In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 300, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 236. In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 300, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 236.

[0072] In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 301, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 236. In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 301, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 236.

[0073] In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 274, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 275. In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 274, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 275.

[0074] In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 264, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 265. In one embodiment, a DLL3 antigen binding portion comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 264, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 265.

[0075] In certain embodiments, the DLL3 antigen-binding portion comprises any one of the combinations of HVR sequences shown in Table 4 below. In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion comprises a domain comprising an antibody variable fragment that competes for binding to DLL3 with any one of the antibody variable regions shown in Table 4, or that competes for binding to DLL3 with any antibody variable fragment comprising HVR sequences identical to the HVR regions of the antibody variable regions shown in Table 4. Table 4. SEQ ID NOs for HVR (CDR) sequences of exemplary DLL3 antigen binding portions TIFF2026031947000005.tif70170

[0076] In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention comprises any one of the following (a1) to (a5): (a1) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 233, heavy chain CDR 2 of SEQ ID NO: 234, heavy chain CDR 3 of SEQ ID NO: 235, light chain CDR 1 of SEQ ID NO: 237, light chain CDR 2 of SEQ ID NO: 238, and light chain CDR 3 of SEQ ID NO: 239; (a2) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 276, heavy chain CDR 2 of SEQ ID NO: 277, heavy chain CDR 3 of SEQ ID NO: 278, light chain CDR 1 of SEQ ID NO: 279, light chain CDR 2 of SEQ ID NO: 280, and light chain CDR 3 of SEQ ID NO: 281; (a3) heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO: 285, heavy chain CDR 2 of SEQ ID NO: 286, heavy chain CDR 3 of SEQ ID NO: 287, light chain CDR 1 of SEQ ID NO: 288, light chain CDR 2 of SEQ ID NO: 289, and light chain CDR 3 of SEQ ID NO: 290; (a4) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a3); and (a5) An antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a3). The antibody variable region comprises:

[0077] In some embodiments, the multispecific antigen-binding molecules or DLL3 antigen-binding portions of the present invention also contain post-translational modifications. Examples of post-translational modifications include pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications as pyroglutamylation at the N-terminus and deletion of lysine at the C-terminus have no effect on antibody activity (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0078] In another aspect, the DLL3 antigen binding portion of the present invention can be used in a new chimeric antigen receptor (CAR) (DLL3 CAR) incorporating a DLL3 binding domain. In certain embodiments, the DLL3 binding domain (and DLL3 CAR) of the present invention comprises an scFv construct, which in preferred embodiments comprises heavy and light chain variable regions as disclosed herein. In other preferred embodiments, the DLL3 binding domain (and DLL3 CAR) of the present invention comprises an scFv construct or a fragment thereof comprising the heavy and light chain variable regions disclosed herein. In preferred embodiments, the disclosed chimeric antigen receptors are useful for treating or preventing proliferative diseases and any recurrence or metastasis thereof.

[0079] In certain embodiments, the DLL3 protein is expressed on tumor-initiating cells. DLL3 CARs are expressed on cytotoxic lymphocytes (preferably autologous cytotoxic lymphocytes) through genetic modification (e.g., transduction), resulting in DLL3-sensitive lymphocytes that can be used to target and kill DLL3-positive tumor cells. As broadly discussed herein, the CARs of the present invention typically include an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and include a DLL3-binding domain that activates certain lymphocytes and generates an immune response against DLL3-positive tumor cells. Selected embodiments of the present invention include immunologically active host cells that display the disclosed CARs, as well as various polynucleotide sequences and vectors encoding the DLL3 CARs of the present invention. Other aspects include methods for enhancing the activity of T lymphocytes or natural killer (NK) cells in an individual by introducing host cells expressing a DLL3 CAR molecule into an individual suffering from cancer and treating the individual. Such aspects include, in particular, lung cancer (e.g., small cell lung cancer) and melanoma.

[0080] antigen As used herein, the term "antigen" refers to the site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a three-dimensional structure composed of a continuous stretch of amino acids or a discrete region of non-contiguous amino acids), forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, the proteins referred to herein as antigens (e.g., CD3, CD137, DLL3) can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is human CD3, human CD137, or human DLL3. When a specific protein is referred to herein, the term encompasses the "full-length," unprocessed protein, as well as any form of the protein produced by processing in the cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0081] In certain embodiments, the multispecific antigen-binding molecules described herein bind to epitopes of CD3, CD137, or DLL3 that are conserved among CD3, CD137, or DLL3 of different species. In certain embodiments, the multispecific antigen-binding molecules of the present application are trispecific antigen-binding molecules, i.e., trispecific antigen-binding molecules that can specifically bind to three different antigens, i.e., can bind to either CD3 or CD137, but not both antigens simultaneously, and can specifically bind to DLL3.

[0082] In certain embodiments, the multispecific antigen-binding molecule specifically binds to all or part of a partial peptide of CD3. In certain embodiments, the CD3 is human CD3 or cynomolgus monkey CD3, most typically human CD3. In certain embodiments, the multispecific antigen-binding molecule is cross-reactive with (i.e., specifically binds to) human CD3 and cynomolgus monkey CD3. In some embodiments, the multispecific antigen-binding molecule can specifically bind to the ε subunit of CD3, particularly the human CD3ε subunit of CD3 set forth in SEQ ID NO: 7 (NP_000724.1) (RefSeq accession number shown in parentheses). In some embodiments, the multispecific antigen-binding molecule can specifically bind to the CD3ε chain expressed on the surface of eukaryotic cells. In some embodiments, the multispecific antigen-binding molecule binds to the CD3ε chain expressed on the surface of T cells.

[0083] In certain embodiments, CD137 is human CD137. In some embodiments, suitable examples of antigen-binding molecules of the present invention include antigen-binding molecules that bind to the same epitope as the human CD137 epitope bound by an antibody selected from the group consisting of: an antibody recognizing a region including the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 21); an antibody recognizing a region containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 35); An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 49), and An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQIC (sequence number: 105) in the human CD137 protein.

[0084] The term "DLL3," as used herein, unless otherwise indicated, refers to any naturally occurring DLL3 (Delta-like 3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed "full-length" DLL3, as well as any form of DLL3 produced by cellular processing. The term also encompasses naturally occurring variants of DLL3, such as splice variants or allelic variants. The amino acid sequence of an exemplary human DLL3 is known as NCBI Reference Sequence (RefSeq) NM_016941.3, the amino acid sequence of an exemplary cynomolgus monkey DLL3 is known as NCBI Reference Sequence XP_005589253.1, and the amino acid sequence of an exemplary mouse DLL3 is known as NCBI Reference Sequence NM_007866.2.

[0085] The human DLL3 protein comprises a transmembrane (TM) region and an intracellular domain at the C-terminus, and a DSL (Notch) domain at the N-terminus (see, for example, Figure 6). Additionally, DLL3 has an EGF domain, comprising six regions, EGF1 to EGF6, from the N-terminus to the C-terminus. In some embodiments, multispecific antigen-binding molecules or DLL3 antigen-binding portions of the invention bind to an epitope within the extracellular domain (ECD), i.e., within a domain from the N-terminus to just before the TM region, but not within the TM region or the C-terminal intracellular domain. Multispecific antigen-binding molecules or DLL3 antigen-binding portions of the invention can bind to an epitope within any of the above-mentioned domains / regions within the ECD. In preferred embodiments, multispecific antigen-binding molecules or DLL3 antigen-binding portions of the invention can bind to an epitope within the region from EGF6 to just before the TM region. More specifically, multispecific antigen-binding molecules or DLL3 antigen-binding portions of the invention can bind to an epitope within the region defined by SEQ ID NO: 89 in human DLL3. In some embodiments, the molecules / antibodies of the invention bind to an epitope within the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region, or the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region.

[0086] In human DLL3, the above-mentioned domains / regions have the following amino acid residues (see, for example, http: / / www.uniprot.org / uniprot / Q9NYJ7 or WO2013 / 126746): Extracellular domain (ECD): amino acid residues at positions 1–492; DSL domain: amino acid residues at positions 176–215; EGF domain: amino acid residues at positions 216–465; EGF1 region: amino acid residues at positions 216–249; EGF2 region: amino acid residues at positions 274–310; EGF3 region: amino acid residues at positions 312 to 351; EGF4 region: amino acid residues at positions 353 to 389; EGF5 region: amino acid residues at positions 391–427; EGF6 region: amino acid residues at positions 429–465; The region from EGF6 to just before the TM region: amino acid residues at positions 429 to 492; TM region: amino acid residues at positions 493 to 513; and C-terminal intracellular domain: amino acid residues at positions 516–618 (or 516–587 in some isoforms). The amino acid positions described above also refer to the amino acid positions in the amino acid sequence shown in SEQ ID NO: 90. Accordingly, multispecific antigen-binding molecules or DLL3 antigen-binding portions of the present invention can bind to the above-mentioned regions / domains having amino acid residues at the above-mentioned positions in human DLL3. That is, multispecific antigen-binding molecules or DLL3 antigen-binding portions of the present invention can bind to epitopes within the above-mentioned regions / domains having amino acid residues at the above-mentioned positions in human DLL3.

[0087] The DLL3 protein used in the present invention may be a DLL3 protein having the sequence described above, or may be a modified protein having a sequence derived from the sequence described above by modifying one or more amino acids. Examples of modified proteins having a sequence derived from the sequence described above by modifying one or more amino acids may include polypeptides having 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more identity to the amino acid sequence described above. Alternatively, partial peptides of these DLL3 proteins may be used.

[0088] The DLL3 protein used in the present invention is not limited by its origin, and is preferably a human or cynomolgus monkey DLL3 protein.

[0089] In some embodiments, a DLL3 ECD fragment protein (or ECD variant) can be used as the DLL3 protein. Depending on the cleavage site, the fragment / variant may include, from the N-terminus to the C-terminus, the DSL domain to EGF6, EGF1 to EGF6, EGF2 to EGF6, EGF3 to EGF6, EGF4 to EGF6, EGF5 and EGF6, or EGF6. The fragment / variant may further include a region extending from immediately after the EGF6 region to immediately before the TM region. A Flag tag may be attached to the C-terminus of the fragment / variant using techniques well known in the art.

[0090] antigen-binding domain The term "antigen-binding domain" refers to a portion of an antibody comprising the area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, the antigen-binding domain comprises 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-domain antibody or VHH," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," and "F(ab')2."

[0091] Variable region The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody usually have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6 th(See, e.g., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by using a VH or VL domain from an antibody that binds that antigen to screen a complementary library of VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0092] HVR or CDR As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity-determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contacts"). Hypervariable regions (HVRs) are also referred to as "complementarity-determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable domain that form the antigen-binding region. Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), 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).

[0093] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0094] HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are also referred to as "H-CDR1," "H-CDR2," "H-CDR3," "L-CDR1," "L-CDR2," and "L-CDR3," respectively.

[0095] Able to bind to CD3 and CD137, but does not bind to CD3 and CD137 simultaneously Whether an antibody variable region of the present invention is "capable of binding to CD3 and CD137" can be determined by methods known in the art.

[0096] This can be determined, for example, by electrochemiluminescence (ECL) (BMC Research Notes 2011, 4:281).

[0097] Specifically, for example, a region of a biotin-labeled test antigen-binding molecule capable of binding to CD3 and CD137, such as a small antibody composed of the Fab region, or a monovalent antibody thereof (an antibody lacking one of the two Fab regions of a normal antibody), is mixed with CD3 or CD137 labeled with a sulfo-tag (Ru complex), and the mixture is added to a streptavidin-immobilized plate. During this procedure, the biotin-labeled test antigen-binding molecule binds to the streptavidin on the plate. Light is generated from the sulfo-tag, and the luminescence signal is detected using a Sector Imager 600 or 2400 (MSD KK), etc., thereby confirming the binding of the above-mentioned region of the test antigen-binding molecule to CD3 or CD137.

[0098] Alternatively, the assay may be performed by ELISA, FACS (fluorescence activated cell sorting), ALPHAScreen (amplified luminescence proximity homogeneous assay screen), BIACORE method based on the surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0099] Specifically, the assay can be performed using, for example, Biacore (GE Healthcare Japan Corp.), an interaction analysis instrument based on the surface plasmon resonance (SPR) phenomenon. Biacore analysis instruments include any model, such as the Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any Biacore sensor chip, such as a CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chip, can be used as the sensor chip. Proteins for capturing the antigen-binding molecules of the present invention, such as protein A, protein G, protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human L chain antibody, anti-human Fc antibody, antigen protein, or antigen peptide, are immobilized on the sensor chip by a coupling method such as amine coupling, disulfide coupling, or aldehyde coupling. CD3 or CD137 is injected onto the sensor chip as an analyte, and the interaction is measured to obtain a sensorgram. In this procedure, the concentration of CD3 or CD137 can be selected within the range of several μM to several pM according to the strength of the interaction (eg, KD) of the assay sample.

[0100] Alternatively, CD3 or CD137 may be immobilized on a sensor chip instead of an antigen-binding molecule, and then the antibody sample to be evaluated may be allowed to interact with it. Whether the antibody variable region of the antigen-binding molecule of the present invention has binding activity to CD3 or CD137 can be confirmed based on the dissociation constant (KD) value calculated from the sensorgram of the interaction, or based on the degree of increase in the sensorgram after the action of the antigen-binding molecule sample above the level before the action.

[0101] In some embodiments, the binding activity or affinity of an antibody variable region of the present invention for an antigen of interest (i.e., CD3 or CD137) is evaluated, for example, using a Biacore T200 instrument (GE Healthcare) or a Biacore 8K instrument (GE Healthcare) at 37°C (for CD137) or 25°C (for CD3). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). The antigen-binding molecule or antibody variable region is captured on the anti-Fc sensor surface, and then the antigen (CD3 or CD137) is injected onto the flow cell. The capture level of the antigen-binding molecule or antibody variable region may aim for 200 resonance units (RU). Recombinant human CD3 or CD137 may be injected at 2000 to 125 nM prepared by two-fold serial dilution, followed by dissociation. All antigen-binding molecules or antibody variable regions and analytes are prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The sensor surface is regenerated with 3 M MgCl2 every cycle. Binding affinity is determined by processing data and fitting to a 1:1 binding model, for example, using Biacore Insight Evaluation software, version 2.0 (GE Healthcare) or Biacore 8K Evaluation software (GE Healthcare). To evaluate the specific binding activity or affinity of the antigen-binding domain of the present invention, KD values ​​are calculated.

[0102] ALPHAScreen is implemented using ALPHA technology, which uses two types of beads (donor and acceptor), based on the following principle: luminescence signals are detected only when a biological interaction between a molecule bound to a donor bead and a molecule bound to an acceptor bead brings these two beads into close proximity. A photosensitizer in the donor bead, excited by a laser, converts ambient oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, thereby triggering a chemiluminescent reaction in the bead, ultimately resulting in the emission of light. If there is no interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead will not reach the acceptor bead. Therefore, the chemiluminescent reaction will not occur.

[0103] One of the substances (ligands) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to induce total internal reflection at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (SPR signal) is formed in a portion of the reflected light. The other substance (analyte) whose interaction is to be observed is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, causing a change in the refractive index of the solvent on the sensor chip surface. This change in refractive index causes a shift in the position of the SPR signal (conversely, when the bound molecule dissociates, the signal returns to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the ordinate, and displays the time-dependent change in mass as assay data (sensorgram). The amount of analyte bound to the ligand captured on the sensor chip surface (the amount of change in response on the sensorgram before and after analyte interaction) can be determined from the sensorgram. However, because the amount of binding also depends on the amount of ligand, comparisons must be performed under conditions using substantially the same amount of ligand. Kinetics, i.e., association rate constant (ka) and dissociation rate constant (kd) can be determined from the curve of the sensorgram, while affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also suitable for use in the BIACORE method. An example of inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0104] The terms "does not bind to CD3 and CD137 (4-1BB) simultaneously" or "does not bind to CD3 and CD137 (4-1BB) simultaneously" mean that an antigen-binding portion or antibody variable region of the present invention cannot bind to CD137 when bound to CD3, and conversely, an antigen-binding portion or antibody variable region cannot bind to CD3 when bound to CD137. Here, the phrase "does not bind to CD3 and CD137 simultaneously" also includes not cross-linking cells expressing CD3 with cells expressing CD137, or not simultaneously binding to CD3 and CD137 expressed on different cells. This phrase also includes cases where CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or when both are present on the same cell, and the variable region can simultaneously bind to both CD3 and CD137 but cannot simultaneously bind to CD3 and CD137 expressed on different cells. Such antibody variable regions are not particularly limited as long as they retain these functions. Examples include variable regions derived from IgG-type antibody variable regions in which some of the amino acids have been modified so that they bind to a desired antigen. The modified amino acids are selected from, for example, amino acids in antibody variable regions that bind to CD3 or CD137, so that the modification does not abolish antigen binding.

[0105] Here, the phrase "expressed on different cells" simply means that the antigens are expressed on separate cells, and such cell pairs may be of the same type, such as a T cell and another T cell, or may be of different types, such as a T cell and an NK cell.

[0106] Whether an antigen-binding molecule of the present invention "does not simultaneously bind to CD3 and CD137" can be confirmed by confirming that the antigen-binding molecule has binding activity to both CD3 and CD137; then, pre-binding either CD3 or CD137 to an antigen-binding molecule containing a variable region having this binding activity; and then determining the presence or absence of its binding activity to the other by the above-mentioned method. Alternatively, this can also be confirmed by determining whether the binding of the antigen-binding molecule to either CD3 or CD137 immobilized on an ELISA plate or sensor chip is inhibited by the addition of the other to the solution. In some embodiments, the binding of the antigen-binding molecule of the present invention to either CD3 or CD137 is inhibited by the binding of the antigen-binding molecule to the other by at least 50%, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more.

[0107] In one aspect, while one antigen (e.g., CD3) is immobilized, inhibition of binding of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by a method known in the prior art (i.e., ELISA, BIACORE, etc.). In another aspect, while CD137 is immobilized, inhibition of binding of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is performed, if binding is inhibited by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more, it is determined that the antigen-binding molecule of the present invention does not bind to CD3 and CD137 simultaneously.

[0108] In some embodiments, the concentration of the antigen injected as the analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen that is immobilized.

[0109] In a preferred mode, the concentration of the antigen injected as analyte is 100 times higher than the concentration of the other antigen to be immobilized, and binding is inhibited by at least 80%.

[0110] In one embodiment, the ratio of the KD value for the CD3 (analyte)-binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized)-binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the CD137 (immobilized) concentration by the KD value ratio (KD(CD3) / KD(CD137)) can be used for the above-mentioned competitive measurement. (For example, if the KD value ratio is 0.1, a concentration 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Furthermore, if the KD value ratio is 10, a concentration 100-fold, 500-fold, 1000-fold, or 2000-fold higher can be selected.)

[0111] In one aspect, while one antigen (e.g., CD3) is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by a method known in the prior art (i.e., ELISA, ECL, etc.). In another aspect, while CD137 is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is performed, if the binding signal is attenuated by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more, it is determined that the antigen-binding molecule of the present invention does not simultaneously bind to CD3 and CD137.

[0112] In some embodiments, the concentration of the antigen injected as the analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen that is immobilized.

[0113] In a preferred mode, the concentration of the antigen injected as analyte is 100 times higher than the concentration of the other antigen to be immobilized, and binding is inhibited by at least 80%.

[0114] In one embodiment, the ratio of the KD value for the CD3 (analyte)-binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized)-binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the CD137 (immobilized) concentration by this KD value ratio (KD(CD3) / KD(CD137)) can be used for the above measurement. (For example, if the KD value ratio is 0.1, a concentration 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Furthermore, if the KD value ratio is 10, a concentration 100-fold, 500-fold, 1000-fold, or 2000-fold higher can be selected.)

[0115] Specifically, for example, when using the ECL method, a biotin-labeled test antigen-binding molecule, CD3 labeled with a sulfo-tag (Ru complex), and unlabeled CD137 are prepared. If the test antigen-binding molecule can bind to both CD3 and CD137 but does not simultaneously bind to both CD3 and CD137, the mixture of the test antigen-binding molecule and labeled CD3 is added to a streptavidin-immobilized plate, and the luminescence signal of the sulfo-tag is detected in the absence of unlabeled CD137 by subsequent light emission. In contrast, the luminescence signal decreases in the presence of unlabeled CD137. The decrease in the luminescence signal can be quantified to determine the relative binding activity. This analysis can be similarly performed using labeled CD137 and unlabeled CD3.

[0116] In ALPHAScreen, a test antigen-binding molecule interacts with CD3 in the absence of competing CD137, generating a signal at 520-620 nm. Untagged CD137 competes with CD3 for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or similar techniques is known in the art. For example, CD3 can be tagged with GST by any suitable method, including fusing a polynucleotide encoding CD3 in frame with a polynucleotide encoding GST; expressing the resulting fusion gene in cells carrying a vector capable of expressing it; and then purifying it using a glutathione column. The resulting signal is preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego), which is fitted with a one-site competition model based on nonlinear regression analysis. This analysis can be performed similarly using tagged CD137 and untagged CD3.

[0117] Alternatively, a method using fluorescence resonance energy transfer (FRET) may be used. FRET is a phenomenon in which excitation energy is directly transferred between two closely spaced fluorescent molecules due to electronic resonance. When FRET occurs, the excitation energy of the donor (a fluorescent molecule in an excited state) is transferred to the acceptor (another fluorescent molecule located near the donor), causing the fluorescence emitted from the donor to be quenched (more precisely, the fluorescence lifetime is shortened), and instead, fluorescence is emitted from the acceptor. This phenomenon can be used to analyze whether an antibody simultaneously binds to CD3 and CD137. For example, when CD3 containing a fluorescent donor and CD137 containing a fluorescent acceptor simultaneously bind to a test antigen-binding molecule, the fluorescence of the donor is quenched, while fluorescence is emitted from the acceptor. Therefore, a change in fluorescence wavelength is observed. Such an antibody is confirmed to simultaneously bind to CD3 and CD137. On the other hand, if mixing CD3, CD137, and the test antigen-binding molecule does not change the fluorescence wavelength of the fluorescent donor bound to CD3, the test antigen-binding molecule can be considered to be an antigen-binding domain that can bind to CD3 and CD137 but does not bind to CD3 and CD137 simultaneously.

[0118] For example, a biotin-labeled test antigen-binding molecule is bound to streptavidin on donor beads, while glutathione S-transferase (GST)-tagged CD3 is bound to acceptor beads. The test antigen-binding molecule interacts with CD3 in the absence of a competing second antigen, generating a signal at 520-620 nm. The untagged second antigen competes with CD3 for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or similar is known in the art. For example, CD3 can be tagged with GST by any suitable method, including fusing a polynucleotide encoding CD3 in frame with a polynucleotide encoding GST; expressing the resulting fusion gene in cells carrying a vector capable of expressing it; and then purifying it using a glutathione column. The resulting signals are preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) fitted to a one-site competition model based on nonlinear regression analysis.

[0119] Tagging is not limited to GST tagging, and may be performed with any tag, including, but not limited to, histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc. Binding of the test antigen-binding molecule to the donor beads is not limited to binding using biotin-streptavidin reaction. In particular, when the test antigen-binding molecule contains Fc, possible methods include binding the test antigen-binding molecule via an Fc-recognizing protein such as protein A or protein G on the donor beads.

[0120] In addition, when CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or when both are present on the same cell, the ability of the variable region to simultaneously bind to CD3 and CD137, but not to CD3 and CD137 expressed on different cells, can also be assayed by methods known in the art.

[0121] Specifically, a test antigen-binding molecule that has been confirmed to be positive in ECL-ELISA for detecting simultaneous binding to CD3 and CD137 is also mixed with cells expressing CD3 and cells expressing CD137. Unless the antigen-binding molecule and these cells simultaneously bind to each other, it can be shown that the test antigen-binding molecule cannot simultaneously bind to CD3 and CD137 expressed on different cells. This assay can be performed, for example, by cell-based ECL-ELISA. CD3-expressing cells are immobilized on a plate in advance. After the test antigen-binding molecule binds to it, CD137-expressing cells are added to the plate. A different antigen expressed only on CD137-expressing cells is detected using an antibody labeled with a sulfo-tag against this antigen. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a signal is observed. If the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed.

[0122] Alternatively, this assay can be carried out by the ALPHAScreen method. Test antigen-binding molecule is mixed with the cells expressing CD3 bound to donor beads and the cells expressing CD137 bound to acceptor beads. When the antigen-binding molecule simultaneously binds to the two antigens expressed on the two cells, respectively, a signal is observed. When the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed.

[0123] Alternatively, this assay can be performed using Octet interaction analysis. First, cells expressing peptide-tagged CD3 are bound to a biosensor that recognizes the peptide tag. CD137-expressing cells and a test antigen-binding molecule are placed in a well and analyzed for interaction. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a large wavelength shift is observed due to the binding of the test antigen-binding molecule and the CD137-expressing cells to the biosensor. If the antigen-binding molecule does not simultaneously bind to these antigens, a small wavelength shift is observed due to the binding of only the test antigen-binding molecule to the biosensor.

[0124] Instead of these binding activity-based methods, biological activity-based assays can be performed. For example, CD3-expressing cells and CD137-expressing cells are mixed and cultured with a test antigen-binding molecule. When the antigen-binding molecule simultaneously binds to the two antigens, the two antigens expressed on the two cells are mutually activated via the test antigen-binding molecule. Therefore, changes in activation signals, such as increases in the phosphorylation levels downstream of each antigen, can be detected. Alternatively, cytokine production is induced as a result of activation. Therefore, the amount of cytokine produced can be measured, thereby confirming simultaneous binding to the two cells. Alternatively, cytotoxic activity against CD137-expressing cells can be induced as a result of activation. Alternatively, reporter gene expression can be induced by a promoter activated downstream of the CD137 or CD3 signaling pathway as a result of activation. Therefore, the cytotoxic activity or the amount of reporter protein produced can be measured, thereby confirming simultaneous binding to the two cells.

[0125] At least one disulfide bond In one aspect of the present invention, each of the first and second antigen-binding moieties comprises (via mutation, substitution, or insertion) at least one cysteine ​​residue, preferably in the CH1 region, which is capable of forming at least one disulfide bond between the first and second antigen-binding moieties. In certain embodiments, the cysteine ​​residue is present in the CH1 region of the antibody heavy chain constant region, for example, at a position selected from the group consisting of: 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 165, 167, 174, 176, 177, 178, 190, 191, 192, 194, 195, 197, 213, and 214 according to EU numbering in the CH1 region. In one embodiment, the first antigen-binding moiety and the second antigen-binding moiety each comprise one cysteine ​​residue at position 191 (by way of mutation, substitution, or insertion) according to EU numbering in the CH1 region, which is capable of forming one disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety.

[0126] In some embodiments of the above aspects, the "at least one bond" formed links the first and second antigen-binding moieties described above and can hold the two antigen-binding moieties (i.e., the first and second antigen-binding moieties as described above) in a spatially close position. Due to the link between the first and second antigen-binding moieties via a disulfide bond, the antigen-binding molecule of the present invention can hold the two antigen-binding moieties in a closer position than a control antigen-binding molecule that differs from the antigen-binding molecule of the present invention only in that it does not have an additional bond introduced between the two antigen-binding moieties. In some embodiments, the term "spatially close position" or "closer position" includes the meaning that the first and second antigen-binding domains described above are held at a shorter distance and / or with less mobility.

[0127] As a result, the two antigen-binding portions of the antigen-binding molecules of the present invention (i.e., the first and second antigen-binding portions described above) bind to antigens expressed on the same single cell. In other words, the two antigen-binding portions of the antigen-binding molecules of the present invention (i.e., the first and second antigen-binding portions described above) do not bind to antigens expressed on different cells, resulting in cross-linking of the different cells. In the present application, such an antigen-binding mode of the antigen-binding molecules of the present invention can be referred to as "cis-binding," whereas the antigen-binding mode of the antigen-binding molecules in which each of the two antigen-binding portions of the antigen-binding molecules binds to antigens expressed on different cells, resulting in cross-linking of the different cells, can be referred to as "trans-binding." In some embodiments, the antigen-binding molecules of the present invention primarily bind to antigens expressed on the same single cell in a "cis-binding" manner.

[0128] In some embodiments of the above aspects, due to the disulfide bond between the first and second antigen-binding moieties via the disulfide bond described above, the antigen-binding molecules of the present invention can reduce and / or prevent undesired cross-linking and activation of immune cells (e.g., T cells, NK cells, or DC cells, etc.). That is, in some embodiments of the present invention, the first antigen-binding moiety of the antigen-binding molecule of the present invention binds to any signaling molecule (e.g., a first antigen) expressed on immune cells such as T cells, and the second antigen-binding domain of the antigen-binding molecule of the present invention also binds to any signaling molecule (e.g., a first antigen, or a second antigen different from the first antigen) expressed on immune cells such as T cells. Thus, the first and second antigen-binding domains of the antigen-binding molecules of the present invention can bind to either a first or a second signaling molecule expressed on the same single immune cell, such as a T cell (i.e., in a cis-binding manner), or on different immune cells, such as a T cell (i.e., in a trans-binding manner). When the first antigen-binding domain and the second antigen-binding domain bind to signaling molecules expressed on different immune cells, e.g., T cells, in a trans-binding manner, the different immune cells, e.g., T cells, are cross-linked, and in certain circumstances, such cross-linking of immune cells, e.g., T cells, may cause undesired activation of immune cells, e.g., T cells.

[0129] On the other hand, in another embodiment of the antigen-binding molecule of the present invention, i.e., the antigen-binding molecule comprising a first antigen-binding moiety and a second antigen-binding moiety that are linked to each other via at least one disulfide bond in the CH1 region (position 191 according to EU numbering), both the first antigen-binding moiety and the second antigen-binding moiety can bind to a signaling molecule expressed on the same single immune cell, for example, a T cell, in a "cis-binding" manner, so that cross-linking of different immune cells, for example, T cells, via the antigen-binding molecule is reduced and undesired activation of the immune cell can be avoided.

[0130] In the present application, the above-mentioned characteristic of at least one disulfide bond in the CH1 region (e.g., position 191 according to EU numbering) linking the first and second antigen-binding moieties can be referred to by the abbreviation "LINC." Using this abbreviation, in some embodiments, the above-mentioned antigen-binding molecules of the present invention having at least one disulfide bond can be represented, for example, as "LINC format," "Dual / LINC," or "DLL3-Dual / LINC." Similarly, antigen-binding molecules of a first and second antigen-binding moiety that are not / are not yet linked to each other via at least one disulfide bond in the CH1 region (e.g., position 191 according to EU numbering) can be represented by the abbreviation "UnLINC."

[0131] Fab molecules A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of a light chain (a "Fab light chain").

[0132] to be fused "Fused" means that the components (eg, a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0133] "Crossover" Fab A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which either the variable or constant regions of the Fab heavy and Fab light chains have been exchanged; i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable region and a heavy chain constant region, and a peptide chain composed of a heavy chain variable region and a light chain constant region. For clarity, in a crossover Fab molecule in which the variable regions of the Fab light and Fab heavy chains have been exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant regions of the Fab light and Fab heavy chains have been exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0134] "Traditional" Fab In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain (VH-CH1) composed of the variable and constant regions of the heavy chain, and a light chain (VL-CL) composed of the variable and constant regions of the light chain. The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has, from N- to C-terminus, a variable region (VH), also known as the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also known as the heavy chain constant region. Similarly, each light chain has, from N- to C-terminus, a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a constant light (CL) domain, also known as the light chain constant region. Immunoglobulin heavy chains may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0135] affinity "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antigen-binding molecule or antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities may involve different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0136] Methods for determining affinity In certain embodiments, the antigen-binding molecules or antibodies provided herein have a binding affinity to their antigen of 1 μM or less, 120 nM or less, 100 nM or less, 80 nM or less, 70 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, 10 -8 M~10 -13 M, 10 -9 M~10 -13 In certain embodiments, the KD value of the antibody / antigen-binding molecule for CD3, CD137, or DLL3 falls within the range of 1 to 40, 1 to 50, 1 to 70, 1 to 80, 30 to 50, 30 to 70, 30 to 80, 40 to 70, 40 to 80, or 60 to 80 nM.

[0137] In one embodiment, KD is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured at the lowest concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0138] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. 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. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of protein binding. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C and a flow rate of approximately 25 μl / min. The association rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on The on-rate is calculated as a ratio of 10 to 10. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0139] Following the above-described methods for measuring the affinity of an antigen-binding molecule or antibody, those skilled in the art can measure the affinity of other antigen-binding molecules or antibodies for various antigens.

[0140] antibody The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0141] antibody fragment An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); in addition, WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting increased in vivo half-life. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). A single-domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0142] Antibody classes The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may 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 α, δ, ε, γ, and μ, respectively.

[0143] Unless otherwise indicated, amino acid residues in the light chain constant region are numbered herein according to Kabat et al., and numbering of amino acid residues in the heavy chain constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0144] Framework "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0145] Human Consensus Framework A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0146] chimeric antibodies The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy and / or light chain variable region is derived from a particular source or species, while the remainder of the heavy and / or light chain variable region is derived from a different source or species.

[0147] humanized antibodies A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0148] Human antibodies A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. A "human antibody variable region" refers to the variable region of a human antibody.

[0149] Polynucleotides (nucleic acids) "Polynucleotide" or "nucleic acid," used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can include modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps," substitutions of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralens, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present on the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to generate additional linkages to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by: P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0150] Isolated (nucleic acid) An "isolated" nucleic acid molecule is one that is separated from a component of its original environment. Isolated nucleic acid molecules further include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0151] vector As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. Some vectors are capable of conferring expression of a nucleic acid to which they are operatively linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells using viruses or electroporation. However, vector introduction is not limited to in vitro methods. For example, vectors can also be introduced directly into a subject using in vivo methods.

[0152] In another aspect of the present invention, a vector containing a nucleic acid molecule encoding an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure that can bind to CD3 and CD137 but not simultaneously, or an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure, can be introduced into a subject so that the antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure can be directly expressed in the subject. An example of a vector that can be used is, but is not limited to, an adenovirus. A nucleic acid molecule encoding an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure can be directly administered to a subject, or a nucleic acid molecule encoding an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure can be transferred to a subject via electroporation, or cells containing a nucleic acid molecule encoding an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure that is to be expressed and secreted can be administered to a subject, allowing the antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure to be continuously expressed and secreted in the subject.

[0153] host cell The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0154] specificity "Specific" means that a molecule that specifically binds to one or more binding partners does not exhibit any significant binding to molecules other than the partners. Furthermore, "specific" is also used when an antigen-binding site is specific to a particular epitope among multiple epitopes contained in an antigen. When an antigen-binding molecule specifically binds to an antigen, it is also described as "the antigen-binding molecule has / exhibits specificity for / for the antigen." When the epitope to which the antigen-binding site binds is contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding site can bind to various antigens that have the epitope.

[0155] antibody fragment "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0156] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein.

[0157] Variable fragment (Fv) As used herein, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding site consisting of a pair of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). In the Fv prepared from the periplasmic fraction, the VH and VL are associated in such a manner that they bind to antigens.

[0158] scFv, single chain antibodies, and sc(Fv) 2 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 heavy and light chains but no constant region. Generally, single-chain antibodies further contain a polypeptide linker between the VH and VL domains that allows the formation of the desired structure that will enable antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)." See also International Publication WO 1988 / 001649, U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies can be bispecific and / or humanized.

[0159] scFv is a single-chain low molecular weight antibody in which the VH and VL that form the Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). The peptide linker can hold the VH and VL in close proximity.

[0160] sc(Fv)2 is a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). The two VHs and two VLs may be derived from different monoclonal antibodies. Suitable examples of such sc(Fv)2 include bispecific sc(Fv)2s that recognize two epitopes present in a single antigen, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2s can be produced by methods known to those skilled in the art. For example, sc(Fv)2s can be produced by linking scFvs with a linker such as a peptide linker.

[0161] As used herein, sc(Fv)2 comprises two VH units and two VL units arranged in the following order, starting from the N-terminus of the single-chain polypeptide: VH, VL, VH, VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). The order of the two VH units and two VL units is not limited to the above configuration and may be arranged in any order. Examples of configurations are listed below. [VL]-linker-[VH]-linker-[VH]-linker-[VL] [VH]-linker-[VL]-linker-[VL]-linker-[VH] [VH]-linker-[VH]-linker-[VL]-linker-[VL] [VL]-linker-[VL]-linker-[VH]-linker-[VH] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0162] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352. Those skilled in the art can follow these descriptions to appropriately prepare the desired sc(Fv)2 for producing the polypeptide complexes disclosed herein.

[0163] Furthermore, the antigen-binding molecules or antibodies of the present disclosure may be conjugated with carrier polymers such as PEG or organic compounds such as anticancer drugs. Alternatively, a glycosylation sequence is suitably inserted into the antigen-binding molecules or antibodies so that the sugar chains exert the desired effect.

[0164] Linkers used to link antibody variable regions include any peptide linker that can be introduced by genetic engineering, synthetic linkers, and linkers such as those disclosed in Protein Engineering, 9 (3), 299-305, 1996. However, in the present disclosure, peptide linkers are preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The length is preferably 5 amino acids or more (although not particularly limited, the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, the lengths of these linkers may all be the same or different.

[0165] For example, such peptide linkers include: Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 91), Ser-Gly-Gly-Gly (SEQ ID NO: 92), Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 93), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 94), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 95), Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 96), Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 97), Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 98), (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 93)), and (Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 94))n. Here, n is an integer equal to or greater than 1. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0166] Synthetic linkers (chemical cross-linkers) are commonly used to cross-link peptides, examples of which include: N-hydroxysuccinimide (NHS), Disuccinimidyl suberate (DSS), Bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), Dithiobis(sulfosuccinimidyl propionate) (DTSSP), Ethylene glycol bis(succinimidyl succinate) (EGS), Ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), Disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES) These crosslinking agents are commercially available.

[0167] Three linkers are usually required to link four antibody variable regions, and the linkers used may be of the same type or different types.

[0168] Fab, F(ab') 2 , and Fab' "Fab" consists of one light chain and the CH1 domain and variable region of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0169] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, digesting the immunoglobulin (monoclonal antibody) near the disulfide bond between the hinge regions of the two heavy chains. For example, papain cleaves IgG upstream of the disulfide bond between the hinge regions of the two heavy chains, producing two homologous antibody fragments in which an L chain containing a VL (light chain variable region) and a CL (light chain constant region) is linked by a disulfide bond at their C-terminal regions to an H chain fragment containing a VH (heavy chain variable region) and a CHγ1 (γ1 region of the heavy chain constant region). Each of these two homologous antibody fragments is called Fab'.

[0170] "F(ab')2" consists of two light chains and two heavy chains containing constant regions, such as the CH1 domain and a portion of the CH2 domain, such that disulfide bonds are formed between the two heavy chains. The F(ab')2 disclosed herein can be conveniently produced as follows: a monoclonal whole antibody or the like containing the desired antigen-binding site is partially digested with a protease such as pepsin, and the Fc fragment is removed by adsorption onto a protein A column. The protease used is not particularly limited, as long as it can selectively cleave the whole antibody to yield F(ab')2 under appropriately set enzymatic reaction conditions, such as pH. Examples of such proteases include pepsin and ficin.

[0171] Single Domain Antibodies As used herein, the term "single-domain antibody" is not limited by its structure, as long as the domain can exhibit antigen-binding activity by itself. While typical antibodies, such as IgG antibodies, exhibit antigen-binding activity when their variable regions are formed by pairing VH and VL, it is known that the domain structure of a single-domain antibody can exhibit antigen-binding activity by itself, without pairing with another domain. Single-domain antibodies typically have a relatively low molecular weight and exist in the form of a monomer.

[0172] Examples of single domain antibodies include, but are not limited to, antigen-binding molecules such as camelid VHH and shark VNAR, which naturally lack light chains, and antibody fragments containing the entire or a portion of an antibody VH domain or the entire or a portion of an antibody VL domain. Examples of single domain antibodies, which are antibody fragments containing the entire or a portion of an antibody VH domain or an antibody VL domain, include, but are not limited to, artificially prepared single domain antibodies derived from human antibody VH or human antibody VL, such as those described in U.S. Patent No. 6,248,516 B1. In some embodiments of the present invention, a single domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0173] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies or by immunization of animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals carrying genes capable of producing single-domain antibodies. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals carrying genes capable of producing single-domain antibodies include, but are not limited to, the transgenic animals described in International Publication No. WO2015 / 143414 and U.S. Patent Publication No. US2011 / 0123527 A1. The framework sequences of single-domain antibodies obtained from such animals may be converted to human germline sequences or sequences similar thereto to obtain humanized single-domain antibodies. Humanized single-domain antibodies (e.g., humanized VHHs) are also an embodiment of the single-domain antibodies of the present invention.

[0174] Alternatively, single domain antibodies can be obtained by ELISA, panning, or the like from a polypeptide library containing single domain antibodies. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78); and Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764: 8 (1307-1319)), antibody libraries obtained by immunization of various animals (e.g., Journal of Applied Microbiology 2014 117: 2 (528-536)), and synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21: 1 (35-43); Journal of Biological Chemistry 2016 291:24 (12641-12657); and AIDS 2016 30: 11 (1691-1701)). Fc area As used herein, the term "Fc region" or "Fc domain" refers to a region of an antibody molecule comprising a hinge or a portion thereof, and a fragment consisting of the CH2 and CH3 domains. The Fc region of an IgG class refers, for example, but is not limited to, the region from cysteine ​​226 (EU numbering, also referred to herein as the EU index) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus. The Fc region can be obtained, for example, by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, followed by re-elution of the fraction adsorbed to a protein A or protein G column. The protease is not particularly limited, as long as it can digest a full-length antibody to form Fab or F(ab')2 under appropriately selected enzyme reaction conditions (e.g., pH). Examples include pepsin and papain.

[0175] In the present invention, for example, an Fc region derived from a native IgG can be used as the "Fc region" of the present invention. Here, native IgG refers to a polypeptide that contains the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by the immunoglobulin γ gene. Native human IgG refers to, for example, native human IgG1, native human IgG2, native human IgG3, or native human IgG4. Native IgG also includes naturally occurring variants thereof. Multiple allotype sequences based on genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, as constant region sequences for human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of these can be used in the present invention. In particular, the sequence of human IgG1 may have DEL or EEM as the amino acid sequence at positions 356 to 358 (EU numbering).

[0176] In some embodiments, the Fc domain of a multispecific antigen-binding molecule is composed of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule.For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises CH2 and CH3 IgG heavy chain constant domains.The two subunits of an Fc domain can stably associate with each other.In one embodiment, the multispecific antigen-binding molecule described herein comprises no more than one Fc domain.

[0177] In one embodiment described herein, the Fc domain of the multispecific antigen-binding molecule is an IgG Fc domain. In another embodiment, the Fc domain is an IgG1 Fc domain. In a further specific embodiment, the Fc domain is a human IgG1 Fc region.

[0178] In certain embodiments, the Fc domain of the multispecific antigen-binding molecule is composed of a first and a second Fc region subunit capable of stable association, and the Fc domain exhibits reduced binding affinity for human Fcγ receptors compared to native human IgG1 Fc domains.

[0179] In certain embodiments, the Fc domain of the multispecific antigen-binding molecule described herein comprises a modification that promotes association of the first and second subunits of the Fc domain. In certain embodiments, the modification is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain, as described in more detail below.

[0180] In a particular embodiment, the Fc domain is composed of a first and a second Fc region subunit capable of stable association and exhibits reduced binding affinity for human Fcγ receptors compared to a native human IgG1 Fc domain, wherein the first Fc region subunit is selected from the group consisting of: (a1) Fc region polypeptide containing the mutations L234A, L235A; (a2) Fc region polypeptide containing mutations L234A, L235A, and N297A; (a3) Fc region polypeptide containing mutations L234A, L235A, N297A, S354C, and T366W and The second Fc region polypeptide may comprise: (a4) Fc region polypeptide containing mutations L234A, L235A; (a5) an Fc region polypeptide comprising the mutations L234A, L235A, and N297A; and (a6) Fc region polypeptide containing the mutations L234A, L235A, N297A, Y349C, T366S, L368A, and Y407V (amino acid positions are numbered using EU index numbering).

[0181] In certain embodiments, the Fc domain of the multispecific antigen-binding molecule described herein exhibits enhanced FcRn-binding activity under acidic pH conditions (e.g., pH 5.8) compared to that of the Fc region of native IgG. Such an Fc domain comprises, for example, Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, according to EU numbering. In some embodiments, the Fc domain comprises Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering. In some embodiments, the Fc domain further comprises Ile or Leu at position 428; and / or Ile, Leu, Val, Thr, or Phe at position 436, according to EU numbering. In some embodiments, the Fc domain comprises the following, according to EU numbering: (a)N434A / Q438R / S440E; (b) N434A / Q438R / S440D; (c)N434A / Q438K / S440E; (d)N434A / Q438K / S440D; (e)N434A / Y436T / Q438R / S440E; (f)N434A / Y436T / Q438R / S440D; (g)N434A / Y436T / Q438K / S440E; (h)N434A / Y436T / Q438K / S440D; (i)N434A / Y436V / Q438R / S440E; (j)N434A / Y436V / Q438R / S440D; (k)N434A / Y436V / Q438K / S440E; (l)N434A / Y436V / Q438K / S440D; (m)N434A / R435H / F436T / Q438R / S440E; (n)N434A / R435H / F436T / Q438R / S440D; (o)N434A / R435H / F436T / Q438K / S440E; (p)N434A / R435H / F436T / Q438K / S440D; (q)N434A / R435H / F436V / Q438R / S440E; (r)N434A / R435H / F436V / Q438R / S440D; (s)N434A / R435H / F436V / Q438K / S440E; (t)N434A / R435H / F436V / Q438K / S440D; (u)M428L / N434A / Q438R / S440E; (v)M428L / N434A / Q438R / S440D; (w)M428L / N434A / Q438K / S440E; (x)M428L / N434A / Q438K / S440D; (y)M428L / N434A / Y436T / Q438R / S440E; (z)M428L / N434A / Y436T / Q438R / S440D; (aa)M428L / N434A / Y436T / Q438K / S440E; (ab)M428L / N434A / Y436T / Q438K / S440D; (ac)M428L / N434A / Y436V / Q438R / S440E; (ad)M428L / N434A / Y436V / Q438R / S440D; (ae)M428L / N434A / Y436V / Q438K / S440E; (af)M428L / N434A / Y436V / Q438K / S440D; (ag)L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (ah)L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E The amino acid substitutions include a combination of amino acid substitutions selected from the group consisting of:

[0182] In some embodiments, the Fc domain of the multispecific antigen-binding molecule comprises the following amino acid substitution combination: M428L / N434A / Q438R / S440E. In some embodiments, the Fc domain is an IgG Fc domain, preferably a human IgG Fc domain, more preferably a human IgG1 Fc domain. In certain embodiments, the Fc domain of the multispecific antigen-binding molecule comprises either: (a) a first Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 100 and a second Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 111; and (b) a first Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 99 and a second Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 109.

[0183] Fc region with reduced Fcγ receptor binding activity As used herein, "reduced Fcγ receptor-binding activity" means that the competitive activity of a test antigen-binding molecule or antibody is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, and particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the competitive activity of a control antigen-binding molecule or antibody, for example, based on the above-mentioned analytical methods.

[0184] Antigen-binding molecules or antibodies containing the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be suitably used as a control antigen-binding molecule or antibody. The Fc domain structures are shown in SEQ ID NO: 85 (A is added to the N-terminus of RefSeq Accession No. AAC82527.1), SEQ ID NO: 86 (A is added to the N-terminus of RefSeq Accession No. AAB59393.1), SEQ ID NO: 87 (A is added to the N-terminus of RefSeq Accession No. CAA27268.1), and SEQ ID NO: 88 (A is added to the N-terminus of RefSeq Accession No. AAB59394.1). Furthermore, when antigen-binding molecules or antibodies containing Fc domain variants of a specific antibody isotype are used as test substances, the effect of the mutations of the variants on Fcγ receptor binding activity can be evaluated using an antigen-binding molecule or antibody containing an Fc domain of the same isotype as a control. As described above, an antigen-binding molecule or antibody comprising an Fc domain mutant whose Fcγ receptor binding activity has been determined to be reduced is suitably prepared.

[0185] Such known mutants include, for example, a mutant having a deletion of amino acids 231A to 238S (EU numbering) (WO 2009 / 011941), as well as the mutants C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54); C226S, C229S, E233P, L234V, and L235A (Blood (2007) 109, 1185-1192).

[0186] Specifically, preferred antigen-binding molecules or antibodies include those comprising an Fc domain having at least one amino acid mutation (e.g., substitution) selected from the following amino acid positions forming the Fc domain of an antibody of a particular isotype: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering). The antibody isotype from which the Fc domain is derived is not particularly limited, and an appropriate Fc domain derived from a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be used. Preferably, an Fc domain derived from an IgG1 antibody is used.

[0187] Preferred antigen-binding molecules or antibodies include, for example, any one of the following substitutions, whose positions are designated by EU numbering in amino acids forming the Fc domain of an IgG1 antibody (each number represents the position of an amino acid residue in EU numbering; and the one-letter amino acid code before the number represents the amino acid residue before substitution, and the one-letter amino acid code after the number represents the amino acid residue after substitution): (a) L234F, L235E, P331S; (b) C226S, C229S, P238S; (c) C226S, C229S; or (d)C226S, C229S, E233P, L234V, L235A and those having an Fc domain with a deletion of the amino acid sequence at positions 231 to 238.

[0188] Furthermore, preferred antigen-binding molecules or antibodies include any one of the following substitutions, whose positions are designated by EU numbering in the amino acids forming the Fc domain of IgG2 antibodies: (e) H268Q, V309L, A330S, and P331S; (f)V234A; (g)G237A; (h) V234A and G237A; (i) A235E and G237A; or (j) V234A, A235E, and G237A Each number represents the position of an amino acid residue according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution, and the single-letter amino acid code following the number represents the amino acid residue after substitution.

[0189] Furthermore, preferred antigen-binding molecules or antibodies include any one of the following substitutions, whose positions are designated by EU numbering in the amino acids forming the Fc domain of IgG3 antibodies: (k)F241A; (l) D265A; or (m)V264A Each number represents the position of an amino acid residue according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution, and the single-letter amino acid code following the number represents the amino acid residue after substitution.

[0190] Furthermore, preferred antigen-binding molecules or antibodies include any one of the following substitutions, whose positions are designated by EU numbering in the amino acids forming the Fc domain of IgG4 antibodies: (n) L235A, G237A, and E318A; (o) L235E; or (p)F234A and L235A Each number represents the position of an amino acid residue according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution, and the single-letter amino acid code following the number represents the amino acid residue after substitution.

[0191] Other preferred antigen-binding molecules or antibodies include, for example, those comprising an Fc domain in which any amino acid at position 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) in the amino acids forming the Fc domain of an IgG1 antibody has been substituted with an amino acid at the corresponding position (EU numbering) in the corresponding IgG2 or IgG4.

[0192] Preferred antigen-binding molecules or antibodies also include, for example, those comprising an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 (EU numbering) in the amino acid sequence that forms the Fc domain of an IgG1 antibody have been substituted with other amino acids. The type of substituted amino acid is not particularly limited; however, antigen-binding molecules or antibodies comprising an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 have been substituted with alanine are particularly preferred.

[0193] Preferred antigen-binding molecules or antibodies also include, for example, those comprising an Fc domain in which the amino acid at position 265 (EU numbering) of the amino acids forming the Fc domain of an IgG1 antibody has been substituted with another amino acid. The type of substituted amino acid is not particularly limited; however, antigen-binding molecules or antibodies comprising an Fc domain in which the amino acid at position 265 has been substituted with alanine are particularly preferred.

[0194] Fc receptors The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0195] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

[0196] In vivo binding to human FcRn and plasma half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with variant Fc regions are administered. WO2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0197] Fcγ receptor Fcγ receptor refers to a receptor that can bind to the Fc domain of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody, and includes all members of a family of proteins substantially encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); as well as unidentified human Fcγ receptors, Fcγ receptor isoforms, and all their allotypes. However, Fcγ receptors are not limited to these examples. Fcγ receptors include, but are not limited to, those derived from humans, mice, rats, rabbits, and monkeys. Fcγ receptors may be derived from any organism. Mouse Fcγ receptors include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as unidentified mouse Fcγ receptors, Fcγ receptor isoforms, and allotypes thereof. Preferred Fcγ receptors include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16).The polynucleotide and amino acid sequences of FcγRI are set forth in RefSeq accession numbers NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIA are set forth in RefSeq accession numbers BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIB are set forth in RefSeq accession numbers BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIA are set forth in RefSeq accession numbers BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIB are set forth in RefSeq accession numbers BC128562.1 and AAI28563.1, respectively. In addition to the FACS and ELISA formats described above, whether an Fcγ receptor has binding activity to the Fc domain of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be assessed by ALPHA screens (amplified luminescence proximity homogeneous assays), surface plasmon resonance (SPR)-based BIACORE methods, and others (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0198] On the other hand, "Fc ligand" or "effector ligand" refers to a molecule, preferably a polypeptide, that binds to an antibody Fc domain to form an Fc / Fc ligand complex. The molecule may be derived from any organism. Binding of an Fc ligand to Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, Fcγ receptors, Fcα receptors, Fcβ receptors, FcRn, C1q, and C3, mannan-binding lectin, mannose receptor, Staphylococcus protein A, Staphylococcus protein G, and viral Fcγ receptors. Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors homologous to Fcγ receptors (Davis et al., (2002) Immunological Reviews 190, 123-136). Fc ligands also include unidentified molecules that bind to Fc.

[0199] Fcγ receptor binding activity Impaired binding activity of the Fc domain to any of the Fcγ receptors, FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB, can be assessed using the FACS and ELISA formats described above, as well as ALPHA screens (amplified luminescence proximity homogeneous assays) and surface plasmon resonance (SPR)-based BIACORE methods (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0200] The ALPHA screen is performed using ALPHA technology, which uses two types of beads: donor beads and acceptor beads, and is based on the following principle: A luminescent signal is detected only if the molecule linked to the donor bead biologically interacts with the molecule linked to the acceptor bead and the two beads are located in close proximity. A photosensitizer in the donor bead is excited by laser light and converts oxygen around the bead into excited singlet oxygen. When the singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, it induces a chemiluminescent reaction in the acceptor bead. This reaction ultimately produces light. If the molecule linked to the donor bead does not interact with the molecule linked to the acceptor bead, the singlet oxygen generated by the donor bead will not reach the acceptor bead, and the chemiluminescent reaction will not occur.

[0201] For example, a biotin-labeled antigen-binding molecule or antibody is immobilized on donor beads, and an Fcγ receptor tagged with glutathione S-transferase (GST) is immobilized on acceptor beads. In the absence of an antigen-binding molecule or antibody containing a competitive mutant Fc domain, the Fcγ receptor interacts with an antigen-binding molecule or antibody containing a wild-type Fc domain, resulting in a signal at 520–620 nm. The antigen-binding molecule or antibody containing an untagged mutant Fc domain competes with the antigen-binding molecule or antibody containing the wild-type Fc domain for interaction with the Fcγ receptor. Relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from competition. Methods for biotinylating antigen-binding molecules or antibodies, such as antibodies, using sulfo-NHS-biotin are well known. A suitable method for adding a GST tag to an Fcγ receptor involves fusing a polypeptide encoding an Fcγ receptor and a polypeptide encoding GST in frame, expressing the gene using cells transfected with a vector carrying the fusion gene, and then purifying the gene using a glutathione column. The induced signal can be preferably analyzed by fitting to a one-site competition model based on nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad; San Diego).

[0202] One of the substances to be observed for interaction is immobilized on a thin gold film on a sensor chip as a ligand. When light is shone on the back of the sensor chip so that total reflection occurs at the interface between the thin gold film and the glass, the intensity of the reflected light is partially reduced at a specific site (SPR signal). The other substance to be observed for interaction is injected onto the surface of the sensor chip as an analyte. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases. This changes the refractive index of the solvent on the sensor chip surface. This change in refractive index causes a shift in the position of the SPR signal (conversely, upon dissociation, the signal shifts back to its original position). In the Biacore system, the amount of this shift (i.e., the change in mass on the sensor chip surface) is plotted on the vertical axis, and the change in mass over time is displayed as measurement data (sensorgram). Kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the sensorgram curve, and affinity (KD) is determined from the ratio of these two constants. Inhibition assays are preferably used in the BIACORE method. An example of such an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0203] Production and purification of multispecific antibodies The multispecific antigen-binding molecules described herein comprise two antigen-binding moieties (e.g., a "first antigen-binding moiety" and a "second antigen-binding moiety") with different binding specificities, both capable of binding to CD3 and CD137, and a "third antigen-binding moiety" capable of binding to another antigen, each of which is ultimately fused to one or the other of two subunits of the Fc domain; thus, the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization provides the possibility of multiple combinations of the two polypeptides. Therefore, to improve the yield and purity of multispecific antigen-binding molecules during recombinant production, it is advantageous to introduce modifications into the Fc domain of the multispecific antigen-binding molecule that promote the association of the desired polypeptides.

[0204] Therefore, in certain embodiments, the Fc domain of the multispecific antigen-binding molecule described herein comprises a modification that promotes the association of the first subunit and the second subunit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0205] In a particular embodiment, the modification is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0206] Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, so that the protrusion ("knob") can be positioned in the cavity ("hole"), promoting heterodimer formation and preventing homodimer formation. The protrusion is constructed by replacing small amino acid side chains in the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0207] Thus, in a particular embodiment, in the CH3 domain of a first subunit of the Fc domain of a multispecific antigen-binding molecule, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned in a cavity in the CH3 domain of a second subunit, and in the CH3 domain of a second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned.

[0208] The protrusions and cavities can be made by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis, or by peptide synthesis.

[0209] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0210] In yet a further embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced with a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0211] In other embodiments, other techniques for promoting the association of desired combinations of H chains and L chains can be applied to the multispecific antigen-binding molecules of the present invention.

[0212] For example, in the association of multispecific antibodies, a technique can be applied that suppresses undesired association of antibody heavy chains by introducing electrostatic repulsion at the interface of the second or third constant region (CH2 or CH3) of the antibody heavy chain (WO2006 / 106905).

[0213] In the technique for suppressing unintended H chain association by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues that contact the interface of the other H chain constant region include regions corresponding to residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in the CH3 region.

[0214] More specifically, examples include antibodies comprising two types of H chain CH3 regions, in which one to three pairs of amino acid residues selected from the pairs of amino acid residues shown in (1) to (3) below in the first H chain CH3 region have the same electric charge: (1) the amino acid residues at EU numbering positions 356 and 439 in the H chain CH3 region, (2) the amino acid residues at EU numbering positions 357 and 370 in the H chain CH3 region, and (3) the amino acid residues at EU numbering positions 399 and 409 in the H chain CH3 region.

[0215] Furthermore, the antibody may be an antibody in which pairs of amino acid residues in a second H chain CH3 region different from the first H chain CH3 region are selected from the pairs of amino acid residues (1) to (3) above, and one to three pairs of amino acid residues corresponding to the pairs of amino acid residues (1) to (3) with the same charge in the first H chain CH3 region have charges opposite to those of the corresponding amino acid residues in the first H chain CH3 region.

[0216] The amino acid residues shown in (1) to (3) above are close to each other when associated. Those skilled in the art can find the positions corresponding to the amino acid residues shown in (1) to (3) above in the desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can appropriately modify the amino acid residues at these positions.

[0217] In the above-described antibody, the "charged amino acid residue" is preferably selected from, for example, amino acid residues included in any one of the following groups: (a) glutamic acid (E) and aspartic acid (D), and (b) Lysine (K), arginine (R), and histidine (H).

[0218] In the above-mentioned antibodies, the phrase "having the same charge" means, for example, that two or more amino acid residues are all selected from amino acid residues included in either one of the above groups (a) and (b). The phrase "having opposite charges" means, for example, that when at least one amino acid residue among two or more amino acid residues is selected from amino acid residues included in either one of the above groups (a) and (b), the remaining amino acid residue is selected from amino acid residues included in the other group.

[0219] In a preferred embodiment, the above-mentioned antibody may have the first H chain CH3 region and the second H chain CH3 region cross-linked by a disulfide bond.

[0220] In the present invention, the amino acid residues to be modified are not limited to those in the antibody variable region or constant region described above. Those skilled in the art can identify amino acid residues that form an interface in a mutant polypeptide or heteromultimer by homology modeling using commercially available software, and then modify the amino acid residues at these positions to control association.

[0221] In addition, other known techniques can also be used to form the multispecific antibodies of the present invention. A strand-exchange engineered domain CH3 can be generated by substituting a portion of one antibody H chain CH3 with a corresponding IgA-derived sequence and then introducing the corresponding IgA-derived sequence into the complementary portion of the other antibody H chain CH3. This allows efficient induction of association between polypeptides with different sequences through complementary association of CH3s (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently form the desired multispecific antibodies.

[0222] In addition, for the formation of multispecific antigen-binding molecules, various techniques can be used, including antibody production techniques utilizing the association of antibody CH1 and CL and VH and VL, such as those described in WO2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb;32(2):191-8; techniques for producing bispecific antibodies by combining separately prepared monoclonal antibodies (Fab Arm Exchange), such as those described in WO2008 / 119353 and WO2011 / 131746; techniques for controlling the association between antibody heavy chain CH3s, such as those described in WO2012 / 058768 and WO2013 / 063702; techniques for producing multispecific antibodies composed of two types of light chains and one type of heavy chain, such as those described in WO2012 / 023053; and techniques for producing multispecific antibodies composed of two types of light chains and one type of heavy chain, such as those described in Christoph et al. (Nature Biotechnology Vol. 31, pp. 753-758). Alternatively, a technique for producing a multispecific antibody using two bacterial cell lines each expressing one half of an antibody chain, including one H chain and one L chain, as described by [Schmidt et al. (2013)], may be used.

[0223] Alternatively, even if the desired multispecific antibody cannot be efficiently formed, it can still be obtained by separating and purifying the desired multispecific antibody from the produced antibodies. For example, a method has been reported in which amino acid substitutions are introduced into the variable regions of two types of H chains to impart a difference in isoelectric point, thereby enabling the purification of two types of homoantibodies and the desired heteroantibody by ion exchange chromatography (WO2007114325). Previously reported methods for purifying heteroantibodies include using Protein A to purify a heterodimerized antibody comprising a mouse IgG2a H chain that binds to Protein A and a rat IgG2b H chain that does not bind to Protein A (WO98050431 and WO95033844). Furthermore, by using H chains in which the amino acid residues at EU numbering positions 435 and 436, which are the binding sites between IgG and Protein A, are substituted with amino acids such as Tyr and His that confer different Protein A affinities, or by using H chains with different Protein A affinities, the interaction between each H chain and Protein A can be changed, and then using a Protein A column, it is possible to efficiently purify only the heterodimerized antibody.

[0224] Furthermore, Fc regions with reduced C-terminal heterogeneity can be used as appropriate as Fc regions of the present invention. More specifically, the present invention provides Fc regions generated by deleting glycine at position 446 and lysine at position 447 (EU numbering) in the amino acid sequences of two polypeptides that constitute the Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0225] Multispecific antigen-binding molecules prepared as described herein may be purified by techniques known in the art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size-exclusion chromatography. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. Affinity chromatography purification can use an antibody, ligand, receptor, or antigen to which the multispecific antigen-binding molecule binds. For example, affinity chromatography purification of the multispecific antigen-binding molecules of the present invention can use a matrix with Protein A or Protein G. Sequential Protein A or G affinity chromatography and size-exclusion chromatography can be used to isolate the multispecific antigen-binding molecules. The purity of the multispecific antigen-binding molecules can be determined by any of a variety of well-known analytical methods, including gel electrophoresis and high-pressure liquid chromatography.

[0226] Antibody-dependent cell-mediated cytotoxicity "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcR) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0227] Complement-dependent cytotoxicity "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to the corresponding antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased C1q binding ability are described, for example, in U.S. Patent No. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0228] T cell-dependent cytotoxicity "T cell-dependent cytotoxicity" or "TDCC" refers to a form of cytotoxicity in which an antigen-binding molecule binds to both an antigen expressed on a target cell and another antigen expressed on a T cell, redirecting the T cell to the vicinity of the target cell, so that cytotoxicity against the target cell is induced by the T cell. An in vitro TDCC assay, a method for assessing T cell-dependent cytotoxicity, is also described in the "Measurement of T cell-dependent cytotoxicity" section of this specification.

[0229] Measurement of T cell-dependent cytotoxicity In embodiments in which an antigen-binding molecule binds to both DLL3 and CD3 / CD137, the following methods are preferably used to assess or determine T cell-dependent cytotoxicity (TDCC) induced by contacting an antigen-binding molecule of the present disclosure with a DLL3-expressing cell to which the antigen-binding site of the antigen-binding molecule of the present disclosure binds. In vitro methods for assessing or determining cytotoxic activity include methods for determining the activity of cytotoxic T cells, etc. Whether an antigen-binding molecule of the present disclosure has the activity to induce T cell-mediated cytotoxicity can be determined by known methods (see, for example, Current Protocols in Immunology, Chapter 7. Immunologic Studies in Humans, Editor, John E., Coligan et al., John Wiley & Sons, Inc., (1993)). In cytotoxicity assays, an antigen-binding molecule capable of binding to an antigen different from DLL3 and not expressed in cells, and to CD3 / CD137, is used as a control antigen-binding molecule. The control antigen-binding molecule is assayed in the same manner. Activity is then evaluated by testing whether the antigen-binding molecules of the present disclosure exhibit stronger cytotoxic activity than that of a control antigen-binding molecule.

[0230] Meanwhile, in vivo anti-tumor efficacy can be evaluated or determined, for example, by the following method: Cells expressing an antigen to which the antigen-binding site of an antigen-binding molecule of the present disclosure binds are intradermally or subcutaneously implanted into a non-human animal subject. Then, starting from the day of implantation or thereafter, a test antigen-binding molecule is administered intravenously or intraperitoneally daily or at intervals of several days. Tumor size is measured over time. The difference in change in tumor size can be defined as cytotoxic activity. As in the in vitro assay, a control antigen-binding molecule is administered. If the tumor size is smaller in the group administered with the antigen-binding molecule of the present disclosure than in the group administered with the control antigen-binding molecule, it can be determined that the antigen-binding molecule of the present disclosure has cytotoxic activity.

[0231] To evaluate or determine the effect of contact with the antigen-binding molecule of the present disclosure on inhibiting the proliferation of cells expressing an antigen to which the antigen-binding site of the antigen-binding molecule binds, the MTT method and measurement of intracellular isotope-labeled thymidine incorporation are preferably used.On the other hand, to evaluate or determine the activity of inhibiting cell proliferation in vivo, preferably, the same method described above for evaluating or determining in vivo cytotoxic activity can be used.

[0232] The TDCC of an antibody or antigen-binding molecule of the present disclosure can be assessed by any suitable method known in the art. For example, TDCC can be measured by a lactate dehydrogenase (LDH) release assay. In this assay, target cells (e.g., DLL3-expressing cells) are incubated with T cells (e.g., PBMCs) in the presence of a test antibody or antigen-binding molecule, and the LDH activity released from target cells killed by the T cells is measured using an appropriate reagent. Typically, cytotoxic activity is calculated as the ratio of LDH activity generated by incubation with the antibody or antigen-binding molecule to LDH activity generated by 100% killing of target cells (e.g., lysed by treatment with Triton-X). If the cytotoxic activity calculated as described above is higher, the test antibody or antigen-binding molecule is determined to have a higher TDCC.

[0233] Additionally or alternatively, TDCC can be measured by a real-time cell proliferation inhibition assay. In this assay, target cells (e.g., DLL3-expressing cells) are incubated with T cells (e.g., PBMCs) in the presence of a test antibody or antigen-binding molecule in a 96-well plate, and target cell proliferation is monitored by methods known in the art, for example, by using an appropriate analytical instrument (e.g., the xCELLigence real-time cell analyzer). The cell proliferation inhibition rate (CGI:%) is determined from the cell index according to the formula: CGI (%) = 100 - (CIAb x 100 / CINoAb). "CIAb" represents the cell index of wells with the antibody or antigen-binding molecule at a specific experimental time, and "CINoAb" represents the average cell index of wells without the antibody or antigen-binding molecule. If the CGI rate of an antibody or antigen-binding molecule is high, i.e., has a significantly positive value, the antibody or antigen-binding molecule can be said to have TDCC activity.

[0234] In one aspect, the antibody or antigen-binding molecule of the present disclosure has T cell activation activity. T cell activation can be assayed by methods known in the art, such as using a modified T cell line (e.g., Jurkat / NFAT-RE reporter cell line (T cell activation bioassay, Promega)) that expresses a reporter gene (e.g., luciferase) in response to its activation. In this method, target cells (e.g., DLL3-expressing cells) are cultured with T cells in the presence of a test antibody or antigen-binding molecule, and the level or activity of the reporter gene expression product is then measured by an appropriate method as an indicator of T cell activation. When the reporter gene is a luciferase gene, luminescence generated by the reaction of luciferase with its substrate can be measured as an indicator of T cell activation. If the T cell activation measured as described above is higher, the test antibody or antigen-binding molecule is determined to have higher T cell activation activity.

[0235] Pharmaceutical Composition In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule or antibody of the present disclosure. In certain embodiments, the pharmaceutical composition of the present disclosure induces T cell-dependent cytotoxicity, in other words, the pharmaceutical composition of the present disclosure is a therapeutic agent for inducing cytotoxicity. In certain embodiments, the pharmaceutical composition of the present disclosure is a pharmaceutical composition used for the treatment and / or prevention of cancer. In certain embodiments, the pharmaceutical composition of the present disclosure is a pharmaceutical composition used for the treatment and / or prevention of DLL3-positive or DLL3-expressing cancer, including lung cancer (including small cell lung cancer) and melanoma. In certain embodiments, the pharmaceutical composition of the present disclosure is a cytostatic agent. In certain embodiments, the pharmaceutical composition of the present disclosure is an anticancer agent.

[0236] The pharmaceutical compositions, therapeutic agents for inducing cytotoxicity, cell growth inhibitors, or anticancer agents of the present disclosure can be formulated with various types of antigen-binding molecules or antibodies, as needed. For example, a cocktail of multiple antigen-binding molecules or antibodies of the present disclosure can enhance the cytotoxic effect against cells expressing the antigen.

[0237] Pharmaceutical compositions of the antigen-binding molecules or antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antigen-binding molecules or antibodies having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, the following: buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.; small (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs) (e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of use thereof (including rHuPH20) are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0238] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0239] The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. Such active ingredients are present in suitable combinations and in amounts that are effective for the purpose intended.

[0240] If desired, the antigen-binding molecules or antibodies of the present disclosure may be encapsulated in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], etc.) or may be components of colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remington's Pharmaceutical Sciences 16 th edition, Oslo Ed. (1980)). Furthermore, methods for preparing drugs as sustained-release drugs are also known, and these can be applied to the antigen-binding molecules of the present disclosure (J. Biomed. Mater. Res. (1981) 15, 267-277; Chemtech. (1982) 12, 98-105; U.S. Pat. No. 3,773,719; European Patent Application (EP) Nos. EP58481 and EP133988; Biopolymers (1983) 22, 547-556).

[0241] The pharmaceutical composition, cytostatic agent, or anticancer agent of the present disclosure can be administered to a patient either orally or parenterally. Parenteral administration is preferred. Specific administration methods include injection, intranasal administration, pulmonary administration, and transdermal administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition, therapeutic agent for inducing cytotoxicity, cytostatic agent, or anticancer agent of the present disclosure can be administered locally or systemically by injection. Furthermore, an appropriate administration method can be selected depending on the patient's age and symptoms. The administration dose can be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dose can be selected, for example, from the range of 0.001 mg to 100,000 mg per patient. However, the dose of the pharmaceutical composition of the present disclosure is not limited to these doses.

[0242] Preferably, the pharmaceutical composition of the present disclosure comprises an antigen-binding molecule or antibody as described herein. In one aspect, the composition is a pharmaceutical composition for use in inducing cytotoxicity. In another aspect, the composition is a pharmaceutical composition for use in treating or preventing cancer. Preferably, the cancer is lung cancer (including small cell lung cancer) and melanoma. The pharmaceutical composition of the present disclosure can be used to treat or prevent cancer. Thus, the present disclosure provides a method for treating or preventing cancer, in which an antigen-binding molecule or antibody described herein is administered to a patient in need thereof.

[0243] The present disclosure also provides a method for damaging or inhibiting cell proliferation of DLL3-expressing cells by contacting the cells with an antigen binding molecule of the present disclosure that binds to DLL3. The cells to which the antigen binding molecule of the present disclosure binds are not particularly limited, as long as they express DLL3. Specifically, in the present disclosure, preferred DLL3-expressing cells include lung cancer (including small cell lung cancer) and melanoma.

[0244] In the present disclosure, "contact" can be performed, for example, by adding an antigen-binding molecule of the present disclosure to the culture medium of cells expressing DLL3 cultured in vitro. In this case, the antigen-binding molecule to be added can be used in an appropriate form, such as a solution or a solid prepared by lyophilization or the like. When the antigen-binding molecule of the present disclosure is added as an aqueous solution, the solution may be a pure aqueous solution containing the antigen-binding molecule alone, or a solution containing, for example, the above-mentioned surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. The addition concentration is not particularly limited; however, the final concentration in the culture medium is preferably in the range of 1 pg / ml to 1 g / ml, more preferably 1 ng / ml to 1 mg / ml, and even more preferably 1 μg / ml to 1 mg / ml.

[0245] In another embodiment of the present disclosure, "contacting" can also be performed by administering to a non-human animal transplanted with DLL3-expressing cells in vivo or to an animal with cancer cells that endogenously express DLL3. The administration method can be oral or parenteral. Parenteral administration is particularly preferred. Specifically, parenteral administration methods include injection, intranasal administration, pulmonary administration, and transdermal administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition, therapeutic agent for inducing cell damage, cytostatic agent, or anticancer agent of the present disclosure can be administered locally or systemically by injection. Furthermore, an appropriate administration method can be selected depending on the age and symptoms of the animal subject. When an antigen-binding molecule is administered as an aqueous solution, the solution may be a pure aqueous solution containing the antigen-binding molecule alone, or a solution containing, for example, the above-mentioned surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. The administration dose can be selected, for example, from the range of 0.0001 to 1,000 mg per kg of body weight per administration. Alternatively, the dose can be selected, for example, from the range of 0.001 to 100,000 mg per patient. However, the dose of the antigen-binding molecule of the present disclosure is not limited to these examples.

[0246] The present disclosure also provides kits for use in the methods of the present disclosure, which contain the antigen-binding molecules of the present disclosure or antigen-binding molecules produced by the methods of the present disclosure. The kits may be packaged together with additional pharmaceutically acceptable carriers or vehicles, or instructions for use of the kit.

[0247] In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Preferred containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. The article of manufacture may further comprise: (a) a first container with a composition comprising an antibody of the invention contained therein; and (b) a second container with a composition comprising an additional cytotoxic or otherwise therapeutic agent contained therein. The article of manufacture in this aspect of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other equipment desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0248] Attached document The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product, which contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0249] Pharmaceutical preparations The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in a form such that the biological activity of the active ingredients contained therein can be effective, and which does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0250] Pharmaceutically acceptable carrier A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0251] treatment As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed for prophylaxis or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antigen-binding molecules or antibodies of the present disclosure are used to delay the onset of disease or slow the progression of disease.

[0252] cancer The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0253] In certain embodiments, the cancer is a DLL3-expressing or DLL3-positive cancer, including lung cancer (including small cell lung cancer) and melanoma.

[0254] tumor The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0255] In preferred embodiments, the cancer is a cancer (including cancer tissue or cancer cells) that expresses DLL3. In some embodiments, the cancer is lung cancer, small cell lung cancer (SCLC), or melanoma.

[0256] Other Agents and Treatments The multispecific antigen-binding molecules described herein may be administered in combination with one or more other therapeutic agents. For example, the multispecific antigen-binding molecules described herein may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredient suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis-inducing factors. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule-disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

[0257] Such other agents are present in suitable combination in amounts that are effective for the intended purpose. The effective amount of such other agents depends on the amount of multispecific antigen-binding molecule used, the type of disorder or treatment, and other factors discussed above. The multispecific antigen-binding molecule is generally used in the same dosages and by the same routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0258] Such combination therapy as described above encompasses combined administration (two or more therapeutic agents contained in the same or separate compositions) and separate administration, in which the administration of the multispecific antigen-binding molecules described herein can occur prior to, simultaneously with, and / or subsequent to the administration of the additional therapeutic agent and / or adjuvant. The multispecific antigen-binding molecules described herein can also be used in combination with radiation therapy.

[0259] All documents cited herein are hereby incorporated by reference.

[0260] The following are examples of the methods and compositions of the present disclosure. In light of the above general description, it will be understood that various other embodiments may be practiced. [Example]

[0261] [Example 1]

[0262] Screening of affinity matured variants of the parent Dual-Fab H183L072 for improved in vitro cytotoxic activity against tumor cells 1.1 Sequences of affinity matured variants The concept of providing an immunoglobulin variable (Fab) region (Dual-Fab) that binds to CD3 and CD137 but does not simultaneously bind to CD3 and CD137 is disclosed in WO2019111871 (incorporated herein by reference). To increase the binding affinity of the parent Dual-Fab H183L072 (heavy chain: SEQ ID NO: 1; light chain: SEQ ID NO: 57) disclosed in WO2019111871, more than 1,000 Dual-Fab variants were generated by introducing single or multiple mutations into the variable region using H183L072 as a template. The antibodies were expressed using Expi293 (Invitrogen) and purified by Protein A purification, followed by gel filtration if necessary. The sequences of 22 representative Dual-Fab variants with multiple mutations are listed in Table 6 and Tables 8-1 to 8-6, and their binding affinities and kinetics to CD3 and CD137 were assessed at 25°C and / or 37°C using a Biacore T200 instrument (GE Healthcare), as described below in Example 1.2.2 (Table 9).

[0263] 1.2 Binding kinetics information of affinity matured variants 1.2.1 Expression and purification of human CD3 and CD137 The γ and ε subunits of the human CD3 complex (human CD3eg linker) were linked by a 29-mer linker, and a Flag tag was fused to the C-terminus of the γ subunit (SEQ ID NO: 102, Tables 5 and 7). This construct was transiently expressed using the FreeStyle293F cell line (Thermo Fisher). Culture supernatants expressing the human CD3eg linker were concentrated using a column packed with Q HP resin (GE Healthcare) and then applied to FLAG-tag affinity chromatography. Fractions containing the human CD3eg linker were collected and subsequently applied to a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1×D-PBS. The fractions containing the human CD3eg linker were then pooled. Human CD137 extracellular domain (ECD) (SEQ ID NO: 103, Tables 5 and 7) bearing a hexahistidine (His-tag) and biotin acceptor peptide (BAP) at its C-terminus was transiently expressed using the FreeStyle293F cell line (Thermo Fisher). Culture supernatant expressing human CD137 ECD was applied to a HisTrap HP column (GE Healthcare) and eluted with a buffer containing imidazole (Nacalai). Fractions containing human CD137 ECD were collected and subsequently applied to a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1x D-PBS. Fractions containing human CD137 ECD were then pooled and stored at -80°C.

[0264] 1.2.2 Affinity measurement for human CD3 and CD137 The binding affinity of Dual-Fab antibodies (Dual-Ig) to human CD3 was evaluated at 25°C using a Biacore 8K instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). The antibody was captured on the anti-Fc sensor surface, and then recombinant human CD3 or CD137 was injected onto the flow cell. All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The sensor surface was regenerated with 3 M MgCl2 after each cycle. Binding affinity was determined by processing the data and fitting to a 1:1 binding model using Biacore Insight Evaluation software, version 2.0 (GE Healthcare). The CD137 binding affinity assay was performed under the same conditions, except that the assay temperature was set to 37°C. The binding affinities of the Dual-Fab antibodies to recombinant human CD3 and CD137 are shown in Tables 9-1 and 9-2 (K on value, K off The term E used to represent the KD value means "10 to the power of ____", e.g., 3.54E+04=3.54 * 10 4 As shown in Tables 9-1 and 9-2, the Dual Fab variants exhibited different binding kinetics to CD3 and CD137 compared to H183 / L072. Table 5: Annotation of SEQ ID NOs in Table 7 (SEQ ID NOs for human CD3 and CD137 antigens used in affinity measurements in Table 9) TIFF2026031947000006.tif36170 (Table 6) Annotation of SEQ ID NOs in Tables 8-1 to 8-6 (antibody names and SEQ ID NOs of variable regions including CDRs 1, 2, and 3) TIFF2026031947000007.tif242166 (Table 7) Full-length amino acid sequence of the antigen TIFF2026031947000008....

Claims

[Claim 1] The invention described herein.

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