Method for producing multispecific antigen-binding molecule

Abstract

To provide methods for producing preparations of multispecific antigen-binding molecules, and to provide the preparations.SOLUTION: A method comprises a multispecific antigen-binding molecule comprising: (a) a first antigen-binding portion and a second antigen-binding portion, each capable of binding to a first antigen and a second antigen different from the first antigen, but not simultaneously binding to both antigens; and (b) a third antigen-binding portion capable of binding to a third antigen different from the first and second antigens, preferably an antigen expressed on cancer cells / tissues. Each of the first and second antigen-binding portions comprises at least one cysteine residue that is not in the hinge region, preferably at least one cysteine is located in the CH1 region, at least one cysteine residue, preferably in the CH1 region, is capable of forming at least one disulfide bond between the first antigen-binding portion and the second antigen-binding portion, and the method comprises contacting the preparation with a reducing reagent.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to multispecific antigen-binding molecules comprising two or more antigen-binding moieties that can be linked to each other via at least one disulfide bond, and methods for producing such multispecific antigen-binding molecules. More particularly, the present invention relates to methods for increasing or concentrating preferred forms of multispecific antibody proteins, and methods for removing disulfide heterogeneity of such recombinant antibody proteins. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Among the many therapeutic antibodies, some types of antibodies require effector cells to exert antitumor responses. Antibody-dependent cellular cytotoxicity (ADCC) is a cytotoxicity that effector cells exert against antibody-bound cells via binding of the Fc region of the antibody 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 treating cancer (Nat. Biotechnol. (2005) 23, 1073-1078).

[0003] 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 Europe 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).

[0004] 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 antigen-independent induction of various cytokine expression. Systemic administration of trifunctional antibodies is thought to cause cytokine storm-like side effects as a result of the induction of such 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.

[0005] Recently, improved antibodies have been provided that induce T cell-mediated cytotoxicity while avoiding adverse reactions by using an Fc region with reduced binding activity to FcγR (Patent Document 1). However, even such antibodies, given their molecular structure, are unable to bind to cancer antigens while acting on two immune receptors, i.e., CD3ε and FcγR. No known antibodies are yet known that exert both T cell-mediated cytotoxicity and cytotoxicity mediated by cells other than T cells in a cancer antigen-specific manner while avoiding adverse reactions.

[0006] On the other hand, unlike catumaxomab, bispecific sc(Fv)2 format molecules (BiTEs) lack Fcγ receptor binding sites and therefore do not crosslink receptors expressed on T cells, NK cells, macrophages, and other cells in a cancer antigen-independent manner. However, because bispecific sc(Fv)2s are engineered low-molecular-weight antibody molecules lacking an Fc region, their serum half-life after administration to patients is significantly shorter than that of IgG antibodies commonly used as therapeutic antibodies. In fact, the serum half-life of bispecific sc(Fv)2s administered in vivo has been reported to be approximately several hours (Non-Patent Documents 9 and 10). Blinatumomab, an sc(Fv)2 molecule that binds to CD19 and CD3, has been approved for the treatment of acute lymphoblastic leukemia. The serum half-life of blinatumomab has been shown to be less than 2 hours in patients (Non-Patent Document 11). In clinical trials of blinatumomab, it was administered by continuous intravenous infusion using a minipump. This administration method is not only extremely inconvenient for patients, but also carries the risk of medical accidents due to device malfunctions. Therefore, such an administration method cannot be said to be desirable.

[0007] T cells play an important role in tumor immunity and are known to be activated by two signals: 1) T cell receptor (TCR) binding to antigen peptides presented by major histocompatibility complex (MHC) class I molecules and TCR activation; and 2) costimulatory molecules on the surface of T cells binding to ligands on antigen-presenting cells and activation of costimulatory molecules. Furthermore, activation of molecules belonging to the tumor necrosis factor (TNF) superfamily and the TNF receptor superfamily, such as CD137 (4-1BB) on the surface of T cells, has been described as important for T cell activation (Non-Patent Document 12). In this regard, CD137 agonist antibodies have already been demonstrated to exhibit antitumor effects, which have been experimentally demonstrated primarily through the activation of CD8-positive T cells and NK cells (Non-Patent Document 13). T cells engineered to carry chimeric antigen receptor molecules (CAR-T cells) consisting of a tumor antigen-binding domain as the extracellular domain and CD3 and CD137 signaling domains as the intracellular domain can enhance the durability of efficacy (Porter, N ENGL J MED, 2011, 365;725-733 (Non-Patent Document 14)). However, the side effects of such CD137 agonist antibodies due to their nonspecific hepatotoxicity are a clinical and non-clinical problem, preventing progress in drug development (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22 (Non-Patent Document 15)). It has been suggested that the main cause of side effects is related to antibody binding to Fcγ receptors via the antibody constant region (Schabowsky, Vaccine, 2009, 28, 512-22 (Non-Patent Document 16)). Furthermore, it has been reported that antibody cross-linking by Fcγ receptor-expressing cells (FcγRII-expressing cells) is necessary for agonist antibodies targeting receptors belonging to the TNF receptor superfamily to exert their agonist activity in vivo (Li, Proc Natl Acad Sci USA. 2013, 110(48), 19501-6 (Non-Patent Document 17)).WO2015 / 156268 (Patent Document 2) describes that a bispecific antibody having a binding domain with CD137 agonist activity and a binding domain for a tumor-specific antigen can exert CD137 agonist activity and activate immune cells only in the presence of cells expressing the tumor-specific antigen.

[0008] Trispecific antibodies containing a tumor-specific antigen (EGFR)-binding domain, a CD137-binding domain, and a CD3-binding domain have already been reported ( WO2014116846 ). However, because antibodies with such molecular formats can simultaneously bind to three different antigens, it was speculated that these trispecific antibodies might simultaneously bind to CD3 and CD137, thereby forming a bridge between CD3ε-expressing T cells and CD137-expressing cells (e.g., T cells, B cells, NK cells, DCs, etc.). In this context, no antibody has yet been known that specifically exerts both T cell-mediated cytotoxicity and CD137-mediated activation of T cells and other immune cells while avoiding adverse reactions.

[0009] For antibodies with multiple disulfide bonds, structural heterogeneity between antibody preparations has been observed, but the reasons behind this heterogeneity remain unclear. For example, U.S. Patent Application Publication No. 2005 / 0161399, Dillon et al., discusses a reversed-phase LC / MS method for analyzing high molecular weight proteins, including antibodies. In addition, U.S. Patent Application Publication No. 2006 / 194280, Dillon et al., describes a method for transiently enriching specific IgG isoforms by subjecting a preparation of recombinant IgG protein to a reduction / oxidation coupling reagent and, optionally, a chaotropic agent. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2012 / 073985 [Patent Document 2] WO2015 / 156268

Patent document 3

Non-licensed literature

[0011] [Non-licensed document 1] Nat. Biotechnol. (2005) 23, 1073-1078 [Non-licensed document 2] Nature. 1985 Apr 18-24;314(6012):628-31. [Non-licensed document 3] Int J Cancer. 1988 Apr 15;41(4):609-15.

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Non-licensed Document 7

Non-licensed literature 9

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Non-licensed Document 11

[0012] No antibody has yet been known that specifically exerts both immune cell (e.g., T cell)-mediated cytotoxic activity and costimulatory molecule (e.g., CD137)-mediated activation of T cells and / or other immune cells while avoiding adverse reactions. The object of the present invention is to provide an antigen-binding molecule that exhibits effective target-specific cell-killing effect mediated by immune cells (e.g., T cells) while reducing or minimizing side effects.

[0013] Another object of the present invention is to provide methods for producing multispecific antigen-binding molecules, methods for increasing or concentrating preferred forms of multispecific antibody proteins, and methods for removing disulfide heterogeneity of such recombinant antibody proteins. [Means for solving the problem]

[0014] Antigen-binding molecules are provided that can bind to multiple different antigens (e.g., CD3 on T cells, and / or CD137 on T cells, NK cells, DC cells, etc.) but do not nonspecifically crosslink two or more immune cells, such as T cells. Such multispecific antigen-binding molecules can regulate and / or activate immune responses while avoiding crosslinking between different cells (e.g., different T cells) that occurs when conventional multispecific antigen-binding molecules bind to antigens expressed on different cells, which is thought to be responsible for adverse reactions when using multispecific antigen-binding molecules as drugs.

[0015] In one aspect, the antigen-binding molecules of the present invention provide novel antigen-binding molecules with highly unique structural formats that improve or enhance the efficacy of multispecific antigen-binding molecules. Novel antigen-binding molecules with unique structural formats provide an increased number of antigen-binding domains, resulting in increased valency and / or specificity for each antigen on effector cells and target cells, with reduced undesirable adverse effects.

[0016] In a further aspect, one of the antigen-binding molecules of the present invention having such a new unique structural format comprises at least two first and second antigen-binding moieties (e.g., Fab domains) that are linked to each other (e.g., via an Fc, disulfide bond, or linker, etc.) and each binds to a first and / or second antigen on an effector cell (e.g., an immune cell such as a T cell, NK cell, or DC cell), and further comprises a third (and optionally a fourth) antigen-binding domain that is linked to either the first or second antigen-binding moiety and binds to a third antigen on a target cell (e.g., a tumor cell).

[0017] In a further aspect, one of the antigen-binding molecules of the present invention having such a novel unique structural format comprises at least a first antigen-binding portion and a second antigen-binding portion (e.g., Fab domain) that are linked to each other (e.g., via an Fc, disulfide bond, or linker, etc.) and each binds to a first and / or second antigen on an effector cell (e.g., an immune cell such as a T cell, NK cell, or DC cell), and further comprises a third (and optionally a fourth) antigen-binding portion that is linked to either the first or second antigen-binding portion and binds to a third antigen on a target cell (e.g., a tumor cell), wherein the first antigen and and / or the first and second antigen-binding moieties (e.g., Fab domains) capable of binding to a second antigen each contain at least one amino acid mutation that creates a disulfide bond between the first and second antigen-binding moieties, maintaining them in close proximity to each other, and promotes cis-antigen binding to the same single effector cell, for example, as a result of steric hindrance or short distance between the two Dual-Fabs, thereby improving the safety profile of the trispecific antibody (triaBb) 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 contains at least one cysteine ​​residue (via mutation, substitution, or insertion) in the CH1 region, which can form 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, the first antigen-binding portion and the second antigen-binding portion each comprise (via mutation, substitution, or insertion) a cysteine ​​residue at position 191 according to EU numbering in the CH1 region that can form a disulfide bond between the CH1 region of the first antigen-binding portion and the CH1 region of the second antigen-binding portion.

[0018] Antigen-binding molecules with 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 different cells (e.g., effector cells such as T cells). In one 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, thereby more efficiently inducing T cell-dependent cytotoxicity while avoiding potentially harmful toxicity concerns or side effects associated with other multispecific antigen-binding molecules. The present invention provides multispecific antigen-binding molecules and pharmaceutical compositions capable of treating various cancers, particularly DLL3-associated cancers such as DLL3-positive tumors, by comprising the antigen-binding molecule as an active ingredient.

[0019] In another aspect, the present invention relates to a method for producing a novel format of multispecific antigen-binding molecule comprising one or more disulfide bonds between a first antigen-binding portion and a second antigen-binding portion (e.g., in the CH1 region); a method for increasing or enriching a preferred form of a multispecific antibody protein having said at least one disulfide bond; and a method for removing disulfide heterogeneity of such a recombinant antibody protein by contacting an antibody preparation with a reducing agent under conditions that allow efficient and appropriate formation of said at least one disulfide bond (e.g., in the CH1 region). In a further aspect, the present invention relates to a conformation-specific antibody that specifically recognizes a preferred form of a multispecific antibody protein, and the use of the conformation-specific antibody in the purification, analysis, or quantification of an antibody-containing sample.

[0020] In one particular aspect, 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 selected from 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 according to any one of [1] to [1A], comprising an antibody variable region comprising any one of the following: [3] The first antigen-binding portion and the second antigen-binding portion are each selected from 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; 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 (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 according to any one of [1] and [2], comprising an antibody variable region comprising any one of: [4] The multispecific antigen-binding molecule according to any one of [1] to [3], wherein each of the first and second antigen-binding moieties is a Fab molecule and comprises at least one disulfide bond formed between the first and second antigen-binding moieties, and preferably, the at least one disulfide bond is formed between amino acid residues (cysteines) that are not present in the hinge region, preferably between amino acid residues (cysteines) in the CH1 region of each antigen-binding moiety. [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 (cysteine) at position 191 according to 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 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 of the Fab heavy chain (CH1) 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) (EU numbering). [9] The 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) (EU numbering).

[10] The third antigen-binding moiety capable of binding to DLL3 is selected from 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 according to any one of [1] to [9], comprising an antibody variable region comprising any one of the following:

[11] The third antigen-binding moiety capable of binding to DLL3 is selected from 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 according to any one of [1] to

[10] , comprising an antibody variable region comprising any one of the following:

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

[11] , further comprising an Fc domain. [12A] The 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 for human Fcγ receptors compared to a native human IgG1 Fc domain. [12C] The multispecific antigen-binding molecule according to any one of

[12] to [12A], 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 Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, according to EU numbering. [12E] The multispecific antigen-binding molecule according to [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 The multispecific antigen-binding molecule according to any one of [12C] to [12F], comprising a combination of amino acid substitutions selected from the group consisting of: [12H] The multispecific antigen-binding molecule of any one of [12C] to [12G], wherein the Fc domain comprises a combination of amino acid substitutions of M428L / N434A / Q438R / S440E. [12I] The multispecific antigen-binding molecule according to 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; or (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. The multispecific antigen-binding molecule according to any one of

[12] to [12I], comprising any one of: [12K] The multispecific antigen-binding molecule of any one of

[12] to [12J], wherein each of the first and second antigen-binding moieties is 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chain 4 & chain 5) each containing the amino acid sequence of SEQ ID NO: 215 and Preferably, the multispecific antigen-binding molecule according to any one of [1] to [12L], wherein the five polypeptide chains (chain 1 to chain 5) are connected to and / or associated with each other in the orientation shown in Figure 1(a).

[0021] Another aspect of the present invention relates to: [1] A method for (i) producing a preparation of multispecific antigen-binding molecules (recombinantly produced by mammalian cells), (ii) purifying a multispecific antigen-binding molecule having a desired conformation, or (iii) improving the homogeneity of a preparation of multispecific antigen-binding molecules, comprising: the multispecific antigen-binding molecule comprises a first antigen-binding portion and a second antigen-binding portion, each of which is a Fab and can bind to the first antigen and a second antigen different from the first antigen, but does not bind to both antigens simultaneously; Each of the first antigen-binding portion and the second antigen-binding portion comprises (via mutation, substitution, or insertion) at least one cysteine ​​residue that is not within the hinge region; preferably, the at least one cysteine ​​residue is located in the CH1 region; and the at least one cysteine ​​residue, preferably in the CH1 region, is capable of forming at least one disulfide bond between the first antigen-binding portion and the second antigen-binding portion; contacting the preparation with a reducing reagent; The method. [2] each of the first antigen-binding portion and the second antigen-binding portion is a cysteine ​​residue at position 191 (EU numbering) in the CH1 region that can form a disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety; The method according to [1], comprising (via mutation, substitution, or insertion) [3] The method according to [1] or [2], wherein the step of contacting the preparation with a reducing reagent allows and / or promotes the formation of at least one disulfide bond formed between an amino acid residue located in the CH1 region or at position 191 (EU numbering) in the CH1 region. [4] The method of [3], wherein the multispecific antigen-binding molecule preparation (before contacting with a reducing agent) comprises two or more structural isoforms that differ by at least one disulfide bond formed between amino acid residues located in the CH1 region or at position 191 (EU numbering) in the CH1 region, and wherein the step of contacting with a reducing agent preferentially enriches or increases the population of structural isoforms having at least one disulfide bond formed between amino acid residues located in the CH1 region or at position 191 (EU numbering) in the CH1 region. [5] The method according to any one of [1] to [4], wherein the reducing reagent to be contacted with the multispecific antigen-binding molecule has a pH of about 3 to about 10. [6] The method according to [5], wherein the reducing reagent contacted with the multispecific antigen-binding molecule has a pH of about 6, 7, or 8. [7] The method according to [6], wherein the reducing reagent contacted with the multispecific antigen-binding molecule has a pH of about 7. [8] The method according to [5], wherein the reducing reagent contacted with the multispecific antigen-binding molecule has a pH of about 3. [9] The method according to any one of [1] to [8], wherein the reducing agent is selected from the group consisting of TCEP, 2-MEA, DTT, cysteine, GSH, and Na2SO3.

[10] The method according to [9], wherein the reducing agent is TCEP, preferably 0.25 mM TCEP.

[11] The method according to any one of [1] to [9], wherein the concentration of the reducing agent is from about 0.01 mM to about 100 mM.

[12] The method according to

[11] , wherein the concentration of the reducing agent is about 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, or 100 mM, preferably about 0.25 mM.

[13] The method according to any one of [1] to

[12] , wherein the contacting step is carried out for at least 30 minutes.

[14] The method according to any one of [1] to

[12] , wherein the contacting step is carried out for about 10 minutes to about 48 hours.

[15] The method according to any one of [1] to

[12] , wherein the contacting step is carried out for about 2 hours or about 18 hours.

[16] The method according to any one of [1] to

[15] , wherein the contacting step is carried out at a temperature of about 4°C to 37°C, preferably 23°C to 25°C.

[17] The method according to any one of [1] to

[16] , wherein the multispecific antigen-binding molecule is at least partially purified before the step of contacting with the reducing agent.

[18] The method according to

[17] , wherein the multispecific antigen-binding molecule is partially purified by affinity chromatography (preferably protein A chromatography) prior to the contacting step.

[19] The method according to any one of [1] to

[18] , wherein the concentration of the multispecific antigen-binding molecule is from about 0.1 mg / ml to about 50 mg / ml or more.

[20] The method according to

[19] , wherein the concentration of the multispecific antigen-binding molecule is about 10 mg / ml or about 20 mg / ml.

[21] The method according to any one of [1] to

[20] , further comprising a step of promoting reoxidation of cysteine ​​disulfide bonds, preferably by removing the reducing agent by dialysis or buffer exchange.

[22] The method according to any one of [1] to

[21] , wherein each of the first antigen-binding portion and the second antigen-binding portion can bind to CD3 and CD137, but does not simultaneously bind to both CD3 and CD137.

[23] The first antigen-binding portion and the second antigen-binding portion are each selected from 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).

[22] The method described in

[22] , wherein the antibody variable region comprises any one of:

[24] The first antigen-binding portion and the second antigen-binding portion are each selected from 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; 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. (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).

[23] The method described in

[23] , wherein the antibody variable region comprises any one of:

[25] The method according to any one of [1] to

[24] , wherein the multispecific antigen-binding molecule further comprises a third antigen-binding moiety capable of binding to a third antigen different from the first and second antigens, preferably an antigen expressed on cancer cells / tissues.

[26] The multispecific antigen-binding molecule according to

[25] , wherein the third antigen-binding portion is fused to either the first antigen-binding portion or the second antigen-binding portion.

[27] The method according to any one of

[25] to

[26] , wherein the third antigen-binding portion is Fab or scFv.

[28] The method according to any one of

[25] to

[27] , 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 of the Fab heavy chain (CH1) 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.

[29] The method according to

[28] , wherein the peptide linker is selected from the group consisting of the amino acid sequences of SEQ ID NO: 248, SEQ ID NO: 249, or SEQ ID NO: 259.

[30] The method according to any one of [1] to

[29] , wherein the first antigen-binding portion is identical to the second antigen-binding portion. [30A] The method according to any one of

[25] to

[30] , 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. [30B] The method of any one of

[25] to [30A], 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) (EU numbering). [30C] The method according to [30B], 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) (EU numbering).

[31] The method according to any one of [1] to

[30] , wherein the third antigen-binding moiety is capable of binding to DLL3, preferably human DLL3.

[32] The method of

[31] , wherein the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region comprising 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.

[33] The method described in

[32] , wherein the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region comprising 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.

[34] The method according to any one of [1] to

[33] , wherein the multispecific antigen-binding molecule further comprises an Fc domain.

[35] The method according to

[34] , 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.

[36] The multispecific antigen-binding molecule comprises any one of the following (a1) to (a15): (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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chain 4 & chain 5) each containing 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).

[37] The method according to any one of [1] to

[36] , wherein the fourth polypeptide (chain 4) and the fifth polypeptide (chain 5) are identical.

[38] A preparation of multispecific antigen-binding molecules prepared according to the method of any one of [1] to

[37] , which comprises a homogeneous population of multispecific antigen-binding molecules having at least one disulfide bond in the CH1 region (position 191 according to EU numbering).

[39] A preparation of multispecific antigen-binding molecules prepared according to the method of any one of [1] to

[37] , comprising multispecific antigen-binding molecules having at least one disulfide bond in the CH1 region (position 191 according to EU numbering) in a molar ratio of at least 50%, 60%, 70%, 80%, or 90%, preferably at least 95%.

[0022] Yet another aspect of the present invention relates to: [1] A method for producing a multispecific antigen-binding molecule, comprising: a first antigen-binding portion and a second antigen-binding portion, each of which is a Fab and is capable of binding to the first antigen and a second antigen different from the first antigen, but does not bind to both antigens simultaneously; and a third antigen-binding moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), capable of binding to a third antigen different from the first and second antigens, preferably an antigen expressed on cancer cells / tissues; wherein the method comprises: (a) providing one or more nucleic acids encoding: i. a first polypeptide comprising (from N-terminus to C-terminus) a VH or VL of a third antigen-binding portion, optionally a heavy chain constant region (CH1); and a VH or VL of a first antigen-binding portion, a heavy chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3); ii. a second polypeptide comprising (from N-terminus to C-terminus) a third antigen-binding portion, VH or VL, and optionally a light chain constant region (CL); iii. A third polypeptide comprising (from N-terminus to C-terminus) a second antigen-binding portion, VH or VL, a heavy chain constant region (CH1); and optionally a hinge region and / or Fc region (CH2 and CH3); iv. a fourth polypeptide comprising (from N-terminus to C-terminus) the VH or VL of a second antigen-binding portion, optionally a light chain constant region (CL); and v. a fifth polypeptide comprising (from N-terminus to C-terminus) the VH or VL of the first antigen-binding portion, optionally a light chain constant region (CL); (b) introducing the nucleic acid(s) produced in (a) into a host cell; (c) culturing a host cell to express the polypeptide in (i) to (v); and (d) collecting multispecific antigen-binding molecules comprising the five polypeptides in (i) to (v) from the culture medium of the cells cultured in step (c). and optionally, the polypeptides in (iv)-(v) are identical; and each of the first antigen-binding moiety and the second antigen-binding moiety comprises (via mutation, substitution, or insertion) at least one cysteine ​​residue that is not in the hinge region, and preferably, the at least one cysteine ​​is located in the CH1 region; and the at least one cysteine ​​residue, preferably in the CH1 region, is capable of forming at least one disulfide bond between the first antigen-binding moiety and the second antigen-binding moiety; The method, wherein the method comprises contacting the preparation with a reducing reagent. [2] each of the first antigen-binding portion and the second antigen-binding portion is a cysteine ​​residue at position 191 (EU numbering) in the CH1 region that can form a disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety; The method according to [1], comprising (via mutation, substitution, or insertion) [3] The method according to any one of [1] to [2], further comprising step (e) of contacting the multispecific antigen-binding molecule (multispecific antigen-binding molecule) preparation collected from step (d) with a reducing reagent under reducing conditions that allow the cysteine ​​in the CH1 region (position 191 according to EU numbering) to form one or more disulfide bonds. [4] The method of [3], wherein the multispecific antigen-binding molecule preparation collected from step (d) (prior to contacting with a reducing agent) comprises two or more structural isoforms that differ by at least one disulfide bond formed between amino acid residues located in the CH1 region or at position 191 (EU numbering) in the CH1 region, and wherein step (e) of contacting with a reducing agent preferentially enriches or increases the population of multispecific antigen-binding molecule structural isoforms having at least one disulfide bond formed between amino acid residues located in the CH1 region or at position 191 (EU numbering) in the CH1 region. [5] The method according to any one of [3] to [4], wherein the reducing reagent to be contacted with the multispecific antigen-binding molecule has a pH of about 3 to about 10. [6] The method according to [5], wherein the reducing reagent contacted with the multispecific antigen-binding molecule has a pH of about 6, 7, or 8. [7] The method according to [6], wherein the reducing reagent contacted with the multispecific antigen-binding molecule has a pH of about 7. [8] The method according to [5], wherein the reducing reagent contacted with the multispecific antigen-binding molecule has a pH of about 3. [9] The method according to any one of [3] to [8], wherein the reducing agent is selected from the group consisting of TCEP, 2-MEA, DTT, cysteine, GSH, and Na2SO3.

[10] The method according to [9], wherein the reducing agent is TCEP, preferably 0.25 mM TCEP.

[11] The method according to any one of [3] to [9], wherein the concentration of the reducing agent is from about 0.01 mM to about 100 mM.

[12] The method according to

[11] , wherein the concentration of the reducing agent is about 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, or 100 mM, preferably about 0.25 mM.

[13] The method according to any one of [3] to

[12] , wherein the contacting step is carried out for at least 30 minutes.

[14] The method according to any one of [3] to

[12] , wherein the contacting step is carried out for about 10 minutes to about 48 hours.

[15] The method according to any one of [3] to

[12] , wherein the contacting step is carried out for about 2 hours or about 18 hours.

[16] The method according to any one of [3] to

[15] , wherein the contacting step is carried out at a temperature of about 4°C to 37°C, preferably 23°C to 25°C.

[17] The method according to any one of [3] to

[16] , wherein the multispecific antigen-binding molecule is at least partially purified before the step of contacting with the reducing agent.

[18] The method according to

[17] , wherein the multispecific antigen-binding molecule is partially purified by affinity chromatography (preferably protein A chromatography) prior to the contacting step.

[19] The method according to any one of [3] to

[18] , wherein the concentration of the multispecific antigen-binding molecule is from about 0.1 mg / ml to about 50 mg / ml or more.

[20] The method according to

[19] , wherein the concentration of the multispecific antigen-binding molecule is about 10 mg / ml or about 20 mg / ml.

[21] The method according to any one of [3] to

[20] , further comprising a step of promoting reoxidation of cysteine ​​disulfide bonds, preferably by removing the reducing agent by dialysis or buffer exchange.

[22] A preparation of multispecific antigen-binding molecules prepared according to the method of any one of [3] to

[21] , which comprises a homogeneous population of multispecific antigen-binding molecules having at least one disulfide bond in the CH1 region (position 191 according to EU numbering).

[23] A preparation of multispecific antigen-binding molecules prepared according to the method of any one of [3] to

[21] , comprising multispecific antigen-binding molecules having at least one disulfide bond in the CH1 region (position 191 according to EU numbering) in a molar ratio of at least 50%, 60%, 70%, 80%, or 90%, preferably at least 95%.

[24] the third antigen-binding portion is a conventional Fab; and (a) the first polypeptide comprises (from N-terminus to C-terminus) a VH, heavy chain constant region (CH1) of a third antigen-binding portion; and a VH, heavy chain constant region (CH1) of a first antigen-binding portion; and optionally a hinge region and / or an Fc region (CH2 and CH3); (b) the second polypeptide comprises (from N-terminus to C-terminus) a third antigen-binding portion VL, and a light chain constant region (CL); (c) a third polypeptide comprising (from N-terminus to C-terminus) a VH of a second antigen-binding portion, a heavy chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3); (d) the fourth polypeptide comprises (from N-terminus to C-terminus) the VL of the second antigen-binding portion, and a light chain constant region (CL); and (e) the fifth polypeptide comprises (from N-terminus to C-terminus) the VL of the first antigen-binding portion, and a light chain constant region (CL); The method according to any one of [1] to

[21] .

[25] The third antigen-binding portion is a VH / VL crossover Fab (in which the variable regions of the Fab light chain and the Fab heavy chain are swapped), and (a) the first polypeptide comprises (from N-terminus to C-terminus) a VL, heavy chain constant region (CH1) of a third antigen-binding portion; and a VH, heavy chain constant region (CH1) of a first antigen-binding portion; and optionally a hinge region and / or an Fc region (CH2 and CH3); (b) the second polypeptide comprises (from N-terminus to C-terminus) a VH of a third antigen-binding portion, and a light chain constant region (CL); (c) a third polypeptide comprising (from N-terminus to C-terminus) a VH of a second antigen-binding portion, a heavy chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3); (d) the fourth polypeptide comprises (from N-terminus to C-terminus) the VL of the second antigen-binding portion, and a light chain constant region (CL); and (e) the fifth polypeptide comprises (from N-terminus to C-terminus) the VL of the first antigen-binding portion, and a light chain constant region (CL); The method according to any one of [1] to

[21] .

[26] The method according to

[25] , wherein the amino acids at positions 123 and 124 in the CL 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 CH1 constant domain of the heavy chain of each of the first and second antigen-binding moieties are glutamic acid (E) (EU numbering). [26-2] The method described in any one of [1] to

[21] , wherein in step (a)(i), the first polypeptide further comprises a peptide linker between the third antigen-binding portion and the VH or VL of the first antigen-binding portion.

[27] The method described in [26-2], wherein the peptide linker is selected from the group consisting of the amino acid sequences of SEQ ID NO: 248, SEQ ID NO: 249, or SEQ ID NO: 259.

[28] The method according to any one of [1] to

[27] , wherein each of the first antigen-binding portion and the second antigen-binding portion can bind to CD3 and CD137, but does not simultaneously bind to both CD3 and CD137.

[29] The first antigen-binding portion and the second antigen-binding portion are each selected from 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] The method described in

[28] , wherein the antibody variable region comprises any one of:

[30] The first antigen-binding portion and the second antigen-binding portion are each selected from 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; 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; (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).

[29] The method described in

[29] , wherein the antibody variable region comprises any one of:

[31] The method according to any one of [1] to

[30] , wherein the third antigen-binding moiety is capable of binding to DLL3, preferably human DLL3.

[32] The method of

[31] , wherein the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region comprising 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.

[33] The method of

[32] , wherein the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region comprising 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.

[34] The method according to any one of [1] to

[33] , wherein the multispecific antigen-binding molecule further comprises an Fc domain.

[35] The method according to

[34] , 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.

[36] The multispecific antigen-binding molecule comprises any one of the following (a1) to (a15): (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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chain 4 & chain 5) each containing the amino acid sequence of SEQ ID NO: 215 and Preferably, the method according to any one of [1] to

[35] , wherein 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).

[37] The method according to any one of [1] to

[36] , wherein the fourth polypeptide (chain 4) and the fifth polypeptide (chain 5) are identical.

[38] The method of any one of [1] or

[37] , wherein only one nucleic acid, or two, three, four or five different nucleic acids, encode and express the first, second, third, fourth and fifth polypeptides.

[0023] In yet another aspect, the present invention relates to: [1] A method for capturing and / or removing a target antibody from an antibody preparation, comprising the steps of: a) contacting an antibody preparation containing a target antibody with an antigen-binding molecule immobilized on a support; and b) capturing the target antibody by specific binding to the antigen-binding molecule Including, the antibody comprises at least two Fabs derived from IgG (preferably human IgG or human IgG1), and the antibody preparation comprises two antibody structural isoforms that differ only by a disulfide bond formed between the two Fabs in the CH1 domain; and The above method, wherein the antigen-binding molecule specifically binds to and captures a target antibody that does not contain a disulfide bond. [2] The method according to [1], wherein the antigen-binding molecule binds to a target antibody at an epitope that is accessible to the antigen-binding molecule only when the target antibody does not have a disulfide bond. [3] The method according to [1] or [2], wherein the disulfide bond is a disulfide bond formed between two Fabs of the antibody at position 191 according to EU numbering in the CH1 domain. [4] The antigen-binding molecule is selected from the group consisting of: (a1) heavy chain CDR1 of SEQ ID NO: 166, heavy chain CDR2 of SEQ ID NO: 170, heavy chain CDR3 of SEQ ID NO: 174, light chain CDR1 of SEQ ID NO: 182, light chain CDR2 of SEQ ID NO: 186, and light chain CDR3 of SEQ ID NO: 190; (a2) heavy chain CDR1 of SEQ ID NO: 167, heavy chain CDR2 of SEQ ID NO: 171, heavy chain CDR3 of SEQ ID NO: 175, light chain CDR1 of SEQ ID NO: 183, light chain CDR2 of SEQ ID NO: 187, and light chain CDR3 of SEQ ID NO: 191; (a3) heavy chain CDR1 of SEQ ID NO: 168, heavy chain CDR2 of SEQ ID NO: 172, heavy chain CDR3 of SEQ ID NO: 176, light chain CDR1 of SEQ ID NO: 184, light chain CDR2 of SEQ ID NO: 188, and light chain CDR3 of SEQ ID NO: 192; (a4) heavy chain CDR1 of SEQ ID NO: 169, heavy chain CDR2 of SEQ ID NO: 173, heavy chain CDR3 of SEQ ID NO: 177, light chain CDR1 of SEQ ID NO: 185, light chain CDR2 of SEQ ID NO: 189, and light chain CDR3 of SEQ ID NO: 193; (a5) heavy chain CDR1 of SEQ ID NO: 166, heavy chain CDR2 of SEQ ID NO: 170, heavy chain CDR3 of SEQ ID NO: 174, light chain CDR1 of SEQ ID NO: 115, light chain CDR2 of SEQ ID NO: 124, and light chain CDR3 of SEQ ID NO: 134; (a6) heavy chain CDR1 of SEQ ID NO: 167, heavy chain CDR2 of SEQ ID NO: 171, heavy chain CDR3 of SEQ ID NO: 175, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 125, and light chain CDR3 of SEQ ID NO: 135; (a7) heavy chain CDR1 of SEQ ID NO: 168, heavy chain CDR2 of SEQ ID NO: 172, heavy chain CDR3 of SEQ ID NO: 176, light chain CDR1 of SEQ ID NO: 118, light chain CDR2 of SEQ ID NO: 128, and light chain CDR3 of SEQ ID NO: 137; (a8) an antibody that binds to the same epitope as an antibody comprising any one of (a1) to (a7); and (a9) An antibody that competes with the binding of an antibody comprising any one of (a1) to (a7). The method according to any one of [1] to [3], wherein the antibody comprises any one selected from the group consisting of: [5] The antigen-binding molecule is any one of the following: (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 178; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 179; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 180; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 165, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 181; (a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 196; (a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 197; (a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 198; (a8) an antibody that binds to the same epitope as an antibody comprising any one of (a1) to (a7); and (a9) An antibody that competes with the binding of an antibody comprising any one of (a1) to (a7). The method according to any one of [1] to [3], wherein the antibody comprises any one selected from the group consisting of: [5A] The target antibody is one of the following (a1) to (a15): (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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chain 4 & chain 5) each containing the amino acid sequence of SEQ ID NO: 215 and Preferably, the method according to any one of [1] to [5], wherein 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). [6] The following: (a1) heavy chain CDR1 of SEQ ID NO: 166, heavy chain CDR2 of SEQ ID NO: 170, heavy chain CDR3 of SEQ ID NO: 174, light chain CDR1 of SEQ ID NO: 182, light chain CDR2 of SEQ ID NO: 186, and light chain CDR3 of SEQ ID NO: 190; (a2) heavy chain CDR1 of SEQ ID NO: 167, heavy chain CDR2 of SEQ ID NO: 171, heavy chain CDR3 of SEQ ID NO: 175, light chain CDR1 of SEQ ID NO: 183, light chain CDR2 of SEQ ID NO: 187, and light chain CDR3 of SEQ ID NO: 191; (a3) heavy chain CDR1 of SEQ ID NO: 168, heavy chain CDR2 of SEQ ID NO: 172, heavy chain CDR3 of SEQ ID NO: 176, light chain CDR1 of SEQ ID NO: 184, light chain CDR2 of SEQ ID NO: 188, and light chain CDR3 of SEQ ID NO: 192; (a4) heavy chain CDR1 of SEQ ID NO: 169, heavy chain CDR2 of SEQ ID NO: 173, heavy chain CDR3 of SEQ ID NO: 177, light chain CDR1 of SEQ ID NO: 185, light chain CDR2 of SEQ ID NO: 189, and light chain CDR3 of SEQ ID NO: 193; (a5) heavy chain CDR1 of SEQ ID NO: 166, heavy chain CDR2 of SEQ ID NO: 170, heavy chain CDR3 of SEQ ID NO: 174, light chain CDR1 of SEQ ID NO: 115, light chain CDR2 of SEQ ID NO: 124, and light chain CDR3 of SEQ ID NO: 134; (a6) heavy chain CDR1 of SEQ ID NO: 167, heavy chain CDR2 of SEQ ID NO: 171, heavy chain CDR3 of SEQ ID NO: 175, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 125, and light chain CDR3 of SEQ ID NO: 135; (a7) heavy chain CDR1 of SEQ ID NO: 168, heavy chain CDR2 of SEQ ID NO: 172, heavy chain CDR3 of SEQ ID NO: 176, light chain CDR1 of SEQ ID NO: 118, light chain CDR2 of SEQ ID NO: 128, and light chain CDR3 of SEQ ID NO: 137; (a8) an antibody that binds to the same epitope as an antibody comprising any one of (a1) to (a7); and (a9) An antibody that competes with the binding of an antibody comprising any one of (a1) to (a7). An antigen-binding molecule comprising any one selected from the group consisting of: [7] The following: (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 178; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 179; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 180; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 165, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 181; (a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 196; (a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 197; (a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 198; (a8) an antibody that binds to the same epitope as an antibody comprising any one of (a1) to (a7); and (a9) An antibody that competes with the binding of an antibody comprising any one of (a1) to (a7). An antigen-binding molecule comprising any one selected from the group consisting of: [8] An antigen-binding molecule of [6] or [7] that specifically binds to CH1 of human IgG1. [9] The antigen-binding molecule according to [8], which does not specifically bind to CH1 of human IgG1 when a disulfide bond is formed between the CH1 domains of two Fabs of human IgG1.

[10] The antigen-binding molecule according to [9], wherein the disulfide bond is a disulfide bond formed between two IgG1 Fabs at position 191 according to EU numbering in the CH1 domain.

[11] The antigen-binding molecule of any one of [8] to

[10] , which does not bind to CH1 of human IgG4.

[12] Use of the antigen-binding molecule according to any one of [6] to

[11] in the purification, analysis, or quantification of an antibody sample. [Brief explanation of the drawings]

[0024] [Figure 1] Illustrating various antibody formats. Annotation of each Fv region in Table 2. (a) A (1+2) trivalent antibody that has been applied with LINC technology, designated 1+2 Dual / LINC ("LINC" refers to engineered disulfide bonds, e.g., in the CH1 region); and (b) a schematic diagram showing a (1+2) format trivalent antibody without engineered disulfide bonds. [Figure 2a] Figure 1(b) is an illustration showing that LINC-Ig technology in a 1+2 format can reduce toxicity. LINC-Ig ("LINC," i.e., containing an engineered disulfide bond, e.g., in the CH1 region) can restrict antigen binding of the antibody shown in Figure 1(a) primarily to a cis-like manner, i.e., binding to antigens present on the same immune cell. In contrast, the (1+2) trivalent format without an engineered disulfide bond shown in Figure 1(b) can result in a trans-like manner of antigen binding, i.e., binding of the antibody in Figure 1(b) to antigens present on two different immune cells. This may cause cross-linking of two immune cells independent of tumor antigen binding, which may increase toxicity. [Figure 2b]Schematic diagram (left) showing that the engineered disulfide-free (1+2) trivalent antibody in Figure 1(b), which has unpaired surface cysteines, can form disulfide bonds with molecules containing free thiol groups, e.g., free cysteines or glutathione, in antibody preparations, resulting in capping of the unpaired cysteines on the antibody that prevents LINC formation. Treating such capped antibodies with a reducing agent can help decapping the surface cysteines (center), and further reoxidation of the decapped antibody (e.g., removal of the reducing agent by buffer exchange) promotes disulfide bond formation between the decapped cysteines, facilitating LINC formation (right). (For simplicity, native disulfide bonds, such as those between the hinge regions of the antibody and between the heavy chain CH1 and light chain CL, are not shown.) [Figure 3] Non-reducing SDS-PAGE analysis of trivalent (1+2) antibodies with and without LINC engineering (with or without the S191C mutation for engineered disulfide bond formation) is shown. A single protein band (lanes 2 and 5) was observed in the (1+2) trivalent format without the S191C mutation. In contrast, two protein bands were detected in the (1+2) Dual / LINC antibody variant, with the slower band exhibiting an electrophoretic mobility similar to that of the (1+2) trivalent format without the LINC mutation. This suggests that the faster band is Dual / LINC-Ig. The percentage of Dual-LINC-Ig (unLINC format) with unpaired cysteines in an antibody sample can be calculated by dividing the intensity of the slower / upper band corresponding to the "UnLINC" format by the sum of the intensities of the two bands corresponding to the "LINC" and "UnLINC" structures. [Figure 4] Non-reducing SDS-PAGE of Dual-LINC-Ig after treatment with various reducing agents is shown. "-" indicates "without CuSO4 addition." "+" indicates the addition of 25 μM / 50 μM CuSO4 during overnight (O / N) reoxidation. [Figure 5]1 shows non-reducing SDS-PAGE of Dual-LINC-Ig after TCEP treatment at various concentrations of Dual-LINC-Ig. [Figure 6] Figure 1 shows non-reducing SDS-PAGE of Dual-LINC-Ig after TCEP treatment for various incubation periods. The percentage of Dual-LINC-Ig (unLINC format) with unpaired cysteines in an antibody sample can be calculated by dividing the intensity of the late / upper band corresponding to the "UnLINC" format by the sum of the two bands corresponding to the "LINC" and "UnLINC" structures. [Figure 7] FIG. 1 is a schematic diagram illustrating the concept of a conformation-specific antibody (e.g., a conformation-specific anti-CH1 antibody) that binds to a target antibody (e.g., an epitope within the CH1 region) only when the antibody does not have an engineered disulfide bond, e.g., in the CH1 region ("unpaired cysteine" configuration), where the epitope is inaccessible to the conformation-specific antibody when the target antibody has an engineered disulfide bond ("paired cysteine" configuration), e.g., due to steric hindrance or short distance between the two Fabs caused by the engineered disulfide bond. [Figure 8]1 illustrates a Dual / LINC(1+2) antibody format comprising three Fabs, where two of the Fabs (Fab B and Fab C, composed of chain 1-chain 5 and chain 3-chain 4, respectively) each contain an engineered cysteine ​​(capable of forming an engineered disulfide bond linking both Fabs and thus existing in either the "unpaired cysteine" or "paired cysteine" form), and one Fab (Fab A, composed of chain 1-chain 2) does not contain an engineered cysteine ​​(exists only in the "paired cysteine" form). (a) CH1 of Fab A is in the "unpaired cysteine" or "unLINC" form / conformation, and CH1 of Fab B and Fab C are in the "paired cysteine" or "LINC" form / conformation. (b) The conformation-specific anti-IgG1 CH1 antibody can only bind to the CH1 of IgG1 in the "unpaired cysteine" or "unLINC" form / conformation. The CH1 of Fab A was engineered to have the IgG4 CH1 sequence. As a result, the conformation-specific anti-IgG1 CH1 antibody only binds to Dual / LINC(1+2) antibodies in the "unpaired cysteine" or "unLINC" form / conformation, but not to antibody species with the "paired cysteine" or "LINC" form / conformation. [Figure 9a] Illustrative diagram of various tool antibodies with different antibody formats for screening of conformation-specific anti-CH1 antibodies. [Figure 9b] The SEQ ID NOs of the amino acid sequences for each of the polypeptide chains of the tool antibody are shown. [Figure 10a] 1 shows the chromatographic profile of affinity purification of DLL3-DualAE05 / DualAE05-FF056 using the conformation-specific anti-CH1 antibody FAB0133Hh / FAB0133L0001 affinity column. [Figure 10b]1 shows non-reducing SDS-PAGE analysis of antibodies eluted in affinity purification of DLL3-DualAE05 / DualAE05-FF056 using conformation-specific anti-CH1 antibody FAB0133Hh / FAB0133L0001 affinity columns. Specifically, the flow-through fraction contains highly purified "paired cysteine" or "LINC" forms of DualAE05 / DualAE05-FF056, as indicated by a single predominant protein band that migrates faster in non-reducing SDS-PAGE analysis (lanes 1 to 13) (flow-through: white bars); the wash fraction contains a mixture of "unpaired cysteine" and "paired cysteine" forms of DualAE05 / DualAE05-FF056 (wash: gray bars); and the elution fraction contains predominantly "unpaired cysteine" or "unLINC" forms of DualAE05 / DualAE05-FF056, as indicated by a single predominant protein band that migrates slower in non-reducing SDS-PAGE analysis (lanes 20 to 23) (50 mM HCl acid eluate: black bars). The purity of the antibody samples was determined by densitometry of the bands in non-reducing SDS-PAGE. Images of unstained gels shown herein were captured using ChemiDoc Imaging Systems (Bio-Rad). Densitometry analysis of the protein bands of the unLINC and LINC forms of the LINC-Ig antibody was performed using Image Lab Software (Bio-Rad). The unLINC form migrated slightly slower than the LINC form due to differences in conformation. To obtain a more pure antibody, a protein sample containing 30-40% of the unLINC form (labeled input) was applied to the anti-CH1 column. DETAILED DESCRIPTION OF THE INVENTION

[0025] Description of Aspects 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).

[0026] The following definitions and detailed description are provided to facilitate understanding of the disclosure described herein.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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 against 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 against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the 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.

[0033] Recombination methods and constructs For example, as described in U.S. Patent No. 4,816,567, antibodies and antigen-binding molecules can be produced using recombinant methods and constructs. 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).

[0034] 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).

[0035] 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.

[0036] 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).

[0037] 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.

[0038] 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).

[0039] 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).

[0040] 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.

[0041] 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.

[0042] 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 certain embodiments, 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., that can bind to either CD3 or CD137, but not both antigens simultaneously, and that can specifically bind to DLL3.

[0043] In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: 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; The present invention provides a multispecific antigen-binding molecule comprising:

[0044] In one aspect, the present disclosure provides a method for producing a cellular membrane comprising: 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; The present invention provides a multispecific antigen-binding molecule comprising:

[0045] In one aspect, the first antigen-binding portion and the second antigen-binding portion each have one of the following structures (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 any one of:

[0046] In one aspect, the first antigen-binding portion and the second antigen-binding portion each have one of the following structures (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; 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. (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 any one of:

[0047] In one aspect, each of the first and second antigen-binding moieties is a Fab molecule and comprises at least one disulfide bond formed between the first and second antigen-binding moieties, preferably, the at least one disulfide bond is formed between amino acid residues (cysteines) that are not in the hinge region, preferably between amino acid residues (cysteines) in the CH1 region of each antigen-binding moiety.

[0048] In one aspect, 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 (cysteine) according to EU numbering in the CH1 region of each of the first antigen-binding portion and the second antigen-binding portion.

[0049] In some aspects, the third antigen-binding moiety is fused to either the first antigen-binding moiety or the second antigen-binding moiety.

[0050] In one aspect, the third antigen-binding portion is a Fab or scFv.

[0051] In one aspect, 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 of the Fab heavy chain (CH1) to the N-terminus of the Fab heavy chain of either the first or second antigen-binding moiety, optionally via a peptide linker.

[0052] In one aspect, the peptide linker is selected from the group consisting of the amino acid sequence of SEQ ID NO:248, SEQ ID NO:249, or SEQ ID NO:259.

[0053] In one aspect, the first antigen-binding portion is identical to the second antigen-binding portion.

[0054] In one aspect, the third antigen-binding moiety is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain have been exchanged, and each of the first and second antigen-binding moieties is a conventional Fab molecule.

[0055] In one aspect, 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 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 acid at position 147 and / or 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (EU numbering).

[0056] In one aspect, 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 (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 213 are glutamic acid (E) (EU numbering).

[0057] In one aspect, the third antigen-binding moiety capable of binding to DLL3 is 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 any one of:

[0058] In one aspect, the third antigen-binding moiety capable of binding to DLL3 is 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 antibody variable region comprises any one of:

[0059] In one aspect, the multispecific antigen-binding molecule of the present invention further comprises an Fc domain.

[0060] In one aspect, 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 native human IgG1 Fc domain.

[0061] In one aspect, the Fc domain exhibits enhanced FcRn-binding activity under acidic pH conditions (eg, pH 5.8) compared to that of the Fc region of native IgG.

[0062] In one aspect, the Fc domain comprises Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, according to EU numbering.

[0063] In one aspect, 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.

[0064] In one aspect, 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.

[0065] In one aspect, 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:

[0066] In one aspect, the Fc domain comprises the following combination of amino acid substitutions: M428L / N434A / Q438R / S440E.

[0067] In one aspect, the Fc domain is an IgG Fc domain, preferably a human IgG Fc domain, more preferably a human IgG1 Fc domain.

[0068] In one aspect, the Fc domain comprises: (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; or (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 Contains any of the following.

[0069] In one aspect, 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.

[0070] In one aspect, 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.

[0071] In one aspect, the multispecific antigen-binding molecule of the present invention comprises the following (a1) to (a15): (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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chain 4 & chain 5) each containing 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).

[0072] The components of the multispecific antigen-binding molecules of the invention can be fused to each other in a variety of configurations. Exemplary configurations are illustrated in Figure 1(a) read in conjunction with Table 2.

[0073] 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.

[0074] 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).

[0075] In some embodiments, the multispecific antigen-binding molecules 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, 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).

[0076] 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.

[0077] 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.

[0078] an antigen-binding moiety capable of binding to CD3 and CD137 but not simultaneously binding to CD3 and CD137 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 ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab"). In some embodiments, each of the two Dual antigen-binding moieties ("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").

[0079] 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") is a domain comprising antibody light and heavy chain variable regions (VL and VH). Suitable examples of such domains comprising antibody light and heavy chain variable regions include "single-chain Fv (scFv)," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," "F(ab')2," etc. In certain embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" 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 portion is cross-reactive with (i.e., specifically binds to) human and cynomolgus CD3. In some embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") 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 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 CD3ε chain expressed on the surface of a eukaryotic cell. In some embodiments, the first antigen-binding moiety binds to the CD3ε chain expressed on the surface of a T cell. 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:

[0080] 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 1A 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 1A.

[0081] Table 1A: 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") TIFF2025172866000002.tif132170

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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 1B below.

[0086] (Table 1B) SEQ ID NOs of HVR (CDR) sequences of dual antigen-binding moieties ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") TIFF2025172866000003.tif130170

[0087] In some embodiments, the Dual antigen-binding moiety ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") is each 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:

[0088] In some embodiments, the multispecific antigen-binding molecule or Dual antigen-binding portion of the present invention ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") also contains 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).

[0089] An antigen-binding moiety capable of binding to DLL3 The multispecific antigen-binding molecules described herein comprise at least one antigen-binding moiety capable of binding to Delta-like 3 (DLL3) (also referred to herein as a "DLL3 antigen-binding moiety" or "third antigen-binding moiety"). In certain embodiments, the multispecific antigen-binding molecule comprises one antigen-binding moiety capable of binding to DLL3. In certain embodiments, the multispecific antigen-binding molecule comprises two antigen-binding moieties capable of binding to DLL3 ("DLL3 antigen-binding moieties"). In certain such embodiments, each of these antigen-binding moieties specifically binds to the same epitope of DLL3. In even more specific embodiments, all of these "DLL3 antigen-binding moieties" are identical. In one embodiment, the multispecific antigen-binding molecule comprises an immunoglobulin molecule capable of specifically binding to DLL3 ("DLL3 antigen-binding moiety"). In one embodiment, the multispecific antigen-binding molecule comprises no more than two antigen-binding moieties capable of binding to DLL3 ("DLL3 antigen-binding moieties").

[0090] 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.

[0091] 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.

[0092] In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion binds to an epitope within the extracellular domain (ECD), i.e., the domain 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 a previously reported anti-DLL3 antibody (e.g., WO2019131988 and WO2011093097) in which the DLL3 epitope that it binds has been characterized.

[0093] 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 1C. 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 1C.

[0094] Table 1C: SEQ ID NOs of exemplary variable regions of DLL3 antigen binding sites TIFF2025172866000004.tif193170

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] In certain embodiments, the DLL3 antigen-binding portion comprises any one of the combinations of HVR sequences shown in Table ID below. In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion is a domain comprising an antibody variable fragment that competes for binding to DLL3 with any one of the antibody variable regions shown in Table ID, 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 ID.

[0101] Table 1D. SEQ ID NOs for exemplary HVR (CDR) sequences of DLL3 antigen binding sites TIFF2025172866000005.tif100170

[0102] In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention comprises 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 any one of:

[0103] In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention also includes 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, p. 24-39). 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 will comprise an scFv construct, and in preferred embodiments, will comprise and contain heavy and light chain variable regions as disclosed herein. In other preferred embodiments, the DLL3 binding domain (and DLL3 CAR) of the present invention will comprise an scFv construct or a fragment thereof comprising the heavy and light chain variable regions described herein. In preferred embodiments, the disclosed chimeric antigen receptors are useful for treating or preventing proliferative disorders and any recurrence or metastasis thereof. 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.

[0104] Methods for producing multispecific antigen-binding molecules The present disclosure provides methods of producing any of the multispecific antigen-binding molecules described herein. In one aspect, the present disclosure provides a method for producing a multispecific antigen-binding molecule, the multispecific antigen-binding molecule comprising: a first antigen-binding portion and a second antigen-binding portion, each of which is a Fab and is capable of binding to the first antigen and a second antigen different from the first antigen, but does not bind to both antigens simultaneously; and a third antigen-binding moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), capable of binding to a third antigen different from the first and second antigens, preferably an antigen expressed on cancer cells / tissues; The method comprises the steps of: (a) providing one or more nucleic acids encoding: i. a first polypeptide comprising (from N-terminus to C-terminus) a VH or VL of a third antigen-binding portion, optionally a heavy chain constant region (CH1); and a VH or VL of a first antigen-binding portion, a heavy chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3); ii. a second polypeptide comprising (from N-terminus to C-terminus) a third antigen-binding portion, VH or VL, and optionally a light chain constant region (CL); iii. A third polypeptide comprising (from N-terminus to C-terminus) a second antigen-binding portion, VH or VL, a heavy chain constant region (CH1); and optionally a hinge region and / or Fc region (CH2 and CH3); iv. a fourth polypeptide comprising (from N-terminus to C-terminus) the VH or VL of a second antigen-binding portion, optionally a light chain constant region (CL); and v. a fifth polypeptide comprising (from N-terminus to C-terminus) the VH or VL of the first antigen-binding portion, optionally a light chain constant region (CL); (b) introducing one or more nucleic acids produced in (a) into a host cell; (c) culturing a host cell to express the polypeptide in (i) to (v); and (d) collecting multispecific antigen-binding molecules comprising the five polypeptides in (i) to (v) from the culture medium of the cells cultured in step (c). and optionally, the polypeptides in (iv)-(v) are identical; and Each of the first antigen-binding moiety and the second antigen-binding moiety comprises (via mutation, substitution, or insertion) at least one cysteine ​​residue that is not in the hinge region, and preferably, the at least one cysteine ​​is located in the CH1 region; the at least one cysteine ​​residue, preferably in the CH1 region, is capable of forming at least one disulfide bond between the first antigen-binding moiety and the second antigen-binding moiety; The method includes contacting the preparation with a reducing reagent.

[0105] In one aspect, the first antigen-binding moiety and the second antigen-binding moiety each contain (via mutation, substitution, or insertion) a cysteine ​​residue at position 191 according to EU numbering in the CH1 region that can form a disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety.

[0106] In one aspect, the method further comprises step (e) of contacting the multispecific antigen-binding molecule (multispecific antigen-binding molecule) preparation collected from step (d) with a reducing reagent under reducing conditions that allow the cysteine ​​in the CH1 region (position 191 according to EU numbering) to form one or more disulfide bonds.

[0107] In one aspect, the multispecific antigen-binding molecule preparation collected from step (d) (prior to contacting with the reducing agent) comprises two or more structural isoforms that differ by at least one disulfide bond formed between amino acid residues located in or at position 191 (EU numbering) in the CH1 region, and contacting with the reducing agent in step (e) preferentially enriches or increases the population of multispecific antigen-binding molecule structural isoforms having at least one disulfide bond formed between amino acid residues located in or at position 191 (EU numbering) in the CH1 region.

[0108] In one aspect, the pH of the reducing reagent contacted with the multispecific antigen-binding molecule is from about 3 to about 10. In one aspect, the pH of the reducing reagent contacted with the multispecific antigen-binding molecule is about 6, 7, or 8. In one aspect, the pH of the reducing reagent contacted with the multispecific antigen-binding molecule is about 7. In one aspect, the pH of the reducing reagent contacted with the multispecific antigen-binding molecule is about 3.

[0109] In some aspects, the reducing agent is selected from the group consisting of TCEP, 2-MEA, DTT, cysteine, GSH, and Na2SO3. In one aspect, the reducing agent is TCEP, preferably 0.25 mM TCEP.

[0110] In one aspect, the concentration of the reducing agent is from about 0.01 mM to about 100 mM. In some aspects, the concentration of the reducing agent is about 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100 mM, preferably about 0.25 mM.

[0111] In one aspect, the contacting step is carried out for at least 30 minutes. In one aspect, the contacting step is carried out for about 10 minutes to about 48 hours. In some aspects, the contacting step is carried out for about 2 hours or about 18 hours. In one aspect, the contacting step is carried out at a temperature of about 4°C to 37°C, preferably 23°C to 25°C.

[0112] In some aspects, the multispecific antigen-binding molecule is at least partially purified prior to the step of contacting with the reducing agent. In one aspect, the multispecific antigen-binding molecule is partially purified by affinity chromatography (preferably Protein A chromatography) prior to the contacting step.

[0113] In one aspect, the concentration of the multispecific antigen-binding molecule is from about 0.1 mg / ml to about 50 mg / ml or more. In one aspect, the concentration of the multispecific antigen-binding molecule is about 10 mg / ml or about 20 mg / ml.

[0114] In one aspect, the method further comprises the step of promoting reoxidation of cysteine ​​disulfide bonds, preferably by removing the reducing agent, preferably by dialysis or buffer exchange.

[0115] In one aspect, the third antigen-binding portion is a conventional Fab, and (a) the first polypeptide comprises (from N-terminus to C-terminus) a VH, heavy chain constant region (CH1) of a third antigen-binding portion; and a VH, heavy chain constant region (CH1) of a first antigen-binding portion; and optionally a hinge region and / or an Fc region (CH2 and CH3); (b) the second polypeptide comprises (from N-terminus to C-terminus) a VL of a third antigen-binding portion, and a light chain constant region (CL); (c) a third polypeptide comprising (from N-terminus to C-terminus) a VH, a heavy chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3) of a second antigen-binding portion; (d) the fourth polypeptide comprises (from N-terminus to C-terminus) the VL of the second antigen-binding portion, and a light chain constant region (CL); and (e) The fifth polypeptide comprises (from N-terminus to C-terminus) the VL of the first antigen-binding portion, and a light chain constant region (CL).

[0116] In one aspect, the third antigen-binding portion is a VH / VL crossover Fab (in which the variable regions of the Fab light chain and Fab heavy chain are swapped), and (a) the first polypeptide comprises (from N-terminus to C-terminus) a VL, heavy chain constant region (CH1) of a third antigen-binding portion; and a VH, heavy chain constant region (CH1) of a first antigen-binding portion; and optionally a hinge region and / or an Fc region (CH2 and CH3); (b) a second polypeptide comprising (from N-terminus to C-terminus) a VH of a third antigen-binding portion, and a light chain constant region (CL); (c) a third polypeptide comprising (from N-terminus to C-terminus) a VH, a heavy chain constant region (CH1); and optionally a hinge region and / or an Fc region (CH2 and CH3) of a second antigen-binding portion; (d) the fourth polypeptide comprises (from N-terminus to C-terminus) the VL of the second antigen-binding portion, and a light chain constant region (CL); and (e) The fifth polypeptide comprises (from N-terminus to C-terminus) the VL of the first antigen-binding portion, and a light chain constant region (CL).

[0117] In one aspect, in the CL 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 (Kabat numbering), and in the CH1 constant domain 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) (EU numbering).

[0118] In one aspect, in step (a)(i), the first polypeptide further comprises a peptide linker between the third antigen-binding moiety and the VH or VL of the first antigen-binding moiety.

[0119] In one aspect, the peptide linker is selected from the group consisting of the amino acid sequence of SEQ ID NO:248, SEQ ID NO:249, or SEQ ID NO:259.

[0120] In one aspect, each of the first antigen-binding moiety and the second antigen-binding moiety is capable of binding to CD3 and CD137, but does not bind to both CD3 and CD137 simultaneously.

[0121] In one aspect, the first antigen-binding portion and the second antigen-binding portion each have one of the following structures (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 any one of:

[0122] In one aspect, the first antigen-binding portion and the second antigen-binding portion each have one of the following structures (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; 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. (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 any one of:

[0123] In one aspect, the third antigen-binding moiety is capable of binding to DLL3, preferably human DLL3.

[0124] In one aspect, the third antigen binding portion capable of binding to DLL3 comprises an antibody variable region comprising 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.

[0125] In one aspect, the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region comprising 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.

[0126] In one aspect, the multispecific antigen-binding molecule further comprises an Fc domain.

[0127] In one aspect, 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 native human IgG1 Fc domain.

[0128] In one aspect, the multispecific antigen-binding molecule comprises any one of the following (a1) to (a15): (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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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).

[0129] In one aspect, the fourth polypeptide (chain 4) and the fifth polypeptide (chain 5) are identical.

[0130] In one aspect, only one nucleic acid, or two, three, four, or five different nucleic acids, encode and express the first, second, third, fourth, and fifth polypeptides.

[0131] Contact with reducing reagents "Contacting" means subjecting or exposing to a solution. The antibody, protein, or polypeptide can be contacted with a reducing reagent while bound to a solid support (e.g., an affinity column or a chromatography matrix). Preferably, the solution is buffered. To maximize the yield of an antibody / protein having a desired conformation, the pH of the solution is selected to protect the stability of the antibody / protein and optimize disulfide exchange. In practicing the present invention, the pH of the solution is preferably not strongly acidic. Thus, some pH ranges are above pH 5, preferably from about pH 6 to about pH 11, more preferably from about pH 7 to about pH 10, and even more preferably from about pH 6 to about pH 8. In one non-limiting embodiment of the present invention, the optimal pH was found to be about pH 7. However, the optimal pH for a particular embodiment of the present invention can be easily determined experimentally by one skilled in the art. While not wishing to be bound by the following theory, we believe that the presence of UnLINC formats (i.e., trivalent 1+2 antibodies without engineered disulfide bonds or "paired cysteines") may be due to unpaired Cys residues, which often form disulfide bonds with molecules containing free thiol groups, such as cysteinylation and glutathionylation, which "cap" the unpaired Cys residues and prevent LINC formation (engineered disulfide bond formation). As shown in Figure 2(b), to remove unpaired cysteine-capped molecules, a reducing agent can help decap the surface cysteines, and further reoxidation of the decapped antibody (e.g., removal of the reducing reagent by buffer exchange) can promote disulfide bond formation between the decapped cysteines for LINC formation. Therefore, removal of cysteinylation from unpaired sulfhydryls in the UnLINC format by reduction and reoxidation can remove the UnLINC format and improve antibody homogeneity.

[0132] The terms "reducing reagent" and "reducing agent" are used interchangeably. In some embodiments, the reducing agent is a free thiol. The reducing reagent is preferably comprised of a compound from the group consisting of glutathione (GSH), dithiothreitol (DTT), 2-mercaptoethanol, 2-aminoethanethiol (2-MEA), TCEP (tris(2-carboxyethyl)phosphine), dithionitrobenzoate, cysteine, and Na2SO3. In some embodiments, TCEP, 2-MEA, DTT, cysteine, GSH, or Na2SO3 can be used. In some preferred embodiments, 2-MEA can be used. In some preferred embodiments, TCEP can be used.

[0133] The reducing agent may be added to the fermentation medium in which the cells producing the recombinant protein are grown. In additional embodiments, the reducing agent may be added to the LC mobile phase during the LC separation step to separate the recombinant protein. In certain embodiments, the protein is immobilized on the stationary phase of the LC column, and the reducing agent is part of the mobile phase. In certain embodiments, intact IgG antibodies may be eluted as a heterogeneous mixture, as indicated by the number of peaks. The use of a reduction / oxidation coupling reagent results in a simpler and more uniform peak pattern. It is contemplated that this more uniform peak of interest may be isolated as a more homogeneous preparation of IgG.

[0134] The reducing agent is present at a concentration sufficient to increase the relative proportion of a desired conformation (e.g., an antibody in a "paired cysteine" form with one or more engineered disulfide bonds formed between the two Fabs of the antibody, e.g., between amino acid residues not in the hinge region). The optimal absolute concentration and molar ratio of the reducing agent will depend on the concentration of total IgG and, in some circumstances, the specific IgG subclass. When used to prepare IgG1 molecules, it will also depend on the number and accessibility of unpaired cysteines in the protein. Generally, the concentration of free thiol from the reducing agent can be about 0.05 mM to about 100 mM, more preferably about 0.1 mM to about 50 mM, and even more preferably about 0.2 mM to about 20 mM. In some preferred embodiments, the concentration of the reducing agent is 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, or 100 mM. In some preferred embodiments, 0.05 mM to 1 mM of 2-MEA can be used. In some preferred embodiments, 0.01 mM to 25 mM TCEP can be used.

[0135] The contacting of the recombinant protein preparation with the reducing agent is carried out for a time sufficient to increase the relative proportion of the desired conformation. Any relative increase in proportion is desirable, including, for example, conversion of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or even 80% or 90% of the protein having the undesired conformation to protein having the desired conformation. The contacting may be carried out by providing the reducing agent to the fermentation medium from which the protein is produced. Alternatively, the contacting is carried out during partial purification of the protein from the cell culture from which it is produced. In yet other embodiments, the contacting is carried out after the protein has been eluted from a chromatography column but before further processing. Essentially, the contacting can be carried out at any stage during antibody preparation, purification, storage, or formulation. In some embodiments, partial purification by affinity chromatography (e.g., Protein A chromatography) can be carried out prior to the contacting.

[0136] The contacting may be performed by an antibody attached to the stationary phase of a chromatography column, with the reducing agent being part of the mobile phase; in this case, the contacting may be performed as part of a chromatographic purification procedure. Representative chromatographic refolding processes include size exclusion (SEC); solvent exchange during reversible adsorption on a Protein A column; hydrophobic interaction chromatography (HIC); immobilized metal affinity chromatography (IMAC); reversed-phase chromatography (RPC); and the use of immobilized folding catalysts, such as GroE1, GroES, or other proteins with folding properties. On-column refolding is attractive because it can be easily automated using commercially available preparative chromatography systems. On-column refolding of recombinant proteins produced in microbial cells was recently reviewed in (Li et al., 2004).

[0137] When the contacting step is performed on a partially or highly purified preparation of recombinant protein, the contacting step can be performed for as little as about 1 hour to about 4 hours and as long as about 6 hours to about 4 days. Contacting steps of about 2 to about 48 hours, or about 16 hours, have been found to work well. The contacting step can also be performed on a solid phase during another step, for example, filtration or any other step in the purification.

[0138] The methods of the invention can be carried out over a wide temperature range. For example, the methods of the invention have been successfully carried out at temperatures from about 4° C. to about 37° C., although best results have been achieved at lower temperatures. Typical temperatures for contacting partially or fully purified preparations of recombinant protein are from about 4° C. to about 25° C. (ambient temperature), or preferably 23° C., although lower and higher temperatures can also be used.

[0139] Additionally, it is contemplated that the method can be performed at high pressure. High hydrostatic pressure (1000-2000 bar) combined with low, non-denaturing concentrations of guanidine hydrochloride (less than 1 M) has previously been used to disaggregate (solubilize) and refold several denatured proteins produced by Escherichia coli as inclusion bodies, including human growth hormone and lysozyme, as well as b-lactamase (St John et al., Proc Natl Acad Sci USA, 96:13029-13033 (1999)). b-lactamase was refolded to high yields of active protein, even in the absence of added GdmHCl. In another study (Seefeldt et al., Protein Sci, 13:2639-2650 (2004)), the refolding yield of the mammalian cell-produced protein bikunin obtained by high-pressure-regulated refolding at 2000 bar was 70% by RP-HPLC, significantly higher than the 55% value (by RP-HPLC) obtained by typical guanidine hydrochloride "dilution refolding." These findings indicate that high hydrostatic pressure promotes the disruption of intermolecular and intramolecular interactions, leading to protein unfolding and disaggregation. The interaction of high pressure with proteins is similar to the interaction of proteins with chaotropic agents. Therefore, the method of the present invention contemplates the use of high pressure instead of chaotropic agents for protein unfolding. Of course, a combination of high pressure and chaotropic agents can also be used in some instances.

[0140] The recombinant antibody / protein preparation can be contacted with the reducing agent in various amounts, as needed. For example, the methods of the present invention have been successfully performed at analytical laboratory scales (1-50 mL), preparative scales (50 mL-10 L), and manufacturing scales (10 L or more). The methods of the present invention can be performed reproducibly on both small and large scales. Thus, the antibody concentration can be in industrial quantities (gram weight units) (e.g., industrial quantities of a particular IgG) or milligram amounts. In certain embodiments, the concentration of the recombinant antibody in the reaction mixture is about 1 mg / ml to about 50 mg / ml, more specifically, 10 mg / ml, 15 mg / ml, or 20 mg / ml. Recombinant IgG1 molecules at these concentrations are specifically contemplated.

[0141] In certain embodiments, proteins produced using a medium containing a reducing agent are further treated with a separation treatment step utilizing a chaotropic denaturant, such as sodium dodecyl sulfate (SDS), urea, or guanidinium hydrochloride (GuHCl). A significant amount of chaotropic agent is required to observe detectable unfolding. In some embodiments, the treatment step uses between 0.1 M and 2 M of a chaotrope, which produces an effect equivalent to the use of 0.1 M to 2 M of guanidinium hydrochloride. In certain embodiments, oxidative refolding is achieved in the presence of approximately 1.0 M guanidinium hydrochloride, or an amount of other chaotropic agent that produces the same or similar amount of refolding as 1 M guanidinium hydrochloride. In some embodiments, the method uses between about 1.5 M and 0.5 M of a chaotrope. The amount of chaotropic agent used is based on the structural stability of the protein in the presence of the chaotrope. The chaotrope must be present enough to disrupt the local tertiary and / or quaternary structure of the protein's domain interactions, but less than that required to completely unfold the secondary structure of the molecule and / or individual domains. To determine the point at which a protein begins to unfold by equilibrium denaturation, one skilled in the art can titrate a chaotrope into a solution containing the protein and monitor the structure using techniques such as circular dichroism or fluorescence. There are other parameters that can be used instead of chaotropes to unfold or slightly disrupt the structure of a protein. Temperature and pressure are two basic parameters that have been used to change the structure of proteins and can be used instead of chaotropic agents during contact with redox agents. The inventors contemplate that any parameter shown to denature or disrupt the structure of a protein can be used by those skilled in the art instead of chaotropic agents.

[0142] Disulfide exchange can be quenched by any method known to those skilled in the art.For example, the reducing agent can be removed or its concentration can be reduced through a purification step, and / or it can be chemically inactivated, for example, by acidifying the solution.Typically, when the reaction is quenched by acidification, the pH of the solution containing the reducing agent is reduced to less than pH 7.In some embodiments, the pH is reduced to less than pH 6.Generally, the pH is reduced to between about pH 2 and about pH 6.

[0143] In some embodiments, removal of the reducing agent may be carried out by dialysis, buffer exchange, or any of the chromatographic methods described herein.

[0144] Preferential enrichment (or increase) The term "preferentially enrich (or enriched)" refers to an increase in the relative abundance of a desired form, or an increase in the relative proportion of a desired form, or an increase in the population of a desired form (structural isoform). In some embodiments, the methods described herein increase the relative abundance of an antibody structural isoform, such as an antibody having at least one disulfide bond formed between amino acid residues outside the hinge region. In one embodiment, the at least one disulfide bond is formed between the amino acid residue at position 191 (EU numbering) in the CH1 region of each of the first and second antigen-binding domains. In certain embodiments, the methods produce a homogeneous antibody preparation having at least 50%, 60%, 70%, 80%, 90%, and preferably at least 95% molar ratio of the antibody having at least one disulfide bond formed outside the hinge region.

[0145] uniformity A "homogeneous" population of antibodies refers to an antibody population that primarily comprises antibodies of a single form, e.g., at least 50%, 60%, 70%, 80% or more, preferably at least 90%, 95%, 96%, 97%, 99%, or 100%, of the antibodies in a solution or composition are in a properly folded form. Similarly, a "homogeneous" population of antibodies having at least one disulfide bond formed outside the hinge region refers to a population of such antibodies that primarily comprises a single properly folded form, e.g., at least 50%, 60%, 70%, 80% or more, preferably at least 90%, 95%, 96%, 97%, 99%, or 100% molar ratio of such antibodies having at least one disulfide bond formed outside the hinge region. In a preferred embodiment, the "homogeneous" population of antibodies comprises at least one disulfide bond formed between the amino acid residue at position 191 according to EU numbering in the CH1 region of each of the first and second antigen-binding domains (i.e., the "paired cysteines" at position 191 according to EU numbering in the CH1 region).

[0146] In a preferred embodiment, the methods of the present invention produce a homogeneous antibody population or homogeneous antibody preparation by the steps described herein.

[0147] Determining whether an antibody population is homogeneous and the relative abundance or proportion of protein / antibody conformations in a mixture can be performed using any of a variety of analytical and / or qualitative techniques. If two conformations are differentially resolved during a separation technique, such as chromatography, electrophoresis, filtration, or other purification technique, the relative proportions of the conformations in the mixture can be determined using such a purification technique. For example, at least two different conformations of a recombinant IgG can be separated by hydrophobic interaction chromatography. Furthermore, because far-ultraviolet circular dichroism has been used to predict the secondary structural organization of proteins (Perczel et al., 1991, Protein Engrg. 4:669-679), such a technique can determine whether alternative conformations of a protein exist. Yet another technique used to determine conformation is fluorescence spectroscopy, which can be used to identify complementary differences in tertiary structure that can be assigned to tryptophan and tyrosine fluorescence. Other techniques that can be used to determine conformational differences and therefore the relative proportions of conformations are online SEC to measure aggregation state, differential scanning calorimetry to measure melting transitions (Tm) and component enthalpies, and chaotropic unfolding. Yet another technique that can be used to determine conformational differences and therefore the relative proportions of conformations is LC / MS detection to determine protein heterogeneity.

[0148] Alternatively, if differences in activity exist between antibody / protein conformations, determining the relative proportions of conformations in the mixture can be done by activity assays (e.g., binding to a ligand, enzymatic activity, biological activity, etc.). The biological activity of the protein can also be used. Alternatively, binding assays can be used in which activity is expressed as activity units / mg protein.

[0149] In some embodiments, described in detail herein below, the present invention uses IEC chromatography to determine antibody / protein heterogeneity. In such cases, the antibody is purified or considered "homogeneous," meaning that no polypeptide peaks or fractions corresponding to other polypeptides are detected upon analysis by IEC chromatography. In certain embodiments, the antibody is purified or considered "homogeneous," meaning that no polypeptide bands corresponding to other polypeptides are detected upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Those skilled in the relevant art will recognize that multiple bands corresponding to a polypeptide can be visualized by SDS-PAGE due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptides of the present invention are purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, and / or (if the polypeptide is radiolabeled) autoradiography.

[0150] Herein, examples of conditions for SDS-PAGE analysis are as follows: Non-reducing SDS-PAGE was performed using 4-20% Mini-PROTEAN® TGX Stain-Free™ Precast Gels (Bio-Rad) with 1x Tris / glycine / SDS running buffer (Bio-Rad). Monoclonal antibody samples were heated at 70°C for 10 minutes. 0.2 micrograms were loaded, and electrophoresis was performed at 200 V for 90 minutes. Proteins were visualized using a Chemidoc Imaging System (Bio-Rad). The percentage of individual bands was analyzed using Image Lab software version 6.0 (Bio-Rad). The percent intensity of each band (e.g., fast-migrating (lower band) and slow-migrating (upper band) bands) was calculated by dividing the band intensity by the sum of the two bands. The gel may then be stained with CBB, a gel image may be captured, and the bands may be quantified using an imaging device. In the gel image, multiple bands, e.g., two bands, i.e., an "upper band" and a "lower band," may be observed in the antibody variant sample. In this case, the molecular weight of the upper band may correspond to that of the parent antibody (before modification). The cysteine ​​substitution may cause structural changes, such as cross-linking via disulfide bonds in the Fab, which may result in changes in electrophoretic mobility. In this case, the lower band may be considered to correspond to the antibody with one or more engineered disulfide bonds formed between the CH1 regions. An antibody variant sample with additional cysteine ​​substitutions may exhibit a higher ratio of lower band to upper band compared to the control sample. The additional cysteine ​​residue may enhance / promote disulfide bond cross-linking of the Fab; increase the percentage or structural uniformity of antibody preparations with engineered disulfide bonds formed at the mutated positions; or decrease the percentage of antibody preparations without engineered disulfide bonds formed at the mutated positions.

[0151] Methods for capturing and / or removing target antibodies from antibody preparations The present disclosure provides methods for capturing and / or removing target antibodies from antibody preparations. In one aspect, the present disclosure provides a method for producing a cellular membrane comprising the steps of: a) contacting an antibody preparation containing a target antibody with an antigen-binding molecule immobilized on a support; and b) capturing the target antibody by specific binding to the antigen-binding molecule 1. A method for capturing and / or removing a target antibody from an antibody preparation, comprising: the antibody comprises at least two Fabs derived from IgG (preferably human IgG or human IgG1), and the antibody preparation comprises two antibody structural isoforms that differ only by a disulfide bond formed between the two Fabs in the CH1 domain; and The antigen-binding molecule specifically binds to and captures a target antibody that does not contain a disulfide bond. The method is provided.

[0152] In one aspect, the antigen-binding molecule binds to the target antibody at an epitope that is accessible to the antigen-binding molecule only when the target antibody does not have disulfide bonds.

[0153] In one aspect, the disulfide bond is a disulfide bond formed between two Fabs of an antibody at position 191 according to EU numbering in the CH1 domain.

[0154] In one aspect, the antigen-binding molecule that specifically binds to the target antibody is one of the following: (a1) heavy chain CDR1 of SEQ ID NO: 166, heavy chain CDR2 of SEQ ID NO: 170, heavy chain CDR3 of SEQ ID NO: 174, light chain CDR1 of SEQ ID NO: 182, light chain CDR2 of SEQ ID NO: 186, and light chain CDR3 of SEQ ID NO: 190; (a2) heavy chain CDR1 of SEQ ID NO: 167, heavy chain CDR2 of SEQ ID NO: 171, heavy chain CDR3 of SEQ ID NO: 175, light chain CDR1 of SEQ ID NO: 183, light chain CDR2 of SEQ ID NO: 187, and light chain CDR3 of SEQ ID NO: 191; (a3) heavy chain CDR1 of SEQ ID NO: 168, heavy chain CDR2 of SEQ ID NO: 172, heavy chain CDR3 of SEQ ID NO: 176, light chain CDR1 of SEQ ID NO: 184, light chain CDR2 of SEQ ID NO: 188, and light chain CDR3 of SEQ ID NO: 192; (a4) heavy chain CDR1 of SEQ ID NO: 169, heavy chain CDR2 of SEQ ID NO: 173, heavy chain CDR3 of SEQ ID NO: 177, light chain CDR1 of SEQ ID NO: 185, light chain CDR2 of SEQ ID NO: 189, and light chain CDR3 of SEQ ID NO: 193; (a5) heavy chain CDR1 of SEQ ID NO: 166, heavy chain CDR2 of SEQ ID NO: 170, heavy chain CDR3 of SEQ ID NO: 174, light chain CDR1 of SEQ ID NO: 115, light chain CDR2 of SEQ ID NO: 124, and light chain CDR3 of SEQ ID NO: 134; (a6) heavy chain CDR1 of SEQ ID NO: 167, heavy chain CDR2 of SEQ ID NO: 171, heavy chain CDR3 of SEQ ID NO: 175, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 125, and light chain CDR3 of SEQ ID NO: 135; (a7) heavy chain CDR1 of SEQ ID NO: 168, heavy chain CDR2 of SEQ ID NO: 172, heavy chain CDR3 of SEQ ID NO: 176, light chain CDR1 of SEQ ID NO: 118, light chain CDR2 of SEQ ID NO: 128, and light chain CDR3 of SEQ ID NO: 137; (a8) an antibody that binds to the same epitope as an antibody comprising any one of (a1) to (a7); and (a9) An antibody that competes with the binding of an antibody comprising any one of (a1) to (a7). The antibody comprises any one selected from the group consisting of:

[0155] In one aspect, the antigen-binding molecule that specifically binds to the target antibody is one of the following: (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 178; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 179; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 180; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 165, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 181; (a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 196; (a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 197; (a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 198; (a8) an antibody that binds to the same epitope as an antibody comprising any one of (a1) to (a7); and (a9) An antibody that competes with the binding of an antibody comprising any one of (a1) to (a7). The antibody comprises any one selected from the group consisting of:

[0156] In one aspect, the target antibody is one of the following (a1) to (a15): (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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & 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 & chain 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) containing the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) containing the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chain 4 & chain 5) each containing 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).

[0157] Conformation-specific antibodies The present disclosure provides conformation-specific antibodies that specifically bind to a target antibody only when the target antibody does not have an engineered disulfide bond between the two Fabs, e.g., in the CH1 region ("unpaired cysteine" configuration). In some aspects, the epitope is inaccessible to the conformation-specific antibody when the target antibody has an engineered disulfide bond ("paired cysteine" configuration), e.g., due to steric hindrance or the short distance between the two Fabs caused by the engineered disulfide bond.

[0158] In one aspect, a conformation-specific antibody (an antigen-binding molecule that specifically binds to a target antibody) is one of the following: (a1) heavy chain CDR1 of SEQ ID NO: 166, heavy chain CDR2 of SEQ ID NO: 170, heavy chain CDR3 of SEQ ID NO: 174, light chain CDR1 of SEQ ID NO: 182, light chain CDR2 of SEQ ID NO: 186, and light chain CDR3 of SEQ ID NO: 190; (a2) heavy chain CDR1 of SEQ ID NO: 167, heavy chain CDR2 of SEQ ID NO: 171, heavy chain CDR3 of SEQ ID NO: 175, light chain CDR1 of SEQ ID NO: 183, light chain CDR2 of SEQ ID NO: 187, and light chain CDR3 of SEQ ID NO: 191; (a3) heavy chain CDR1 of SEQ ID NO: 168, heavy chain CDR2 of SEQ ID NO: 172, heavy chain CDR3 of SEQ ID NO: 176, light chain CDR1 of SEQ ID NO: 184, light chain CDR2 of SEQ ID NO: 188, and light chain CDR3 of SEQ ID NO: 192; (a4) heavy chain CDR1 of SEQ ID NO: 169, heavy chain CDR2 of SEQ ID NO: 173, heavy chain CDR3 of SEQ ID NO: 177, light chain CDR1 of SEQ ID NO: 185, light chain CDR2 of SEQ ID NO: 189, and light chain CDR3 of SEQ ID NO: 193; (a5) heavy chain CDR1 of SEQ ID NO: 166, heavy chain CDR2 of SEQ ID NO: 170, heavy chain CDR3 of SEQ ID NO: 174, light chain CDR1 of SEQ ID NO: 115, light chain CDR2 of SEQ ID NO: 124, and light chain CDR3 of SEQ ID NO: 134; (a6) heavy chain CDR1 of SEQ ID NO: 167, heavy chain CDR2 of SEQ ID NO: 171, heavy chain CDR3 of SEQ ID NO: 175, light chain CDR1 of SEQ ID NO: 116, light chain CDR2 of SEQ ID NO: 125, and light chain CDR3 of SEQ ID NO: 135; (a7) heavy chain CDR1 of SEQ ID NO: 168, heavy chain CDR2 of SEQ ID NO: 172, heavy chain CDR3 of SEQ ID NO: 176, light chain CDR1 of SEQ ID NO: 118, light chain CDR2 of SEQ ID NO: 128, and light chain CDR3 of SEQ ID NO: 137; (a8) an antibody that binds to the same epitope as an antibody comprising any one of (a1) to (a7); and (a9) An antibody that competes with the binding of an antibody comprising any one of (a1) to (a7). The present invention includes any one selected from the group consisting of:

[0159] In one aspect, a conformation-specific antibody (an antigen-binding molecule that specifically binds to a target antibody) is one of the following: (a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 178; (a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 179; (a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 180; (a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 165, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 181; (a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 162, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 196; (a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 197; (a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 198; (a8) an antibody that binds to the same epitope as an antibody comprising any one of (a1) to (a7); and (a9) An antibody that competes with the binding of an antibody comprising any one of (a1) to (a7). The present invention includes any one selected from the group consisting of:

[0160] In one aspect, the conformation-specific antibody (antigen-binding molecule that specifically binds to a target antibody) specifically binds to CH1 of human IgG1. In one aspect, a conformation-specific antibody (an antigen-binding molecule that specifically binds to a target antibody) does not specifically bind to the CH1 of human IgG1 when a disulfide bond is formed between the CH1 domains of two Fabs of human IgG1. In a further aspect, the disulfide bond is a disulfide bond formed between two Fabs of IgG1 at position 191 (EU numbering) in the CH1 domain. In one aspect, the conformation-specific antibody (antigen-binding molecule that specifically binds to a target antibody) does not bind to CH1 of human IgG4.

[0161] The present disclosure provides for the use of conformation-specific antibodies (antigen-binding molecules that specifically bind to a target antibody) in the purification, analysis, or quantification of antibody-containing samples.

[0162] antigen As used herein, the term "antigen" refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a three-dimensional structure composed of a contiguous 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).

[0163] The "first antigen" or "second antigen" to which the first antigen-binding moiety and / or second antigen-binding moiety binds is preferably, for example, an immune cell surface molecule (e.g., a T cell surface molecule, an NK cell surface molecule, a dendritic cell surface molecule, a B cell surface molecule, an NKT cell surface molecule, an MDSC cell surface molecule, and a macrophage surface molecule), or an antigen expressed not only on tumor cells, tumor blood vessels, and stromal cells, but also on normal tissues (integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF, etc.).

[0164] In a combination of a "first antigen" and a "second antigen," preferably, one of the first antigen and the second antigen is, for example, a molecule specifically expressed on T cells, and the other antigen is a molecule expressed on the surface of T cells or any other immune cells. In another embodiment of a combination of a "first antigen" and a "second antigen," preferably, one of the first antigen and the second antigen is, for example, a molecule specifically expressed on T cells, and the other antigen is a molecule expressed on immune cells, and is different from the preselected antigen.

[0165] Specific examples of molecules specifically expressed on T cells include CD3 and T cell receptors. CD3 is particularly preferred. For example, in the case of human CD3, the site of CD3 to which the antigen-binding molecule of the present invention binds can be any epitope present in the sequence of the γ chain, δ chain, or ε chain that constitutes human CD3. In particular, an epitope present in the extracellular region of the ε chain in the human CD3 complex is preferred. The polynucleotide sequences of the γ chain, δ chain, and ε chain structures that constitute CD3 are NM_000073.2, NM_000732.4, and NM_000733.3, and their polypeptide sequences are NP_000064.1, NP_000723.1, and NP_000724.1 (RefSeq accession numbers). Examples of other antigens include Fcγ receptors, TLRs, lectins, IgA, immune checkpoint molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules.

[0166] In one embodiment, the first antigen is a molecule specifically expressed on T cells, preferably a T cell receptor complex molecule such as CD3, more preferably human CD3. In another embodiment, the second antigen is a molecule expressed on T cells or any other immune cells, preferably a cell surface regulator on immune cells, more preferably a costimulatory molecule expressed on T cells, even more preferably a protein of the "TNF superfamily" or "TNF receptor superfamily," including but not limited to human CD137 (4-1BB), CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL. In one preferred embodiment, the first antigen is CD3, and the second antigen is CD137. Herein, the terms "first antigen" and "second antigen" are defined interchangeably.

[0167] As used herein, the term "CD137," also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. Examples of factors belonging to the TNF superfamily or TNF receptor superfamily include CD137, CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL.

[0168] In some embodiments of the present invention, the antigen-binding molecule of the present invention further comprises a third antigen-binding moiety that binds to a "third antigen" different from the above-mentioned "first antigen" and "second antigen." The third antigen-binding domain that binds to the third antigen of the present invention may be an antigen-binding moiety that recognizes any antigen. The third antigen-binding moiety that binds to the third antigen of the present invention may be an antigen-binding moiety that recognizes a molecule specifically expressed in cancer tissue.

[0169] In the present invention, the third antigen-binding portion of the antigen-binding molecule of the present invention binds to a "third antigen" that is different from the "first antigen" and the "second antigen." In some embodiments, the third antigen is derived from a human, mouse, rat, monkey, rabbit, or dog. In some embodiments, the third antigen is a molecule that is specifically expressed on cells or organs derived from a human, mouse, rat, monkey, rabbit, or dog. The third antigen is preferably a molecule that is not systemically expressed on cells or organs. The third antigen is preferably, for example, a tumor cell-specific antigen, and these include antigens that are expressed in association with the malignant transformation of cells, as well as abnormal sugar chains that appear on cell surfaces or protein molecules during malignant transformation of cells. Specific examples include the ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate-specific antigen (PSA), melanoma-associated antigen p97, melanoma antigen gp75, high-molecular-weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigens (e.g., CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1, and LEA), and Burkitt's lymphoma antigen 38.13, CD19, human B lymphoma antigen CD20, CD33, melanoma-specific antigens (e.g., ganglioside GD2, ganglioside GD3, ganglioside GM2, and ganglioside GM3), tumor-specific transplantation antigen (TSTA), T antigen, virus-induced tumor antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), colon CEA, carcinoembryonic antigen α-fetoprotein (e.g., carcinoembryonic trophoblast glycoprotein 5T4 and carcinoembryonic bladder tumor antigen), differentiation antigens (e.g., human lung cancer antigens L6 and L20), fibrosarcoma antigen, human T-cell leukemia-associated antigen Gp37, neoglycoprotein, sphingolipid, breast cancer antigen (e.g., EGFR (epidermal growth factor receptor)), NY-BR-16, NY-BR-16, and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen APO-1, differentiation antigens such as I antigen found in fetal erythrocytes, early endoderm I antigen found in adult erythrocytes, I (Ma) found in embryos before implantation or gastric cancer, M18, M39 found in mammary epithelium, SSEA-1, VEP8, VEP9, Myl, VIM-D5 found in bone marrow cells, D156-22 found in colorectal cancer, TRA-1-85 (blood type H), SCP-1 found in testicular and ovarian cancer, C14 found in colon cancer, F3 found in lung cancer, AH6 found in gastric cancer, Y hapten, Ley found in embryonic carcinoma cells, TL5 (blood type A) ), EGF receptor found in A431 cells, E1 series (blood type B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric cancer antigen, CO-514 (blood type Lea) found in adenocarcinoma, NS-10 and CO-43 (blood type Leb) found in adenocarcinoma, G49 found in EGF receptor of A431 cells, MH2 (blood type ALeb / Ley) found in colon cancer, 19.9 found in colon cancer, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2, GD3, and D1 found in embryonal carcinoma cells.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8, SSEA-3 and SSEA-4 found in 4-cell to 8-cell embryos, cutaneous T-cell lymphoma-associated antigen, MART-1 antigen, sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, paraneoplastic-associated brain-testis cancer antigen (tumor neural antigen MA2 and paraneoplastic neural antigen), neuro-oncological ventral antigen 2 (NOVA2), blood cell cancer antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigens MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, and MAGE-4b These include MAGE-X2, cancer-testis antigen (NY-EOS-1), YKL-40, and any fragments of these polypeptides, as well as modified structures thereof (such as the aforementioned modified phosphate groups and sugar chains), EpCAM, EREG, CA19-9, CA15-3, sialyl SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A.

[0170] In one preferred embodiment, the third antigen is glypican-3 (GPC3). In yet another embodiment, the third antigen is DLL3 (Delta-like 3). The term "DLL3," as used herein, unless otherwise specified, 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 and 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.

[0171] The human DLL3 protein contains a transmembrane (TM) region and an intracellular domain at the C-terminus, and a DSL (Notch) domain at the N-terminus. Additionally, DLL3 has six regions from the N-terminus to the C-terminus, the EGF domains including EGF1 to EGF6. In some embodiments, multispecific antigen-binding molecules or DLL3 antigen-binding portions of the present invention bind to an epitope within the extracellular domain (ECD), i.e., within the 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 present 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 present invention 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 present invention can bind to an epitope within the region of human DLL3 defined by SEQ ID NO: 89. 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.

[0172] In human DLL3, the above-mentioned domains / regions are represented by the following amino acid residues (see, for example, http: / / www.uniprot.org / uniprot / Q9NYJ7 or WO2013 / 126746): Extracellular domain (ECD): amino acid residues located at positions 1 to 492; DSL domain: amino acid residues located at positions 176 to 215; EGF domain: amino acid residues located at positions 216 to 465; EGF1 region: amino acid residues at positions 216 to 249; EGF2 region: amino acid residues at positions 274 to 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 to 427; EGF6 region: amino acid residues at positions 429 to 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 to 618 (or 516 to 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.

[0173] Therefore, the 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, i.e., the 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.

[0174] 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 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. The DLL3 protein used in the present invention is not limited by its origin, and is preferably a human or cynomolgus monkey DLL3 protein.

[0175] In some embodiments, DLL3 ECD fragment proteins (or ECD variants) can be used as DLL3 proteins. Depending on the truncation site, the fragment / variant can include, from N-terminal to C-terminal, DSL domain to EGF6, EGF1 to EGF6, EGF2 to EGF6, EGF3 to EGF6, EGF4 to EGF6, EGF5 and EGF6, or EGF6. The fragment / variant can also include a region extending from immediately after the EGF6 region to immediately before the TM region. A Flag tag can be attached to the C-terminus of the fragment / variant using techniques well known in the art.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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 (via mutation, substitution, or insertion) one cysteine ​​residue at position 191 according to EU numbering in the CH1 region that can form one disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety.

[0180] In some embodiments of the above aspects, the "at least one bond" formed linking the first and second antigen-binding moieties described above 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 molecules 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 terms "spatially close position" or "closer position" include the meaning that the first and second antigen-binding domains described above are held at a shorter distance and / or with reduced mobility.

[0181] 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.

[0182] 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 first or second signaling molecules expressed on the same single immune cell, e.g., T cells (i.e., in a cis-binding manner) or on different immune cells, e.g., T cells (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.

[0183] 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 domain and the second antigen-binding domain can bind to a signaling molecule expressed on the same single immune cell, for example, a T cell, in a "cis-binding" manner, thereby reducing cross-linking of different immune cells, for example, T cells, via the antigen-binding molecule, and thereby avoiding undesired activation of the immune cell.

[0184] 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 may be referred to by the abbreviation "LINC." Using this abbreviation, in some embodiments, the above-mentioned antigen-binding molecules of the present invention may be represented, for example, as "Dual / LINC," "DLL3-Dual / LINC," "paired cysteine ​​form," or "GPC3-Dual / Dual(linc)." 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) may be represented by the abbreviation "UnLINC" or "Dual-LINC-Ig with unpaired cysteine," etc.

[0185] Hinge Area The term "hinge region" refers to the portion of the antibody heavy chain polypeptide in a wild-type antibody heavy chain that connects the CH1 domain and the CH2 domain, e.g., from about position 216 to about position 230 according to the EU numbering system, or from about position 226 to about position 243 according to the Kabat numbering system. In native IgG antibodies, the cysteine ​​residue at position 220 according to EU numbering in the hinge region is known to form a disulfide bond with the cysteine ​​residue at position 214 in the antibody light chain. Furthermore, it is also known that disulfide bonds are formed between the cysteine ​​residues at positions 226 and 229 according to EU numbering in the hinge region between two antibody heavy chains. Generally, the "hinge region" is defined as extending from positions 216 to 238 (EU numbering) or from positions 226 to 251 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions: the upper hinge, the middle hinge, and the lower hinge. In human IgG1 antibodies, these regions are generally defined as follows: Upper hinge: 216-225 (EU numbering) or 226-238 (Kabat numbering), Central hinge: 226-230 (EU numbering) or 239-243 (Kabat numbering), Lower hinge: 231-238 (EU numbering) or 244-251 (Kabat numbering).

[0186] Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by positioning the first and last cysteine ​​residues that form inter-heavy chain S—S bonds at the same positions (see, for example, Brekke et al., 1995, Immunol (Table 1 of Today 16: 85-90)). Hinge regions herein include wild-type hinge regions as well as variants in which amino acid residues in the wild-type hinge region have been altered by substitution, addition, or deletion.

[0187] The term "disulfide bond formed between amino acids not within the hinge region" (or "disulfide bond formed between amino acids outside the hinge region") refers to a disulfide bond formed by, connected, or linked by amino acids located in any antibody region outside the "hinge region" as defined above. For example, such a disulfide bond is formed by, connected, or linked by amino acids at any position in an antibody other than the hinge region (e.g., from about position 216 to about position 230 according to the EU numbering system, or from about position 226 to about position 243 according to the Kabat numbering system). In some embodiments, such a disulfide bond is formed by, connected, or linked by amino acids located in the CH1 region, CL region, VL region, VH region, and / or VHH region. In some embodiments, such disulfide bonds are formed by, connected, or linked by amino acids located at positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, and 201 to 214 (EU numbering) in the CH1 region. In some embodiments, such disulfide bonds are formed by, connected, or linked by amino acids located at positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164 (EU numbering) in the CH1 region. In some embodiments, the disulfide bond is formed, connected, or linked by amino acids located at positions 165, 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, and 214 (EU numbering) in the CH1 region.In one preferred embodiment, such a disulfide bond is formed by, connected to, or linked by the amino acid located at position 191 according to EU numbering in the CH1 region.

[0188] 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-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," and "F(ab')2."

[0189] 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 heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., 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 screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0190] 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 contact 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). 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. 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.

[0191] 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. This can be determined, for example, by electrochemiluminescence (ECL) (BMC Research Notes 2011, 4:281).

[0192] 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.

[0193] 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).

[0194] 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, 8K, 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.

[0195] 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.

[0196] 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 degrees Celsius (°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 400 to 25 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 T200 Evaluation software, version 2.0 (GE Healthcare) or Biacore Insight 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.

[0197] ALPHAScreen is implemented using ALPHA technology, which uses two types of beads (donor and acceptor), based on the following principle: a luminescent signal is detected only when a molecule bound to a donor bead and a molecule bound to an acceptor bead are brought into close proximity due to a biological interaction between these two beads. 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 accesses 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 access the acceptor bead. Therefore, no chemiluminescent reaction will occur.

[0198] 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.

[0199] 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. 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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%.

[0204] 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.)

[0205] 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.

[0206] 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.

[0207] 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%.

[0208] 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.)

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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").

[0220] 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.

[0221] "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.

[0222] "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.

[0223] 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).

[0224] 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, ≦120 nM, ≦100 nM, ≦80 nM, ≦70 nM, ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, ≦10 nM, ≦2 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (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.

[0225] 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 bloc...

Claims

1. A method for producing a preparation of multispecific antigen-binding molecules, the method comprising: (a) a first antigen-binding moiety and a second antigen-binding moiety, each capable of binding to a first antigen and a second antigen different from the first antigen, but not simultaneously binding to both antigens; and (b) a third antigen-binding moiety capable of binding to a third antigen different from the first and second antigens, preferably an antigen expressed on cancer cells / tissues; wherein each of the first antigen-binding moiety and the second antigen-binding moiety comprises (via mutation, substitution, or insertion) at least one cysteine ​​residue that is not in the hinge region, and preferably the at least one cysteine ​​is located in the CH1 region; and the at least one cysteine ​​residue, preferably in the CH1 region, is capable of forming at least one disulfide bond between the first antigen-binding moiety and the second antigen-binding moiety; The method, wherein the method comprises contacting the preparation with a reducing reagent.

2. each of the first antigen-binding moiety and the second antigen-binding moiety a cysteine ​​residue at position 191 (EU numbering) in the CH1 region that can form a disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety; 2. The method of claim 1, comprising (via mutation, substitution, or insertion):

3. The method of claim 2, wherein the multispecific antigen-binding molecule preparation (before contacting with a reducing agent) comprises two or more structural isoforms that differ by at least one disulfide bond formed between amino acid residues located in or at position 191 (EU numbering) in the CH1 region, and wherein contacting with a reducing agent preferentially enriches or increases the population of structural isoforms having at least one disulfide bond formed between amino acid residues located in or at position 191 (EU numbering) in the CH1 region.

4. The method of any one of claims 1 to 3, wherein the reducing reagent contacted with the multispecific antigen-binding molecule has a pH of about 3 to about 10, preferably pH 6 to 8.

5. Reducing agents include TCEP, 2-MEA, DTT, cysteine, GSH, and Na 2 SO 3 5. The method according to any one of claims 1 to 4, wherein the hydroxybenzoate is selected from the group consisting of , preferably TCEP.

6. 6. The method of any one of claims 1 to 5, wherein the concentration of the reducing agent is from about 0.01 mM to about 100 mM.

7. The method of any one of claims 1 to 6, wherein the concentration of the multispecific antigen-binding molecule is from about 0.1 mg / ml to about 50 mg / ml, preferably about 10 mg / ml.

8. promoting reoxidation of cysteine ​​disulfide bonds, preferably by removing the reducing agent, preferably by dialysis or buffer exchange; 8. The method of any one of claims 1 to 7, further comprising:

9. 9. The method of any one of claims 1 to 8, wherein each of the first antigen-binding moiety and the second antigen-binding moiety is capable of binding to CD3 and CD137, but does not bind to both CD3 and CD137 simultaneously.

10. The first antigen-binding portion and the second antigen-binding portion are each (a1) to (a17) below: (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 of the antibody variable fragments selected from (a1) to (a15). The method of claim 9, wherein the antibody variable region comprises any one of:

11. The first antigen-binding portion and the second antigen-binding portion are each (a1) to (a17) below: (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. (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 of the antibody variable fragments selected from (a1) to (a15). The method of claim 10, wherein the antibody variable region comprises any one of:

12. The method of any one of claims 1 to 11, wherein the third antigen-binding moiety is capable of binding to DLL3, preferably human DLL3.

13. 13. The method of claim 12, wherein the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region comprising 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.

14. The method of claim 13, wherein the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region comprising 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.

15. The method of any one of claims 1 to 14, wherein the multispecific antigen-binding molecule further comprises an Fc domain.

16. A preparation of multispecific antigen-binding molecules prepared according to the method of any one of claims 1 to 15, comprising a homogeneous population of multispecific antigen-binding molecules having at least one disulfide bond in the CH1 region (position 191 according to EU numbering).

17. 16. A preparation of multispecific antigen-binding molecules prepared according to the method of any one of claims 1 to 15, comprising at least 80%, 90%, preferably at least 95% molar ratio of multispecific antigen-binding molecules having at least one disulfide bond in the CH1 region (position 191 according to EU numbering).

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