Multispecific antigen-binding molecules targeting DLL3 and their applications

Abstract

This disclosure provides a multispecific antigen-binding molecule comprising a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion. The first and second antigen-binding portions are each capable of binding CD3 and CD137, but not simultaneously. The third antigen-binding portion is capable of binding DLL3, preferably human DLL3. This multispecific antigen-binding molecule more effectively induces T-cell-dependent cytotoxicity while avoiding the adverse toxicity problems or side effects that may exist with other multispecific antigen-binding molecules. This invention provides a multispecific antigen-binding molecule and pharmaceutical compositions that, by comprising the antigen-binding molecule as an active ingredient, can treat various cancers, particularly DLL3-related cancers.

Description

Technical Field

[0001] This invention relates to a multispecific antigen-binding molecule comprising a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion, wherein the first and second antigen-binding portions are each capable of binding CD3 and CD137, but not simultaneously, and the third antigen-binding portion is capable of binding DLL3. The invention also relates to nucleic acids encoding such antigen-binding molecules; methods for preparing such antigen-binding molecules; host cells expressing or capable of expressing such antigen-binding molecules; compositions comprising such antigen-binding molecules; and uses of such antigen-binding molecules or compositions, particularly for therapeutic purposes in the field of cancer. Background Technology

[0002] Cancer is one of the leading causes of death worldwide. With the exception of certain cancers, tumors are often inoperable when discovered. Conventional cancer treatments include radiation therapy, chemotherapy, and immunotherapy. These treatments are often insufficiently effective, eventually leading to cancer recurrence or metastasis after treatment. The lack of tumor specificity is one of the factors limiting maximum efficacy; therefore, more tumor-specific molecularly targeted therapies have emerged as another viable option in cancer treatment.

[0003] Antibodies have attracted attention as drugs due to their high stability in plasma and minimal side effects. Among various therapeutic antibodies, some types require effector cells to generate an antitumor response. Antibody-dependent cell-mediated cytotoxicity (ADCC) is the cytotoxic effect exhibited by effector cells against antibody-binding cells by binding to Fc receptors present on NK cells and macrophages via the Fc region of the antibody. To date, several therapeutic antibodies that can induce ADCC to exert antitumor effects have been developed as drugs for treating cancer (NPL 1). Therapies using conventional therapeutic antibodies targeting tumor-specific expressed antigens have shown excellent antitumor activity, but the administration of these antibodies does not always produce satisfactory results.

[0004] Besides antibodies that employ ADCC by recruiting NK cells or macrophages as effector cells, T cell recruitment antibodies (TR antibodies) that employ cytotoxicity by recruiting T cells as effector cells have been known since the 1980s (NPL2 to 4). TR antibodies are bispecific antibodies that recognize and bind to either subunit of the T cell receptor complex formed on T cells, particularly the CD3ε chain and antigens on cancer cells. Several TR antibodies are currently under development. Catuxomab, a TR antibody against EpCAM, has been approved in the EU for the treatment of malignant ascites. Furthermore, a TR antibody called a “bispecific T cell adaptor (BiTE)” has recently been found to exhibit potent antitumor activity (NPL5 and 6). Blinatumomab, a BiTE molecule targeting CD19, was first approved by the FDA in 2014. Compared to rituximab, bonatetumab has been shown to exhibit stronger cytotoxic activity against CD19 / CD20 positive cancer cells in vitro, inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (NPL7).

[0005] However, it is well known that trifunctional antibodies bind to both T cells and other cells (e.g., NK cells or macrophages) in a manner independent of cancer antigens, resulting in cross-linked receptors expressed on cells and inducing the expression of various cytokines in a cancer antigen-independent manner. Due to this induction of cytokine expression, systemic administration of trifunctional antibodies is thought to cause cytokine storm-like side effects. Indeed, in a Phase I clinical trial, a very low dose of 5 μg / body was reported to be the maximum tolerated dose of systemic caputoxumab in non-small cell lung cancer patients, while higher doses resulted in various serious side effects (NPL 8). When administered at such low doses, caputoxumab never reaches effective blood levels. That is, administration of caputoxumab at such low doses cannot achieve the expected antitumor effect.

[0006] Unlike caputoxumab, BiTE lacks an Fcγ receptor binding site, thus preventing cancer antigen-independent cross-linking between receptors expressed on T cells and cells such as NK cells and macrophages. Therefore, BiTE has been shown not to induce cancer antigen-independent cytokine induction observed with caputoxumab. However, because BiTE is a modified low-molecular-weight antibody molecule without an Fc region, its blood half-life after administration is significantly shorter than that of IgG antibodies commonly used as therapeutic antibodies. In fact, the blood half-life of BiTE administered in vivo has been reported to be approximately several hours (NPL9 and 10). In clinical trials of bonatomab, it was administered via continuous intravenous infusion using a micropump. This method of administration not only causes significant inconvenience to patients but also carries the potential risk of medical accidents due to equipment malfunction. Therefore, such an administration method cannot be considered ideal.

[0007] δ-like 3 (DLL3) is a type I membrane protein belonging to the Notch ligand family. DLL3 is essential for normal somnogenesis and pattern development. Mutations in DLL3 result in rib defects or vertebral detachment in patients with autosomal recessive vertebral rib dysplasia (NPL11 and 12). Previous studies have reported chromosomal amplification of the DLL3 gene and increased expression of this gene in cancer cell lines (NPL 13), and increased expression of DLL3 in some glioma cases (NPL 14). Furthermore, in addition to SCLC, DLL3 has previously been proposed for use in the diagnosis and treatment of gliomas, in addition to the use of ADCC-enhanced antibodies, antibody-drug conjugates (ADCs), and bispecific T-cell connective molecules (PTL1, 2, and 3) in the BiTE-Fc format.

[0008] Reference List

[0009] Patent documents

[0010] [PTL 1]WO 2011 / 093097

[0011] [PTL 2]WO 2013 / 126746

[0012] [PTL 3]WO 2017 / 021349

[0013] Non-patent literature

[0014] [NPL 1]Clin Cancer Res.2010Jan 1;16(1):11-20.

[0015] [NPL 2]Nature.1985Apr 18-24;314(6012):628-31.

[0016] [NPL 3]Int J Cancer. 1988 Apr 15; 41(4): 609 - 15.

[0017] [NPL 4]Proc Natl Acad Sci U S A. 1986 Mar; 83(5): 1453 - 7.

[0018] [NPL 5]Proc Natl Acad Sci U S A. 1995 Jul 18; 92(15): 7021 - 5.

[0019] [NPL 6]Drug Discov Today. 2005 Sep 15; 10(18): 1237 - 44.

[0020] [NPL 7]Int J Cancer. 2002 Aug 20; 100(6): 690 - 7.

[0021] [NPL 8]Cancer Immunol Immunother (2007) 56(10), 1637 - 44

[0022] [NPL 9]Cancer Immunol Immunother. (2006) 55(5), 503 - 14

[0023] [NPL 10]Cancer Immunol Immunother. (2009) 58(1), 95 - 109

[0024] [NPL 11]Bulman, M.P. et al. (2000) Nat Genet 24, 438 - 441.

[0025] [NPL 12]Turnpenny, P.D. et al. (2003) J Med Genet 40, 333 - 339.

[0026] [NPL 13]Phillips, H.S. (2006) Cancer Cell 9, 157 - 173.

[0027] [NPL 14]Mulledndore, M.E. (2009) Clin Cancer Res 15, 2291 - 2301. Summary of the Invention

[0028] Technical Problem

[0029] The present invention aims to provide a multispecific antigen-binding molecule capable of treating cancer by recruiting T cells near cells expressing DLL3 and utilizing the cytotoxicity of T cells against DLL3-expressing cancer cells, a method for generating the multispecific antigen-binding molecule, and a therapeutic agent comprising such a multispecific antigen-binding molecule as an active ingredient for inducing cytotoxicity of cells. Another object of the present invention is to provide a pharmaceutical composition for treating or preventing various cancers, comprising one of the aforementioned antigen-binding molecules as an active ingredient, and a treatment method using the pharmaceutical composition.

[0030] Solution to the problem

[0031] This invention relates to a multispecific antigen-binding molecule comprising a first antigen-binding portion, a second antigen-binding portion, and a third antigen-binding portion. The first and second antigen-binding portions are each capable of binding CD3 and CD137, but not simultaneously (i.e., capable of binding to CD3 and CD137, but not simultaneously). The third antigen-binding portion is capable of binding DLL3, preferably human DLL3. This multispecific antigen-binding molecule more effectively induces T-cell-dependent cytotoxicity while avoiding the adverse toxicity problems or side effects that may exist with other multispecific antigen-binding molecules. This invention provides a multispecific antigen-binding molecule and pharmaceutical compositions that, by comprising the antigen-binding molecule as an active ingredient, can treat various cancers, particularly DLL3-related cancers, such as DLL3-positive tumors.

[0032] On the one hand, the multispecific antigen-binding molecule of the present invention has a very unique structural form, which improves or enhances the efficacy of multispecific antigen-binding molecules. The novel antigen-binding molecule with a unique structural form provides an increased number of antigen-binding domains, thereby increasing the titer and / or specificity of individual antigens on effector cells and target cells, while reducing unwanted adverse side effects.

[0033] In one particular aspect, the present invention relates to a multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion; and a third antigen-binding portion, wherein the first and second antigen-binding portions are each capable of binding CD3 and CD137, but not simultaneously (i.e., capable of binding to CD3 and CD137, but not simultaneously), and the third antigen-binding portion is capable of binding DLL3, preferably human DLL3, wherein the multispecific antigen-binding molecule effectively induces T cell-dependent cytotoxicity while avoiding the adverse toxicity problems or side effects that may exist with other multispecific antigen-binding molecules. In one aspect, each of the first and second antigen-binding moieties contains at least one amino acid mutation, such as cysteine ​​insertion / substitution / mutation, which creates a disulfide bond between the first and second antigen-binding moieties to bring them close to each other and, for example, facilitates cis-antigen binding to antigens (CD3 and / or CD137) on the same single effector cell due to steric hindrance or a shorter distance between the two dual-Fab moieties. This improves the safety of the trispecific antibody by preventing unwanted cross-linking of two CD3 / CD137-expressing immune cells mediated by the two dual-Fab moieties in a DLL3-independent manner. In a specific aspect, each of the first and second antigen-binding moieties is a Fab moiety and contains at least one cysteine ​​residue (through mutation, substitution, or insertion) in the CH1 region, which is capable of forming at least one disulfide bond between the CH1 regions of the first and second antigen-binding moieties. In another specific aspect, the first antigen-binding portion and the second antigen-binding portion each contain a cysteine ​​residue (by mutation, substitution or insertion) at position 191 according to EU number in the CH1 region, the cysteine ​​residue being capable of forming a disulfide bond between the CH1 regions of the first antigen-binding portion and the CH1 regions of the second antigen-binding portion.

[0034] Surprisingly, antigen-binding molecules with this unique structural form have been found to exhibit superior efficacy compared to other forms of multispecific antibodies (e.g., BiTE), while showing reduced or minimal off-target side effects caused by unwanted cross-linking between different cells (e.g., effector cells, such as T cells).

[0035] More specifically, this disclosure provides the following:

[0036] [1] Multispecific antigen-binding molecules, which include:

[0037] The first antigen-binding region and the second antigen-binding region can each bind CD3 and CD137, but not simultaneously; and

[0038] The third antigen-binding portion is capable of binding to a third antigen, preferably an antigen expressed on cancer cells / tissues.

[0039] [1A] A multispecific antigen-binding molecule comprising:

[0040] The first antigen-binding region and the second antigen-binding region can each bind CD3 and CD137, but not simultaneously; and

[0041] The third antigen-binding part can bind to DLL3, preferably human DLL3.

[0042] The multispecific antigen-binding molecule described in any one of [2][1] to [1A], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region, the antibody variable region comprising any one of the following (a1) to (a17):

[0043] (a1) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:17, CDR 2 of the heavy chain of SEQ ID NO:31, CDR 3 of the heavy chain of SEQ ID NO:45, CDR 1 of the light chain of SEQ ID NO:64, CDR 2 of the light chain of SEQ ID NO:69 and CDR 3 of the light chain of SEQ ID NO:74;

[0044] (a2) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:18, CDR 2 of the heavy chain in SEQ ID NO:32, CDR 3 of the heavy chain in SEQ ID NO:46, CDR 1 of the light chain in SEQ ID NO:63, CDR 2 of the light chain in SEQ ID NO:68 and CDR 3 of the light chain in SEQ ID NO:73;

[0045] (a3) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:19, CDR 2 of the heavy chain in SEQ ID NO:33, CDR 3 of the heavy chain in SEQ ID NO:47, CDR 1 of the light chain in SEQ ID NO:63, CDR 2 of the light chain in SEQ ID NO:68 and CDR 3 of the light chain in SEQ ID NO:73;

[0046] (a4) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:19, CDR 2 of the heavy chain of SEQ ID NO:33, CDR 3 of the heavy chain of SEQ ID NO:47, CDR 1 of the light chain of SEQ ID NO:65, CDR 2 of the light chain of SEQ ID NO:70 and CDR 3 of the light chain of SEQ ID NO:75;

[0047] (a5) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:20, CDR 2 of the heavy chain of SEQ ID NO:34, CDR 3 of the heavy chain of SEQ ID NO:48, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0048] (a6) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:22, CDR 2 of the heavy chain of SEQ ID NO:36, CDR 3 of the heavy chain of SEQ ID NO:50, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0049] (a7) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:23, CDR 2 of the heavy chain of SEQ ID NO:37, CDR 3 of the heavy chain of SEQ ID NO:51, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0050] (a8) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:23, CDR 2 of the heavy chain of SEQ ID NO:37, CDR 3 of the heavy chain of SEQ ID NO:51, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0051] (a9) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:24, CDR 2 of the heavy chain of SEQ ID NO:38, CDR 3 of the heavy chain of SEQ ID NO:52, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0052] (a10) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:25, CDR 2 of the heavy chain of SEQ ID NO:39, CDR 3 of the heavy chain of SEQ ID NO:53, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0053] (a11) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:26, CDR 2 of the heavy chain of SEQ ID NO:40, CDR 3 of the heavy chain of SEQ ID NO:54, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0054] (a12) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:26, CDR 2 of the heavy chain of SEQ ID NO:40, CDR 3 of the heavy chain of SEQ ID NO:54, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0055] (a13) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:27, CDR 2 of the heavy chain of SEQ ID NO:41, CDR 3 of the heavy chain of SEQ ID NO:55, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0056] (a14) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:28, CDR 2 of the heavy chain of SEQ ID NO:42, CDR 3 of the heavy chain of SEQ ID NO:56, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0057] (a15) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:82, CDR 2 of the heavy chain of SEQ ID NO:83, CDR 3 of the heavy chain of SEQ ID NO:84, CDR 1 of the light chain of SEQ ID NO:65, CDR 2 of the light chain of SEQ ID NO:70 and CDR 3 of the light chain of SEQ ID NO:75;

[0058] (a16) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a15); and

[0059] (a17) is an antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a15).

[0060] The multispecific antigen-binding molecule described in any one of [3][1] to [2], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region, the antibody variable region comprising any one of the following (a1) to (a17):

[0061] (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;

[0062] (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;

[0063] (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;

[0064] (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;

[0065] (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;

[0066] (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;

[0067] (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;

[0068] (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;

[0069] (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;

[0070] (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;

[0071] (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;

[0072] (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;

[0073] (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;

[0074] (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;

[0075] (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;

[0076] (a16) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a15); and

[0077] (a17) is an antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a15).

[0078] The multispecific antigen-binding molecule described in any one of [3A][1] to [3], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region, the antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:20, the heavy chain CDR 2 of SEQ ID NO:34, the heavy chain CDR 3 of SEQ ID NO:48, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73.

[0079] [3B][3A] The multispecific antigen-binding molecule, wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region, wherein the antibody variable region comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:6 and a light chain variable region containing the amino acid sequence of SEQ ID NO:58.

[0080] The multispecific antigen-binding molecule of any one of [4][1] to [3B], wherein the first antigen-binding portion and the second antigen-binding portion are each Fab, and include at least one disulfide bond formed between the CH1 region of the first antigen-binding portion and the CH1 region of the second antigen-binding portion.

[0081] [4A][4] The multispecific antigen-binding molecule, wherein the first antigen-binding portion and the second antigen-binding portion are each Fab, and contain a disulfide bond formed between amino acid residues at position 191 according to EU number in the CH1 region of each of the first antigen-binding portion and the second antigen-binding portion.

[0082] The multispecific antigen-binding molecule described in any one of [5][1] to [4A], wherein the third antigen-binding portion is fused with either the first antigen-binding portion or the second antigen-binding portion.

[0083] [5A][5] The multispecific antigen-binding molecule, wherein the third antigen-binding portion is Fab or scFv.

[0084] The multispecific antigen-binding molecule of any one of [6][5] to [5A], wherein the first, second and third antigen-binding portions are each Fab molecules, wherein the third antigen-binding portion is optionally fused at the C-terminus (CH1) of the Fab heavy chain to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion via a peptide linker.

[0085] The multispecific antigen-binding molecule described in any one of [6A][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 or SEQ ID NO:259.

[0086] The multispecific antigen-binding molecule described in any one of [6B][1] to [6A], wherein the first antigen-binding portion is the same as the second antigen-binding portion.

[0087] The multispecific antigen-binding molecule of any one of [7][1] to [6B], wherein the third antigen-binding portion is a cross-Fab molecule, wherein the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and wherein the first and second antigen-binding portions are each conventional Fab molecules.

[0088] [8][7] The multispecific antigen-binding molecule wherein, in the constant structural domain CL of the respective light chain of the first and second antigen-binding moieties, the amino acids at positions 123 and / or 124 are independently lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and wherein, in the constant structural domain CH1 of the respective heavy chain of the first and second antigen-binding moieties, the amino acid at position 147 and / or the amino acid at position 213 are independently glutamic acid (E) or aspartic acid (D) (according to EU numbering).

[0089] [9][8] The multispecific antigen-binding molecule, wherein, in the constant structural domain CL of the respective light chain of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 are arginine (R) and lysine (K) (according to Kabat numbering), respectively, and wherein, in the constant structural domain CH1 of the respective heavy chain of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 are glutamic acid (E) (according to EU numbering).

[0090] The multispecific antigen-binding molecule described in any one of

[10] [1] to [9], wherein the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region, said antibody variable region comprising any one of the following (a1) to (a5):

[0091] (a1) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:233, CDR 2 of the heavy chain of SEQ ID NO:234, CDR 3 of the heavy chain of SEQ ID NO:235, CDR 1 of the light chain of SEQ ID NO:237, CDR 2 of the light chain of SEQ ID NO:238 and CDR 3 of the light chain of SEQ ID NO:239;

[0092] (a2) Complementary determinant region (CDR) 1 of the heavy chain of SEQ ID NO:276, CDR 2 of the heavy chain of SEQ ID NO:277, CDR 3 of the heavy chain of SEQ ID NO:278, CDR 1 of the light chain of SEQ ID NO:279, CDR 2 of the light chain of SEQ ID NO:280 and CDR 3 of the light chain of SEQ ID NO:281;

[0093] (a3) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:285, CDR 2 of the heavy chain in SEQ ID NO:286, CDR 3 of the heavy chain in SEQ ID NO:287, CDR 1 of the light chain in SEQ ID NO:288, CDR 2 of the light chain in SEQ ID NO:289 and CDR 3 of the light chain in SEQ ID NO:290;

[0094] (a4) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a3); and

[0095] (a5) An antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a3).

[0096] The multispecific antigen-binding molecule described in any one of

[11] [1] to

[10] , wherein the third antigen-binding portion capable of binding to DLL3 comprises an antibody variable region, said antibody variable region comprising any one of the following (a1) to (a6):

[0097] (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;

[0098] (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;

[0099] (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;

[0100] (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;

[0101] (a5) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a4); and

[0102] (a6) An antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a4).

[0103] [11A]

[10] or

[11] of the multispecific antigen-binding molecule, wherein the third antigen-binding portion comprises an antibody variable region, the antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:233, the heavy chain CDR 2 of SEQ ID NO:234, the heavy chain CDR 3 of SEQ ID NO:235, the light chain CDR 1 of SEQ ID NO:237, the light chain CDR 2 of SEQ ID NO:238 and the light chain CDR 3 of SEQ ID NO:239.

[0104] [11B][11A] The multispecific antigen-binding molecule, wherein the third antigen-binding portion comprises an antibody variable region, the antibody variable region comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO:232, and a light chain variable region containing the amino acid sequence of SEQ ID NO:236.

[0105] The multispecific antigen-binding molecule described in any one of

[12] [1] to [11B] further comprises an Fc domain.

[0106] [12A]

[12] The multispecific antigen-binding molecule, wherein the Fc domain is composed of first and second Fc subunits capable of stable association, and wherein the Fc domain exhibits reduced binding affinity for human Fcγ receptors compared to the natural human IgG1 Fc domain.

[0107] [12B][12A] The multispecific antigen-binding molecule, wherein the first Fc region subunit is selected from the group consisting of:

[0108] (a1) contains the Fc region polypeptide of Ala at position 234 and Ala at position 235;

[0109] (a2) A polypeptide containing the Fc region of Ala at position 234, Ala at position 235, and Ala at position 297;

[0110] (a3) An Fc region polypeptide comprising Ala at position 234, Ala at position 235, Ala at position 297, Cys at position 354, and Trp at position 366; and

[0111] The second Fc region polypeptide is selected from the group consisting of:

[0112] (a4) contains the Fc region polypeptide of Ala at position 234 and Ala at position 235;

[0113] (a5) contains the Fc region polypeptide of Ala at position 234, Ala at position 235 and Ala at position 297;

[0114] (a6) contains the Fc region polypeptide of Ala at position 234, Ala at position 235, Ala at position 297, Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407; and

[0115] The amino acid positions are numbered using EU index numbers.

[0116] The multispecific antigen-binding molecule described in any one of [12C]

[12] to [12B], wherein the Fc domain exhibits enhanced FcRn binding activity under acidic pH conditions (e.g., pH 5.8) compared to the Fc region of natural IgG.

[0117] [12D][12C] The multispecific antigen-binding molecule, wherein the Fc domain comprises Ala at position 434 according to EU number; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440.

[0118] The multispecific antigen-binding molecule described in [12E][12D], wherein the Fc domain comprises Ala at position 434 according to EU number; Arg or Lys at position 438; and Glu or Asp at position 440.

[0119] The multispecific antigen-binding molecule described in [12F][12E] further comprises Ile or Leu at position 428 according to EU number; and / or Ile, Leu, Val, Thr or Phe at position 436.

[0120] The multispecific antigen-binding molecule of any one of [12G], [12C] to [12F], wherein the Fc domain comprises a combination of amino acid substitutions selected from the group consisting of:

[0121] According to EU number (a) N434A / Q438R / S440E;

[0122] (b)N434A / Q438R / S440D;

[0123] (c)N434A / Q438K / S440E;

[0124] (d)N434A / Q438K / S440D;

[0125] (e)N434A / Y436T / Q438R / S440E;

[0126] (f)N434A / Y436T / Q438R / S440D;

[0127] (g)N434A / Y436T / Q438K / S440E;

[0128] (h)N434A / Y436T / Q438K / S440D;

[0129] (i)N434A / Y436V / Q438R / S440E;

[0130] (j)N434A / Y436V / Q438R / S440D;

[0131] (k)N434A / Y436V / Q438K / S440E;

[0132] (l)N434A / Y436V / Q438K / S440D;

[0133] (m)N434A / R435H / F436T / Q438R / S440E;

[0134] (n)N434A / R435H / F436T / Q438R / S440D;

[0135] (o)N434A / R435H / F436T / Q438K / S440E;

[0136] (p)N434A / R435H / F436T / Q438K / S440D;

[0137] (q)N434A / R435H / F436V / Q438R / S440E;

[0138] (r)N434A / R435H / F436V / Q438R / S440D;

[0139] (s)N434A / R435H / F436V / Q438K / S440E;

[0140] (t)N434A / R435H / F436V / Q438K / S440D;

[0141] (u)M428L / N434A / Q438R / S440E;

[0142] (v)M428L / N434A / Q438R / S440D;

[0143] (w)M428L / N434A / Q438K / S440E;

[0144] (x)M428L / N434A / Q438K / S440D;

[0145] (y)M428L / N434A / Y436T / Q438R / S440E;

[0146] (z)M428L / N434A / Y436T / Q438R / S440D;

[0147] (aa)M428L / N434A / Y436T / Q438K / S440E;

[0148] (ab)M428L / N434A / Y436T / Q438K / S440D;

[0149] (ac)M428L / N434A / Y436V / Q438R / S440E;

[0150] (ad)M428L / N434A / Y436V / Q438R / S440D;

[0151] (ae)M428L / N434A / Y436V / Q438K / S440E;

[0152] (af)M428L / N434A / Y436V / Q438K / S440D;

[0153] (ag)L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and

[0154] (ah)L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E.

[0155] The multispecific antigen-binding molecule according to any one of [12H][12C] to [12G], wherein the Fc domain comprises a combination of amino acid substitutions of M428L / N434A / Q438R / S440E.

[0156] The multispecific antigen-binding molecule described in any one of [12I]

[12] to [12H], wherein the Fc domain is an IgG Fc domain, preferably a human IgG Fc domain, and more preferably a human IgG1 Fc domain.

[0157] The multispecific antigen-binding molecule described in any one of [12J]

[12] to [12I], wherein the Fc domain comprises any one of the following:

[0158] (a) A first Fc subunit comprising the amino acid sequence shown in SEQ ID NO: 100 and a second Fc subunit comprising the amino acid sequence shown in SEQ ID NO: 111; and

[0159] (b) A first Fc subunit containing the amino acid sequence shown in SEQ ID NO:99 and a second Fc subunit containing the amino acid sequence shown in SEQ ID NO:109.

[0160] A multispecific antigen-binding molecule of any one of [12K]

[12] to [12J], wherein the first and second antigen-binding portions are each Fab, wherein the first antigen-binding portion 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain.

[0161] The multispecific antigen-binding molecule described in [12L][12K], wherein the third antigen-binding moiety is optionally fused at the C-terminus to the N-terminus of the Fab heavy chain of one of the first antigen-binding moiety or the second antigen-binding moiety via a peptide linker.

[0162] The multispecific antigen-binding molecule described in any one of

[13] [1] to [12L] comprises five polypeptide chains selected from any combination of the following (a1) to (a15):

[0163] (a1) A polypeptide chain containing the amino acid sequence of SEQ ID NO:201 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:208 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0164] (a2) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:203, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0165] (a3) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:204, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0166] (a4) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:205, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0167] (a5) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:216, 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:229, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0168] (a6) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:217, 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:210, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0169] (a7) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:219, 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:211, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0170] (a8) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:220, 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:211, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0171] (a9) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:221, 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:211, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0172] (a10) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:222, 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:230, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0173] (a11) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:223, 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:212, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0174] (a12) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:225, 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:213, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0175] (a13) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:226, 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:213, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0176] (a14) a polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:227, 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:213, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215; and

[0177] (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 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0178] Preferably, the five polypeptide chains (chain 1 to chain 5) are arranged according to... Figure 1 (a) indicates that the directions are interconnected and / or associated.

[0179]

[14] A single polynucleotide or multiple polynucleotides encoding a multispecific antigen-binding molecule as described in any one of [1] to

[13] .

[0180]

[15] A vector that encodes the polynucleotide or multiple polynucleotides described in

[14] .

[0181]

[16] A host cell comprising the polynucleotide or multiple polynucleotides described in

[14] , or the carrier described in

[15] .

[0182] A method for preparing a multispecific antigen-binding molecule as described in any one of

[17] [1] to

[13] , comprising the following steps: a) culturing the host cells described in

[16] under conditions suitable for the expression of the antigen-binding molecule and b) recovering the antigen-binding molecule.

[0183] [17A] A multispecific antigen-binding molecule, which is prepared by the method described in

[17] .

[0184]

[18] A pharmaceutical composition comprising any one of [1] to

[13] a multispecific antigen-binding molecule and a pharmaceutically acceptable carrier.

[0185] The multispecific antigen-binding molecule described in any one of

[19] [1] to

[13] or the pharmaceutical composition described in

[18] induces cytotoxicity, preferably T-cell-dependent cytotoxicity.

[0186] The multispecific antigen-binding molecule described in any one of

[20] [1] to

[13] or the pharmaceutical composition described in

[18] , which is used as a drug.

[0187] The multispecific antigen-binding molecule described in any one of

[21] [1] to

[13] or the pharmaceutical composition described in

[18] , for use in treating or preventing disease in individuals in need.

[0188]

[22]

[21] The multispecific antigen-binding molecules or pharmaceutical compositions described herein are used to treat / prevent diseases, wherein the disease is cancer.

[0189] [22A]

[22] The multispecific antigen-binding molecule or pharmaceutical composition described herein is used to treat / prevent disease, wherein the cancer is a cancer expressing DLL3 or a DLL3-positive cancer.

[0190] The multispecific antigen-binding molecules or pharmaceutical compositions described in [22B]

[22] or [22A] are used to treat / prevent diseases, wherein the cancer is lung cancer (including small cell lung cancer) and melanoma.

[0191] Use of any of the multispecific antigen-binding molecules described in

[23] [1] to

[13] or the pharmaceutical composition described in

[18] in the preparation of a medicament for the treatment or prevention of a disease in an individual in need.

[0192]

[24] A method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising any one of [1] to

[13] a multispecific antigen-binding molecule or

[18] a pharmaceutical composition.

[0193]

[25]

[23] the use described or

[24] the method described, wherein the disease is cancer, preferably DLL3 positive cancer or cancer expressing DLL3.

[0194] [25A]

[25] The use or method described herein, wherein the cancer is a DLL3-expressing cancer or a DLL3-positive cancer.

[0195] The uses or methods described in [25B][25A], wherein the cancer is lung cancer (including small cell lung cancer) or melanoma.

[0196]

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

[13] or the pharmaceutical composition described in

[18] in the presence of T cells.

[0197]

[27] A kit comprising the composition described in

[18] ; and a packaging insert containing instructions for administration to a subject to treat or delay the progression of cancer, preferably DLL3-positive cancer or cancer expressing DLL3.

[0198] [27A]

[27] The kit described herein, wherein the cancer is lung cancer (including small cell lung cancer) and melanoma.

[0199] Another aspect of the present invention relates to:

[0200]

[28] Multispecific antigen-binding molecules, comprising:

[0201] It can bind to both CD3 and CD137, but not simultaneously to the antigen-binding regions of both CD3 and CD137; and

[0202] It can bind to DLL3, preferably the antigen-binding portion of human DLL3.

[0203]

[29]

[28] The multispecific antigen-binding molecule, wherein the antigen-binding portion capable of binding CD3 and CD137 but not simultaneously binding CD3 and CD137 comprises an antibody variable region, said antibody variable region comprising any one of the following (a1) to (a17):

[0204] (a1) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:17, CDR 2 of the heavy chain of SEQ ID NO:31, CDR 3 of the heavy chain of SEQ ID NO:45, CDR 1 of the light chain of SEQ ID NO:64, CDR 2 of the light chain of SEQ ID NO:69 and CDR 3 of the light chain of SEQ ID NO:74;

[0205] (a2) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:18, CDR 2 of the heavy chain in SEQ ID NO:32, CDR 3 of the heavy chain in SEQ ID NO:46, CDR 1 of the light chain in SEQ ID NO:63, CDR 2 of the light chain in SEQ ID NO:68 and CDR 3 of the light chain in SEQ ID NO:73;

[0206] (a3) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:19, CDR 2 of the heavy chain in SEQ ID NO:33, CDR 3 of the heavy chain in SEQ ID NO:47, CDR 1 of the light chain in SEQ ID NO:63, CDR 2 of the light chain in SEQ ID NO:68 and CDR 3 of the light chain in SEQ ID NO:73;

[0207] (a4) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:19, CDR 2 of the heavy chain of SEQ ID NO:33, CDR 3 of the heavy chain of SEQ ID NO:47, CDR 1 of the light chain of SEQ ID NO:65, CDR 2 of the light chain of SEQ ID NO:70 and CDR 3 of the light chain of SEQ ID NO:75;

[0208] (a5) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:20, CDR 2 of the heavy chain of SEQ ID NO:34, CDR 3 of the heavy chain of SEQ ID NO:48, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0209] (a6) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:22, CDR 2 of the heavy chain of SEQ ID NO:36, CDR 3 of the heavy chain of SEQ ID NO:50, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0210] (a7) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:23, CDR 2 of the heavy chain of SEQ ID NO:37, CDR 3 of the heavy chain of SEQ ID NO:51, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0211] (a8) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:23, CDR 2 of the heavy chain of SEQ ID NO:37, CDR 3 of the heavy chain of SEQ ID NO:51, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0212] (a9) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:24, CDR 2 of the heavy chain of SEQ ID NO:38, CDR 3 of the heavy chain of SEQ ID NO:52, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0213] (a10) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:25, CDR 2 of the heavy chain of SEQ ID NO:39, CDR 3 of the heavy chain of SEQ ID NO:53, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0214] (a11) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:26, CDR 2 of the heavy chain of SEQ ID NO:40, CDR 3 of the heavy chain of SEQ ID NO:54, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0215] (a12) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:26, CDR 2 of the heavy chain of SEQ ID NO:40, CDR 3 of the heavy chain of SEQ ID NO:54, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0216] (a13) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:27, CDR 2 of the heavy chain of SEQ ID NO:41, CDR 3 of the heavy chain of SEQ ID NO:55, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0217] (a14) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:28, CDR 2 of the heavy chain of SEQ ID NO:42, CDR 3 of the heavy chain of SEQ ID NO:56, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0218] (a15) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:82, CDR 2 of the heavy chain of SEQ ID NO:83, CDR 3 of the heavy chain of SEQ ID NO:84, CDR 1 of the light chain of SEQ ID NO:65, CDR 2 of the light chain of SEQ ID NO:70 and CDR 3 of the light chain of SEQ ID NO:75;

[0219] (a16) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a15); and

[0220] (a17) is an antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a15).

[0221] The multispecific antigen-binding molecule described in any one of

[30]

[28] to

[29] , wherein the antigen-binding portion capable of binding CD3 and CD137 but not simultaneously binding CD3 and CD137 comprises an antibody variable region, said antibody variable region comprising any one of the following (a1) to (a17):

[0222] (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;

[0223] (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;

[0224] (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;

[0225] (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;

[0226] (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;

[0227] (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;

[0228] (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;

[0229] (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;

[0230] (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;

[0231] (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;

[0232] (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;

[0233] (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;

[0234] (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;

[0235] (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;

[0236] (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;

[0237] (a16) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a15); and

[0238] (a17) is an antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a15).

[0239] Another aspect of the present invention relates to:

[0240]

[31] A multispecific antigen-binding molecule comprising five polypeptide chains selected from any combination of the following (a1) to (a15):

[0241] (a1) A polypeptide chain containing the amino acid sequence of SEQ ID NO:201 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:208 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0242] (a2) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:203, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0243] (a3) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:204, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0244] (a4) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:205, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0245] (a5) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:216, 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:229, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0246] (a6) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:217, 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:210, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0247] (a7) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:219, 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:211, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0248] (a8) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:220, 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:211, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0249] (a9) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:221, 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:211, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0250] (a10) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:222, 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:230, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0251] (a11) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:223, 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:212, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0252] (a12) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:225, 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:213, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0253] (a13) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:226, 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:213, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0254] (a14) a polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:227, 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:213, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215; and

[0255] (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 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0256] Furthermore, preferably, the five polypeptide chains (chain 1 to chain 5) are arranged in accordance with... Figure 1 (a) indicates that the directions are interconnected and / or associated.

[0257] Another aspect of the present invention relates to:

[0258]

[32] An antigen-binding molecule capable of binding DLL3, comprising an antibody variable region, said antibody variable region comprising any one of the following (a1) to (a5):

[0259] (a1) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:233, CDR 2 of the heavy chain in SEQ ID NO:234, CDR 3 of the heavy chain in SEQ ID NO:235, CDR 1 of the light chain in SEQ ID NO:237, CDR 2 of the chain in SEQ ID NO:238, and CDR 3 of the light chain in SEQ ID NO:239;

[0260] (a2) Complementary determinant region (CDR) 1 of the heavy chain of SEQ ID NO:276, CDR 2 of the heavy chain of SEQ ID NO:277, CDR 3 of the heavy chain of SEQ ID NO:278, CDR 1 of the light chain of SEQ ID NO:279, CDR 2 of the light chain of SEQ ID NO:280 and CDR 3 of the light chain of SEQ ID NO:281;

[0261] (a3) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:285, CDR 2 of the heavy chain in SEQ ID NO:286, CDR 3 of the heavy chain in SEQ ID NO:287, CDR 1 of the light chain in SEQ ID NO:288, CDR 2 of the light chain in SEQ ID NO:289 and CDR 3 of the light chain in SEQ ID NO:290;

[0262] (a4) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a3); and

[0263] (a5) An antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a3).

[0264]

[33] An antigen-binding molecule capable of binding DLL3, comprising an antibody variable region, said antibody variable region comprising any one of the following (a1) to (a6):

[0265] (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;

[0266] (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;

[0267] (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;

[0268] (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;

[0269] (a5) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a4); and

[0270] (a6) An antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a4).

[0271] Another aspect of the present invention relates to:

[0272] [2-1] Multispecific antigen-binding molecules, which include:

[0273] (a) A first antigen-binding region and a second antigen-binding region, each binding to human CD3 and containing antibody variable regions that may be the same or different and independently selected from the group consisting of:

[0274] (a1) Antibody variable region comprising 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;

[0275] (a2) The antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:18, the heavy chain CDR 2 of SEQ ID NO:32, the heavy chain CDR 3 of SEQ ID NO:46, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73;

[0276] (a3) Antibody variable region comprising 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;

[0277] (a4) Antibody variable region comprising 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;

[0278] (a5) Antibody variable region comprising heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:20, heavy chain CDR 2 of SEQ ID NO:34, heavy chain CDR 3 of SEQ ID NO:48, light chain CDR 1 of SEQ ID NO:63, light chain CDR 2 of SEQ ID NO:68 and light chain CDR 3 of SEQ ID NO:73;

[0279] (a6) Antibody variable region comprising 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;

[0280] (a7) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:23, the heavy chain CDR 2 of SEQ ID NO:37, the heavy chain CDR 3 of SEQ ID NO:51, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73;

[0281] (a8) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:23, the heavy chain CDR 2 of SEQ ID NO:37, the heavy chain CDR 3 of SEQ ID NO:51, the light chain CDR 1 of SEQ ID NO:66, the light chain CDR 2 of SEQ ID NO:71 and the light chain CDR 3 of SEQ ID NO:76;

[0282] (a9) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:24, the heavy chain CDR 2 of SEQ ID NO:38, the heavy chain CDR 3 of SEQ ID NO:52, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73;

[0283] (a10) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:25, the heavy chain CDR 2 of SEQ ID NO:39, the heavy chain CDR 3 of SEQ ID NO:53, the light chain CDR 1 of SEQ ID NO:66, the light chain CDR 2 of SEQ ID NO:71, and the light chain CDR 3 of SEQ ID NO:76;

[0284] (a11) Antibody variable region comprising 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;

[0285] (a12) Antibody variable region comprising 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;

[0286] (a13) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:27, the heavy chain CDR 2 of SEQ ID NO:41, the heavy chain CDR 3 of SEQ ID NO:55, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73;

[0287] (a14) An antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:28, the heavy chain CDR 2 of SEQ ID NO:42, the heavy chain CDR 3 of SEQ ID NO:56, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73; and

[0288] (a15) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:82, the heavy chain CDR 2 of SEQ ID NO:83, the heavy chain CDR 3 of SEQ ID NO:84, the light chain CDR 1 of SEQ ID NO:65, the light chain CDR 2 of SEQ ID NO:70, and the light chain CDR 3 of SEQ ID NO:75;

[0289] and

[0290] (b) A third antigen-binding portion that binds to human delta-like 3 (DLL3) and includes an antibody variable region comprising a heavy chain CDR1 containing SEQ ID NO:233, a heavy chain CDR2 containing SEQ ID NO:234, a heavy chain CDR3 containing SEQ ID NO:235, a light chain CDR1 containing SEQ ID NO:237, a light chain CDR2 containing SEQ ID NO:238, and a light chain CDR3 containing SEQ ID NO:239.

[0291] [2-2][2-1] The multispecific antigen-binding molecule, wherein the first and second antigen-binding portions each comprise an antibody variable region that may be the same or different and is independently selected from the group consisting of:

[0292] (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;

[0293] (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;

[0294] (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;

[0295] (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;

[0296] (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;

[0297] (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;

[0298] (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;

[0299] (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;

[0300] (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;

[0301] (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;

[0302] (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;

[0303] (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;

[0304] (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;

[0305] (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

[0306] (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.

[0307] The multispecific antigen-binding molecule described in any one of [2-2A], [2-1] to [2-2], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region, the antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:20, the heavy chain CDR 2 of SEQ ID NO:34, the heavy chain CDR 3 of SEQ ID NO:48, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73.

[0308] [2-2B][2-2A] The multispecific antigen-binding molecule, wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region, wherein the antibody variable region comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:6 and a light chain variable region containing the amino acid sequence of SEQ ID NO:58.

[0309] The multispecific antigen-binding molecule of any one of [2-2C][2-1] to [2-2B], wherein the third antigen-binding portion comprises an antibody variable region, the antibody variable region comprising a VH containing SEQ ID NO:232 and a VL containing SEQ ID NO:236.

[0310] The multispecific antigen-binding molecule described in any one of [2-3][2-1] to [2-2C], wherein the first and second antigen-binding portions are each Fabs having a cysteine ​​residue at position 191 (EU number), and wherein a disulfide bond connecting the two cysteine ​​residues is present.

[0311] [2-4][2-3] The multispecific antigen-binding molecule, wherein the first, second and third antigen-binding moieties are each Fab, the Fab comprising a heavy chain containing VH and CH1 domains and a light chain containing VL and light chain constant (CL) domains, and wherein the C-terminus of the heavy chain of the third antigen-binding moieties is fused directly or via a peptide linker to the N-terminus of the Fab heavy chain of either the first or second antigen-binding moieties.

[0312] [2-5][2-4] The multispecific antigen-binding molecule, wherein the C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the Fab heavy chain of either the first or second antigen-binding portion via a peptide linker, and wherein the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO:248, SEQ ID NO:249 and SEQ ID NO:259.

[0313] [2-6][2-5] The multispecific antigen-binding molecule, wherein, in the respective CL domains of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 (Kabat number) are arginine and lysine, respectively; and wherein, in the respective CHl domains of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 (EU number) are glutamic acid.

[0314] The multispecific antigen-binding molecule described in [2-7][2-6] further comprises an Fc domain.

[0315] [2-8][2-7] The multispecific antigen-binding molecule, wherein the Fc domain comprises first and second Fc subunits, the first Fc subunit being selected from the group consisting of:

[0316] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0317] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0318] The Fc region polypeptide contains alanine at positions 234, 235, and 297, cysteine ​​at position 354, and tryptophan at position 366; and

[0319] The second Fc region subunit is selected from the group consisting of:

[0320] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0321] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0322] The Fc region polypeptide contains alanine at positions 234, 235, and 297, cysteine ​​at position 349, serine at position 366, alanine at position 368, and valine at position 407.

[0323] All positions are numbered according to EU.

[0324] Another aspect of the present invention relates to:

[0325] [3-1] Multispecific antigen-binding molecules, which include:

[0326] (a) A first antigen-binding region and a second antigen-binding region, each binding to human CD137 and containing the same or different antibody variable regions independently selected from the group consisting of:

[0327] (a1) Antibody variable region comprising 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;

[0328] (a2) The antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:18, the heavy chain CDR 2 of SEQ ID NO:32, the heavy chain CDR 3 of SEQ ID NO:46, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73;

[0329] (a3) Antibody variable region comprising 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;

[0330] (a4) Antibody variable region comprising 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;

[0331] (a5) Antibody variable region comprising heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:20, heavy chain CDR 2 of SEQ ID NO:34, heavy chain CDR 3 of SEQ ID NO:48, light chain CDR 1 of SEQ ID NO:63, light chain CDR 2 of SEQ ID NO:68 and light chain CDR 3 of SEQ ID NO:73;

[0332] (a6) Antibody variable region comprising 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;

[0333] (a7) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:23, the heavy chain CDR 2 of SEQ ID NO:37, the heavy chain CDR 3 of SEQ ID NO:51, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73;

[0334] (a8) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:23, the heavy chain CDR 2 of SEQ ID NO:37, the heavy chain CDR 3 of SEQ ID NO:51, the light chain CDR 1 of SEQ ID NO:66, the light chain CDR 2 of SEQ ID NO:71 and the light chain CDR 3 of SEQ ID NO:76;

[0335] (a9) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:24, the heavy chain CDR 2 of SEQ ID NO:38, the heavy chain CDR 3 of SEQ ID NO:52, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73;

[0336] (a10) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:25, the heavy chain CDR 2 of SEQ ID NO:39, the heavy chain CDR 3 of SEQ ID NO:53, the light chain CDR 1 of SEQ ID NO:66, the light chain CDR 2 of SEQ ID NO:71, and the light chain CDR 3 of SEQ ID NO:76;

[0337] (a11) Antibody variable region comprising 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;

[0338] (a12) Antibody variable region comprising 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;

[0339] (a13) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:27, the heavy chain CDR 2 of SEQ ID NO:41, the heavy chain CDR 3 of SEQ ID NO:55, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73;

[0340] (a14) An antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:28, the heavy chain CDR 2 of SEQ ID NO:42, the heavy chain CDR 3 of SEQ ID NO:56, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73; and

[0341] (a15) Antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:82, the heavy chain CDR 2 of SEQ ID NO:83, the heavy chain CDR 3 of SEQ ID NO:84, the light chain CDR 1 of SEQ ID NO:65, the light chain CDR 2 of SEQ ID NO:70, and the light chain CDR 3 of SEQ ID NO:75;

[0342] and

[0343] (b) A third antigen-binding portion that binds to human delta-like 3 (DLL3) and includes an antibody variable region comprising a heavy chain CDR1 containing SEQ ID NO:233, a heavy chain CDR2 containing SEQ ID NO:234, a heavy chain CDR3 containing SEQ ID NO:235, a light chain CDR1 containing SEQ ID NO:237, a light chain CDR2 containing SEQ ID NO:238, and a light chain CDR3 containing SEQ ID NO:239.

[0344] [3-2][3-1] The multispecific antigen-binding molecule, wherein the first and second antigen-binding portions each comprise the same or different antibody variable regions independently selected from the group consisting of:

[0345] (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;

[0346] (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;

[0347] (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;

[0348] (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;

[0349] (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;

[0350] (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;

[0351] (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;

[0352] (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;

[0353] (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;

[0354] (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;

[0355] (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;

[0356] (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;

[0357] (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;

[0358] (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

[0359] (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.

[0360] The multispecific antigen-binding molecule described in any one of [3-2A], [3-1] to [3-2], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region, the antibody variable region comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:20, the heavy chain CDR 2 of SEQ ID NO:34, the heavy chain CDR 3 of SEQ ID NO:48, the light chain CDR 1 of SEQ ID NO:63, the light chain CDR 2 of SEQ ID NO:68, and the light chain CDR 3 of SEQ ID NO:73.

[0361] [3-2B][3-2A] The multispecific antigen-binding molecule, wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region, wherein the antibody variable region comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:6 and a light chain variable region containing the amino acid sequence of SEQ ID NO:58.

[0362] The multispecific antigen-binding molecule of any one of [3-2C], [3-1] to [3-2B], wherein the third antigen-binding portion comprises an antibody variable region, the antibody variable region comprising a VH containing SEQ ID NO:232 and a VL containing SEQ ID NO:236.

[0363] The multispecific antigen-binding molecule described in any one of [3-3][3-1] to [3-2C], wherein the first and second antigen-binding portions are each Fabs having a cysteine ​​residue at position 191 (EU number), and wherein a disulfide bond connecting the two cysteine ​​residues is present.

[0364] [3-4][3-3] The multispecific antigen-binding molecule, wherein the first, second and third antigen-binding moieties are each Fab, wherein the Fab comprises a heavy chain containing VH and CH1 domains and a light chain containing VL and light chain constant (CL) domains, and wherein the C-terminus of the heavy chain of the third antigen-binding moieties is fused directly or via a peptide linker to the N-terminus of the Fab heavy chain of either the first or second antigen-binding moieties.

[0365] [3-5][3-4] The multispecific antigen-binding molecule wherein the C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the Fab heavy chain of either the first or second antigen-binding portion via a peptide linker, and wherein the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO:248, SEQ ID NO:249 and SEQ ID NO:259.

[0366] [3-6][3-5] The multispecific antigen-binding molecule, wherein, in the CL domain of each of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 (Kabat number) are arginine and lysine, respectively; and wherein, in the CH1 domain of each of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 (EU number) are glutamic acid.

[0367] The multispecific antigen-binding molecules described in [3-7][3-6] further include an Fc domain.

[0368] [3-8][3-7] The multispecific antigen-binding molecule, wherein the Fc domain comprises first and second Fc subunits, the first Fc subunit being selected from the group consisting of:

[0369] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0370] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0371] The Fc region polypeptide contains alanine at positions 234, 235, and 297, cysteine ​​at position 354, and tryptophan at position 366; and

[0372] The second Fc region subunit is selected from the group consisting of:

[0373] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0374] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0375] The Fc region polypeptide contains alanine at positions 234, 235, and 297, cysteine ​​at position 349, serine at position 366, alanine at position 368, and valine at position 407.

[0376] All positions are numbered according to EU.

[0377] Another aspect of the present invention relates to:

[0378] [4-1] Multispecific antigen-binding molecules, which include:

[0379] (a) A first antigen-binding region and a second antigen-binding region, each binding to human CD3 and containing the same or different antibody variable regions independently selected from the group consisting of (a1) to (a15):

[0380] (a1) An antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:17, a heavy chain CDR 2 containing SEQ ID NO:31, a heavy chain CDR 3 containing SEQ ID NO:45, a light chain CDR 1 containing SEQ ID NO:64, a light chain CDR 2 containing SEQ ID NO:69, and a light chain CDR 3 containing SEQ ID NO:74;

[0381] (a2) The antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:18, a heavy chain CDR 2 containing SEQ ID NO:32, a heavy chain CDR 3 containing SEQ ID NO:46, a light chain CDR 1 containing SEQ ID NO:63, a light chain CDR 2 containing SEQ ID NO:68, and a light chain CDR 3 containing SEQ ID NO:73;

[0382] (a3) The antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:19, a heavy chain CDR 2 containing SEQ ID NO:33, a heavy chain CDR 3 containing SEQ ID NO:47, a light chain CDR 1 containing SEQ ID NO:63, a light chain CDR 2 containing SEQ ID NO:68, and a light chain CDR 3 containing SEQ ID NO:73;

[0383] (a4) The antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:19, a heavy chain CDR 2 containing SEQ ID NO:33, a heavy chain CDR 3 containing SEQ ID NO:47, a light chain CDR 1 containing SEQ ID NO:65, a light chain CDR 2 containing SEQ ID NO:70, and a light chain CDR 3 containing SEQ ID NO:75;

[0384] (a5) An antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:20, a heavy chain CDR 2 containing SEQ ID NO:34, a heavy chain CDR 3 containing SEQ ID NO:48, a light chain CDR 1 containing SEQ ID NO:63, a light chain CDR 2 containing SEQ ID NO:68, and a light chain CDR 3 containing SEQ ID NO:73;

[0385] (a6) An antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:22, a heavy chain CDR 2 containing SEQ ID NO:36, a heavy chain CDR 3 containing SEQ ID NO:50, a light chain CDR 1 containing SEQ ID NO:63, a light chain CDR 2 containing SEQ ID NO:68, and a light chain CDR 3 containing SEQ ID NO:73;

[0386] (a7) An antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:23, a heavy chain CDR 2 containing SEQ ID NO:37, a heavy chain CDR 3 containing SEQ ID NO:51, a light chain CDR 1 containing SEQ ID NO:63, a light chain CDR 2 containing SEQ ID NO:68, and a light chain CDR 3 containing SEQ ID NO:73;

[0387] (a8) An antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:23, a heavy chain CDR 2 containing SEQ ID NO:37, a heavy chain CDR 3 containing SEQ ID NO:51, a light chain CDR 1 containing SEQ ID NO:66, a light chain CDR 2 containing SEQ ID NO:71, and a light chain CDR 3 containing SEQ ID NO:76;

[0388] (a9) Antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:24, a heavy chain CDR 2 containing SEQ ID NO:38, a heavy chain CDR 3 containing SEQ ID NO:52, a light chain CDR 1 containing SEQ ID NO:63, a light chain CDR 2 containing SEQ ID NO:68, and a light chain CDR 3 containing SEQ ID NO:73;

[0389] (a10) Antibody variable region, comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:25, a heavy chain CDR 2 containing SEQ ID NO:39, a heavy chain CDR 3 containing SEQ ID NO:53, a light chain CDR 1 containing SEQ ID NO:66, a light chain CDR 2 containing SEQ ID NO:71, and a light chain CDR 3 containing SEQ ID NO:76;

[0390] (a11) Antibody variable region, comprising heavy chain complementarity determination region (CDR) 1 containing SEQ ID NO:26, heavy chain CDR 2 containing SEQ ID NO:40, heavy chain CDR 3 containing SEQ ID NO:54, light chain CDR 1 containing SEQ ID NO:66, light chain CDR 2 containing SEQ ID NO:71 and light chain CDR 3 containing SEQ ID NO:76;

[0391] (a12) Antibody variable region, comprising heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:26, heavy chain CDR 2 containing SEQ ID NO:40, heavy chain CDR 3 containing SEQ ID NO:54, light chain CDR 1 containing SEQ ID NO:63, light chain CDR 2 containing SEQ ID NO:68, and light chain CDR 3 containing SEQ ID NO:73;

[0392] (a13) Antibody variable region, comprising heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:27, heavy chain CDR 2 containing SEQ ID NO:41, heavy chain CDR 3 containing SEQ ID NO:55, light chain CDR 1 containing SEQ ID NO:63, light chain CDR 2 containing SEQ ID NO:68 and light chain CDR 3 containing SEQ ID NO:73;

[0393] (a14) An antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:28, a heavy chain CDR 2 containing SEQ ID NO:42, a heavy chain CDR 3 containing SEQ ID NO:56, a light chain CDR 1 containing SEQ ID NO:63, a light chain CDR 2 containing SEQ ID NO:68, and a light chain CDR 3 containing SEQ ID NO:73; and

[0394] (a15) Antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 containing SEQ ID NO:82, a heavy chain CDR 2 containing SEQ ID NO:83, a heavy chain CDR 3 containing SEQ ID NO:84, a light chain CDR 1 containing SEQ ID NO:65, a light chain CDR 2 containing SEQ ID NO:70, and a light chain CDR 3 containing SEQ ID NO:75;

[0395] and

[0396] (b) A third antigen-binding portion that binds to human delta-like 3 (DLL3) and includes an antibody variable region comprising a heavy chain CDR1 containing SEQ ID NO:233, a heavy chain CDR2 containing SEQ ID NO:234, a heavy chain CDR3 containing SEQ ID NO:235, a light chain CDR1 containing SEQ ID NO:237, a light chain CDR2 containing SEQ ID NO:238, and a light chain CDR3 containing SEQ ID NO:239.

[0397] [4-2] Multispecific antigen-binding molecules, which include:

[0398] (a) A first antigen-binding portion and a second antigen-binding portion, each comprising an antibody variable region, the antibody variable regions being identical or different and independently selected from the group consisting of:

[0399] (a1) Antibody variable region, which includes a heavy chain variable region (VH) containing SEQ ID NO:3 and a light chain variable region (VL) containing SEQ ID NO:59;

[0400] (a2) An antibody variable region comprising a VH containing SEQ ID NO:4 and a VL containing SEQ ID NO:58;

[0401] (a3) Antibody variable region, which includes VH containing SEQ ID NO:5 and VL containing SEQ ID NO:58;

[0402] (a4) An antibody variable region comprising a VH containing SEQ ID NO:5 and a VL containing SEQ ID NO:60;

[0403] (a5) Antibody variable region, which includes VH containing SEQ ID NO:6 and VL containing SEQ ID NO:58;

[0404] (a6) Antibody variable region, which includes VH containing SEQ ID NO:8 and VL containing SEQ ID NO:58;

[0405] (a7) Antibody variable region, which includes VH containing SEQ ID NO:9 and VL containing SEQ ID NO:58;

[0406] (a8) An antibody variable region comprising a VH containing SEQ ID NO:9 and a VL containing SEQ ID NO:61;

[0407] (a9) Antibody variable region, which includes VH containing SEQ ID NO:10 and VL containing SEQ ID NO:58;

[0408] (a10) Antibody variable region, which includes VH containing SEQ ID NO:11 and VL containing SEQ ID NO:61;

[0409] (a11) Antibody variable region, which includes VH containing SEQ ID NO:12 and VL containing SEQ ID NO:61;

[0410] (a12) Antibody variable region, which includes VH containing SEQ ID NO:12 and VL containing SEQ ID NO:58;

[0411] (a13) Antibody variable region, which includes VH containing SEQ ID NO:13 and VL containing SEQ ID NO:58;

[0412] (a14) An antibody variable region comprising VH containing SEQ ID NO:14 and VL containing SEQ ID NO:58; and

[0413] (a15) Antibody variable region, which includes VH containing SEQ ID NO:81 and VL containing SEQ ID NO:60;

[0414] and

[0415] (b) A third antigen-binding portion comprising an antibody variable region, said antibody variable region comprising a VH containing SEQ ID NO:232 and a VL containing SEQ ID NO:236;

[0416] [4-3][4-1] The multispecific antigen-binding molecule, wherein the antibody variable regions of the first and second antigen-binding portions are identical.

[0417] [4-4][4-2] The multispecific antigen-binding molecule wherein the antibody variable regions of the first and second antigen-binding portions are identical.

[0418] [4-5][4-3] The multispecific antigen-binding molecule, wherein the first and second antigen-binding portions are each Fabs having a cysteine ​​residue at position 191 (EU number), and wherein the two cysteine ​​residues are linked by a disulfide bond.

[0419] [4-6][4-4] The multispecific antigen-binding molecule, wherein the first and second antigen-binding portions are each Fabs having a cysteine ​​residue at position 191 (EU number), and wherein the two cysteine ​​residues are linked by disulfide bonds.

[0420] [4-7][4-5] The multispecific antigen-binding molecule, wherein the first, second and third antigen-binding moieties are each in the form of Fab, wherein the Fab comprises VH, VL, CH1 domains and a light chain constant (CL) domain, and wherein the C-terminus of the CH1 domain of the third antigen-binding moieties is fused directly or via a peptide linker to the N-terminus of the VH of either the first or second antigen-binding moieties.

[0421] [4-8][4-6] The multispecific antigen-binding molecule, wherein the first, second and third antigen-binding moieties are each in the form of Fab, wherein the Fab comprises a CH1 domain and a light chain constant (CL) domain, and wherein the C-terminus of the CH1 domain of the third antigen-binding moieties is fused directly or via a peptide linker to the N-terminus of the VH of either the first or second antigen-binding moieties.

[0422] [4-9][4-7] The multispecific antigen-binding molecule, wherein the fusion is performed via a peptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO:248, SEQ ID NO:249 and SEQ ID NO:259.

[0423] [4-10][4-8] The multispecific antigen-binding molecule, wherein the fusion is performed via a peptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO:248, SEQ ID NO:249 and SEQ ID NO:259.

[0424] [4-11][4-9] The multispecific antigen-binding molecule, wherein the third antigen-binding portion is a cross-Fab in which VH is connected to the CL domain and VL is connected to the CH1 domain, and wherein the first and second antigen-binding portions are each conventional Fab in which VH is connected to the CH1 domain and VL is connected to the CL domain.

[0425] [4-12][4-10] The multispecific antigen-binding molecule, wherein the third antigen-binding portion is a cross-fabricated Fab in which VH is connected to the CL domain and VL is connected to the CH1 domain, and wherein the first and second antigen-binding portions are each conventional Fab in which VH is connected to the CH1 domain and VL is connected to the CL domain.

[0426] [4-13][4-11] The multispecific antigen-binding molecule, wherein, in the CL domain of each of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 (Kabat number) are arginine and lysine, respectively; and wherein, in the CHl domain of each of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 (EU number) are glutamic acid.

[0427] [4-14][4-12] The multispecific antigen-binding molecule, wherein, in the respective CL domains of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 (Kabat number) are arginine and lysine, respectively; and wherein, in the respective CHl domains of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 (EU number) are glutamic acid.

[0428] The multispecific antigen-binding molecule described in [4-15][4-13] further comprises an Fc domain.

[0429] The multispecific antigen-binding molecule described in [4-16][4-14] further comprises an Fc domain.

[0430] The multispecific antigen-binding molecules described in [4-17][4-15],

[0431] The Fc structural domain comprises first and second Fc region subunits.

[0432] The first Fc region subunit is selected from the group including:

[0433] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0434] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0435] The Fc region polypeptide contains alanine at positions 234, 235 and 297, cysteine ​​at position 354, and tryptophan at position 366.

[0436] The second Fc region subunit is selected from the group consisting of:

[0437] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0438] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0439] Fc region polypeptides containing alanine at positions 234, 235, and 297, cysteine ​​at position 349, serine at position 366, alanine at position 368, and valine at position 407; and

[0440] All locations are numbered using EU codes.

[0441] The multispecific antigen-binding molecules described in [4-18][4-16],

[0442] The Fc structural domain comprises first and second Fc region subunits.

[0443] The first Fc region subunit is selected from the group including:

[0444] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0445] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0446] The Fc region polypeptide contains alanine at positions 234, 235 and 297, cysteine ​​at position 354, and tryptophan at position 366.

[0447] The second Fc region subunit is selected from the group consisting of:

[0448] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0449] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0450] Fc region polypeptides containing alanine at positions 234, 235, and 297, cysteine ​​at position 349, serine at position 366, alanine at position 368, and valine at position 407; and

[0451] All positions are numbered according to EU.

[0452] [4-19] A multispecific antigen-binding molecule comprising five polypeptide chains selected from the group consisting of (A) to (O):

[0453] (A) A polypeptide chain containing the amino acid sequence of SEQ ID NO:201 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:208 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0454] (B) A polypeptide chain containing the amino acid sequence of SEQ ID NO:203 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:209 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0455] (C) A polypeptide chain containing the amino acid sequence of SEQ ID NO:204 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:209 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0456] (D) A polypeptide chain containing the amino acid sequence of SEQ ID NO:205 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:209 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0457] (E) A polypeptide chain containing the amino acid sequence of SEQ ID NO:216 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:229 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0458] (F) A polypeptide chain containing the amino acid sequence of SEQ ID NO:217 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:210 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0459] (G) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:219, 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:211, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0460] (H) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:220, 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:211, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0461] (I) A polypeptide chain containing the amino acid sequence of SEQ ID NO:221 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:211 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0462] (J) a polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:222, 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:230, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0463] (K) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:223, 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:212, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0464] (L) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:225, 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:213, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0465] (M) A polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:226, 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:213, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0466] (N) A polypeptide chain containing the amino acid sequence of SEQ ID NO:227 (chain 1), a polypeptide chain containing the amino acid sequence of SEQ ID NO:206 (chain 2), a polypeptide chain containing the amino acid sequence of SEQ ID NO:213 (chain 3), and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0467] (O) 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 and chain 5) each containing the amino acid sequence of SEQ ID NO:215.

[0468] [4-20][4-19] The multispecific antigen-binding molecule comprises a polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:203, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0469] [4-21][4-19] The multispecific antigen-binding molecule comprises a polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:204, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0470] [4-22][4-19] The multispecific antigen-binding molecule comprises a polypeptide chain (chain 1) containing the amino acid sequence of SEQ ID NO:205, 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:209, and two polypeptide chains (chain 4 and chain 5) each containing the amino acid sequence of SEQ ID NO:214.

[0471] [4-23] Multispecific antigen-binding molecules, comprising:

[0472] (b) A first antigen-binding portion and a second antigen-binding portion, each binding to human CD3 and comprising an antibody variable region, wherein the antibody variable region comprises a heavy chain CDR1 containing SEQ ID NO:20, a heavy chain CDR2 containing SEQ ID NO:34, a heavy chain CDR3 containing SEQ ID NO:48, a light chain CDR1 containing SEQ ID NO:63, a light chain CDR2 containing SEQ ID NO:68, and a light chain CDR3 containing SEQ ID NO:73;

[0473] and

[0474] (b) A third antigen-binding portion that binds to human DLL3 and includes an antibody variable region, said antibody variable region comprising a heavy chain CDR1 containing SEQ ID NO:233, a heavy chain CDR2 containing SEQ ID NO:234, a heavy chain CDR3 containing SEQ ID NO:235, a light chain CDR1 containing SEQ ID NO:237, a light chain CDR2 containing SEQ ID NO:238, and a light chain CDR3 containing SEQ ID NO:239.

[0475] [4-24][4-23] The multispecific antigen-binding molecule, wherein the antibody variable region of each of the first and second antigen-binding portions comprises a VH containing SEQ ID NO:6 and a VL containing SEQ ID NO:58; and the antibody variable region of the third antigen-binding portion comprises a VH containing SEQ ID NO:232 and a VL containing SEQ ID NO:236.

[0476] [4-25][4-24] The multispecific antigen-binding molecule, wherein the first and second antigen-binding portions are each Fabs having a cysteine ​​residue at position 191 (EU number), and wherein the two cysteine ​​residues are linked by disulfide bonds.

[0477] [4-26][4-25] The multispecific antigen-binding molecule, wherein the first, second and third antigen-binding portions are each in the form of Fab, the Fab comprising VH, VL, CH1 and CL domains, and wherein the C-terminus of the CH1 domain of the third antigen-binding portion is fused directly or via a peptide linker to the N-terminus of the VH of either the first or second antigen-binding portion.

[0478] [4-27][4-26] The multispecific antigen-binding molecule, wherein the fusion is performed via a peptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO:248, SEQ ID NO:249 and SEQ ID NO:259.

[0479] [4-28][4-27] The multispecific antigen-binding molecule, wherein the third antigen-binding portion is a cross-fabricated Fab in which VH is connected to the CL domain and VL is connected to the CH1 domain, and wherein the first and second antigen-binding portions are each conventional Fab in which VH is connected to the CH1 domain and VL is connected to the CL domain.

[0480] [4-29][4-28] The multispecific antigen-binding molecule, wherein, in the CL domain of each of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 (Kabat number) are arginine and lysine, respectively; and wherein, in the CH1 domain of each of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 (EU number) are glutamic acid.

[0481] The multispecific antigen-binding molecule described in [4-30][4-29] further comprises an Fc domain.

[0482] The multispecific antigen-binding molecules described in [4-31][4-30],

[0483] The Fc structural domain comprises first and second Fc region subunits.

[0484] The first Fc region subunit is selected from the group including:

[0485] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0486] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0487] The Fc region polypeptide contains alanine at positions 234, 235 and 297, cysteine ​​at position 354, and tryptophan at position 366.

[0488] The second Fc region subunit is selected from the group consisting of:

[0489] Fc region polypeptides containing alanine at positions 234 and 235 respectively;

[0490] Fc region polypeptides containing alanine at positions 234, 235, and 297; and

[0491] Fc region polypeptides containing alanine at positions 234, 235, and 297, cysteine ​​at position 349, serine at position 366, alanine at position 368, and valine at position 407; and

[0492] All positions are numbered according to EU. Attached Figure Description

[0493] [ Figure 1 ] Figure 1 The design and naming rules for trivalent antibodies in the DUAL / LINC(1+2) format, bispecific antibodies in the standard Ab format, and bispecific antibodies in the BiTE format are shown.

[0494] [ Figure 2 ] Figure 2 The antibody's TDCC activity against the SK-MEL30 cell line was demonstrated. a) Comparison of TDCC between the DUAL / LINC and CD3 bispecific formats. b) Effect of adapter length on cytotoxicity.

[0495] [ Figure 3 ] Figure 3 This demonstrates the in vivo efficacy of the antibody against the NCI-H1436 xenograft in the huNOG mouse model. The Y-axis represents tumor volume (mm). 3 The X-axis represents the number of days after tumor implantation.

[0496] [ Figure 4 ] Figure 4 The results of the analysis of CD8 T cell infiltration are shown. Tumors were harvested at specified time points after antibody injection, and T cell infiltration was analyzed using flow cytometry.

[0497] [ Figure 5 ] Figure 5 The results of analyzing exhaustion markers on CD8 T cells are shown. Tumors were harvested on day 7 after antibody injection, and the expression of exhaustion markers was analyzed using flow cytometry.

[0498] [ Figure 6 ] Figure 6 This is a schematic diagram showing the structure of the full-length human DLL3 and the human DLL3 ECD fragment protein. It also shows the epitopes recognized by each anti-DLL3 antibody. From the N-terminus to the C-terminus, the EGF domain has six regions, EGF1 to EGF6. Detailed Implementation

[0499] The techniques and procedures described or cited herein are generally well understood by those skilled in the art and are typically used with conventional methodologies, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual 3dedition* (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (edited by F.M. Usubel et al., (2003)); Enzymatic Methods Series (Academic Press, Inc.): PCR 2: Practical Methods (edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor, (1995)); Antibodies, Laboratory Manuals and Animal Cell Culture (edited by R.R. Freshney, (1987)); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual (edited by J.E.C. Lis, 1998) Academic Press; Animal Cell Culture (edited by R.R. Freshney, 1987); Introduction to Cell and Tissue Culture (edited by J.M. Pather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J.B. G. R ... Leigh and Sons, 1999; Immunobiology (CA. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRL Publishing, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); Antibodies (M. Zanetti and JD.Capra (ed., Harwood Academic Publishers, 1995); and Cancer: Principles and Practices of Oncology (VT DeVita et al., eds., JBLippincott Company, 1993).

[0500] The following definitions and detailed descriptions are provided to aid in understanding this disclosure.

[0501] definition

[0502] amino acids

[0503] In this article, amino acids are described by single-letter codes, three-letter codes, or both, such as 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.

[0504] Changes in amino acids

[0505] For amino acid alterations in the amino acid sequence of antigen-binding molecules (also referred to as “amino acid substitutions” or “amino acid mutations” in this specification), 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 used. In addition, several known methods can also be used as amino acid alteration methods to replace non-natural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100(11), 6353-6357). For example, a cell-free translation system containing tRNA (Clover Direct (Protein Express)) is suitable, containing a non-natural amino acid that complements the UAG codon (amber codon), one of the stop codons, to inhibit tRNA binding.

[0506] In this specification, when describing sites of amino acid alteration, the term "and / or" means each combination of "and" and "or" in appropriate combinations. Specifically, for example, "amino acids at positions 33, 55, and / or 96 are substituted" includes variations of the following amino acid alterations: (a) position 33, (b) position 55, (c) position 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96, and (g) amino acids at positions 33, 55, and 96.

[0507] Furthermore, in this document, as a way to indicate amino acid changes, it is appropriate to use a code that displays one or three letters before and after the number indicating a specific position, representing the amino acid before and after the change. For example, the change N100bL or Asn100bLeu used when substituting an amino acid contained in the variable region of an antibody indicates that Leu replaces Asn at position 100b (according to the Kabat number). That is, the number indicates the amino acid position according to the Kabat number, the one or three-letter amino acid code before the number indicates the amino acid before substitution, and the one or three-letter amino acid code after the number indicates the amino acid after substitution. Similarly, the change P238D or Pro238Asp used when substituting an amino acid in the Fc region contained in the constant region of an antibody indicates that Pro is replaced by Asp at position 238 (according to the EU number). That is, the number indicates the amino acid position according to the EU number, the one or three-letter amino acid code before the number indicates the amino acid before substitution, and the one or three-letter amino acid code after the number indicates the amino acid after substitution.

[0508] polypeptide

[0509] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, not a product of a specific length. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" may be used in place of any of these terms, or used interchangeably with any of these terms. The term "polypeptide" also means a product of post-expression modifications of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modifications of amino acids that do not occur naturally. Polypeptides may be derived from natural biological sources or produced through recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be produced in any manner, including through chemical synthesis. The size of polypeptides as described herein can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily need to have such a structure. Polypeptides with a defined three-dimensional structure are called folded, while polypeptides that do not have a defined three-dimensional structure but can take on a large number of different conformations are called unfolded.

[0510] Percentage (%) amino acid sequence identity

[0511] The "percentage (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence after the sequences have been aligned and, where necessary, vacancies have been introduced to obtain the maximum percentage sequence identity, and no conserved substitutions have been considered as part of the sequence identity. Alignments used to determine percentage amino acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, and Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this document, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington, D.C., 20559, U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and will not change. When using ALIGN-2 for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A relative to, and / or with respect to, a given amino acid sequence B (which can be alternatively stated as a given amino acid A having or including a specific % amino acid sequence identity relative to, and / or with respect to, a given amino acid sequence B) is calculated as follows:

[0512] 100 times the fraction X / Y

[0513] Where X is the number of amino acid residues that the sequence alignment program ALIGN-2 scores as identical matches in the alignments 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, then the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0514] Recombination Method and Composition

[0515] Antibody and antigen-binding molecules can be generated using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed with): (1) a vector comprising 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 a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp2 / O cells). In one embodiment, a method for preparing the multispecific antigen-binding molecule of the present invention is provided, wherein the method comprises culturing a host cell containing a nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0516] For the recombinant production of the antibodies described herein, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody.

[0517] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be produced in bacteria, for example, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*.) Antibodies can be isolated from bacterial cell paste in soluble fractions and can be further purified.

[0518] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," thereby producing antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0519] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells.

[0520] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES for producing antibodies in transgenic plants). TM technology).

[0521] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney lines (such as 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK); mouse supporting cells (TM4 cells, e.g., described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells, such as those described in Mather et al., Annals. As described in NYAcad.Sci.383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0522] The recombinant production of the antigen-binding molecule described herein can be carried out using methods similar to those described above, by using host cells containing (e.g., already transformed) one or more vectors, the vectors containing nucleic acids encoding an amino acid sequence that comprises the entire antigen-binding molecule or a portion thereof.

[0523] Antigen-binding molecules and multispecific antigen-binding molecules

[0524] As used herein, the term "antigen-binding molecule" refers to any molecule containing an antigen-binding site or any molecule that is active in binding to an antigen, and may further refer to such molecules, for example, peptides or proteins having a length of about five or more amino acids. Peptides and proteins are not limited to those derived from organisms; for example, they can be polypeptides produced from artificially designed sequences. They can also be any naturally occurring polypeptide, synthetic polypeptide, recombinant polypeptide, etc. A scaffold molecule containing a known stable conformational structure such as an α / β barrel as a scaffold, wherein a portion of the molecule is configured to form an antigen-binding site, is also an embodiment of the antigen-binding molecule described herein.

[0525] A "multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to more than one antigen. 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 some embodiments, the multispecific antigen-binding molecule of this application is a trispecific antigen-binding molecule, meaning it can specifically bind to three different antigens—it can bind to either CD3 or CD137 but not both simultaneously, and it can specifically bind to DLL3.

[0526] In a first aspect, this disclosure provides a multispecific antigen-binding molecule comprising: a first antigen-binding portion and a second antigen-binding portion, each capable of binding CD3 and CD137, but not simultaneously binding CD3 and CD137; and a third antigen-binding portion capable of binding a third antigen, preferably an antigen expressed on cancer cells / tissues. In some embodiments, the third antigen bound by the third antigen-binding portion is DLL3, preferably human DLL3.

[0527] The first and second antigen-binding moieties can be "dual antigen-binding moieties" capable of binding to both CD3 and CD137, but not simultaneously, which will be described in more detail below. The third antigen-binding moieties can be "DLL3 antigen-binding moieties," which will also be described in more detail below.

[0528] In some embodiments, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules and contain at least one disulfide bond formed between the CH1 regions of the first antigen-binding portion and the CH1 regions of the second antigen-binding portion. The disulfide bond may be formed between amino acid residues at position 191 according to EU numbering within the respective CH1 regions of the first and second antigen-binding portions.

[0529] In some embodiments, the third antigen-binding moiety of Fab or scFv may be fused to either the first or second antigen-binding moiety. When the first, second, and third antigen-binding moieties are each Fab molecules, the third antigen-binding moiety may optionally be fused via a peptide linker to the C-terminus (CH1) of the Fab heavy chain and the N-terminus of the Fab heavy chain of either the first or second antigen-binding moiety. Representative peptide linkers include those consisting of the amino acid sequences of SEQ ID NO:248, SEQ ID NO:249, or SEQ ID NO:259. In some embodiments, the first antigen-binding moiety is identical to the second antigen-binding moiety.

[0530] In some embodiments, the third antigen-binding moiety is a cross-Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and in which the first and second antigen-binding moieties are each conventional Fab molecules.

[0531] In some embodiments, in the constant domain CL of the respective light chains 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) (according to Kabat designations), and wherein in the constant domain CH1 of the respective heavy chains 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) (according to EU designations). In other embodiments, in the constant domain CL of the respective light chains of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 are arginine (R) and lysine (K), respectively (according to Kabat designations), and wherein in the constant domain CH1 of the respective heavy chains of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 are glutamic acid (E) (according to EU designations).

[0532] Multispecific antigen-binding molecules may further include an Fc domain, which will be described in detail below. Where the first and second antigen-binding moieties are each Fab, the first antigen-binding moieties may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding moieties may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain. In some embodiments, a third antigen-binding moieties may optionally be fused at the C-terminus of the N-terminus of the Fab heavy chain of either the first or second antigen-binding moieties via a peptide linker.

[0533] In another aspect of the invention, this disclosure provides a multispecific antigen-binding molecule comprising: an antigen-binding portion capable of binding CD3 and CD137 but not simultaneously; and an antigen-binding portion capable of binding DLL3, preferably human DLL3. In some embodiments, the antigen-binding portion capable of binding CD3 and CD137 but not simultaneously is a "dual antigen-binding portion" capable of binding CD3 and CD137 but not simultaneously, which will be described in detail below.

[0534] The components of the multispecific antigen-binding molecule of the present invention can be fused together in multiple configurations. Figure 1 Exemplary configurations are depicted in a) and should be read in conjunction with Tables 10-1 to 10-3.

[0535] According to any of the above embodiments, components of the multispecific antigen-binding molecule (e.g., antigen-binding moiety, Fc domain) can be fused directly or via various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, which are described herein or are 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 typically a number between 1 and 10, and typically 2 to 4.

[0536] Pyroglutamylation

[0537] It is known that antibodies undergo post-translational modifications when expressed in cells. Examples of post-translational modifications include cleavage of the C-terminal lysine of the heavy chain by carboxypeptidase; modification of the N-terminal glutamine or glutamate of the heavy and light chains to pyroglutamic acid via 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, pp. 2426-2447).

[0538] In some embodiments, the multispecific antigen-binding molecule of the present invention further 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 a lysine residue at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications resulting from N-terminal pyroglutamylation and C-terminal lysine deletion have no effect on antibody activity (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0539] antigen-binding portion

[0540] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigen. In one embodiment, the antigen-binding moiety is capable of directing its attached entity to a target site, such as to a specific type of tumor cell expressing a cancer antigen (DLL3). In another embodiment, the antigen-binding moiety is capable of activating signal transduction via its target antigen, such as a T-cell receptor complex antigen (particularly CD3) and / or a co-stimulatory receptor (CD137). Antigen-binding moieties include antibodies and fragments thereof as further defined herein. Specific antigen-binding moieties include antigen-binding domains or variable regions of an antibody, including antibody heavy chain variable regions and antibody light chain variable regions. In some embodiments, the antigen-binding moiety may include antibody constant regions as further defined herein and known in the art. Useful heavy chain constant regions include any of five isoforms: α, δ, ε, γ, or μ. Useful light chain constant regions include any of two isoforms: κ and λ.

[0541] As used herein, the terms “first,” “second,” and “third” relating to antigen-binding moieties, etc., are used for ease of distinction when there is more than one of each type of moieties. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order or direction to multispecific antigen-binding molecules.

[0542] On the other hand, the antigen-binding portions of the present invention disclosed herein can be used in novel chimeric antigen receptors (CARs) comprising one or more of the antigen-binding portions disclosed herein. In some embodiments, the CAR of the present invention will comprise an scFv construct, and in a preferred embodiment, will comprise or comprise heavy and light chain variable regions as disclosed herein. In a preferred embodiment, the disclosed chimeric antigen receptor can be used to treat or prevent proliferative diseases and any recurrence or metastasis thereof.

[0543] Antigen-binding moieties that can bind to CD3 and CD137 but not simultaneously.

[0544] The multispecific antigen-binding molecules described herein comprise at least one antigen-binding moiety capable of binding to both CD3 and CD137, but not simultaneously (also referred to herein as a "dual antigen-binding moiety," "first antigen-binding moiety," "dual Fab," or "dual Ig"). In certain embodiments, the multispecific antigen-binding molecule comprises two dual antigen-binding moieties ("first antigen-binding moiety" and "second antigen-binding moiety," each of which may be referred to as "dual Fab"). In some embodiments, the two dual antigen-binding moieties ("first antigen-binding moiety," "second antigen-binding moiety," or "dual Fab") each provide monovalent binding to either CD3 or CD137, but not simultaneously. In certain embodiments, the multispecific antigen-binding molecule comprises no more than two dual antigen-binding moieties ("first antigen-binding moiety," "second antigen-binding moiety," or "dual Fab").

[0545] In some embodiments, the dual antigen-binding moiety (“first antigen-binding moiety” or “second antigen-binding moiety” or “dual Fab”) is typically a Fab molecule, particularly a conventional Fab molecule. In some embodiments, the dual antigen-binding moiety (“first antigen-binding moiety” or “second antigen-binding moiety” or “dual Fab”) is a domain comprising variable regions (VL and VH) of the antibody light chain and heavy chain. Suitable examples of such domains comprising variable regions of the antibody light chain and heavy chain include “single-chain Fv (scFv)”, “single-chain antibody”, “Fv”, “single-chain Fv 2 (scFv2)”, “Fab”, “F(ab')2”, etc.

[0546] In some embodiments, the dual antigen-binding portion (“first antigen-binding portion” or “second antigen-binding portion” or “double Fab”) specifically binds all or part of a portion of the CD3 peptide. In a particular embodiment, CD3 is human CD3 or cynomolgus monkey CD3, most particularly human CD3. In a particular embodiment, the first antigen-binding portion is cross-reactive (i.e., specifically binds) to human and cynomolgus monkey CD3. In some embodiments, the first antigen-binding portion is capable of specifically binding to the ε subunit of CD3, particularly the human CD3 ε subunit of CD3 shown in SEQ ID NO:7 (NP_000724.1) (RefSeq registration number shown in parentheses). In some embodiments, the dual antigen-binding portion (“first antigen-binding portion” or “second antigen-binding portion” or “double Fab”) is capable of specifically binding to the CD3 ε chain expressed on the surface of eukaryotic cells. In some embodiments, the dual antigen-binding portion (“first antigen-binding portion” or “second antigen-binding portion” or “double Fab”) binds to the CD3 ε chain expressed on the surface of T cells.

[0547] In some embodiments, CD137 is human CD137. In some embodiments, advantageous examples of the antigen-binding molecule of the present invention comprise a dual antigen-binding portion (“first antigen-binding portion” or “second antigen-binding portion” or “double Fab”) that binds to the same epitope as the human CD137 epitope bound by an antibody selected from the group consisting of:

[0548] Identify entries containing SPCPPNSFSSAGGQRTCD

[0549] Antibody against the region of the ICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC sequence (SEQ ID NO:21),

[0550] Antibodies that recognize the region containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO:35)

[0551] Antibodies that recognize the region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO:49), and

[0552] Antibodies that recognize the region of the human CD137 protein containing the LQDPCSNCPAGTFCDNNRNQIC sequence (SEQ ID NO:105).

[0553] In a specific embodiment, the dual antigen-binding portion (“first antigen-binding portion”, “second antigen-binding portion”, or “double Fab”) comprises any one of the antibody variable region sequences shown in Table 1 below. In a specific embodiment, the dual antigen-binding portion (“first antigen-binding portion”, “second antigen-binding portion”, or “double Fab”) comprises any combination of heavy chain variable regions and light chain variable regions shown in Table 1.

[0554] (Table 1)

[0555] SEQ ID NO of the variable region of the dual antigen-binding portion (“first antigen-binding portion” or “second antigen-binding portion” or “dual Fab”)

[0556]

[0557]

[0558] In one embodiment, the dual antigen-binding region (“first antigen-binding region”, “second antigen-binding region”, or “dual Fab”) comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:6, and the light chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:58. In another embodiment, the dual antigen-binding region (“first antigen-binding region”, “second antigen-binding region”, or “dual Fab”) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:58.

[0559] In one embodiment, the dual antigen-binding region (“first antigen-binding region”, “second antigen-binding region”, or “dual Fab”) comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:14, and the light chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:58. In another embodiment, the dual antigen-binding region (“first antigen-binding region”, “second antigen-binding region”, or “dual Fab”) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:14, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:58.

[0560] In one embodiment, the dual antigen-binding region (“first antigen-binding region”, “second antigen-binding region”, or “dual Fab”) comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:81, and the light chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:58. In another embodiment, the dual antigen-binding region (“first antigen-binding region”, “second antigen-binding region”, or “dual Fab”) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:81, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:58.

[0561] In a specific implementation, the dual antigen-binding portion (“first antigen-binding portion” or “second antigen-binding portion” or “dual Fab”) comprises any combination of the HVR sequences shown in Table 2 below.

[0562] (Table 2)

[0563] SEQ ID NO of the HVR(CDR) sequence of the dual antigen-binding portion (“first antigen-binding portion” or “second antigen-binding portion” or “Dual-Fab”)

[0564]

[0565] In some embodiments, each of the dual antigen-binding portions (“first antigen-binding portion” or “second antigen-binding portion” or “dual Fab”) comprises an antibody variable region, said antibody variable region comprising any one of the following (a1) to (a17):

[0566] (a1) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:17, CDR 2 of the heavy chain of SEQ ID NO:31, CDR 3 of the heavy chain of SEQ ID NO:45, CDR 1 of the light chain of SEQ ID NO:64, CDR 2 of the light chain of SEQ ID NO:69 and CDR 3 of the light chain of SEQ ID NO:74;

[0567] (a2) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:18, CDR 2 of the heavy chain in SEQ ID NO:32, CDR 3 of the heavy chain in SEQ ID NO:46, CDR 1 of the light chain in SEQ ID NO:63, CDR 2 of the light chain in SEQ ID NO:68 and CDR 3 of the light chain in SEQ ID NO:73;

[0568] (a3) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:19, CDR 2 of the heavy chain in SEQ ID NO:33, CDR 3 of the heavy chain in SEQ ID NO:47, CDR 1 of the light chain in SEQ ID NO:63, CDR 2 of the light chain in SEQ ID NO:68 and CDR 3 of the light chain in SEQ ID NO:73;

[0569] (a4) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:19, CDR 2 of the heavy chain of SEQ ID NO:33, CDR 3 of the heavy chain of SEQ ID NO:47, CDR 1 of the light chain of SEQ ID NO:65, CDR 2 of the light chain of SEQ ID NO:70 and CDR 3 of the light chain of SEQ ID NO:75;

[0570] (a5) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:20, CDR 2 of the heavy chain of SEQ ID NO:34, CDR 3 of the heavy chain of SEQ ID NO:48, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0571] (a6) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:22, CDR 2 of the heavy chain of SEQ ID NO:36, CDR 3 of the heavy chain of SEQ ID NO:50, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0572] (a7) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:23, CDR 2 of the heavy chain of SEQ ID NO:37, CDR 3 of the heavy chain of SEQ ID NO:51, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0573] (a8) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:23, CDR 2 of the heavy chain of SEQ ID NO:37, CDR 3 of the heavy chain of SEQ ID NO:51, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0574] (a9) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:24, CDR 2 of the heavy chain of SEQ ID NO:38, CDR 3 of the heavy chain of SEQ ID NO:52, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0575] (a10) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:25, CDR 2 of the heavy chain of SEQ ID NO:39, CDR 3 of the heavy chain of SEQ ID NO:53, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0576] (a11) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:26, CDR 2 of the heavy chain of SEQ ID NO:40, CDR 3 of the heavy chain of SEQ ID NO:54, CDR 1 of the light chain of SEQ ID NO:66, CDR 2 of the light chain of SEQ ID NO:71 and CDR 3 of the light chain of SEQ ID NO:76;

[0577] (a12) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:26, CDR 2 of the heavy chain of SEQ ID NO:40, CDR 3 of the heavy chain of SEQ ID NO:54, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0578] (a13) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:27, CDR 2 of the heavy chain of SEQ ID NO:41, CDR 3 of the heavy chain of SEQ ID NO:55, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0579] (a14) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:28, CDR 2 of the heavy chain of SEQ ID NO:42, CDR 3 of the heavy chain of SEQ ID NO:56, CDR 1 of the light chain of SEQ ID NO:63, CDR 2 of the light chain of SEQ ID NO:68 and CDR 3 of the light chain of SEQ ID NO:73;

[0580] (a15) Complementary Determinant Region (CDR) 1 of the heavy chain of SEQ ID NO:82, CDR 2 of the heavy chain of SEQ ID NO:83, CDR 3 of the heavy chain of SEQ ID NO:84, CDR 1 of the light chain of SEQ ID NO:65, CDR 2 of the light chain of SEQ ID NO:70 and CDR 3 of the light chain of SEQ ID NO:75;

[0581] (a16) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a15); and

[0582] (a17) is an antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a15).

[0583] In some embodiments, the multispecific antigen-binding molecule or dual antigen-binding moiety of the present invention (“first antigen-binding moiety” or “second antigen-binding moiety” or “double Fab”) further comprises post-translational modifications. Examples of post-translational modifications include pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of a lysine residue at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications resulting from N-terminal pyroglutamylation and C-terminal lysine deletion have no effect on antibody activity (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0584] Antigen-binding moiety that can bind to DLL3

[0585] The multispecific antigen-binding molecules described herein contain at least one antigen-binding moiety capable of binding δ-like 3 (DLL3) (also referred to herein as the “DLL3 antigen-binding moiety”, the “third antigen-binding moiety”, or the “DLL3-binding antigen-binding moiety”).

[0586] In some embodiments, the multispecific antigen-binding molecule includes one antigen-binding moiety capable of binding DLL3. In some embodiments, the multispecific antigen-binding molecule includes two antigen-binding moieties capable of binding DLL3 (“DLL3 antigen-binding moieties”). In particular such embodiments, these antigen-binding moieties each specifically bind to the same epitope of DLL3. In even more specific embodiments, all these “DLL3 antigen-binding moieties” are identical. In one embodiment, the multispecific antigen-binding molecule includes an immunoglobulin molecule capable of specifically binding DLL3 (“DLL3 antigen-binding moieties”). In one embodiment, the multispecific antigen-binding molecule includes no more than two antigen-binding moieties capable of binding DLL3 (“DLL3 antigen-binding moieties”).

[0587] In some embodiments, the DLL3 antigen-binding portion is a cross-Fab molecule, i.e., a DLL3 molecule in which the variable or constant regions of the Fab heavy and light chains are exchanged. In some embodiments, the DLL3 antigen-binding portion is a cross-Fab molecule in which the variable regions of the Fab light and Fab heavy chains are exchanged.

[0588] 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 DLL3 protein expressed on the surface of eukaryotic cells. In some embodiments, the DLL3 antigen-binding moiety binds to DLL3 protein expressed on the surface of cancer cells.

[0589] In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety binds to epitopes within the extracellular domain (ECD), i.e., the domain from the N-terminus to the immediate preceding TM region, but not the intracellular domain of the TM region or C-terminus. The multispecific antigen-binding molecule or DLL3 antigen-binding moiety may bind to epitopes within any of the aforementioned domains / regions within the ECD. In a preferred embodiment, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety binds to epitopes in the region from EGF6 to the immediate preceding TM region. More specifically, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety may bind to epitopes within the region defined in SEQ ID NO:89 of human DLL3. In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety binds to an epitope in the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region, or from EGF6 to the region immediately preceding the TM region of human DLL3, or in the region of EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6, or from EGF6 to the region immediately preceding the TM region of human DLL3. In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety may be derived from previously reported anti-DLL3 antibodies, wherein the bound DLL3 epitope has been characterized (e.g., WO2019131988 and WO2011093097).

[0590] In specific embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety comprises any of the antibody variable region sequences shown in Table 1C below. In specific embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety comprises any combination of heavy chain variable regions and light chain variable regions shown in Table 3. In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety comprises a domain containing an antibody variable fragment that competes with any of the antibody variable regions shown in Table 3 for binding to DLL3.

[0591] (Table 3)

[0592] SEQ ID NO of the variable region of the exemplary DLL3 antigen-binding portion

[0593]

[0594]

[0595] In one embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:232, and the light chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:236. In another embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:232, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:236.

[0596] In one embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:300, and the light chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:236. In another embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:300, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:236.

[0597] In one embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:301, and the light chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:236. In another embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:301, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:236.

[0598] In one embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:274, and the light chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:275. In another embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:274, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:275.

[0599] In one embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the heavy chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:264, and the light chain variable region sequence has at least about 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:265. In another embodiment, the DLL3 antigen-binding region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:264, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:265.

[0600] In specific embodiments, the DLL3 antigen-binding moiety comprises any combination of the HVR sequences shown in Table 4 below. In some embodiments, the multispecific antigen-binding molecule or the DLL3 antigen-binding moiety comprises a domain containing an antibody variable fragment that competes with any antibody variable region shown in Table 4 for binding to DLL3, or with any antibody variable fragment containing the same HVR sequence as the antibody variable regions shown in Table 4 for binding to DLL3.

[0601] (Table 4)

[0602] SEQ ID NO of the exemplary HVR(CDR) sequence of the DLL3 antigen-binding region

[0603]

[0604]

[0605] In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety of the present invention includes an antibody variable region, said antibody variable region comprising any one of the following (a1) to (a5):

[0606] (a1) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:233, CDR 2 of the heavy chain in SEQ ID NO:234, CDR 3 of the heavy chain in SEQ ID NO:235, CDR 1 of the light chain in SEQ ID NO:237, CDR 2 of the chain in SEQ ID NO:238, and CDR 3 of the light chain in SEQ ID NO:239;

[0607] (a2) Complementary determinant region (CDR) 1 of the heavy chain of SEQ ID NO:276, CDR 2 of the heavy chain of SEQ ID NO:277, CDR 3 of the heavy chain of SEQ ID NO:278, CDR 1 of the light chain of SEQ ID NO:279, CDR 2 of the light chain of SEQ ID NO:280 and CDR 3 of the light chain of SEQ ID NO:281;

[0608] (a3) Complementary Determinant Region (CDR) 1 of the heavy chain in SEQ ID NO:285, CDR 2 of the heavy chain in SEQ ID NO:286, CDR 3 of the heavy chain in SEQ ID NO:287, CDR 1 of the light chain in SEQ ID NO:288, CDR 2 of the light chain in SEQ ID NO:289 and CDR 3 of the light chain in SEQ ID NO:290;

[0609] (a4) An antibody variable region that binds to the same epitope of an antibody variable region selected from any one of (a1) to (a3); and

[0610] (a5) An antibody variable fragment that competes with the binding of an antibody variable fragment selected from any of (a1) to (a3).

[0611] In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding moiety of the present invention further comprises post-translational modifications. Examples of post-translational modifications include pyroglutamylation at the N-terminus of the heavy chain variable region and / or deletion of a lysine residue at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications resulting from N-terminal pyroglutamylation and C-terminal lysine deletion have no effect on antibody activity (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0612] In another aspect, the DLL3 antigen-binding portion of the present invention can be used in novel chimeric antigen receptors (CARs) (DLL3 CARs) incorporated into a DLL3-binding domain. In some embodiments, the DLL3-binding domain (and DLL3 CAR) of the present invention will comprise an scFv construct, and in a preferred embodiment, will comprise 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, said scFv construct or fragment thereof comprising the heavy and light chain variable regions disclosed herein. In a preferred embodiment, the disclosed chimeric antigen receptor can be used to treat or prevent proliferative diseases and any recurrence or metastasis thereof.

[0613] In some embodiments, the DLL3 protein is expressed on tumor-initiating cells. The DLL3 CAR is expressed on cytotoxic lymphocytes (preferably autologous cytotoxic lymphocytes) via genetic modification (e.g., transduction) to generate DLL3-sensitive lymphocytes that can be used to target and kill DLL3-positive tumor cells. As will be discussed extensively herein, the CAR of the present invention generally comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain including a DLL3-binding domain that activates certain lymphocytes and generates an immune response of DLL3-positive tumor cells. Selected embodiments of the invention include immunologically active host cells displaying the disclosed CAR, and various polynucleotide sequences and vectors encoding the DLL3 CAR of the present invention. Other aspects include methods for enhancing the activity of T lymphocytes or natural killer (NK) cells in an individual and treating the individual by introducing host cells expressing the DLL3 CAR molecule into an individual with cancer. These aspects particularly include lung cancer (e.g., small cell lung cancer) and melanoma.

[0614] antigen

[0615] As used herein, the term "antigen" refers to a site on a polypeptide macromolecule where an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex (e.g., a continuous segment of amino acids or a conformational configuration consisting of discontinuous regions of amino acids). Useful antigenic determinants may be present, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins referred to herein as antigens (e.g., CD3, CD137, DLL3) may be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is human CD3, human CD137, or human DLL3. When referring to a particular protein herein, the term includes "full-length," unprocessed protein, and any form of protein produced by cellular processing. The term also includes naturally occurring protein variants, such as splice variants or allelic variants.

[0616] In some embodiments, the multispecific antigen-binding molecules described herein bind to conserved CD3, CD137, or DLL3 epitopes from different species. In some embodiments, the multispecific antigen-binding molecules of this application are trispecific antigen-binding molecules, meaning they can specifically bind to three different antigens—capable of binding to either CD3 or CD137 but not both simultaneously, and capable of specifically binding to DLL3.

[0617] In some embodiments, the multispecific antigen-binding molecule specifically binds all or part of a portion of the CD3 peptide. In a particular embodiment, CD3 is human CD3 or cynomolgus monkey CD3, most particularly human CD3. In a particular embodiment, the multispecific antigen-binding molecule exhibits cross-reactivity (i.e., specific binding) to human and cynomolgus monkey CD3. In some embodiments, the multispecific antigen-binding molecule is capable of specifically binding to the ε subunit of CD3, particularly the human CD3 ε subunit of CD3 shown in SEQ ID NO:7 (NP_000724.1) (RefSeq registration number shown in parentheses). In some embodiments, the multispecific antigen-binding molecule is capable of specifically binding to the CD3 ε chain expressed on the surface of eukaryotic cells. In some embodiments, the multispecific antigen-binding molecule binds to the CD3 ε chain expressed on the surface of T cells.

[0618] In some embodiments, CD137 is human CD137. In some embodiments, advantageous examples of the 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:

[0619] Antibodies that recognize regions containing the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO:21)

[0620] Antibodies that recognize the region containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO:35)

[0621] Antibodies that recognize the region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO:49), and

[0622] Antibodies that recognize the region of the human CD137 protein containing the LQDPCSNCPAGTFCDNNRNQIC sequence (SEQ ID NO:105).

[0623] Unless otherwise stated, the term "DLL3" as used herein refers to any naturally occurring DLL3 (δ-like 3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed DLL3 as well as any form of DLL3 produced by cellular processing. The term also includes naturally occurring DLL3 variants, such as splice variants or allelic variants. The amino acid sequence of exemplary human DLL3 is known as the NCBI reference sequence (RefSeq) NM_016941.3, the amino acid sequence of exemplary cynomolgus monkey DLL3 is known as the NCBI reference sequence XP_005589253.1, and the amino acid sequence of exemplary mouse DLL3 is known as the NCBI reference sequence NM_007866.2.

[0624] The human DLL3 protein contains a transmembrane (TM) domain and an intracellular domain at its C-terminus, and a DSL (Notch) domain at its N-terminus (see example). Figure 6Additionally, DLL3 has an EGF domain comprising six regions, EGF1 to EGF6, extending from the N-terminus to the C-terminus. In some embodiments, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention binds to epitopes within the extracellular domain (ECD), i.e., the domain extending from the N-terminus to immediately preceding the TM region, but not to the intracellular domain of the TM region or the C-terminus. The multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention can bind to epitopes within any of the aforementioned domains / regions within the ECD. In a preferred embodiment, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention binds to epitopes within the region extending from EGF6 to immediately preceding the TM region. More specifically, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention can bind to epitopes within the region defined in SEQ ID NO:89 of human DLL3. In some embodiments, the molecules / antibodies of the present invention bind to epitopes in the EGF1, EGF2, EGF3, EGF4, EGF5 or EGF6 region or from EGF6 to the region immediately preceding the TM region of human DLL3, or in the EGF1, EGF2, EGF3, EGF4, EGF5 or EGF6 region or from EGF6 to the region immediately preceding the TM region of human DLL3.

[0625] In human DLL3, the above-mentioned domain / region has the following amino acid residues (see, for example, http: / / www.uniprot.org / uniprot / Q9NYJ7 or WO2013 / 126746):

[0626] Extracellular domain (ECD): amino acid residues at positions 1 to 492;

[0627] DSL domain: amino acid residues located at positions 176 to 215;

[0628] EGF domain: amino acid residues located at positions 216 to 465;

[0629] EGF1 region: amino acid residues located at positions 216 to 249;

[0630] EGF2 region: amino acid residues located at positions 274 to 310;

[0631] EGF3 region: amino acid residues located at positions 312 to 351;

[0632] EGF4 region: amino acid residues located at positions 353 to 389;

[0633] EGF5 region: amino acid residues located at positions 391 to 427;

[0634] EGF6 region: amino acid residues located at positions 429 to 465;

[0635] From EGF6 to the region immediately preceding the TM region: amino acid residues at positions 429 to 492;

[0636] TM region: amino acid residues located at positions 493 to 513; and

[0637] C-terminal intracellular domain: amino acid residues at positions 516 to 618 (or 516 to 587 in some isotypes). These amino acid positions also refer to the amino acid positions in the amino acid sequence shown in SEQ ID NO:90.

[0638] Therefore, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention can bind to the aforementioned region / domain having amino acid residues at the aforementioned positions in human DLL3. That is, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention can bind to epitopes within the aforementioned region / domain having amino acid residues at the aforementioned positions in human DLL3.

[0639] The DLL3 protein used in this invention can be a DLL3 protein having the above-described sequence, or a modified protein having a sequence derived from the above-described sequence through modification with one or more amino acids. Examples of modified proteins having a sequence derived from the above-described sequence through modification with one or more amino acids may include polypeptides having 70% or more, preferably 80% or more, more preferably 90% or more, or even more preferably 95% or more identity with the above-described amino acid sequence. Alternatively, partial peptides of these DLL3 proteins may be used.

[0640] The DLL3 protein used in this invention is not limited by its source, but human or cynomolgus monkey DLL3 protein is preferred.

[0641] In some implementations, for the DLL3 protein, a DLL3 ECD fragment protein (or ECD variant) can be used. Depending on the truncation site, the fragment / variant may include DSL domains from the N-terminus to the C-terminus, to EGF6, EGF1 to EGF6, EGF2 to EGF6, EGF3 to EGF6, EGF4 to EGF6, EGF5 and EGF6, or EGF6. The fragment / variant may also include a region spanning from immediately following the EGF6 region to immediately preceding the TM region. A Flag tag can be attached to the C-terminus of the fragment / variant using techniques known in the art.

[0642] Antigen-binding domain

[0643] The term "antigen-binding domain" refers to a portion of an antibody that contains a region that specifically binds to and complements a portion or all of an antigen. An antigen-binding domain can be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Preferably, the antigen-binding domain comprises both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen-binding domains include, for example, "single-chain Fv (scFv)," "single-domain antibody or VHH," "single-chain antibody," "Fv," "single-chain Fv2 (scFv2)," "Fab," and "F(ab')2."

[0644] Variable region

[0645] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, libraries containing complementary VL or VH domains can be screened separately using VH or VL domains from antibodies binding to antigens, thereby isolating antibodies binding to specific antigens. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0646] HVR or CDR

[0647] As used herein, the term “hypervariant region” or “HVR” refers to each region of an antibody variable domain that is sequence-highly variable (“complementarity-determining region” or “CDR”) and / or forms a structurally defined loop (“hypervariant loop”) and / or contains antigen contact residues (“antigen contact”). The hypervariant region (HVR) is also referred to as the “complementarity-determining region” (CDR), and these terms are used interchangeably herein to refer to the variable region portion that forms the antigen-binding region. Typically, an antibody contains six HVRs: three of the VHs (H1, H2, H3) and three of the VLs (L1, L2, L3).

[0648] Exemplary HVRs in this article include:

[0649] (a) Hypervariable rings appearing 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));

[0650] (b) CDRs appearing 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));

[0651] (c) Antigen contacts appearing 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

[0652] (d) Combinations 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).

[0653] Unless otherwise stated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered in this document according to Kabat et al., see above.

[0654] 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.

[0655] It can bind with CD3 and CD137, but not with both CD3 and CD137 simultaneously.

[0656] Whether the antibody variable region of the present invention is “capable of binding to CD3 and CD137” can be determined by methods known in the art.

[0657] For example, this can be determined by electrochemiluminescence (ECL method) (BMC Research Notes 2011, 4:281).

[0658] Specifically, for example, a low-molecular-weight antibody or a monovalent antibody (lacking one of the two Fab regions typically carried by antibodies) consisting of a biotinylated antigen-binding molecule capable of binding to CD3 and CD137 (e.g., the Fab region) is mixed with CD3 or CD137 labeled with a sulfonyl tag (Ru complex), and the mixture is added to a streptavidin-fixed plate. In this operation, the biotinylated antigen-binding molecule to be tested binds to streptavidin on the plate. Emission from the sulfonyl tag is detected using a Sector Imager 600 or 2400 (MSD KK) or similar instrument, thereby confirming the binding of the aforementioned region of the antigen-binding molecule to CD3 or CD137.

[0659] Alternatively, this assay can be performed using ELISA or FACS (fluorescence activated cell sorting), ALPHAScreen (amplified luminescent proximity homogeneous assay screen), or the BIACORE method based on the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0660] Specifically, for example, this determination can be performed using a Biacore (GE Healthcare Japan Corp.) interaction analyzer based on the surface plasmon resonance (SPR) phenomenon. Biacore analyzers include any model, such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any Biacore sensor chip, such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chips, can be used as the sensor chip. Proteins (e.g., protein A, protein G, protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human L-chain antibody, anti-human Fc antibody, antigenic protein, or antigenic peptide) that capture the antigen-binding molecules of the present invention are immobilized on the sensor chip using coupling methods such as amine coupling, disulfide coupling, or aldehyde coupling. CD3 or CD137 is injected as an analyte onto the chip, and the interaction is measured to obtain a sensor map. In this operation, the concentration of CD3 or CD137 can be selected in the range of several μM to several pM, depending on the interaction strength of the samples being measured (e.g., KD).

[0661] Alternatively, CD3 or CD137 can be immobilized on the sensor chip instead of the antigen-binding molecule, allowing the antibody sample to be evaluated to interact with CD3 or CD137. The degree of dissociation constant (KD) calculated from the interaction sensor map, or the increase in the sensor map after the antigen-binding molecule sample has been treated relative to its initial level, can confirm whether the antibody variable region of the antigen-binding molecule of the present invention has binding activity against CD3 or CD137.

[0662] In some embodiments, the binding activity or affinity of the antibody variable region of the present invention to the target antigen (i.e., CD3 or CD137) is evaluated using, for example, a Biacore T200 instrument (GE Healthcare) or a Biacore 8K instrument (GE Healthcare) at 37°C (for CD137) or 25°C (for CD3). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of the CM4 sensor chip using an amine conjugation kit (e.g., GE Healthcare). The antigen-binding molecule or antibody variable region is captured onto the anti-Fc sensor surface, and then the antigen (CD3 or CD137) is injected into the flow cells. The target capture level of the antigen-binding molecule or antibody variable region can be 200 resonance units (RU). Recombinant human CD3 or CD137 can be injected at doses from 2000 to 125 nM, prepared by serial doubling and subsequent dissociation. All antigen-binding molecules or antibody variable regions and analytes were prepared in ACES at pH 7.4 containing 20 mM MACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The sensor surface was regenerated with 3 M MgCl2 for each cycle. Binding affinity was determined by processing the data and fitting them to a 1:1 binding model using, for example, Biacore Insight Evaluation software version 2.0 (GE Healthcare) or Biacore 8K evaluation software (GE Healthcare). KD values ​​were calculated to assess the specific binding activity or affinity of the antigen-binding domains of the present invention.

[0663] ALPHA screens utilize ALPHA technology, employing two types of beads (donor and acceptor), based on the principle that luminescence is detected only when the two beads are close together through biological interactions between molecules bound to the donor bead and those bound to the acceptor bead. A laser-excited photosensitizer within the donor bead converts surrounding oxygen into excited singlet oxygen. This singlet oxygen diffuses around the donor bead, reaching the acceptor bead near it, triggering a chemiluminescent reaction within the bead and ultimately emitting light. When the molecules bound to the donor and acceptor beads do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead; therefore, no chemiluminescent reaction occurs.

[0664] One of the interacting substances (ligands) is immobilized on a thin gold film of a sensor chip. Light is shone from the back of the sensor chip, causing total internal reflection at the interface between the gold film and the glass. This results in a point where the reflected intensity (SPR signal) decreases within a portion of the reflected light. The other interacting substance (analyte) is then injected onto the surface of the sensor chip. As the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, thereby altering the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation of the bound molecule returns the signal to its original position). The Biacore system plots this displacement, i.e., the mass change on the sensor chip surface, on the ordinate, displaying the time-dependent mass change as measurement data (sensor map). The amount of analyte bound to the ligands trapped on the sensor chip surface can be determined from the sensor map (the change in response on the sensor map before and after the analyte interaction). However, since the binding amount also depends on the amount of ligand, comparisons must be made using substantially the same amount of ligand. The kinetics, namely the association rate constant (ka) and the dissociation rate constant (kd), can be determined from the curves of the sensor plot, and the affinity (KD) can be determined from the ratio between these constants. In the BIACORE method, the inhibition assay is also preferred. An example of the inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0665] 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 the antigen-binding portion or antibody variable region of the present invention cannot bind to CD137 in a CD3-binding state, and conversely, the antigen-binding portion or antibody variable region cannot bind to CD3 in a CD137-binding state. In this document, the phrase "does not bind to CD3 and CD137 simultaneously" also includes cases where cells expressing CD3 and cells expressing CD137 are not cross-linked, or do not simultaneously bind to CD3 and CD137 expressed on different cells. This phrase further includes situations where CD3 and CD137 are not expressed on the cell membrane like soluble proteins, or where both are present on the same cell, the variable region can simultaneously bind to CD3 and CD137, but not simultaneously bind to CD3 and CD137 expressed on different cells. Such antibody variable regions are not particularly limited, as long as the antibody variable region has these functions. Examples of this could include variable regions obtained by altering a portion of the amino acids in the variable region of an IgG-type antibody to bind to a desired antigen. The amino acids to be altered are selected, for example, from the variable region of an antibody that binds to CD3 or CD137, and the alteration does not eliminate the amino acids that bind to the antigen.

[0666] In this article, the phrase "expressed on different cells" refers only to the expression of antigens on separate cells. Such cell combinations can be, for example, cells of the same type, such as T cells with another T cell, or cells of different types, such as T cells and NK cells.

[0667] Whether the antigen-binding molecule of the present invention "does not bind to CD3 and CD137 simultaneously" can be confirmed as follows: Confirm that the antigen-binding molecule has binding activity to both CD3 and CD137, then pre-bind CD3 or CD137 to the antigen-binding molecule containing a variable region with binding activity, and then determine whether it has binding activity to the other using the method described above. Alternatively, this can also be confirmed by determining whether the binding of the antigen-binding molecule to CD3 or CD137 immobilized on an ELISA plate or sensor chip is inhibited by another component added to the solution. In some embodiments, the binding of the antigen-binding molecule of the present invention to 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, further preferably 90% or more, or even more preferably 95% or more.

[0668] On one hand, when an antigen (e.g., CD3) is immobilized, inhibition of binding of the antigen-binding molecule to CD3 in the presence of another antigen (e.g., CD137) can be determined by methods known in the art (i.e., ELISA, BIACORE, etc.). On the other hand, when CD137 is immobilized, inhibition of binding of the antigen-binding molecule to CD137 in the presence of CD3 can also be determined. When either 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, more preferably 90% or more, and even more preferably 95% or more, then it is determined that the antigen-binding molecule of the present invention does not bind to both CD3 and CD137 simultaneously.

[0669] In some implementations, the concentration of the antigen injected as an analyte is at least 1, 2, 5, 10, 30, 50, or 100 times higher than the concentration of other antigens to be immobilized.

[0670] In a preferred manner, the concentration of the antigen injected as an analyte is 100 times higher than the concentration of other antigens to be immobilized, and binding is inhibited by at least 80%.

[0671] In one embodiment, the KD value of the CD3 (analyte) binding activity of the antigen-binding molecule is calculated as the ratio of the KD value of the CD137 (immobilized) binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)), and the concentration of CD3 (analyte) is such that the KD value ratio (KD(CD3) / KD(CD137)) is 10, 50, 100, or 200 times higher than the CD137 (immobilized) concentration, which can be used for the competitive measurement above. (For example, when the KD value ratio is 0.1, concentrations that are 1, 5, 10, or 20 times higher can be selected. Similarly, when the KD value ratio is 10, concentrations that are 100, 500, 1000, or 2000 times higher can be selected.)

[0672] On one hand, when an antigen (e.g., CD3) is immobilized, the attenuation of the binding signal between the antigen-binding molecule and CD3 in the presence of another antigen (e.g., CD137) can be determined using methods known in the art (i.e., ELISA, ECL, etc.). On the other hand, when CD137 is immobilized, the attenuation of the binding signal between the antigen-binding molecule and CD137 in the presence of CD3 can also be determined. When either of the above two aspects is performed, if the binding signal attenuation is at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more, then it is determined that the antigen-binding molecule of the present invention does not bind to both CD3 and CD137 simultaneously.

[0673] In some implementations, the concentration of the antigen injected as an analyte is at least 1, 2, 5, 10, 30, 50, or 100 times higher than the concentration of other antigens to be immobilized.

[0674] In a preferred manner, the concentration of the antigen injected as an analyte is 100 times higher than the concentration of other antigens to be immobilized, and binding is inhibited by at least 80%.

[0675] In one embodiment, the KD value of the CD3 (analyte) binding activity of the antigen-binding molecule is calculated as the ratio of the KD value of the CD137 (immobilized) binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)), where the concentration of CD3 (analyte) is 10, 50, 100, or 200 times higher than the CD137 (immobilized) concentration, which can be used for the above measurement. (For example, when the KD value ratio is 0.1, concentrations that are 1, 5, 10, or 20 times higher can be selected. Similarly, when the KD value ratio is 10, concentrations that are 100, 500, 1000, or 2000 times higher can be selected.)

[0676] Specifically, using, for example, the ECL method, a biotin-labeled antigen-binding molecule to be tested, a sulfonated CD3 (Ru complex), and unlabeled CD137 were prepared. When the antigen-binding molecule to be tested could bind to both CD3 and CD137, but not simultaneously, a mixture of the antigen-binding molecule to be tested and labeled CD3 was added to a plate immobilized with streptavidin, followed by photodevelopment. A luminescent signal from the sulfonated label was detected in the absence of unlabeled CD137. Conversely, the luminescent signal decreased in the presence of unlabeled CD137. This decrease in luminescent signal could be quantified to determine relative binding activity. This analysis could be performed similarly using labeled CD137 and unlabeled CD3.

[0677] In the case of an ALPHAScreen, the analyte-binding molecule interacts with CD3 in the absence of competing CD137, thereby generating a signal at 520 to 620 nm. Unlabeled CD137 competes with CD3 for interaction with the analyte-binding molecule. The decrease in fluorescence due to competition can be quantified, thereby determining the relative binding activity. Biotinylation of peptides using sulfonyl-NHS-biotin, etc., is known in the art. CD3 can be labeled with GST by a suitable method, which includes, for example, fusing a polynucleotide encoding CD3 with a polynucleotide encoding GST within a frame; expressing the resulting fusion gene through cells with a vector capable of expressing it, etc., and then purifying it using a glutathione column. Preferably, the obtained signal is analyzed using, for example, software suitable for a one-site competition model based on nonlinear regression analysis, such as GraphPad Software, Inc., San Diego. This analysis can be performed similarly using labeled CD137 and unlabeled CD3.

[0678] Alternatively, fluorescence resonance energy transfer (FRET) can be used. FRET is a phenomenon where excitation energy is transferred directly between two adjacent fluorescent molecules via electronic resonance. When FRET occurs, the excitation energy of the donor (an excited-state fluorescent molecule) is transferred to the acceptor (another fluorescent molecule located near the donor), causing the fluorescence emitted from the donor to disappear (more precisely, the fluorescence lifetime to shorten), and vice versa. By using this phenomenon, it is possible to analyze whether CD3 and CD137 are bound simultaneously. For example, when CD3 with a fluorescent donor and CD137 with a fluorescent acceptor bind simultaneously to the analyte antigen-binding molecule, the fluorescence of the donor disappears, while fluorescence is emitted from the acceptor. Thus, a change in fluorescence wavelength is observed. This confirms that such an antibody binds both CD3 and CD137 simultaneously. On the other hand, if mixing CD3, CD137, and the analyte antigen-binding molecule does not change the fluorescence wavelength of the fluorescent donor bound to CD3, then the analyte antigen-binding molecule can be considered an antigen-binding domain capable of binding both CD3 and CD137, but not simultaneously.

[0679] For example, a biotin-labeled antigen-binding molecule to be tested binds to streptavidin on donor beads, while CD3 labeled with glutathione S-transferase (GST) binds to recipient beads. In the absence of a competing secondary antigen, the antigen-binding molecule to be tested interacts with CD3 to produce a signal at 520 to 620 nm. An unlabeled secondary antigen competes with CD3 for interaction with the antigen-binding molecule to be tested. The decrease in fluorescence due to competition can be quantified, thereby determining the relative binding activity. Biotinylation of peptides using sulfonyl-NHS-biotin, etc., is known in the art. CD3 can be labeled with GST by a suitable method, which includes, for example, fusing a polynucleotide encoding CD3 with a polynucleotide encoding GST within a frame; expressing the resulting fusion gene through cells with a vector capable of expressing it, etc., and then purifying it using a glutathione column. Preferably, the obtained signal is analyzed using, for example, software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) suitable for a one-site competition model based on nonlinear regression analysis.

[0680] The label is not limited to the GST label and can be any label, such as, but not limited to, histidine tags, MBP, CBP, Flag tags, HA tags, V5 tags, or c-myc tags. The binding of the test antigen-binding molecule to the donor bead is not limited to binding using the biotin-streptavidin reaction. In particular, when the test antigen-binding molecule contains an Fc, possible methods involve causing the test antigen-binding molecule to bind to the donor bead via an Fc recognition protein, such as protein A or protein G.

[0681] Similarly, when CD3 and CD137 are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell, the variable region can bind CD3 and CD137 simultaneously, but not simultaneously bind CD3 and CD137 expressed on different cells. This situation can also be determined by methods known in the art.

[0682] Specifically, in an ECL-ELISA detecting simultaneous binding to CD3 and CD137, a positive result for the test antigen-binding molecule was confirmed by mixing with cells expressing CD3 and cells expressing CD137. Unless the antigen-binding molecule and these cells bind simultaneously, 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 a cell-based ECL-ELISA. Cells expressing CD3 are pre-fixed onto a plate. After the test antigen-binding molecule is bound to the plate, cells expressing CD137 are added to the plate. A sulfonyl-tagged antibody against this antigen is used to detect the different antigens expressed only on cells expressing CD137. A signal is observed when the antigen-binding molecule binds simultaneously to the two antigens expressed on two different cells. No signal is observed when the antigen-binding molecule binds to these antigens separately.

[0683] Alternatively, the assay can be performed using the ALPHAScreen method. The antigen-binding molecule to be tested is mixed with CD3-expressing cells bound to donor beads and CD137-expressing cells bound to recipient beads. A signal is observed when the antigen-binding molecule binds simultaneously to the two antigens expressed on the two cells, respectively. No signal is observed when the antigen-binding molecule binds to these antigens separately.

[0684] Alternatively, this assay can be performed using the Octet interaction analysis method. First, CD3-expressing cells, labeled with a peptide tag, bind to a biosensor that recognizes the peptide tag. CD137-expressing cells and the target antigen-binding molecule are placed in wells, and their interaction is analyzed. When the antigen-binding molecule simultaneously binds to two antigens expressed on two different cells, a large wavelength shift is observed due to the binding of the target antigen-binding molecule and the CD137-expressing cell to the biosensor. When the antigen-binding molecule binds to different antigens simultaneously, a smaller wavelength shift is observed due only to the binding of the target antigen-binding molecule to the biosensor.

[0685] Instead of these binding activity-based methods, bioactivity-based assays can be performed. For example, cells expressing CD3 and cells expressing CD137 are mixed with an antigen-binding molecule and cultured. When the antigen-binding molecule binds to both antigens simultaneously, the two antigens expressed on the two cells respectively are mutually activated by the antigen-binding molecule. Therefore, changes in activation signals can be detected, such as an increase in the corresponding downstream phosphorylation level of the antigen. Alternatively, activation may induce the production of cytokines. Therefore, the amount of cytokines produced can be measured to confirm whether both cells bind simultaneously. Alternatively, activation may induce cytotoxicity against cells expressing CD137. Alternatively, reporter gene expression may be induced by a promoter that is activated downstream of the CD137 or CD3 signaling pathway. Therefore, the amount of cytotoxicity or reporter protein produced can be measured to confirm whether both cells bind simultaneously.

[0686] At least one disulfide bond

[0687] In one aspect of the invention, the first antigen-binding portion and the second antigen-binding portion each contain at least one cysteine ​​residue (through mutation, substitution, or insertion), preferably in the CH1 region, and said at least one cysteine ​​residue is capable of forming at least one disulfide bond between the first antigen-binding portion and the second antigen-binding portion. In some embodiments, the cysteine ​​residue is present in the CH1 region of the antibody heavy chain constant region, for example, it is present in the CH1 region at a position selected from the group consisting of positions 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 numbers. In one embodiment, the first antigen-binding portion and the second antigen-binding portion each contain a cysteine ​​residue (by mutation, substitution, or insertion) at position 191 according to EU number in the CH1 region, which is capable of forming a disulfide bond between the CH1 regions of the first antigen-binding portion and the CH1 regions of the second antigen-binding portion.

[0688] In embodiments described above, the "at least one bond" connecting the first antigen-binding portion and the second antigen-binding portion as described above can maintain the two antigen-binding portions (i.e., the first antigen-binding portion and the second antigen-binding portion as described above) in a spatially proximal position. By means of the disulfide bond connecting the first and second antigen-binding portions, the antigen-binding molecule of the present invention can maintain the two antigen-binding portions in a position closer than that of a control antigen-binding molecule, which differs from the antigen-binding molecule of the present invention only in that the control antigen-binding molecule does not have any additional bonds introduced between the two antigen-binding portions. In some embodiments, the terms "spatially proximal position" or "closer position" include the meaning that the first antigen-binding domain and the second antigen-binding domain as described above are maintained at a shortened distance and / or with reduced flexibility.

[0689] As a result, the two antigen-binding portions of the antigen-binding molecule of the present invention (i.e., the first antigen-binding portion and the second antigen-binding portion as described above) bind to antigens expressed on the same single cell. In other words, the respective two antigen-binding portions of the antigen-binding molecule of the present invention (i.e., the first antigen-binding portion and the second antigen-binding portion as described above) do not bind to antigens expressed on different cells, thereby causing cross-linking of different cells. In this application, this antigen-binding mode of the antigen-binding molecule of the present invention can be referred to as "cis-binding," while the antigen-binding mode of an antigen-binding molecule in which the two antigen-binding portions of the antigen-binding molecule each bind to antigens expressed on different cells, thereby causing cross-linking of different cells, can be referred to as "trans-binding." In some embodiments, the antigen-binding molecule of the present invention primarily binds to antigens expressed on the same single cell in a "cis-binding" manner.

[0690] In embodiments described above, by means of a disulfide bond linking the first antigen-binding portion and the second antigen-binding portion as described above, the antigen-binding molecule of the present invention can reduce and / or prevent undesirable cross-linking and activation of immune cells (e.g., T cells, NK cells, DC cells, etc.). That is, in some embodiments of the present invention, the first antigen-binding portion of the antigen-binding molecule of the present invention binds to any signaling molecule (e.g., a first antigen) expressed on an immune cell, such as a T cell, 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 an immune cell, such as a T cell. Therefore, the first and second antigen-binding domains of the antigen-binding molecule of the present invention can bind to either the first or second signaling molecule expressed on the same single immune cell, such as a T cell (i.e., cis-binding) or on different immune cells (e.g., T cells) (i.e., trans-binding). When the first antigen-binding domain and the second antigen-binding domain bind in a trans-binding manner to signaling molecules expressed on different immune cells, such as T cells, those different immune cells, such as T cells, are cross-linked, and in some cases, such cross-linking of immune cells, such as T cells, may lead to undesirable activation of immune cells, such as T cells.

[0691] On the other hand, in another embodiment of the antigen-binding molecule of the present invention, namely, an antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion and the second antigen-binding portion are linked to each other by at least one disulfide bond in the CH1 region (position 191 according to EU number), and both the first antigen-binding portion and the second antigen-binding portion can bind to signaling molecules expressed on the same single immune cell such as T cells in a "cis-binding" manner, thereby reducing cross-linking of different immune cells such as T cells through the antigen-binding molecule to avoid unwanted activation of immune cells.

[0692] In this application, the aforementioned feature, where at least one disulfide bond connecting the first antigen-binding portion and the second antigen-binding portion in the CH1 region (e.g., position 191 according to EU numbering), can be described using the abbreviation "LINC". Using this abbreviation, in some embodiments, the aforementioned antigen-binding molecule of the present invention having said at least one disulfide bond can be represented, for example, as "LINC format", "double / LINC", or "DLL3-double / LINC", etc. Similarly, antigen-binding molecules whose first antigen-binding portion and second antigen-binding portion are not connected to each other by at least one disulfide bond in the CH1 region (e.g., position 191 according to EU numbering) can be described using the abbreviation "UnLINC".

[0693] Fab molecules

[0694] "Fab molecule" refers to a protein composed of the VH and CH1 domains of the heavy chain ("Fab heavy chain") of an immunoglobulin and the VL and CL domains of the light chain ("Fab light chain").

[0695] Fusion

[0696] "Integrated" means that components (such as Fab molecules and Fc domain subunits) are linked directly by peptide bonds or through one or more peptide linkers.

[0697] " cross Fab

[0698] A “cross-Fab” molecule (also called a “cross-fab”) is a Fab molecule in which the variable or constant regions of the Fab heavy and light chains are exchanged. That is, a cross-Fab molecule contains a peptide chain consisting of a variable region of the light chain and a constant region of the heavy chain, and a peptide chain consisting of a variable region of the heavy chain and a constant region of the light chain. For clarity, in a cross-Fab molecule in which the variable regions of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the constant region of the heavy chain is referred to herein as the “heavy chain” of the cross-Fab molecule. Conversely, in a cross-Fab molecule in which the constant regions of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the variable region of the heavy chain is referred to herein as the “heavy chain” of the cross-Fab molecule.

[0699] "Regular" Fab

[0700] In contrast, a "conventional" Fab molecule refers to the native form of the Fab molecule, which consists of a heavy chain composed of a variable region and a constant region (VH-CH1) and a light chain composed of a variable region and a constant region (VL-CL) of the light chain. The term "immunoglobulin molecule" refers to a protein with a naturally occurring antibody structure. For example, IgG immunoglobulins 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 a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant domains. Similarly, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain, also called a light chain constant domain. The heavy chains of immunoglobulins can be classified into one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Based on the amino acid sequence of their constant domains, the light chains of immunoglobulins can be classified into one of two types, called κ and λ. Immunoglobulins are essentially composed of two Fab molecules linked by an immunoglobulin hinge region and an Fc domain.

[0701] Affinity

[0702] “Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule or antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of molecule X for its partner Y is typically expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Therefore, equivalent affinities can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by methods known in the art, including those described herein. One particular method for measuring affinity is surface plasmon resonance (SPR).

[0703] Methods for determining affinity

[0704] In some embodiments, the dissociation constant (KD) of the antigen-binding molecule or antibody provided herein to its antigen is 1 μM or less, 120 nM or less, 100 nM or less, 80 nM or less, 70 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 nM). -8 M or smaller, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 In some embodiments, the KD value of the antibody / antigen binding molecule against CD3, CD137, or DLL3 is in the range of 1-40, 1-50, 1-70, 1-80, 30-50, 30-70, 30-80, 40-70, 40-80, or 60-80 nM.

[0705] In one embodiment, KD is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab version of the target antibody and its antigen. For example, the solution binding affinity of the Fab to the antigen is determined by measuring the minimum concentration of the unlabeled antigen in the presence of a series of titrations. 125 I) The labeled antigen was balanced with Fab, and then the bound antigen was captured and measured using a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23°C) for 2 to 5 hours. In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125I] The antigen is mixed with serial dilutions of the target Fab (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab is then incubated overnight; however, incubation may be prolonged (e.g., approximately 65 hours) to ensure equilibration. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. Once the plate is dry, 150 μL / well of scintillation agent (MICROSCINT-20) is added. TM Packard), and in TOPCOUNT TM Count the plates on a gamma counter (Packard) for 10 minutes. Select a concentration for each Fab that produces less than or equal to 20% of the maximum binding for the competitive binding assay.

[0706] According to another embodiment, Kd is measured using the BIACORE (registered trademark) surface plasmon resonance assay. For example, the assay is performed at 25°C using a BIACORE (registered trademark)-2000 or BIACORE (registered trademark)-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip at approximately 10 response units (RU). In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted with 10 mM sodium acetate at pH 4.8 to 5 μg / mL (~0.2 μM) and then injected at a flow rate of 5 μL / min to achieve approximately 10 response units (RU) of conjugate protein. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, Fab was serially diluted twice (0.78 nM to 500 nM) in a solution containing 0.05% polysorbate 20 (TWEEN-20). TM Surfactant (PBST) was injected into PBS at a flow rate of approximately 25 μL / min at 25 °C. The association rate (kJ / kb) was calculated using a simple one-to-one Langmuir binding model (BIACORE evaluation software version 3.2) by simultaneously fitting association and dissociation sensor maps. on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated as the ratio k. off / k onSee, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the association rate determined by the above surface plasmon resonance exceeds 10... 6 M -1 s -1 This can be achieved by using 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C, in the presence of increased antigen concentration, as measured in a spectrometer, such as an Aviv Instruments 8000 series SLM-AMINCO spectrophotometer equipped with a stop-flow spectrophotometer or a stirring cuvette. TM In a ThermoSpectronic spectrophotometer, the association rate is determined by fluorescence quenching techniques that measure increases or decreases in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass).

[0707] Based on the above method for determining the affinity of antigen-binding molecules or antibodies, those skilled in the art can determine the affinity of other antigen-binding molecules or antibodies for various antigens.

[0708] Antibody

[0709] The term “antibody” is used in the broadest sense herein and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0710] antibody fragments

[0711] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody and binds to an antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, biantibodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For reviews of some antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For reviews of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046. Biantibodies are antibody fragments having two antigen-binding sites and can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9,129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90,6444-6448 (1993). Triantibodies and tetraantibodies are also described in Hudson et al., Nat Med 9,129-134 (2003). Single-domain antibodies are antibody fragments containing all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1). As described herein, antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production from recombinant host cells (e.g., E. coli or bacteriophages).

[0712] Antibody categories

[0713] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. Antibodies are mainly classified into five classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0714] Unless otherwise stated, amino acid residues in the light chain constant region are numbered according to Kabat et al., and amino acid residues in the heavy chain constant region are numbered according to the EU numbering system, also known as the EU index number, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0715] frame

[0716] "Frame" or "FR" refers to the variable domain residues outside the hypervariable region (HVR). The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences in VH (or VL) generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0717] Human shared framework

[0718] The “human common framework” is a framework representing the most common amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Typically, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Typically, the sequence subgroup is the subgroup described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, this subgroup is subgroup κI of Kabat et al. described above. In one embodiment, for VH, the subgroup is subgroup III of Kabat et al. described above.

[0719] chimeric antibodies

[0720] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy chain and / or light chain variable region originates from a specific source or species, while the remainder of the heavy chain and / or light chain variable region originates from a different source or species.

[0721] Humanized antibodies

[0722] A “humanized” antibody is a chimeric antibody comprising amino acid residues from a nonhuman HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will include substantially all, at least one, and typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those HVRs of the nonhuman antibody, and all or substantially all of the FRs correspond to those FRs of the human antibody. Optionally, the humanized antibody includes at least a portion of the antibody constant region derived from the human antibody. For example, a “humanized form” of a nonhuman antibody refers to an antibody that has been humanized. A “variable region of a humanized antibody” refers to the variable region of the humanized antibody.

[0723] Human antibodies

[0724] "Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody generated by a human or human cell, or derived from a non-human source using a human antibody library or other human antibody encoding sequences. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. "Variable region of human antibody" refers to the variable region of a human antibody.

[0725] Polynucleotides (nucleic acids)

[0726] As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases or through a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may contain post-synthetic modifications, such as tag conjugation. Other types of modifications include, for example, "caps," substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those with uncharged bonds (e.g., methylphosphonates, triphosphates, aminophosphates, carbamates, etc.) and charged bonds (e.g., thiophosphates, dithiophosphates, etc.), those containing overhanging moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified bonds (e.g., α-anomeric nucleic acids, etc.), and unmodified polynucleotides. Furthermore, any hydroxyl groups typically present in sugars can be replaced, for example, with phosphonate groups or phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds for additional nucleotides, or can be conjugated to solid or semi-solid supports. The 5' and 3' terminal OH groups may be phosphorylated or partially replaced by an amine or an organic capping group of 1 to 20 carbon atoms. Other hydroxyl groups may also be derived as standard protecting groups. The polynucleotide may also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lythose, pyranose, furanose, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methylnucleosides. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is replaced by P(O)S (“thioester”), P(S)S (“dithioester”), (O)NR2 (“amic acid ester”), P(O)R, P(O)OR', CO, or CH2 (“formacetal”), wherein each R or R' is independently H or a substituted or unsubstituted alkyl group (1-20C), optionally comprising an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all bonds in a polynucleotide need to be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0727] Isolated (nucleic acid)

[0728] "Isolated" nucleic acid molecules are nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acid molecules also include nucleic acid molecules found in cells that typically contain such molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0729] carrier

[0730] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell to which the vector has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors." Vectors can be introduced into host cells using viruses or electroporation. However, vector introduction is not limited to in vitro methods. For example, vectors can also be introduced into subjects directly using in vivo methods.

[0731] In another aspect of the invention, a vector comprising a nucleic acid molecule encoding an antigen-binding moiety of the present disclosure capable of binding CD3 and CD137 but not simultaneously, an antigen-binding moiety capable of binding DLL3, an antigen-binding molecule, or an antibody may be introduced into a subject to directly express the antigen-binding moiety, antigen-binding molecule, or antibody of the present disclosure in the subject. Examples of vectors that can be used are adenoviruses, but are not limited to adenoviruses. Nucleic acid molecules encoding the antigen-binding moiety, antigen-binding molecule, or antibody of the present disclosure may also be directly administered to the subject, or transferred to the subject via electroporation, or cells comprising nucleic acid molecules encoding the antigen-binding moiety, antigen-binding molecule, or antibody of the present disclosure to be expressed and secreted into the subject may be administered to the subject to continuously express and secrete the antigen-binding moiety, antigen-binding molecule, or antibody of the present disclosure in the subject.

[0732] host cells

[0733] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny may not be identical to parental cells in terms of nucleic acid content, but may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0734] Specificity

[0735] "Specificity" refers to a molecule that specifically binds to one or more binding partners and does not show any significant binding to molecules other than its partners. Additionally, "specificity" is used when the antigen-binding site is specific to a particular epitope among multiple epitopes contained in an antigen. It is also described as "the antigen-binding molecule has / shows specificity to / against an antigen" if the antigen-binding molecule binds specifically to an antigen. When the epitope bound by the antigen-binding site is contained in multiple different antigens, the antigen-binding molecule containing that antigen-binding site can bind to various antigens that have that epitope.

[0736] antibody fragments

[0737] An "antibody fragment" is a molecule other than a complete antibody that contains a portion of the complete antibody and binds to an antigen that the complete antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0738] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing the Fc region as defined herein.

[0739] Variable fragment (Fv)

[0740] In this paper, the term "variable fragment (Fv)" refers to the smallest unit of an antigen-binding site derived from an antibody, consisting of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be prepared from E. coli periplasmic fractions by inserting an antibody gene downstream of a bacterial signal sequence and inducing the expression of that gene in E. coli (Science (1988) 240 (4855), 1038-1041). In Fv prepared from periplasmic fractions, VH associates with VL in a manner that binds to the antigen.

[0741] scFv, single-chain antibodies and sc(Fv)2

[0742] In this document, the terms “scFv,” “single-chain antibody,” and “sc(Fv)2” all refer to antibody fragments containing variable, rather than constant, regions of a single polypeptide chain derived from both the heavy and light chains. Generally, single-chain antibodies also contain a polypeptide linker between the VH and VL domains, which allows for the formation of desired structures that are thought to permit antigen binding. Pluckthun discusses single-chain antibodies in detail in “The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994).” See also International Patent Publication WO1988 / 001649; U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may be bispecific and / or humanized.

[0743] scFv is a single-chain low molecular weight antibody in which the VH and VL that form the Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be kept in very close proximity by the peptide linker.

[0744] sc(Fv)2 is a single-chain antibody in which four variable regions of two VLs and two VHs are linked by a linker, such as a peptide linker, to form a single chain (J Immunol. Methods (1999) 231(1-2), 177-189). The two VHs and two VLs can be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes, for example, bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen, as disclosed in Journal of Immunology (1994) 152(11), 5368-5374. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, sc(Fv)2 can be prepared by linking scFv via a linker, such as a peptide linker.

[0745] In this paper, sc(Fv)2 comprises two VH units and two VL units, which are arranged in the order of VH, VL, VH, and VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) starting from the N-terminus of the single-chain polypeptide. The order of the two VH units and the two VL units is not limited to the above form, and they can be arranged in any order. An example form is listed below.

[0746] [VL]-Connector-[VH]-Connector-[VH]-Connector-[VL],

[0747] [VH]-Connector-[VL]-Connector-[VL]-Connector-[VH],

[0748] [VH]-Connector-[VH]-Connector-[VL]-Connector-[VL],

[0749] [VL]-Connector-[VL]-Connector-[VH]-Connector-[VH],

[0750] [VL]-Connector-[VH]-Connector-[VL]-Connector-[VH].

[0751] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352. Based on these descriptions, those skilled in the art can appropriately prepare the desired sc(Fv)2 to produce the polypeptide complexes disclosed herein.

[0752] Furthermore, the antigen-binding molecules or antibodies disclosed herein can be conjugated with carrier polymers such as PEG or organic compounds such as anticancer agents. Alternatively, it is preferable to insert a glycan addition sequence into the antigen-binding molecule or antibody, such that the glycan produces the desired effect.

[0753] Adapters for linking antibody variable regions include any peptide adapters that can be introduced through genetic engineering, synthetic adapters, and adapters disclosed, for example, in Protein Engineering, 9(3), 299-305, 1996. However, peptide adapters are preferred in this disclosure. The length of the peptide adapter is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The length is preferably 5 amino acids or more (without particular limitation, the upper limit is generally 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide adapters, their lengths may be the same or different.

[0754] For example, such peptide linkers include:

[0755] Ser,

[0756] Gly-Ser

[0757] Gly-Gly-Ser

[0758] Ser-Gly-Gly,

[0759] Gly-Gly-Gly-Ser (SEQ ID NO:91),

[0760] Ser-Gly-Gly-Gly (SEQ ID NO:92),

[0761] Gly-Gly-Gly-Gly-Ser (SEQ ID NO:93),

[0762] Ser-Gly-Gly-Gly-Gly (SEQ ID NO:94),

[0763] Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:95),

[0764] Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:96),

[0765] Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:97),

[0766] Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 98),

[0767] (Gly-Gly-Gly-Gly-Ser(SEQ ID NO:93))n, and

[0768] (Ser-Gly-Gly-Gly-Gly(SEQ ID NO:94))n,

[0769] Where n is an integer of 1 or greater. Those skilled in the art can select the length or sequence of the peptide linker accordingly, depending on the purpose.

[0770] Synthetic linkers (chemical crosslinking agents) are commonly used for crosslinking peptides; examples include:

[0771] N-hydroxysuccinimide (NHS),

[0772] Disuccinimidyl succinate (DSS),

[0773] bis(sulfosuccinimide) octanoate (BS3),

[0774] Dithiobis(succinimide propionate) (DSP)

[0775] Dithiobis(sulfosuccinimide propionate) (DTSSP)

[0776] Ethylene glycol bis(succinimide succinate) (EGS)

[0777] Ethylene glycol bis(sulfosuccinimide succinate) (sulfon-EGS),

[0778] Disuccinimidyl tartrate (DST), disulfonyl tartrate (sulfon-DST)

[0779] bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone (BSOCOES), and

[0780] Bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl] sulfone (sulfon-BSOCOES). These crosslinking agents are commercially available.

[0781] Typically, three adapters are needed to link the four antibody variable regions together. The adapters used can be of the same type or different types.

[0782] Fab, F(ab') 2 and Fab'

[0783] A "Fab" molecule consists of a single light chain and a CH1 domain and variable region derived from a single heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0784] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, and specifically refers to an antibody fragment produced by digesting an immunoglobulin (monoclonal antibody) near a disulfide bond located between the respective hinge regions of the two H chains. For example, papain cleaves IgG upstream of a disulfide bond between the respective hinge regions of the two H chains to produce two homologous antibody fragments. The L chain, containing the VL (variable region of the L chain) and CL (constant region of the L chain), is linked to an H chain fragment containing the VH (variable region of the H chain) and CHγ1 (γ1 region of the constant region of the H chain) via a disulfide bond in its C-terminal region. Each of these two homologous antibody fragments is called Fab'.

[0785] “F(ab′)2” consists of two light chains and two heavy chains, each containing a constant region of the CH1 domain and a portion of the CH2 domain, thereby forming a disulfide bond between the two heavy chains. The F(ab′)2 disclosed herein can preferably be prepared as follows: A complete monoclonal antibody or a monoclonal antibody containing the desired antigen-binding site is partially digested with a protease such as pepsin; the Fc fragment is removed by adsorption onto a protein A column. The protease is not particularly limited, as long as it can selectively cleave the entire antibody to produce F(ab′)2 under suitable enzymatic reaction conditions, such as pH. Such proteases include, for example, pepsin and ficin.

[0786] Single domain antibodies

[0787] In this specification, the term "single-domain antibody" is not limited by its structure, as long as the domain itself can exert antigen-binding activity. It is known that general antibodies, such as IgG antibodies, exhibit antigen-binding activity when the variable region is formed by the pairing of VH and VL domains. In contrast, the domain structure of a single-domain antibody can exert antigen-binding activity on its own without pairing with another domain. Typically, single-domain antibodies have a relatively low molecular weight and exist in monomeric form.

[0788] Examples of single-domain antibodies include, but are not limited to, antigen-binding molecules that congenitally lack a light chain, such as VHH from camels and VNAR from sharks, as well as antibody fragments comprising all or part of the antibody VH domain or the antibody VL domain. Examples of single-domain antibodies comprising all or part of the antibody VH or VL domain include, but are not limited to, artificially prepared single-domain antibodies derived from human antibody VH or human antibody VL, as described in U.S. Patent No. 6,248,516B1, etc. In some embodiments of the invention, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0789] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies or through immunization of animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals carrying genes capable of producing single-domain antibodies. Camelids include camels, lamas, alpacas, dromedary camels, and guanacos. Examples of transgenic animals carrying genes capable of producing single-domain antibodies include, but are not limited to, the transgenic animals described in International Publication No. WO2015 / 143414 and US Patent Publication No. US2011 / 0123527A1. The framework sequence of a single-domain antibody obtained from an animal can be converted into a human germline sequence or a similar sequence to obtain a humanized single-domain antibody. Humanized single-domain antibodies (e.g., humanized VHH) are also an embodiment of the single-domain antibody of the present invention.

[0790] Alternatively, single-domain antibodies can be obtained from peptide libraries containing single-domain antibodies using methods such as ELISA and panning. Examples of peptide libraries containing single-domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911(65-78) and Biochimica et Biophysica Acta-Proteins and Proteomics 2006 1764:8(1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2(528-536)), and synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1(35-43), Journal of Biological Chemistry 2016 291:24(12641-12657) and AIDS 2016). 30:11 (1691-1701)).

[0791] Fc area

[0792] The term "Fc region" or "Fc domain" refers to a region comprising a segment of the hinge or a portion thereof in an antibody molecule and CH2 and CH3 domains. The Fc region of IgG refers to, but is not limited to, the region extending from, for example, cysteine ​​226 (EU number (also referred to herein as the EU index)) to the C-terminus or from proline 230 (EU number) to the C-terminus. The Fc region can preferably be obtained, for example, by partially digesting IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies with a proteolytic enzyme such as pepsin, followed by reelution of the fraction adsorbed onto a protein A column or a protein G column. There are no particular limitations on the proteolytic enzyme, as long as it is capable of digesting full-length antibodies to restrictively form Fab or F(ab')2 under appropriately set enzymatic reaction conditions (e.g., pH). Examples include pepsin and papain.

[0793] The Fc region derived from, for example, naturally occurring IgG can be used as the “Fc region” of this invention. In this document, naturally occurring IgG refers to a polypeptide containing the same amino acid sequence as naturally occurring IgG and belonging to a class of antibodies essentially encoded by the immunoglobulin γ gene. Naturally occurring human IgG refers to, for example, naturally occurring human IgG1, naturally occurring human IgG2, naturally occurring human IgG3, or naturally occurring human IgG4. Naturally occurring IgG also includes variants spontaneously derived therefrom. In NIH Publication No. 91-3242, Sequences of proteins of immunological interest, multiple allomorphic sequences based on genetic polymorphism are described as constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, any of which can be used in this invention. Specifically, the sequence of human IgG1 may have a DEL or EEM as the amino acid sequence at EU number positions 356 to 358.

[0794] In some embodiments, the Fc domain of a multispecific antigen-binding molecule consists of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each subunit containing CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the multispecific antigen-binding molecule described herein contains no more than one Fc domain.

[0795] In one embodiment described herein, the Fc domain of the multispecific antigen-binding molecule is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In a further particular embodiment, the Fc domain is the human IgG1 Fc region.

[0796] In some embodiments, the Fc domain of the multispecific antigen-binding molecule is composed of first and second Fc subunits capable of stable association, and the Fc domain has a reduced binding affinity for the human Fcγ receptor compared to the native human IgG1 Fc domain.

[0797] In some embodiments, the Fc domain of the multispecific antigen-binding molecule described herein includes modifications that promote association between the first and second subunits of the Fc domain. In a specific embodiment, the modification is a so-called "mortar and pestle" modification, comprising a "mortar" modification in one of the two subunits of the Fc domain and a "mortar" modification in the other of the two subunits of the Fc domain, which will be described in more detail below.

[0798] In a specific implementation scheme, compared to the natural human IgG1 Fc domain, in the Fc domain composed of stable associative first and second Fc subunits and exhibiting reduced affinity for human Fcγ receptors, the first Fc subunit is selected from the group consisting of:

[0799] (a1) An Fc region polypeptide containing mutants L234A and L235A;

[0800] (a2) Fc region polypeptide containing mutants L234A, L235A, and N297A;

[0801] (a3) Fc region polypeptides containing mutants L234A, L235A, N297A, S354C, and T366W; and

[0802] The second Fc region polypeptide is selected from the group consisting of:

[0803] (a4) An Fc region polypeptide containing mutants L234A and L235A;

[0804] (a5) A polypeptide containing the Fc region of mutants L234A, L235A, and N297A; and

[0805] (a6) Fc region polypeptides containing mutations L234A, L235A, N297A, Y349C, T366S, L368A, and Y407V (amino acid positions are indicated by EU index numbers).

[0806] In some embodiments, the Fc domain of the multispecific antigen-binding molecule described herein exhibits enhanced FcRn binding activity under acidic pH conditions (e.g., pH 5.8) compared to the Fc region of natural IgG. Such Fc domains contain, for example, Ala at position 434 according to EU numbering; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440. In some embodiments, the Fc domain contains Ala at position 434 according to EU numbering; Arg or Lys at position 438; and Glu or Asp at position 440. In some embodiments, the Fc domain also contains Ile or Leu at position 428 according to EU numbering; and / or Ile, Leu, Val, Thr, or Phe at position 436. In some embodiments, the Fc domain contains a combination of amino acid substitutions selected from the group consisting of:

[0807] According to EU number (a) N434A / Q438R / S440E;

[0808] (b)N434A / Q438R / S440D;

[0809] (c)N434A / Q438K / S440E;

[0810] (d)N434A / Q438K / S440D;

[0811] (e)N434A / Y436T / Q438R / S440E;

[0812] (f)N434A / Y436T / Q438R / S440D;

[0813] (g)N434A / Y436T / Q438K / S440E;

[0814] (h)N434A / Y436T / Q438K / S440D;

[0815] (i)N434A / Y436V / Q438R / S440E;

[0816] (j)N434A / Y436V / Q438R / S440D;

[0817] (k)N434A / Y436V / Q438K / S440E;

[0818] (l)N434A / Y436V / Q438K / S440D;

[0819] (m)N434A / R435H / F436T / Q438R / S440E;

[0820] (n)N434A / R435H / F436T / Q438R / S440D;

[0821] (o)N434A / R435H / F436T / Q438K / S440E;

[0822] (p)N434A / R435H / F436T / Q438K / S440D;

[0823] (q)N434A / R435H / F436V / Q438R / S440E;

[0824] (r)N434A / R435H / F436V / Q438R / S440D;

[0825] (s)N434A / R435H / F436V / Q438K / S440E;

[0826] (t)N434A / R435H / F436V / Q438K / S440D;

[0827] (u) M428L / N434A / Q438R / S440E;

[0828] (v) M428L / N434A / Q438R / S440D;

[0829] (w) M428L / N434A / Q438K / S440E;

[0830] (x) M428L / N434A / Q438K / S440D;

[0831] (y) M428L / N434A / Y436T / Q438R / S440E;

[0832] (z) M428L / N434A / Y436T / Q438R / S440D;

[0833] (aa) M428L / N434A / Y436T / Q438K / S440E;

[0834] (ab) M428L / N434A / Y436T / Q438K / S440D;

[0835] (ac) M428L / N434A / Y436V / Q438R / S440E;

[0836] (ad) M428L / N434A / Y436V / Q438R / S440D;

[0837] (ae) M428L / N434A / Y436V / Q438K / S440E;

[0838] (af) M428L / N434A / Y436V / Q438K / S440D;

[0839] (ag) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and

[0840] (ah) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E.

[0841] In some embodiments, the Fc domain of the multispecific antigen-binding molecule comprises a combination of amino acid substitutions for M428L / N434A / Q438R / S440E. In some embodiments, the Fc domain is an IgG Fc domain, preferably a human IgG Fc domain, more preferably a human IgG1 Fc domain. In some embodiments, the Fc domain of the multispecific antigen-binding molecule comprises any of the following: (a) a first Fc subunit comprising the amino acid sequence shown in SEQ ID NO:100 and a second Fc subunit comprising the amino acid sequence shown in SEQ ID NO:111; and (b) a first Fc subunit comprising the amino acid sequence shown in SEQ ID NO:99 and a second Fc subunit comprising the amino acid sequence shown in SEQ ID NO:109.

[0842] Fc region with reduced Fcγ receptor binding activity

[0843] In this document, "reduced Fcγ receptor binding activity" means, for example, that, based on the above-described analytical method, the competitive activity of the tested antigen-binding molecule or antibody is less than 50%, preferably less than 45%, less than 40%, less than 35%, less than 30%, less than 20%, less than 15%, particularly preferably less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0844] Antigen-binding molecules or antibodies containing the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be suitably used as control antigen-binding molecules or antibodies. The Fc domain structures are shown in SEQ ID NO: 85 (A added to the N-terminus of RefSeq accession number AAC82527.1), 86 (A added to the N-terminus of RefSeq accession number AAB59393.1), 87 (A added to the N-terminus of RefSeq accession number CAA27268.1), and 88 (A added to the N-terminus of RefSeq accession number AAB59394.1). Furthermore, when antigen-binding molecules or antibodies containing Fc domain mutants of a specific isotype antibody are used as the test substance, the effect of the mutation on Fcγ receptor binding activity is assessed using antigen-binding molecules or antibodies containing the same isotype of Fc domain as a control. As described above, antigen-binding molecules or antibodies containing Fc domain mutants whose Fcγ receptor binding activity has been determined to be reduced are suitably prepared.

[0845] Known mutants of this kind include, for example, the mutant with the deletion of amino acids 231A-238S (EU number) (WO2009 / 011941), and mutants C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11); C226S and C229S (Hum. Antibod. Hybridomas (1990) 1 (1), 47-54); C226S, C229S, E233P, L234V and L235A (Blood (2007) 109, 1185-1192).

[0846] Specifically, preferred antigen-binding molecules or antibodies include those comprising an Fc domain having at least one amino acid mutation (e.g., substitution) selected from the following amino acid positions: positions 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU number) in the amino acids forming the Fc domain of a particular isotype antibody. There is no particular limitation on the isotype of the antibody from which the Fc domain originates; suitable Fc domains derived from monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can be used. Fc domains derived from IgG1 antibodies are preferred.

[0847] Preferred antigen-binding molecules or antibodies include, for example, those amino acids forming the Fc domain of an IgG1 antibody that include an Fc domain having any of the substitutions shown below, the positions of which are specified according to EU numbers (each number indicates the position of an amino acid residue in the EU number; and the single-letter amino acid symbol preceding the number represents the unsubstituted amino acid residue, while the single-letter amino acid symbol following the number represents the substituted amino acid residue):

[0848] (a)L234F,L235E,P331S;

[0849] (b)C226S,C229S,P238S;

[0850] (c)C226S,C229S; or

[0851] (d)C226S, C229S, E233P, L234V, L235A;

[0852] And those with an Fc domain having an amino acid sequence deletion at positions 231 to 238.

[0853] In addition, preferred antigen-binding molecules or antibodies also include those Fc domains containing amino acids of the Fc domain that form IgG2 antibodies and have any of the following substitutions, the positions of which are specified according to EU designations:

[0854] (e)H268Q,V309L,A330S andP331S;

[0855] (f)V234A;

[0856] (g)G237A;

[0857] (h)V234A and G237A;

[0858] (i) A235E and G237A; or

[0859] (j) V234A, A235E, and G237A. Each number represents the position of the amino acid residue in the EU number; the single-letter amino acid symbol before the number indicates the amino acid residue before substitution, and the single-letter amino acid symbol after the number indicates the amino acid residue after substitution.

[0860] In addition, preferred antigen-binding molecules or antibodies also include those Fc domains containing amino acids of the Fc domain that form IgG3 antibodies and have any of the following substitutions, the positions of which are specified according to EU designations:

[0861] (k)F241A;

[0862] (l)D265A; or

[0863] (m)V264A. Each number represents the position of an amino acid residue in the EU number; the single-letter amino acid symbol before the number indicates the amino acid residue before substitution, and the single-letter amino acid symbol after the number indicates the amino acid residue after substitution.

[0864] In addition, preferred antigen-binding molecules or antibodies also include those Fc domains containing amino acids of the Fc domain that form IgG4 antibodies and have any of the following substitutions, the positions of which are specified according to EU designations:

[0865] (n)L235A,G237A andE318A;

[0866] (o)L235E; or

[0867] (p)F234A and L235A. Each number represents the position of the amino acid residue in the EU number; the single-letter amino acid symbol before the number indicates the amino acid residue before substitution, and the single-letter amino acid symbol after the number indicates the amino acid residue after substitution.

[0868] Other preferred antigen-binding molecules or antibodies include, for example, those containing an Fc domain, wherein any amino acid at position 233, 234, 235, 236, 237, 327, 330, or 331 (EU number) of the amino acid in the Fc domain forming the IgG1 antibody is replaced by an amino acid at the corresponding position in the EU number of the corresponding IgG2 or IgG4.

[0869] Preferred antigen-binding molecules or antibodies also include, for example, those containing an Fc domain, wherein any one or more amino acids at positions 234, 235, and 297 (EU number) of the Fc domain forming the IgG1 antibody are substituted with other amino acids. There are no particular limitations on the type of substituted amino acids; however, antigen-binding molecules or antibodies containing an Fc domain in which any one or more amino acids at positions 234, 235, and 297 are replaced with alanine are particularly preferred.

[0870] Preferred antigen-binding molecules or antibodies also include, for example, those containing an Fc domain, wherein the amino acid at position 265 (EU number) of the Fc domain forming the IgG1 antibody is substituted with another amino acid. The type of substituted amino acid is not particularly limited; however, antigen-binding molecules or antibodies containing an Fc domain in which the amino acid at position 265 is substituted with alanine are particularly preferred.

[0871] Fc receptor

[0872] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is a receptor that binds to IgG antibodies (γ receptors) and includes subclasses of FcγRI, FcγRII, and FcγRIII, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activating motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). For example, FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term “FcR” in this paper encompasses other FcRs, including those to be identified in the future.

[0873] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.).

[0874] The in vivo binding of human FcRn and the plasma half-life of human FcRn-binding peptides with high affinity can be determined, for example, in transgenic mice or human cell lines transfected with human FcRn, or in primates administered peptides with variant Fc regions. WO 2000 / 42072 (Presta) describes antibody variants that increase or decrease binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0875] Fcγ receptor

[0876] Fcγ receptors are receptors that can bind to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies, including all members of the protein family that are essentially encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including alloforms H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including alloforms V158 and F158) and FcγRIIIb (including alloforms FcγRIIIb-NA1 and FcγRIIIb-NA2); and all unidentified human Fcγ receptors, Fcγ receptor isoforms, and their alloforms. However, Fcγ receptors are not limited to these examples. They include those derived from humans, mice, rats, rabbits, and monkeys. Fcγ receptors can be derived from any organism. Mouse Fcγ receptors include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as all unidentified mouse Fcγ receptors, Fcγ receptor isoforms, and allotropes. Preferred Fcγ receptors include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16). The polynucleotide and amino acid sequences of FcγRI are shown in RefSeq accession numbers NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIA are shown in RefSeq accession numbers BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIB are shown in RefSeq accession numbers BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIA are shown in RefSeq accession numbers BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIB are shown in RefSeq accession numbers BC128562.1 and AAI28563.1, respectively.Whether the Fcγ receptor has binding activity to the Fc domain of monoclonal IgG1, IgG2, IgG3 or IgG4 antibodies can be determined by ALPHA screening (amplified luminescent proximity homogeneous assay), BIACORE method based on surface plasmon resonance (SPR), and other methods besides the above-mentioned FACS and ELISA forms (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0877] Meanwhile, "Fc ligand" or "effect ligand" refers to a molecule, preferably a polypeptide, that binds to the Fc domain of an antibody to form an Fc / Fc ligand complex. This molecule can be derived from any organism. The binding of the Fc ligand to Fc preferably induces one or more effector functions. Such Fc ligands include, but are not limited to, Fc receptors, Fcγ receptors, Fcα receptors, Fcβ receptors, FcRn, Clq and C3, mannan-binding lectins, mannose receptors, staphylococcal protein A, staphylococcal protein G, and viral Fcγ receptors. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136), which are the Fc receptor family homologous to the Fcγ receptor. Fc ligands also include unidentified molecules that bind to Fc.

[0878] Fcγ receptor binding activity

[0879] Impaired binding activity of the Fc domain to any of the Fcγ receptors FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and / or FcγRIIIB can be assessed using the aforementioned FACS and ELISA methods, as well as ALPHA screening (amplified luminescent proximity homogeneous assay) and the BIACORE method based on surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0880] ALPHA screening is performed using two types of beads based on the ALPHA technique: donor beads and acceptor beads. A luminescent signal is detected only when a molecule attached to a donor bead biologically interacts with a molecule attached to an acceptor bead, and the two beads are very close in position. Under laser excitation, a photosensitizer in the donor bead converts oxygen surrounding the bead into excited singlet oxygen. When this singlet oxygen diffuses around the donor bead and reaches a nearby acceptor bead, it triggers a chemiluminescent reaction within the acceptor bead. This reaction ultimately results in luminescence. If the molecule attached to the donor bead does not interact with the molecule attached to the acceptor bead, the singlet oxygen produced by the donor bead will not reach the acceptor bead, and no chemiluminescent reaction will occur.

[0881] For example, biotin-labeled antigen-binding molecules or antibodies are immobilized on donor beads, while glutathione S-transferase (GST)-labeled Fcγ receptors are immobilized on receptor beads. In the absence of antigen-binding molecules or antibodies containing a competing mutant Fc domain, the Fcγ receptor interacts with antigen-binding molecules or antibodies containing a wild-type Fc domain, resulting in a signal at 520 to 620 nm. Antigen-binding molecules or antibodies with an unlabeled mutant Fc domain compete with antigen-binding molecules or antibodies containing a wild-type Fc domain for interaction with the Fcγ receptor. The relative binding affinity can be determined by quantifying the fluorescence reduction caused by competition. Methods for biotinylating antigen-binding molecules or antibodies, such as antibodies, using sulfonyl-NHS-biotin are known. Appropriate methods for adding GST tags to Fcγ receptors include fusing a polypeptide encoding the Fcγ receptor with a GST box, expressing the fusion gene in cells using a gene-carrying vector, and then purifying using a glutathione column. The induced signal can be preferably analyzed, for example, by fitting a single-point competition model to a nonlinear regression analysis using software such as GraphPad (San Diego).

[0882] One of the substances used to observe their interactions is immobilized as a ligand on a thin gold layer of the sensor chip. When light shines on the back of the sensor chip, causing total internal reflection at the interface between the gold layer and the glass, the intensity of the reflected light is partially reduced at a certain location (SPR signal). Another substance used to observe their interactions is injected as an analyte onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases. This changes the refractive index of the solvent on the surface of the sensor chip. The change in refractive index causes a positional shift in the SPR signal (conversely, dissociation shifts the signal back to its original position). In the Biacore system, this displacement (i.e., the mass change on the sensor chip surface) is plotted on the vertical axis, so the change in mass over time is displayed as measurement data (sensor plot). Kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the sensor plot curve, and affinity (KD) is determined by the ratio between these two constants. Suppression determination is preferred in the BIACORE method. An example of this inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0883] Production and purification of multispecific antibodies

[0884] The multispecific antigen-binding molecules described herein comprise two types of antigen-binding moieties with different binding specificities (e.g., a "first antigen-binding moiety" and a "second antigen-binding moiety" capable of binding CD3 and CD137, and a "third antigen-binding moiety" capable of binding different antigens). Each antigen-binding moiety is ultimately fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides result in several possible combinations of the two polypeptides. To improve the yield and purity of multispecific antigen-binding molecules in recombinant production, it is therefore advantageous to introduce modifications into the Fc domain of the multispecific antigen-binding molecule that promote the association of the desired polypeptide.

[0885] Therefore, in a particular embodiment, the Fc domain of the multispecific antigen-binding molecule described herein includes modifications that promote association between the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0886] In a specific implementation, the modification is a so-called "mortar and pestle" modification, which includes a "mortar" modification in one of the two subunits of the Fc domain and a "mortar" modification in the other of the two subunits of the Fc domain.

[0887] The pestle-and-mortar technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001). Typically, this method involves introducing a protrusion (“pestle”) at the interface of a first polypeptide and a corresponding cavity (“mortar”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and inhibit homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing a large amino acid side chain (e.g., alanine or threonine).

[0888] Therefore, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the multispecific antigen-binding molecule, amino acid residues are replaced by amino acid residues with larger side chain volumes, thereby creating a protrusion within the CH3 domain of the first subunit, which can be located in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby creating a cavity within the CH3 domain of the second subunit, in which the protrusion within the CH3 domain of the first subunit can be located.

[0889] Protrusions and cavities can be formed by altering the nucleic acids encoding polypeptides, for example, through site-specific mutagenesis or peptide synthesis.

[0890] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced by a serine residue (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A).

[0891] In another embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced by a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced by a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues leads to the formation of a disulfide bond between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0892] In other embodiments, other techniques for promoting association between H chains having a desired combination and between L and H chains can be applied to the multispecific antigen-binding molecule of the present invention.

[0893] For example, the technique of inhibiting unwanted H-chain association by introducing electrostatic repulsion at the interface of the second or third constant region of the antibody H chain (CH2 or CH3) can be applied to multispecific antibody association (WO2006 / 106905).

[0894] In techniques that suppress unintended H-chain association by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues in interfacial contact at another constant region of the H-chain include residues at positions 356, 439, 357, 370, 399, and 409 in the CH3 region corresponding to EU numbers.

[0895] More specifically, examples include antibodies comprising two types of H-chain CH3 regions, wherein one to three pairs of amino acid residues in the first H-chain CH3 region are selected from the amino acid residue pairs shown in (1) to (3) below, carrying the same type of charge: (1) amino acid residues contained at EU number positions 356 and 439 in the H-chain CH3 region; (2) amino acid residues contained at EU number positions 357 and 370 in the H-chain CH3 region; and (3) amino acid residues contained at EU number positions 399 and 409 in the H-chain CH3 region.

[0896] Furthermore, the antibody may be an antibody in which the amino acid residue pairs in the second H chain CH3 region, which is different from the first H chain CH3 region described above, are selected from the amino acid residue pairs of (1) to (3) described above, wherein 1 to 3 pairs of amino acid residues corresponding to the amino acid residue pairs of (1) to (3) that carry the same type of charge in the first H chain CH3 region described above carry the opposite charge to the corresponding amino acid residues in the first H chain CH3 region mentioned above.

[0897] Each amino acid residue shown in (1) to (3) above is close to each other during association. Those skilled in the art can use commercially available software to find the positions corresponding to the amino acid residues in (1) to (3) above in the desired H chain CH3 region or H chain constant region by homology modeling, and the amino acid residues at these positions can be appropriately modified.

[0898] In the above-mentioned antibodies, the "charged amino acid residues" are preferably selected from, for example, amino acid residues included in any of the following groups:

[0899] (a) Glutamic acid (E) and aspartic acid (D); and

[0900] (b) Lysine (K), arginine (R) and histidine (H).

[0901] In the antibodies described above, the phrase "carrying the same charge" means, for example, that two or more amino acid residues are all selected from amino acid residues contained in any of groups (a) and (b) above. The phrase "carrying opposite charges" means, for example, that when at least one of two or more amino acid residues is selected from amino acid residues contained in any of groups (a) and (b) above, the remaining amino acid residues are selected from amino acid residues contained in other groups.

[0902] In a preferred embodiment, the antibodies described above may have a first H chain CH3 region and a second H chain CH3 region cross-linked by disulfide bonds.

[0903] In this invention, the modified amino acid residues are not limited to the amino acid residues in the antibody variable region or antibody constant region described above. Those skilled in the art can use commercially available software to identify the amino acid residues that form the interface in the mutant peptide or heteropolymer using methods such as homology modeling; then, these amino acid residues can be modified to regulate association.

[0904] Furthermore, other known techniques can also be used to form the multispecific antibodies of this invention. Association of peptides with different sequences can be effectively induced using a chain-exchange engineered CH3 domain via complementary association of CH3, wherein the chain-exchange engineered CH3 domain is generated by altering a portion of the CH3 of the antibody's H chain to a corresponding IgA-derived sequence and introducing the corresponding IgA-derived sequence into the complementary portion of the CH3 of another H chain (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to effectively form targeted multispecific antibodies.

[0905] In addition, antibody production techniques using the association of antibody CH1 and CL, and the association of VH and VL, as described in WO 2011 / 028952, WO2014 / 018572 and Nat Biotechnol. 2014 Feb; 32(2):191-8; techniques for producing bispecific antibodies by combining separately prepared monoclonal antibodies (Fab arm exchange), as described in WO2008 / 119353 and WO2011 / 131746; techniques for regulating the association between antibody heavy chain CH3, as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing multispecific antibodies composed of two types of light chains and one type of heavy chain, as described in WO2012 / 023053; and techniques for producing multispecific antibodies composed of two types of light chains and one type of heavy chain, as described in Christoph et al. (Nature Biotechnology) The technique described in Vol. 31, pp. 753-758 (2013) for producing multispecific antibodies using two bacterial cell lines (each expressing an antibody chain containing a single H chain and a single L chain, respectively); etc., can all be used to form multispecific antibodies.

[0906] Alternatively, even in cases where the target multispecific antibody cannot be effectively formed, the multispecific antibody of the present invention can be obtained by isolating and purifying the target multispecific antibody from the generated antibody. For example, a method has been reported that imparts an isoelectric point difference by introducing amino acid substitutions into the variable regions of the two H chains, thereby enabling the purification of two types of homologous forms and the target heterodimeric antibody by ion exchange chromatography (WO2007114325). To date, as a method for purifying heterodimeric antibodies, methods have been reported for purifying heterodimeric antibodies containing a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A (WO98050431 and WO95033844). Furthermore, heterodimeric antibodies can be effectively purified individually by using H chains containing amino acid residues at EU number positions 435 and 436, which are IgG protein A binding sites, replaced with amino acids such as Tyr and His that produce different affinities for protein A, or by using H chains with different affinities for protein A to modify the interaction of each H chain with protein A, and then using a protein A column for purification.

[0907] Furthermore, an Fc region with improved C-terminal heterogeneity can be suitably used as the Fc region of the present invention. More specifically, the present invention provides an Fc region generated by deleting glycine at position 446 and lysine at position 447, as specified in the EU number, from the amino acid sequences of two polypeptides constituting an Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0908] The multispecific antigen-binding molecules prepared as described herein can be purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion-exchange chromatography (IEC), gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The specific conditions used for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens bound to the multispecific antigen-binding molecules can be used. For example, for affinity chromatography purification of the multispecific antigen-binding molecules of the present invention, a matrix containing protein A or protein G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to separate multispecific antigen-binding molecules. The purity of the multispecific antigen-binding molecules can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-performance liquid chromatography, etc.

[0909] Antibody-dependent cell-mediated cytotoxicity

[0910] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens, subsequently killing the target cells with cytotoxins. Primary cells and NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991) summarizes FcR expression on hematopoietic cells. To assess the ADCC activity of the target molecule, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337 or 6,737,056 (Presta). Useful effector cells for such assays include PBMCs and NK cells. Alternatively, the ADCC activity of the target molecule can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0911] complement-dependent cytotoxicity

[0912] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (appropriate subclasses) that bind to their homologous antigens. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). For example, peptide variants with altered Fc region amino acid sequences (peptides with variant Fc regions) and increased or decreased C1q binding capacity are described in U.S. Patent Nos. 6,194,551 B1 and WO 1999 / 51642. See also, for example, Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0913] T cell-dependent cytotoxicity

[0914] "T-cell-dependent cytotoxicity" or "TDCC" refers to a form of cytotoxicity in which an antigen-binding molecule binds to both an antigen expressed on a target cell and another antigen expressed on a T cell, thereby redirecting the T cell to a location closer to the target cell, since the cytotoxicity against the target cell is induced by the T cell. Methods for assessing T-cell-dependent cytotoxicity, such as the in vitro TDCC assay, are also described in the "T-cell-dependent cytotoxicity measurement" section of this specification.

[0915] Measuring T cell-dependent cytotoxicity

[0916] In embodiments where the antigen-binding molecule binds to both DLL3 and CD3 / CD137, the following method is preferably used as a method for assessing or determining T cell-dependent cytotoxicity (TDCC) induced by contact between the antigen-binding molecule of this disclosure and cells expressing DLL3, wherein the antigen-binding site in the antigen-binding molecule of this disclosure binds to the cells expressing DLL3. Methods for assessing or determining in vitro cytotoxic activity include methods for determining the activity of cytotoxic T cells, etc. Whether the antigen-binding molecule of this disclosure has the activity of inducing T cell-mediated cytotoxicity can be determined by known methods (see, for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993)). In cytotoxicity assays, an antigen-binding molecule capable of binding to an antigen CD3 / CD137 that is different from DLL3 and not expressed in cells is used as a control antigen-binding molecule. The control antigen-binding molecule is measured in the same manner. The activity was then assessed by testing whether the antigen-binding molecule of this disclosure exhibited stronger cytotoxic activity than the control antigen-binding molecule.

[0917] Simultaneously, the in vivo antitumor efficacy can be assessed or determined, for example, through the following procedure: Cells expressing the antigen-binding site of the antigen-binding molecule of this disclosure are transplanted intradermally or subcutaneously into a non-human animal subject. Then, starting from the day of transplantation or thereafter, the test antigen-binding molecule is administered intravenously or intraperitoneally daily or every few days. Tumor size is measured over time. Differences in tumor size changes can be defined as cytotoxic activity. As in in vitro assays, a control antigen-binding molecule is administered. When the tumor size in the group treated with the antigen-binding molecule of this disclosure is smaller than that in the group treated with the control antigen-binding molecule, the antigen-binding molecule of this disclosure can be judged to have cytotoxic activity.

[0918] The MTT method and the measurement of thymidine uptake into cells by isotope labeling are preferably used to assess or determine the effect of contact with the antigen-binding molecule of this disclosure to inhibit the growth of cells expressing antigens, said antigens binding to antigen-binding sites in the antigen-binding molecule. Simultaneously, the same methods described above for assessing or determining in vivo cytotoxic activity can preferably be used to assess or determine the activity inhibiting in vivo cell growth.

[0919] The TDCC of the antibody or antigen-binding molecule disclosed herein can be assessed by any suitable method known in the art. For example, TDCC can be measured by a lactate dehydrogenase (LDH) release assay. In this assay, target cells (e.g., cells expressing DLL3) are incubated with T cells (e.g., PBMCs) in the presence of the test antibody or antigen-binding molecule, and the activity of LDH released from the target cells killed by the T cells is measured using suitable reagents. Typically, cytotoxic activity is calculated as the percentage of LDH activity generated by incubation with the antibody or antigen-binding molecule relative to the LDH activity generated by 100% killing of target cells (e.g., by treatment with Triton-X to induce lysis). If the cytotoxic activity calculated as described above is high, the test antibody or antigen-binding molecule is determined to have a high TDCC.

[0920] Alternatively or alternatively, TDCC can also be measured by a real-time cell growth inhibition assay. In this assay, target cells (e.g., cells expressing DLL3) and T cells (e.g., PBMCs) are incubated in a 96-well plate in the presence of a test antibody or antigen-binding molecule, and the growth of the target cells is monitored by methods known in the art, such as using a suitable analytical instrument (e.g., a xCELLigence real-time cell analyzer). The cell growth inhibition rate (CGI: %) is determined from the cell index value according to the formula given: CGI(%) = 100 - (CIAb × 100 / CINoAb). “CIAb” represents the cell index value of the wells containing the antibody or antigen-binding molecule at a specific experimental time, and “CINoAb” represents the average cell index value of the wells without the antibody or antigen-binding molecule. If the CGI rate of the antibody or antigen-binding molecule is high, i.e., has a significant positive value, then the antibody or antigen-binding molecule can be said to have TDCC activity.

[0921] In one aspect, the antibody or antigen-binding molecule of this disclosure possesses T-cell activation activity. T-cell activation can be determined by methods known in the art, such as using engineered T-cell lines that express a reporter gene (e.g., luciferase) in response to activation (e.g., Jurkat / NFAT-RE reporter cell line (T-cell activation bioassay, Promega)). In this method, target cells (e.g., cells expressing DLL3) and T cells are cultured in the presence of the test antibody or antigen-binding molecule, and the level or activity of the reporter gene expression product is then measured as an indicator of T-cell activation by an appropriate method. When the reporter gene is a luciferase gene, the luminescence caused by the reaction between the luciferase and its substrate can be measured as an index of T-cell activation. If the T-cell activation measured as described above is high, the test antibody or antigen-binding molecule is determined to have high T-cell activation activity.

[0922] Pharmaceutical Composition

[0923] In one aspect, this disclosure provides a pharmaceutical composition comprising the antigen-binding molecule or antibody of this disclosure. In some embodiments, the pharmaceutical composition of this disclosure induces T-cell-dependent cytotoxicity; in other words, the pharmaceutical composition of this disclosure is a therapeutic agent for inducing cytotoxicity of cells. In some embodiments, the pharmaceutical composition of this disclosure is a pharmaceutical composition for treating and / or preventing cancer. In some embodiments, the pharmaceutical composition of this disclosure is a pharmaceutical composition for treating and / or preventing DLL3-positive or DLL3-expressing cancers, including lung cancer (including small cell lung cancer) and melanoma. In some embodiments, the pharmaceutical composition of this disclosure is a cell growth inhibitor. In some embodiments, the pharmaceutical composition of this disclosure is an anticancer agent.

[0924] If desired, the pharmaceutical compositions, therapeutic agents for inducing cytotoxicity of cells, cell growth inhibitors, or anticancer agents disclosed herein can be formulated with different types of antigen-binding molecules or antibodies. For example, cytotoxic effects against cells expressing antigens can be enhanced by a mixture of the various antigen-binding molecules or antibodies disclosed herein.

[0925] The pharmaceutical formulations of the antigen-binding molecules or antibodies described herein are prepared by mixing such antigen-binding molecules or antibodies, having the desired purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), wherein the pharmaceutical composition is in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residuals). (Based on) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0926] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, the latter of which comprises histidine-acetate buffer.

[0927] The formulations described herein may also contain more than one active ingredient required for the specific indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

[0928] If necessary, the antigen-binding molecules or antibodies of the present invention can be encapsulated in microcapsules (microcapsules made of hydroxymethyl cellulose, gelatin, poly[methyl methacrylate], etc.) and prepared as components of colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) (see, for example, "Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980)). Furthermore, methods for preparing reagents as sustained-release agents are known, and these methods can be applied to the antigen-binding molecules of this disclosure (J. Biomed. Mater. Res. (1981) 15, 267-277; Chemtech. (1982) 12, 98-105; US Patent No. 3773719; European Patent Applications Nos. EP58481 and EP133988; Biopolymers (1983) 22, 547-556).

[0929] The pharmaceutical compositions, cell growth inhibitors, or anticancer agents disclosed herein can be administered to patients orally or parenterally. Parenteral administration is preferred. Specifically, such administration methods include injection, nasal administration, pulmonary administration, and percutaneous administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical compositions, therapeutic agents for inducing cytotoxicity of cells, cell growth inhibitors, or anticancer agents disclosed herein can be administered locally or systemically by injection. Furthermore, an appropriate method of administration can be selected based on the patient's age and symptoms. For each administration, the dosage can be selected from, for example, a range of 0.0001 mg to 1,000 mg / kg body weight. Alternatively, the dosage can be selected from, for example, 0.001 mg / body to 100,000 mg / body per patient. However, the dosage of the pharmaceutical compositions disclosed herein is not limited to these dosages.

[0930] Preferably, the pharmaceutical compositions of this disclosure comprise an antigen-binding molecule or antibody as described herein. On one hand, the composition is a pharmaceutical composition for inducing cytotoxicity in cells. On the other hand, the composition is a pharmaceutical composition for treating or preventing cancer. Preferably, the cancer is lung cancer (including small cell lung cancer) and melanoma. The pharmaceutical compositions of this disclosure can be used to treat or prevent cancer. Therefore, this disclosure provides a method for treating or preventing cancer, wherein the antigen-binding molecule or antibody described herein is administered to a patient in need.

[0931] This disclosure also provides a method for damaging or inhibiting the growth of DLL3-expressing cells by contacting DLL3-expressing cells with an antigen-binding molecule of this disclosure that binds to DLL3. There are no particular limitations on the cells to which the antigen-binding molecule of this disclosure binds, as long as they express DLL3. Specifically, in this disclosure, preferred DLL3-expressing cells include lung cancer (including small cell lung cancer) and melanoma.

[0932] In this disclosure, "contact" can be performed, for example, by adding the antigen-binding molecule of this disclosure to the culture medium of cells expressing DLL3 cultured in vitro. In this case, the added antigen-binding molecule can be used in a suitable form, such as a solution or solid prepared by lyophilization. When the antigen-binding molecule of this disclosure is added as an aqueous solution, the solution can be a pure aqueous solution containing only the antigen-binding molecule, or it can be a solution containing, for example, the surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow promoters, and flavoring agents described above. There are no particular limitations on the added concentration; however, the final concentration in the culture medium is preferably in the range of 1 pg / ml to 1 g / ml, more preferably 1 ng / ml to 1 mg / ml, and even more preferably 1 μg / ml to 1 mg / ml.

[0933] In another embodiment of this disclosure, "contact" can also be achieved by administration to a non-human animal transplanted with cells expressing DLL3 or to an animal with cancer cells endogenously expressing DLL3. Administration can be oral or parenteral. Parenteral administration is particularly preferred. Specifically, parenteral administration methods include injection, nasal administration, pulmonary administration, and percutaneous administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical compositions of this disclosure, therapeutic agents for inducing cytotoxicity of cells, cell growth inhibitors, or anticancer agents can be administered locally or systemically by injection. Furthermore, the method of administration can be appropriately selected based on the age and symptoms of the animal subject. When the antigen-binding molecule is administered as an aqueous solution, the solution can be a pure aqueous solution containing only the antigen-binding molecule, or a solution containing, for example, the surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow promoters, and flavoring agents described above. For each administration, the dosage may be selected from, for example, a range of 0.0001 mg to 1,000 mg / kg body weight. Alternatively, for each patient, the dosage may be selected from, for example, 0.001 mg / body to 100,000 mg / body. However, the dosage of the antigen-binding molecule disclosed herein is not limited to these examples.

[0934] This disclosure also provides kits for use in the methods of this disclosure, which contain the antigen-binding molecules of this disclosure or antigen-binding molecules generated by the methods of this disclosure. The kits may be packaged with additional pharmaceutically acceptable carriers or media, or instructions describing how to use the kit.

[0935] In another aspect of the invention, articles containing materials for treating, preventing, and / or diagnosing the aforementioned conditions are provided. The articles include a container and a label on the container or a packaging insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be made of various materials such as glass or plastic. The container contains a composition, either alone or in combination with another composition effective in treating, preventing, and / or diagnosing the condition, and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or packaging insert indicates that the composition is used to treat the selected condition. Furthermore, the manufactured article may include (a) a first container containing the composition, wherein the composition contains the antibody of the present invention; and (b) a second container containing the composition, wherein the composition contains a further cytotoxic agent or other therapeutic agent. The manufactured article in this embodiment of the invention may further include a packaging insert indicating that the composition is used to treat a specific condition. Alternatively, the production article may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other materials required from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0936] Packaging insert

[0937] The term "packaging insert" is used to refer to instructions that are typically included in the commercial packaging of therapeutic products, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings for using such therapeutic products.

[0938] pharmaceutical preparations

[0939] The terms “pharmaceutical formulation” or “pharmaceutical composition” refer to a formulation whose form allows the bioactivity of the active ingredient contained therein to be effective and does not contain any additional ingredients that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0940] Pharmaceutically acceptable carriers

[0941] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation that are non-toxic to the subjects, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. ...

Claims

1. Multispecific antigen-binding molecules, comprising: (a) A first antigen-binding portion and a second antigen-binding portion, each capable of binding human CD3 and human CD137, but not simultaneously binding human CD3 and human CD137; wherein the first antigen-binding portion and the second antigen-binding portion are each Fab molecules and contain antibody variable regions, wherein the antibody variable regions contain the heavy chain complementarity-determining region CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 34, the heavy chain CDR3 of SEQ ID NO: 48, the light chain CDR1 of SEQ ID NO: 63, the light chain CDR2 of SEQ ID NO: 68, and the light chain CDR3 of SEQ ID NO: 73; (b) The third antigen-binding region of human DLL3, comprising an antibody variable region including the heavy chain complementarity-determining region CDR1 of SEQ ID NO: 233, the heavy chain CDR2 of SEQ ID NO: 234, the heavy chain CDR3 of SEQ ID NO: 235, the light chain CDR1 of SEQ ID NO: 237, the light chain CDR2 of SEQ ID NO: 238, and the light chain CDR3 of SEQ ID NO:

239. and (c) At least one disulfide bond formed between the CH1 region of the first antigen-binding portion and the CH1 region of the second antigen-binding portion, comprising a disulfide bond formed between a cysteine ​​residue at position 191 of the CH1 domain of the first antigen-binding portion according to EU number and a cysteine ​​residue at position 191 of the CH1 domain of the second antigen-binding portion according to EU number. The third antigen-binding portion is Fab. The C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the heavy chain of the first antigen-binding portion or the second antigen-binding portion via a peptide linker. The multispecific antigen-binding molecule further includes an Fc domain fused to the first and second antigen-binding moieties, and Compared to the natural human IgG1 Fc domain, the Fc domain exhibits a reduced binding affinity for the human Fc-γ receptor.

2. The multispecific antigen-binding molecule of claim 1, wherein, The first antigen-binding portion and the second antigen-binding portion each include an antibody variable region, the antibody variable region comprising: The heavy chain variable region contains the amino acid sequence of SEQ ID NO:6, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

58.

3. The multispecific antigen-binding molecule of claim 1, 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.

4. The multispecific antigen-binding molecule of claim 1, wherein the third antigen-binding portion is a cross-Fab molecule, wherein the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and wherein the first and second antigen-binding portions are each conventional Fab molecules.

5. The multispecific antigen-binding molecule of claim 4, wherein in the constant domain CL of the respective light chain of the first and second antigen-binding moieties, the amino acid at positions 123 and / or 124 according to Kabat numbering is independently lysine, arginine, or histidine, and wherein in the constant domain CH1 of the respective heavy chain of the first and second antigen-binding moieties, the amino acid at position 147 according to EU numbering and / or the amino acid at position 213 is independently glutamic acid or aspartic acid.

6. The multispecific antigen-binding molecule of claim 5, wherein, In the constant structural domain CL of the light chain of each of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 according to Kabat numbering are arginine and lysine, respectively, and in the constant structural domain CH1 of the heavy chain of each of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 according to EU numbering are glutamic acid.

7. The multispecific antigen-binding molecule of claim 1, wherein the third antigen-binding portion comprises an antibody variable region, the antibody variable region comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO:232, and a light chain variable region containing the amino acid sequence of SEQ ID NO:

236.

8. The multispecific antigen-binding molecule of claim 1, wherein the Fc domain comprises first and second Fc subunits capable of stable association. The first Fc subbase is selected from the following group: (a1) A polypeptide containing the Fc domains of Ala at position 234 and Ala at position 235; (a2) A polypeptide containing the Fc domains of Ala at position 234, Ala at position 235, and Ala at position 297; (a3) A polypeptide containing the Fc domains of Ala at position 234, Ala at position 235, Ala at position 297, Cys at position 354, and Trp at position 366; and The second Fc subbase is selected from the following group: (a4) A polypeptide containing the Fc domains of Ala at position 234 and Ala at position 235; (a5) A polypeptide containing the Fc domains of Ala at position 234, Ala at position 235, and Ala at position 297; (a6) A polypeptide containing the Fc domains of Ala at position 234, Ala at position 235, Ala at position 297, Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407; and The amino acid positions are numbered using EU index numbers.

9. The multispecific antigen-binding molecule of claim 1, wherein, Compared to the Fc domain of natural IgG, the Fc domain exhibits enhanced FcRn binding activity under acidic pH conditions.

10. The multispecific antigen-binding molecule according to claim 9, wherein, The acidic pH condition is pH 5.

8.

11. The multispecific antigen-binding molecule of claim 9, wherein, The Fc domain includes Ala at position 434 according to EU number; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440.

12. The multispecific antigen-binding molecule of claim 11, wherein, The Fc domain contains Ala at position 434 according to EU number; Arg or Lys at position 438; and Glu or Asp at position 440.

13. The multispecific antigen-binding molecule of claim 12, wherein, The Fc structural domain also includes Ile or Leu at position 428 according to EU number; and / or Ile, Leu, Val, Thr or Phe at position 436.

14. The multispecific antigen-binding molecule of claim 9, wherein, The Fc domain contains a combination of amino acid substitutions selected from the group consisting of: According to EU number (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.

15. The multispecific antigen-binding molecule of claim 9, wherein, The Fc domain contains a combination of amino acid substitutions of M428L / N434A / Q438R / S440E.

16. The multispecific antigen-binding molecule of claim 1, wherein, The Fc domain is the IgG Fc domain.

17. The multispecific antigen-binding molecule of claim 8, wherein the Fc domain is an IgG Fc domain.

18. The multispecific antigen-binding molecule of claim 9, wherein the Fc domain is an IgG Fc domain.

19. The multispecific antigen-binding molecule of claim 1, wherein, The Fc domain includes any of the following: (a) A first Fc subunit comprising the amino acid sequence shown in SEQ ID NO: 100 and a second Fc subunit comprising the amino acid sequence shown in SEQ ID NO: 111; and (b) A first Fc subunit containing the amino acid sequence shown in SEQ ID NO:99 and a second Fc subunit containing the amino acid sequence shown in SEQ ID NO:

109.

20. The multispecific antigen-binding molecule of claim 1, wherein the Fc domain comprises first and second Fc subunits capable of stable association, and wherein the first antigen-binding portion 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain.

21. The multispecific antigen-binding molecule of claim 8, wherein the first antigen-binding portion 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain.

22. The multispecific antigen-binding molecule of claim 9, wherein the Fc domain comprises first and second Fc subunits capable of stable association, and wherein the first antigen-binding portion 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain.

23. The multispecific antigen-binding molecule of claim 19, wherein the first antigen-binding portion 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain.

24. A multispecific antigen-binding molecule comprising five polypeptide chains selected from any combination of the following (a1) to (a3): (a1) A polypeptide chain 1 containing the amino acid sequence of SEQ ID NO:201, a polypeptide chain 2 containing the amino acid sequence of SEQ ID NO:206, a polypeptide chain 3 containing the amino acid sequence of SEQ ID NO:208, a polypeptide chain 4 containing the amino acid sequence of SEQ ID NO:214, and a polypeptide chain 5 containing the amino acid sequence of SEQ ID NO:

214. (a2) A polypeptide chain 1 containing the amino acid sequence of SEQ ID NO:203, a polypeptide chain 2 containing the amino acid sequence of SEQ ID NO:206, a polypeptide chain 3 containing the amino acid sequence of SEQ ID NO:209, a polypeptide chain 4 containing the amino acid sequence of SEQ ID NO:214, and a polypeptide chain 5 containing the amino acid sequence of SEQ ID NO:

214. (a3) polypeptide chain 1 containing the amino acid sequence of SEQ ID NO:204, polypeptide chain 2 containing the amino acid sequence of SEQ ID NO:206, polypeptide chain 3 containing the amino acid sequence of SEQ ID NO:209, polypeptide chain 4 containing the amino acid sequence of SEQ ID NO:214, and polypeptide chain 5 containing the amino acid sequence of SEQ ID NO:

214. In this process, polypeptide chains 2 and 5 each associate with polypeptide chain 1; polypeptide chain 4 associates with polypeptide chain 3; and polypeptide chain 1 associates with polypeptide chain 3.

25. The multispecific antigen-binding molecule of claim 24, wherein, In the respective CH1 regions of the two Fabs contained in polypeptide chains 1 and 3, at least one disulfide bond is formed between amino acid residues at EU number position 191.

26. An isolated polynucleotide or multiple polynucleotides encoding a multispecific antigen-binding molecule as described in any one of claims 1 to 25.

27. A vector encoding one or more polynucleotides as described in claim 26.

28. A host cell comprising the polynucleotide or multiple polynucleotides of claim 26 or the vector of claim 27.

29. A method for preparing the multispecific antigen-binding molecule according to any one of claims 1 to 25, comprising the following steps: a) Culturing the host cells of claim 28 under conditions suitable for the expression of the antigen-binding molecule and b) Recovering the antigen-binding molecule.

30. A multispecific antigen-binding molecule, prepared by the method of claim 29.

31. A pharmaceutical composition comprising the multispecific antigen-binding molecule of any one of claims 1-25 and a pharmaceutically acceptable carrier.

32. The multispecific antigen-binding molecule according to any one of claims 1-25 or the pharmaceutical composition according to claim 31, characterized in that... Induces cytotoxicity.

33. The multispecific antigen-binding molecule according to any one of claims 1-25 or the pharmaceutical composition according to claim 31, characterized in that... Used as a medicine.

34. The multispecific antigen-binding molecule according to any one of claims 1-25 or the pharmaceutical composition according to claim 31, characterized in that... Used to treat or prevent disease in individuals who need it.

35. Use of the multispecific antigen-binding molecule of any one of claims 1-25 or the pharmaceutical composition of claim 31 in the preparation of a medicament for the treatment or prevention of cancer expressing DLL3 or DLL3-positive cancer in an individual in need, wherein said cancer is small cell lung cancer or melanoma.

36. A reagent kit, characterized in that, The pharmaceutical composition of claim 31 and a packaging insert comprising instructions for administration to a subject to treat or delay the progression of a DLL3-expressing cancer or a DLL3-positive cancer, wherein the cancer is small cell lung cancer or melanoma.

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