JP7557922B2 - Uses of DLL3-targeting multispecific antigen-binding molecules - Google Patents

Description

The present invention relates to multispecific antigen-binding molecules targeting DLL3, pharmaceutical compositions comprising such antigen-binding molecules, and the use of such antigen-binding molecules or such compositions for therapeutic purposes in the field of cancer diseases.

In cancer therapy, the desired goal is to effectively and selectively destroy tumor cells while leaving healthy cells and tissues intact. There is a need for improved treatments for neoplastic diseases associated with DLL3 overexpression, such as neuroendocrine carcinomas (NECs), neuroendocrine tumors (NETs), and other solid tumors.

Delta-like 3 (DLL3) is a type I membrane protein that belongs to the Notch ligand family members. DLL3 is an atypical Notch ligand that functions cell-autonomously to inhibit Notch signaling by binding to Notch in cis, thereby blocking cell-cell interactions and internalization of Notch in target cells, which are hallmarks of canonical Notch signaling. The primary role of DLL3 is somitogenesis during embryonic development. Mice with DLL3 knockouts show partial defects in axial skeletal and cranial and neural development. Defects in somitogenesis patterning are also observed in humans with certain germline DLL3 mutations, which result in a condition called spondylocostal dysostosis. There have been previous studies reporting amplification of the DLL3 gene on the chromosome and increased expression of this gene in cancer cell lines (Non-Patent Document 1) as well as increased DLL3 expression in some cases of glioma (Non-Patent Document 2). In addition, DLL3 has previously been proposed in methods for diagnosing and treating glioma or small cell lung cancer (SCLC) using ADCC-enhancing antibodies, antibody-drug conjugates (ADCs), and T cell-inducing bispecific molecules using the BiTE-Fc format (Patent Documents 1, 2, and 3).

WO2011/093097 WO2013/126746 WO2017/021349

Phillips, H. S. (2006) Cancer Cell 9, 157-173. Mulledndore, M. E. (2009) Clin Cancer Res 15, 2291-2301.

The object of the present invention is to provide a multispecific antigen-binding molecule capable of treating cancer by recruiting T cells to the vicinity of DLL3-expressing cells and using the cytotoxicity of T cells against DLL3-expressing cancer cells; a method for producing the multispecific antigen-binding molecule; and a therapeutic agent comprising such a multispecific antigen-binding molecule as an active ingredient for inducing cytotoxic activity. Another object of the present invention is to provide a pharmaceutical composition for use in treating or preventing various cancers, comprising one of the above-mentioned antigen-binding molecules as an active ingredient, and a therapeutic method using the pharmaceutical composition.

The present invention relates to a multispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion, each capable of binding to CD3 and CD137 but not simultaneously (i.e., capable of binding to CD3 and CD137 but not simultaneously); and a third antigen-binding portion capable of binding to DLL3, preferably human DLL3, which induces T cell-dependent cytotoxicity more efficiently while avoiding harmful toxicity concerns or side effects that other multispecific antigen-binding molecules may have. The present invention further relates to medical uses of the multispecific antigen-binding molecule and its pharmaceutical composition, which can treat various cancers, particularly cancers associated with DLL3, such as DLL3-expressing or DLL3-positive cancers, by including the antigen-binding molecule as an active ingredient. In one aspect, the DLL3-expressing or DLL3-positive cancer is selected from the group consisting of pancreatic neuroendocrine tumors (PNETs), small cell NEC (SCNEC), large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine cancer, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma (GBM), melanoma, and medullary thyroid cancer.

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

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, each of which can bind to CD3 and CD137 but not simultaneously (i.e., can bind to CD3 and CD137 but not simultaneously); and a third antigen-binding portion capable of binding to DLL3, preferably human DLL3, which efficiently induces T cell-dependent cytotoxicity while avoiding the harmful toxicity concerns or side effects that other multispecific antigen-binding molecules may have. In one aspect, the antigen-binding portion capable of binding to CD3 and CD137 but not simultaneously is an antigen-binding portion capable of binding to human CD3 and human CD137, and the first antigen-binding portion binds to either human CD3 or human CD137. An antigen-binding portion that can bind to CD3 and CD137 but not simultaneously (i.e., can bind to CD3 and CD137 but not simultaneously) may be referred to as a "Dual antigen-binding portion" or in some aspects a "Dual-Fab," as more specifically defined below. In one such aspect, each of the first and second antigen-binding moieties contains at least one amino acid mutation, e.g., a cysteine insertion/substitution/mutation, which creates a disulfide bond between the first and second antigen-binding moieties to keep them close to each other and promote cis antigen binding to antigens (CD3 and/or CD137) on the same single effector cell, e.g., as a result of steric hindrance or shorter distance between the two antigen-binding moieties (e.g., Dual-Fab), thereby improving the safety profile of the trispecific antigen-binding molecule by preventing undesired cross-linking of two CD3/CD137 expressing immune cells mediated in a DLL3-independent manner by the two antigen-binding moieties (which can bind to CD3 and CD137 but not simultaneously). In one particular aspect, each of the first and second antigen-binding moieties is a Fab and includes at least one cysteine residue (through mutation, substitution, or insertion) in the CH1 region, which can form at least one disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety. In another particular aspect, each of the first and second antigen-binding moieties includes at least one cysteine residue (through mutation, substitution, or insertion) at position 191 according to EU numbering in the CH1 region, which can form one disulfide bond between the CH1 region of the first antigen-binding moiety and the CH1 region of the second antigen-binding moiety. Such a disulfide bond at the CH1 region (e.g., position 191 according to EU numbering) that links the first and second antigen-binding moieties may be referred to herein as a "LINC".

Antigen-binding molecules with such a unique structural format, i.e., trivalent trispecific antibodies comprising two monovalent Dual-Fabs and one monovalent DLL3-binding arm, each capable of binding to CD3 and CD137 but not simultaneously, may also be referred to as "(2+1)" (or "1+2"), and have surprisingly been found to exhibit superior efficacy compared to other multispecific antibody formats (e.g., BiTEs) while exhibiting reduced or minimized off-target side effects due to undesired cross-linking between various cells (e.g., effector cells such as T cells).

The present invention relates to:
[A-1] An antibody for use as a medicine; or an antibody for use in treating cancer; or a pharmaceutical formulation comprising an antibody and a pharma- ceutically acceptable carrier; or a pharmaceutical formulation comprising an antibody and a pharma- ceutically acceptable carrier for use as a medicine; or a pharmaceutical formulation comprising an antibody and a pharma- ceutically acceptable carrier for use in treating cancer.
[A-2] The antibody
(a) a first antigen-binding moiety and a second antigen-binding moiety, each of which comprises an antibody variable region, which may be the same or different, and which antibody variable region is selected from the group consisting of:
(a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, a heavy chain CDR 2 of SEQ ID NO: 34, a heavy chain CDR 3 of SEQ ID NO: 48, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a2) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, a heavy chain CDR 2 of SEQ ID NO: 42, a heavy chain CDR 3 of SEQ ID NO: 56, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a3) a first antigen-binding portion and a second antigen-binding portion independently selected from an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 82, heavy chain CDR 2 of SEQ ID NO: 83, heavy chain CDR 3 of SEQ ID NO: 84, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75; and (b) a third antigen-binding portion that binds to human delta-like 3 (DLL3) and comprises an antibody variable region comprising heavy chain CDR 1 comprising SEQ ID NO: 233, heavy chain CDR 2 comprising SEQ ID NO: 234, heavy chain CDR 3 comprising SEQ ID NO: 235, light chain CDR 1 comprising SEQ ID NO: 237, light chain CDR 2 comprising SEQ ID NO: 238, and light chain CDR 3 comprising SEQ ID NO: 239.
[A-3] Each of the first antigen-binding portion and the second antigen-binding portion has the following characteristics:
(i) binds to human CD3;
(ii) binds to human CD137;
(iii) capable of binding to human CD3 and human CD137, and each of the first antigen-binding portion and the second antigen-binding portion binds to either human CD3 or human CD137; and (iv) capable of binding to human CD3 and human CD137 but not simultaneously to human CD3 and human CD137.
[A-4] Each of the first antigen-binding portion and the second antigen-binding portion comprises an antibody variable region, which may be the same or different, and the antibody variable region is selected from the group consisting of:
(a1) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 58;
(a2) VH comprising the amino acid sequence of SEQ ID NO: 14, and VL comprising the amino acid sequence of SEQ ID NO: 58;
(a3) VH comprising the amino acid sequence of SEQ ID NO: 81, and VL comprising the amino acid sequence of SEQ ID NO: 60;
(a4) a VH and VL comprising one or more amino acid substitutions, deletions, additions, and/or insertions in the VH and VL of any one of (a1) to (a3) and having an activity equivalent to that of any of said VH and VL; and (a5) a VH and VL having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of the VH and VL defined in any one of (a1) to (a3).
More independently selected,
Any one of the antibodies or pharmaceutical preparations [A-2] to [A-3].
[A-5] Any one of the antibodies or pharmaceutical preparations of [A-2] to [A-3], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, heavy chain CDR 2 of SEQ ID NO: 34, heavy chain CDR 3 of SEQ ID NO: 48, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73.
[A-6] The antibody or pharmaceutical preparation of [A-4], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.
[A-7] A third antigen-binding moiety,
Any one of the antibodies or pharmaceutical preparations of [A-2] to [A-6], comprising: (a) an antibody variable region comprising a VH comprising SEQ ID NO: 232 and a VL comprising SEQ ID NO: 236; or (b) an antibody variable region comprising a VH having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 232 and a VL having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 236.
[A-8] Any one of the antibodies or pharmaceutical preparations of [A-2] to [A-7], wherein each of the first antigen-binding portion and the second antigen-binding portion is a Fab having a cysteine residue at position 191 (EU numbering), and a disulfide bond is present linking the two cysteine residues.
[A-9] Any one of the antibodies or pharmaceutical preparations of [A-2] to [A-8], wherein each of the first, second, and third antigen-binding moieties is a Fab comprising a heavy chain comprising a VH and CH1 domain and a light chain comprising a VL and a light chain constant (CL) domain, and the C-terminus of the heavy chain of the third antigen-binding moiety is fused, directly or via a peptide linker, to the N-terminus of the Fab heavy chain of either the first antigen-binding moiety or the second antigen-binding moiety.
[A-10-1] The antibody or pharmaceutical preparation of [A-9], wherein the C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion via a peptide linker, and the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259.
[A-10-2] The antibody or pharmaceutical preparation of [A-10-1], wherein the third antigen-binding portion is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and each of the first antigen-binding portion and the second antigen-binding portion is a conventional Fab molecule.
[A-11] The antibody or pharmaceutical preparation of [A-10-2], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding portions are arginine and lysine, respectively; and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding portions are glutamic acid.
[A-12] Any one of the antibodies or pharmaceutical preparations of [A2] to [A-11], further comprising an Fc domain.
[A-13] The Fc domain comprises a first and a second Fc region subunit;
The first Fc region subunit comprises:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
and a second Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 354, and a tryptophan at position 366, and the second Fc region subunit is selected from the group consisting of:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 349, a serine at position 366, an alanine at position 368, and a valine at position 407;
All positions are according to EU numbering,
[A-12] An antibody or pharmaceutical preparation.
[A-14] The antibody is selected from the group consisting of the following (A) to (C):
(A) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:208, and two polypeptide chains (chains 4 and 5), each comprising the amino acid sequence of SEQ ID NO:214;
(B) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214; and (C) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
The antibody or pharmaceutical preparation of [A-2], which contains five polypeptide chains in a combination selected from the following:
[A-15] The antibody comprises the following combination:
A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:208, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
An antibody or pharmaceutical preparation of [A-2], which contains five polypeptide chains.
[A-16] The antibody comprises the following combination:
A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
An antibody or pharmaceutical preparation of [A-2], which contains five polypeptide chains.
[A-17] The antibody comprises the following combination:
A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
An antibody or pharmaceutical preparation of [A-2], which contains five polypeptide chains.
[A-18] Any one of the antibodies or pharmaceutical preparations of [A-14] to [A-17], in which the five polypeptide chains (chain 1 to chain 5) of the antibody are connected and/or bound to each other as shown in Figure 1 (a).
[A-19] Any one of the antibodies or pharmaceutical preparations of [A-14] to [A-17], in which each of polypeptide chain 2 and chain 5 is bound to polypeptide chain 1; polypeptide chain 4 is bound to polypeptide chain 3; and polypeptide chain 1 is bound to polypeptide chain 3.
[A-20] An antibody or pharmaceutical preparation of any one of [A-2] to [A-17] for use in combination with an additional therapeutic agent, preferably a chemotherapeutic agent or an immune checkpoint inhibitor.
[A-21] The antibody or pharmaceutical preparation of [A-20] for use in combination with an immune checkpoint inhibitor which is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody, more preferably atezolizumab.
[A-22] An antibody or pharmaceutical preparation of [A-20] for use in combination with a chemotherapeutic agent selected from the group consisting of microtubule disrupting agents, antimetabolites, topoisomerase inhibitors, DNA intercalating agents, alkylating agents, hormonal therapy, kinase inhibitors, receptor antagonists, activators of tumor cell apoptosis, antiangiogenic agents, etoposide, irinotecan, lurbinectedin, amrubicin, and platinum agents (such as cisplatin and carboplatin).
[A-23] Any one of the antibodies or pharmaceutical preparations of [A-20] to [A-22], wherein an immune checkpoint inhibitor or a chemotherapeutic agent is administered simultaneously with the antibody or pharmaceutical preparation.
[A-24] Any one of the antibodies or pharmaceutical preparations of [A-20] to [A-23], wherein an immune checkpoint inhibitor or a chemotherapeutic agent is administered before or after administration of the antibody or pharmaceutical preparation.
[A-25] Any one of the antibodies or pharmaceutical preparations of [A-2] to [A-24] for use in treating cancer, wherein the cancer is a DLL3-expressing cancer or a DLL3-positive cancer.
[A-26] The antibody or pharmaceutical preparation of [A-25], wherein the cancer is selected from the group consisting of neuroendocrine neoplasms (NENs), neuroendocrine tumors (NETs), neuroendocrine carcinomas (NECs), and other solid tumors of non-neuroendocrine origin.
[A-27] The antibody or pharmaceutical preparation of [A-25], wherein the cancer is selected from the group consisting of pancreatic neuroendocrine tumor (PNET), small cell NEC (SCNEC), large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine carcinoma, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma (GBM), melanoma, and medullary thyroid carcinoma.
[A-28] The antibody or pharmaceutical preparation of [A-25], wherein the cancer is lung cancer, preferably SCLC.
[A-29] The antibody or pharmaceutical preparation of [A-25], wherein the cancer is glioma or glioblastoma (GBM).
[A-30] The antibody or pharmaceutical preparation of [A-25], wherein the cancer is neuroendocrine prostate cancer.
[A-31] An antibody or pharmaceutical preparation of [A-25], wherein the cancer is neuroblastoma.
[A-32] Any one of the antibodies or pharmaceutical preparations of [A-2] to [A-31], wherein the antibody is a multispecific antibody.
[A-33] The antibody or pharmaceutical preparation for use according to [A-21], further combined with a chemotherapeutic agent selected from the group consisting of microtubule disrupting agents, antimetabolites, topoisomerase inhibitors, DNA intercalating agents, alkylating agents, hormonal therapy, kinase inhibitors, receptor antagonists, activators of tumor cell apoptosis, antiangiogenic agents, etoposide, irinotecan, lurbinectedin, amrubicin, and platinum agents (such as cisplatin and carboplatin).
[A-34] The antibody or pharmaceutical preparation of [A-22], wherein the immune checkpoint inhibitor is further combined with an immune checkpoint inhibitor that is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody, more preferably atezolizumab.
[B-1]
(a) a first antigen-binding moiety and a second antigen-binding moiety, each of which comprises an antibody variable region, which may be the same or different, and which antibody variable region is selected from the group consisting of:
(a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, a heavy chain CDR 2 of SEQ ID NO: 34, a heavy chain CDR 3 of SEQ ID NO: 48, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a2) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, a heavy chain CDR 2 of SEQ ID NO: 42, a heavy chain CDR 3 of SEQ ID NO: 56, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a3) a first antigen-binding portion and a second antigen-binding portion independently selected from an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 82, heavy chain CDR 2 of SEQ ID NO: 83, heavy chain CDR 3 of SEQ ID NO: 84, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75; and (b) a third antigen-binding portion that binds human delta-like 3 (DLL3) and comprises an antibody variable region comprising heavy chain CDR 1 comprising SEQ ID NO: 233, heavy chain CDR 2 comprising SEQ ID NO: 234, heavy chain CDR 3 comprising SEQ ID NO: 235, light chain CDR 1 comprising SEQ ID NO: 237, light chain CDR 2 comprising SEQ ID NO: 238, and light chain CDR 3 comprising SEQ ID NO: 239.
[B-2] Each of the first antigen-binding portion and the second antigen-binding portion has the following properties:
(i) binds to human CD3;
(ii) binds to human CD137;
(iii) a multispecific antibody of [B-1] that is capable of binding to both human CD3 and human CD137, and each of the first and second antigen-binding portions binds to either human CD3 or human CD137; and (iv) a multispecific antibody of [B-1] that is capable of binding to both human CD3 and human CD137, but does not bind to both human CD3 and human CD137 simultaneously.
[B-3] Each of the first antigen-binding portion and the second antigen-binding portion comprises an antibody variable region, which may be the same or different, and the antibody variable region is selected from the group consisting of:
(a1) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 58;
(a2) VH comprising the amino acid sequence of SEQ ID NO: 14, and VL comprising the amino acid sequence of SEQ ID NO: 58;
(a3) VH comprising the amino acid sequence of SEQ ID NO: 81, and VL comprising the amino acid sequence of SEQ ID NO: 60;
(a4) a VH and a VL comprising one or more amino acid substitutions, deletions, additions, and/or insertions in the VH and VL of any one of (a1) to (a3) and having an activity equivalent to that of any of said VH and VL; and (a5) a VH and a VL having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of the VH and VL defined in any one of (a1) to (a3).
More independently selected,
Any one of the multispecific antibodies [B-1] to [B-2].
[B-4] Any one of the multispecific antibodies of [B-1] to [B-2], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, heavy chain CDR 2 of SEQ ID NO: 34, heavy chain CDR 3 of SEQ ID NO: 48, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73.
[B-5] The multispecific antibody of [B-3], wherein the first antigen-binding portion and the second antigen-binding portion each comprise an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.
[B-6] A third antigen-binding moiety,
Any one of the multispecific antibodies [B-1] to [B-5], comprising: (a) an antibody variable region comprising a VH comprising SEQ ID NO: 232 and a VL comprising SEQ ID NO: 236; or (b) an antibody variable region comprising a VH having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 232 and a VL having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 236.
[B-7] Any one of the multispecific antibodies of [B-1] to [B-6], in which each of the first antigen-binding portion and the second antigen-binding portion is a Fab having a cysteine residue at position 191 (EU numbering), and a disulfide bond is present linking the two cysteine residues.
[B-8] Any one of the multispecific antibodies of [B-1] to [B-7], wherein each of the first, second, and third antigen-binding moieties is a Fab comprising a heavy chain comprising a VH and CH1 domain and a light chain comprising a VL and a light chain constant (CL) domain, and the C-terminus of the heavy chain of the third antigen-binding moiety is fused, directly or via a peptide linker, to the N-terminus of the Fab heavy chain of either the first antigen-binding moiety or the second antigen-binding moiety.
[B-9-1] A multispecific antibody according to [B-8], in which the C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion via a peptide linker, and the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259.
[B-9-2] The multispecific antibody of [B-9-1], in which the third antigen-binding portion is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and each of the first antigen-binding portion and the second antigen-binding portion is a conventional Fab molecule.
[B-10] The multispecific antibody of [B-9-2], in which the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding portions are arginine and lysine, respectively; and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding portions are glutamic acid.
[B-11] Any one of the multispecific antibodies of [B-1] to [B-10], further comprising an Fc domain.
[B-12] The Fc domain comprises a first and a second Fc region subunit, and the first Fc region subunit is:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
and a second Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 354, and a tryptophan at position 366, and the second Fc region subunit is selected from the group consisting of:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 349, a serine at position 366, an alanine at position 368, and a valine at position 407;
All positions are according to EU numbering,
[B-11] Multispecific antibody.
[B-13] The antibody is selected from the group consisting of the following (A) to (C):
(A) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:208, and two polypeptide chains (chains 4 and 5), each comprising the amino acid sequence of SEQ ID NO:214;
(B) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214; and (C) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
A multispecific antibody [B-1] comprising five polypeptide chains in a combination selected from the following:
[B-14] The antibody comprises the following combination:
A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:208, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
A multispecific antibody [B-1] containing five polypeptide chains.
[B-15] The antibody comprises the following combination:
A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
A multispecific antibody [B-1] containing five polypeptide chains.
[B-16] The antibody comprises the following combination:
A polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
A multispecific antibody [B-1] containing five polypeptide chains.
[B-17] A multispecific antibody selected from any one of [B-13] to [B-16], in which the five polypeptide chains (chain 1 to chain 5) of the antibody are connected and/or bound to each other as shown in Figure 1 (a).
[B-18] Any one of the multispecific antibodies [B-13] to [B-16], in which each of polypeptide chain 2 and chain 5 is bound to polypeptide chain 1; polypeptide chain 4 is bound to polypeptide chain 3; and polypeptide chain 1 is bound to polypeptide chain 3.
[B-19] A multispecific antibody comprising any one of [B-1] to [B-18] for use as a pharmaceutical.
[B-20] A multispecific antibody comprising any one of [B-1] to [B-18] for use in the treatment of cancer.
[C-1] A pharmaceutical formulation comprising any one of the multispecific antibodies [B-1] to [B-18] and a pharma- ceutical acceptable carrier.
[C-2] A pharmaceutical formulation comprising an immune checkpoint inhibitor and a pharma- ceutical acceptable carrier for use in combination with any one of the multispecific antibodies [B-1] to [B-18] for the treatment of cancer.
[C-3] A pharmaceutical formulation comprising a chemotherapeutic agent and a pharma- ceutical acceptable carrier for use in combination with any one of the multispecific antibodies [B-1] to [B-18] for the treatment of cancer.
[C-4] The pharmaceutical formulation of [C-2], wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody, more preferably atezolizumab.
[C-5] The pharmaceutical formulation of [C-3], wherein the chemotherapeutic agent is selected from the group consisting of microtubule disrupting agents, antimetabolites, topoisomerase inhibitors, DNA intercalating agents, alkylating agents, hormone therapy, kinase inhibitors, receptor antagonists, activators of tumor cell apoptosis, antiangiogenic agents, etoposide, irinotecan, lurbinectedin, amrubicin, and platinum agents (such as cisplatin and carboplatin).
[C-6] Any one of the pharmaceutical formulations of [C-2] to [C-5], in which an immune checkpoint inhibitor or a chemotherapeutic agent is administered simultaneously with the multispecific antibody.
[C-7] Any one of the pharmaceutical formulations of [C-2] to [C-5], wherein an immune checkpoint inhibitor or a chemotherapeutic agent is administered before or after administration of the multispecific antibody.
[D-1] Use of any one of the multispecific antibodies [B-1] to [B-18] or any one of the pharmaceutical preparations [C-1] to [C-7] in the treatment of cancer.
[E-1] A method for treating an individual having cancer, comprising administering to the individual an effective amount of any one of the multispecific antibodies [B-1] to [B-18] or any one of the pharmaceutical preparations [C-1] to [C-7].
[E-2] A method for activating or enhancing a sustained immune response against cancer, comprising administering to a subject a therapeutically effective amount of any one of the multispecific antibodies [B-1] to [B-18] or any one of the pharmaceutical preparations [C-1] to [C-7].
[E-3] A method for increasing the efficacy of an immune checkpoint inhibitor, comprising administering to a subject a therapeutically effective amount of any one of the multispecific antibodies [B-1] to [B-18] or any one of the pharmaceutical preparations [C-1] to [C-7] in combination with the immune checkpoint inhibitor.
[E-4] A method for reducing or preventing metastasis in a subject, comprising administering to the subject a therapeutically effective amount of any one of the multispecific antibodies [B-1] to [B-18] or any one of the pharmaceutical preparations [C-1] to [C-7].
[E-5] A method for suppressing tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of any one of the multispecific antibodies [B-1] to [B-18] or any one of the pharmaceutical preparations [C-1] to [C-7].
[E-6] Any one of the methods of [E-1] to [E-5], further comprising the step of administering to the subject an additional therapeutic agent, preferably a chemotherapeutic agent or an immune checkpoint inhibitor.
[E-7] The method of [E-6], wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody, more preferably atezolizumab.
[E-8] The method of [E-6], wherein the chemotherapeutic agent is selected from the group consisting of microtubule disrupting agents, antimetabolites, topoisomerase inhibitors, DNA intercalating agents, alkylating agents, hormone therapy, kinase inhibitors, receptor antagonists, activators of tumor cell apoptosis, antiangiogenic agents, etoposide, irinotecan, lurbinectedin, amrubicin, and platinum agents (such as cisplatin and carboplatin).
[E-9] Any one of the methods of [E-6] to [E-8], wherein an additional therapeutic agent, immune checkpoint inhibitor, or chemotherapeutic agent is administered simultaneously with the multispecific antibody or pharmaceutical formulation.
[E-10] Any one of the methods of [E-6] to [E-8], wherein an additional therapeutic agent, immune checkpoint inhibitor, or chemotherapeutic agent is administered before or after administration of the multispecific antibody or pharmaceutical formulation.
[F-1] Use of any one of the multispecific antibodies [B-1] to [B-18] or any one of the pharmaceutical preparations [C-1] to [C-3] in the manufacture of a medicine for the treatment of cancer.
[F-2] Use of any one of the multispecific antibodies [B-1] to [B-18] or any one of the pharmaceutical preparations [C-1] to [C-3] in combination with an additional therapeutic agent (preferably a chemotherapeutic agent or an immune checkpoint inhibitor) in the manufacture of a medicament for the treatment of cancer.
[F-3] Use of [F-2], wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist, preferably an anti-PD-1 antibody or an anti-PD-L1 antibody, more preferably atezolizumab.
[F-4] Use of [F-2], wherein the chemotherapeutic agent is selected from the group consisting of microtubule disrupting agents, antimetabolites, topoisomerase inhibitors, DNA intercalating agents, alkylating agents, hormonal therapy, kinase inhibitors, receptor antagonists, activators of tumor cell apoptosis, antiangiogenic agents, etoposide, irinotecan, lurbinectedin, amrubicin, and platinum agents (such as cisplatin and carboplatin).
[G-1] Any one of the pharmaceutical preparations [C-1] to [C-7], or any one of the methods or uses [D-1] to [F-4], wherein the cancer is a DLL3-expressing cancer or a DLL3-positive cancer.
[G-2] Any one of the pharmaceutical preparations of [C-1] to [C-7], or any one of the methods or uses of [D-1] to [F-4], wherein the cancer is selected from the group consisting of neuroendocrine neoplasms (NEN), neuroendocrine tumors (NET), neuroendocrine carcinomas (NEC), or other solid tumors of non-neuroendocrine origin.
[G-3] Any one of the pharmaceutical preparations of [C-1] to [C-7], or any one of the methods or uses of [D-1] to [F-4], wherein the cancer is selected from the group consisting of pancreatic neuroendocrine tumor (PNET), small cell neoplasm (SCNEC), large cell neoplasm (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine cancer, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma (GBM), melanoma, and medullary thyroid carcinoma.
[G-4] Any one of the pharmaceutical preparations of [C-1] to [C-7], or any one of the methods or uses of [D-1] to [F-4], wherein the cancer is lung cancer, preferably SCLC.
[G-5] Any one of the pharmaceutical preparations [C-1] to [C-7], or any one of the methods or uses [D-1] to [F-4], wherein the cancer is glioma or glioblastoma (GBM).
[G-6] Any one of the pharmaceutical preparations [C-1] to [C-7], or any one of the methods or uses [D-1] to [F-4], wherein the cancer is neuroendocrine prostate cancer.
[G-7] Any one of the pharmaceutical preparations [C-1] to [C-7], or any one of the methods or uses [D-1] to [F-4], wherein the cancer is neuroblastoma.

In another aspect, the present disclosure provides:
[1] A first antigen-binding portion and a second antigen-binding portion, each of which has the following properties:
(i) binds to human CD3;
(ii) binds to human CD137;
(iii) a first antigen-binding moiety and a second antigen-binding moiety capable of binding to either human CD3 or human CD137, each of which binds to either human CD3 or human CD137; and (iv) a first antigen-binding moiety and a second antigen-binding moiety capable of binding to either human CD3 or human CD137, but not simultaneously to human CD3 and human CD137; and a third antigen-binding moiety capable of binding to a third antigen, preferably an antigen expressed on a cancer cell/tissue, more preferably DLL3, even more preferably human DLL3.
[2] The first antigen-binding portion and the second antigen-binding portion each are any one of the following (a1) to (a17):
(a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 17, a heavy chain CDR 2 of SEQ ID NO: 31, a heavy chain CDR 3 of SEQ ID NO: 45, a light chain CDR 1 of SEQ ID NO: 64, a light chain CDR 2 of SEQ ID NO: 69, and a light chain CDR 3 of SEQ ID NO: 74;
(a2) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 18, a heavy chain CDR 2 of SEQ ID NO: 32, a heavy chain CDR 3 of SEQ ID NO: 46, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a3) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, a heavy chain CDR 2 of SEQ ID NO: 33, a heavy chain CDR 3 of SEQ ID NO: 47, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a4) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, a heavy chain CDR 2 of SEQ ID NO: 33, a heavy chain CDR 3 of SEQ ID NO: 47, a light chain CDR 1 of SEQ ID NO: 65, a light chain CDR 2 of SEQ ID NO: 70, and a light chain CDR 3 of SEQ ID NO: 75;
(a5) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, a heavy chain CDR 2 of SEQ ID NO: 34, a heavy chain CDR 3 of SEQ ID NO: 48, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a6) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 22, a heavy chain CDR 2 of SEQ ID NO: 36, a heavy chain CDR 3 of SEQ ID NO: 50, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a7) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, a heavy chain CDR 2 of SEQ ID NO: 37, a heavy chain CDR 3 of SEQ ID NO: 51, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a8) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, a heavy chain CDR 2 of SEQ ID NO: 37, a heavy chain CDR 3 of SEQ ID NO: 51, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76;
(a9) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 24, a heavy chain CDR 2 of SEQ ID NO: 38, a heavy chain CDR 3 of SEQ ID NO: 52, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a10) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 25, a heavy chain CDR 2 of SEQ ID NO: 39, a heavy chain CDR 3 of SEQ ID NO: 53, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76;
(a11) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, a heavy chain CDR 2 of SEQ ID NO: 40, a heavy chain CDR 3 of SEQ ID NO: 54, a light chain CDR 1 of SEQ ID NO: 66, a light chain CDR 2 of SEQ ID NO: 71, and a light chain CDR 3 of SEQ ID NO: 76;
(a12) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, a heavy chain CDR 2 of SEQ ID NO: 40, a heavy chain CDR 3 of SEQ ID NO: 54, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a13) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 27, a heavy chain CDR 2 of SEQ ID NO: 41, a heavy chain CDR 3 of SEQ ID NO: 55, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a14) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, a heavy chain CDR 2 of SEQ ID NO: 42, a heavy chain CDR 3 of SEQ ID NO: 56, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a15) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 82, a heavy chain CDR 2 of SEQ ID NO: 83, a heavy chain CDR 3 of SEQ ID NO: 84, a light chain CDR 1 of SEQ ID NO: 65, a light chain CDR 2 of SEQ ID NO: 70, and a light chain CDR 3 of SEQ ID NO: 75;
(a16) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a17) an antibody variable region that comprises an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15). The multispecific antigen-binding molecule of [1].
[3] The first antigen-binding portion and the second antigen-binding portion each are any one of the following (a1) to (a18):
(a1) an antibody variable region comprising a VH comprising SEQ ID NO: 3 and a VL comprising SEQ ID NO: 59;
(a2) an antibody variable region comprising a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 58;
(a3) an antibody variable region comprising a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 58;
(a4) an antibody variable region comprising a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 60;
(a5) an antibody variable region comprising a VH comprising SEQ ID NO: 6 and a VL comprising SEQ ID NO: 58;
(a6) an antibody variable region comprising a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 58;
(a7) an antibody variable region comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 58;
(a8) an antibody variable region comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 61;
(a9) an antibody variable region comprising a VH comprising SEQ ID NO: 10 and a VL comprising SEQ ID NO: 58;
(a10) an antibody variable region comprising a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 61;
(a11) an antibody variable region comprising a VH comprising SEQ ID NO: 12 and a VL comprising SEQ ID NO: 61;
(a12) an antibody variable region comprising a VH comprising SEQ ID NO: 12 and a VL comprising SEQ ID NO: 58;
(a13) an antibody variable region comprising a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 58;
(a14) an antibody variable region comprising a VH comprising SEQ ID NO: 14 and a VL comprising SEQ ID NO: 58; and (a15) an antibody variable region comprising a VH comprising SEQ ID NO: 81 and a VL comprising SEQ ID NO: 60;
(a16) an antibody variable region comprising a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6, 8 to 14, and 81, and/or a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 61;
(a17) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a18) an antibody variable region that comprises an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15). Any one of the multispecific antigen-binding molecules of [1] to [2].
[4] The multispecific antigen-binding molecule of any one of [1] to [3], wherein each of the first antigen-binding portion and the second antigen-binding portion is a Fab molecule and comprises at least one disulfide bond formed between the CH1 region of the first antigen-binding portion and the CH1 region of the second antigen-binding portion.
[4A] The multispecific antigen-binding molecule of [4], wherein each of the first antigen-binding portion and the second antigen-binding portion is a Fab molecule and comprises one disulfide bond formed between the amino acid residue at position 191 according to EU numbering in the CH1 region of each of the first antigen-binding portion and the second antigen-binding portion.
[5] The multispecific antigen-binding molecule of any one of [1] to [4A], wherein a third antigen-binding portion is fused to either the first antigen-binding portion or the second antigen-binding portion.
[5A] The multispecific antigen-binding molecule of [5], wherein the third antigen-binding portion is a Fab or scFv.
[6] Any one of the multispecific antigen-binding molecules of [5] to [5A], wherein each of the first, second, and third antigen-binding moieties is a Fab molecule, and the third antigen-binding moiety is fused at the C-terminus (CH1) of the Fab heavy chain to the N-terminus of the Fab heavy chain of either the first antigen-binding moiety or the second antigen-binding moiety, optionally via a peptide linker.
[6A] The multispecific antigen-binding molecule of any one of [5] to [6], wherein the peptide linker is selected from the group consisting of the amino acid sequences of SEQ ID NO: 248, SEQ ID NO: 249, or SEQ ID NO: 259.
[6B] The multispecific antigen-binding molecule of any one of [1] to [6A], wherein the first antigen-binding portion is identical to the second antigen-binding portion.
[7] The multispecific antigen-binding molecule of any one of [1] to [6B], wherein the third antigen-binding portion is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and each of the first and second antigen-binding portions is a conventional Fab molecule.
[8] A multispecific antigen-binding molecule according to [7], wherein in the constant domain CL of the light chain of each of the first and second antigen-binding moieties, the amino acids at positions 123 and/or 124 are independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the heavy chain of each of the first and second antigen-binding moieties, the amino acids at positions 147 and/or 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).
[9] A multispecific antigen-binding molecule comprising the first and second antigen-binding moieties, each of which has a light-chain constant domain CL in which the amino acids at positions 123 and 124 are arginine (R) and lysine (K), respectively (Kabat numbering), and the first and second antigen-binding moieties, each of which has a heavy-chain constant domain CH1 in which the amino acids at positions 147 and 213 are glutamic acid (E), respectively (Kabat EU index numbering).[8]
[10] The third antigen-binding moiety capable of binding to DLL3 is any one of the following (a1) to (a5):
(a1) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 233, heavy chain CDR 2 of SEQ ID NO: 234, heavy chain CDR 3 of SEQ ID NO: 235, light chain CDR 1 of SEQ ID NO: 237, light chain CDR 2 of SEQ ID NO: 238, and light chain CDR 3 of SEQ ID NO: 239;
(a2) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 276, heavy chain CDR 2 of SEQ ID NO: 277, heavy chain CDR 3 of SEQ ID NO: 278, light chain CDR 1 of SEQ ID NO: 279, light chain CDR 2 of SEQ ID NO: 280, and light chain CDR 3 of SEQ ID NO: 281;
(a3) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 285, heavy chain CDR 2 of SEQ ID NO: 286, heavy chain CDR 3 of SEQ ID NO: 287, light chain CDR 1 of SEQ ID NO: 288, light chain CDR 2 of SEQ ID NO: 289, and light chain CDR 3 of SEQ ID NO: 290;
(a4) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a3); and (a5) an antibody variable region that comprises an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a3). Any one of the multispecific antigen-binding molecules of [1] to [9].
[11] The third antigen-binding moiety capable of binding to DLL3 is any one of the following (a1) to (a6):
(a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 232, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 236;
(a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 264, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 265;
(a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 266, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 267;
(a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 268, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 269;
(a5) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a4); and (a6) an antibody variable region that comprises an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a4). Any one of the multispecific antigen-binding molecules of [1] to [10].
[12] Any one of the multispecific antigen-binding molecules of [1] to [11], further comprising an Fc domain.
[12A] A multispecific antigen-binding molecule according to [12], wherein the Fc domain is composed of a first and a second Fc region subunit capable of stable association, and the Fc domain exhibits reduced binding affinity for human Fcγ receptors compared to a native human IgG1 Fc domain.
[12B] The first Fc domain subunit comprises:
(a1) an Fc region polypeptide comprising Ala at position 234 and Ala at position 235;
(a2) an Fc region polypeptide comprising Ala at position 234, Ala at position 235, and Ala at position 297;
(a3) an Fc region polypeptide comprising Ala at position 234, Ala at position 235, Ala at position 297, Cys at position 354, and Trp at position 366; and wherein the second Fc region polypeptide is selected from the group consisting of:
(a4) an Fc region polypeptide comprising Ala at position 234 and Ala at position 235;
(a5) an Fc region polypeptide comprising Ala at position 234, Ala at position 235, and Ala at position 297;
(a6) an Fc region polypeptide comprising Ala at position 234, Ala at position 235, Ala at position 297, Cys at position 349, Ser at position 366, Ala at position 368, and Val at position 407;
A multispecific antigen-binding molecule of [12A], in which amino acid positions are numbered using EU index numbering.
[12C] Any one of the multispecific antigen-binding molecules of [12] to [12B], wherein the Fc domain exhibits enhanced FcRn-binding activity under acidic pH conditions (e.g., pH 5.8) compared to that of the Fc region of native IgG.
[12D] The multispecific antigen-binding molecule of [12C], wherein the Fc domain comprises Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, according to EU numbering.
[12E] The multispecific antigen-binding molecule of [12D], wherein the Fc domain comprises, according to EU numbering, Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440.
[12F] The multispecific antigen-binding molecule of [12E], wherein the Fc domain further comprises Ile or Leu at position 428; and/or Ile, Leu, Val, Thr, or Phe at position 436, according to EU numbering.
[12G] The Fc domain is:
(a) N434A/Q438R/S440E;
(b) N434A/Q438R/S440D;
(c) N434A/Q438K/S440E;
(d) N434A/Q438K/S440D;
(e) N434A/Y436T/Q438R/S440E;
(f) N434A/Y436T/Q438R/S440D;
(g) N434A/Y436T/Q438K/S440E;
(h) N434A/Y436T/Q438K/S440D;
(i) N434A/Y436V/Q438R/S440E;
(j) N434A/Y436V/Q438R/S440D;
(k) N434A/Y436V/Q438K/S440E;
(l) N434A/Y436V/Q438K/S440D;
(m) N434A/R435H/F436T/Q438R/S440E;
(n) N434A/R435H/F436T/Q438R/S440D;
(o) N434A/R435H/F436T/Q438K/S440E;
(p) N434A/R435H/F436T/Q438K/S440D;
(q) N434A/R435H/F436V/Q438R/S440E;
(r)N434A/R435H/F436V/Q438R/S440D;
(s)N434A/R435H/F436V/Q438K/S440E;
(t)N434A/R435H/F436V/Q438K/S440D;
(u)M428L/N434A/Q438R/S440E;
(v) M428L/N434A/Q438R/S440D;
(w) M428L/N434A/Q438K/S440E;
(x)M428L/N434A/Q438K/S440D;
(y) M428L/N434A/Y436T/Q438R/S440E;
(z) M428L/N434A/Y436T/Q438R/S440D;
(aa) M428L/N434A/Y436T/Q438K/S440E;
(ab) M428L/N434A/Y436T/Q438K/S440D;
(ac) M428L/N434A/Y436V/Q438R/S440E;
(ad) M428L/N434A/Y436V/Q438R/S440D;
(ae) M428L/N434A/Y436V/Q438K/S440E;
(af) M428L/N434A/Y436V/Q438K/S440D;
(ag) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (ah) L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E
The multispecific antigen-binding molecule of any one of [12C] to [12F], comprising a combination of amino acid substitutions selected from the group consisting of:
[12H] Any one of the multispecific antigen-binding molecules of [12C] to [12G], wherein the Fc domain contains a combination of amino acid substitutions of M428L/N434A/Q438R/S440E.
[12I] The multispecific antigen-binding molecule of any one of [12] to [12H], wherein the Fc domain is an IgG Fc domain, preferably a human IgG Fc domain, more preferably a human IgG1 Fc domain.
[12J] The Fc domain:
Any one of the multispecific antigen-binding molecules of [12] to [12I], comprising: (a) a first Fc subunit having the amino acid sequence set forth in SEQ ID NO: 100 and a second Fc subunit having the amino acid sequence set forth in SEQ ID NO: 111; and (b) a first Fc subunit having the amino acid sequence set forth in SEQ ID NO: 99 and a second Fc subunit having the amino acid sequence set forth in SEQ ID NO: 109.
[12K] A multispecific antigen-binding molecule of any one of [12] to [12J], wherein each of the first and second antigen-binding moieties is a Fab, the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the remaining subunit of the Fc domain.
[12L] The multispecific antigen-binding molecule of [12K], in which a third antigen-binding portion is fused at the C-terminus to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion, optionally via a peptide linker.
[13] Any combination selected from (a1) to (a15) below:
(a1) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 208, and two polypeptide chains (chain 4 and chain 5), each comprising the amino acid sequence of SEQ ID NO: 214;
(a2) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a3) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a4) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 205, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a5) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 216, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 229, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a6) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 217, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 210, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a7) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 219, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a8) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 220, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a9) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 221, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a10) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 222, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 230, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a11) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 223, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 212, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215;
(a12) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 225, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215;
(a13) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 226, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215;
(a14) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 227, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215.
and
Preferably, five polypeptide chains (chain 1 to chain 5) are connected and/or associated with each other according to the orientation shown in FIG. 1(a) (more specifically, each of polypeptide chain 2 and chain 5 is associated with polypeptide chain 1; polypeptide chain 4 is associated with polypeptide chain 3; and polypeptide chain 1 is associated with polypeptide chain 3),
Multispecific antigen binding molecules.
[14] An isolated polynucleotide or multiple polynucleotides encoding any one of the multispecific antigen-binding molecules of [1] to [13].
[15] A vector encoding the polynucleotide or polynucleotides of [14].
[16] A host cell comprising the polynucleotide or polynucleotides of [14], or the vector of [15].
[17] A method for producing any one of the multispecific antigen-binding molecules of [1] to [13], comprising the steps of: a) culturing the host cell of [16] under conditions suitable for expression of the antigen-binding molecule; and b) recovering the antigen-binding molecule.
[17A] A multispecific antigen-binding molecule produced by the method of [17].
[18] A pharmaceutical composition comprising any one of the multispecific antigen-binding molecules of [1] to [13] and a pharma- ceutical acceptable carrier.
[19] A multispecific antigen-binding molecule according to any one of [1] to [13] or the pharmaceutical composition of [18], which induces cytotoxic activity, preferably T cell-dependent cytotoxic activity.
[20] A multispecific antigen-binding molecule according to any one of [1] to [13] or a pharmaceutical composition according to [18] for use as a medicament.
[21] A multispecific antigen-binding molecule of any one of [1] to [13] or a pharmaceutical composition of [18] for use in treating or preventing a disease in a subject in need thereof.
[22] The multispecific antigen-binding molecule or pharmaceutical composition for use in the treatment/prevention of the disease of [21], wherein the disease is cancer.
[22A] A multispecific antigen-binding molecule or pharmaceutical composition for use in the treatment/prevention of a disease in accordance with [22], wherein the cancer is a DLL3-expressing or DLL3-positive cancer, preferably a neuroendocrine neoplasm (NEN), neuroendocrine tumor (NET), neuroendocrine carcinoma (NEC), or other solid tumor of non-neuroendocrine origin.
[22B] A multispecific antigen-binding molecule or pharmaceutical composition for use in the treatment/prevention of a disease in accordance with [22] or [22A], wherein the cancer is selected from the group consisting of pancreatic neuroendocrine tumors (PNETs), small cell NEC (SCNEC), large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine carcinoma, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma (GBM), melanoma, and medullary thyroid carcinoma.
[23] Use of any one of the multispecific antigen-binding molecules of [1] to [13] or the pharmaceutical composition of [18] for the manufacture of a medicament for the treatment or prevention of a disease in an individual in need thereof.
[24] A method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising any one of the multispecific antigen-binding molecules of [1]-[13] or the pharmaceutical composition of [18].
[25] The use of [23] or the method of [24], wherein the disease is cancer, preferably DLL3-positive cancer or DLL3-expressing cancer.
[25A] The use or method of [25], wherein the cancer is a DLL3-expressing or DLL3-positive cancer, preferably a neuroendocrine neoplasm (NEN), neuroendocrine tumor (NET), neuroendocrine carcinoma (NEC), or other solid tumor of non-neuroendocrine origin.
[25B] The use or method of [25A], wherein the cancer is selected from the group consisting of pancreatic neuroendocrine tumors (PNETs), small cell NEC (SCNEC), large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine carcinoma, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma (GBM), melanoma, and medullary thyroid carcinoma.
[26] A method for inducing lysis of a target cell, comprising contacting the target cell with any one of the multispecific antigen-binding molecules of [1] to [13] or the pharmaceutical composition of [18] in the presence of a T cell.
[27] [18] A kit comprising the composition of claim 1; and a package insert containing instructions for administering the composition to a subject to treat or slow the progression of cancer, preferably a DLL3-positive or DLL3-expressing cancer, preferably a neuroendocrine neoplasm (NEN), neuroendocrine tumor (NET), neuroendocrine carcinoma (NEC), or other solid tumor of non-neuroendocrine origin.
[27A] The kit of [27], wherein the cancer is selected from the group consisting of pancreatic neuroendocrine tumor (PNET), small cell NEC (SCNEC), large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine carcinoma, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma (GBM), melanoma, and medullary thyroid carcinoma.

Another aspect of the present invention relates to:
[28] A first antigen-binding portion, comprising:
(i) binds to human CD3;
(ii) binds to human CD137;
(iii) a first antigen-binding moiety capable of binding to human CD3 and human CD137, wherein the first antigen-binding moiety binds to either human CD3 or human CD137; and (iv) a first antigen-binding moiety capable of binding to human CD3 and human CD137, but not simultaneously to human CD3 and human CD137; and
A multispecific antigen-binding molecule comprising a second antigen-binding portion capable of binding to DLL3, preferably human DLL3.
[29] The first antigen-binding portion is any one of the following (a1) to (a17):
(a1) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 17, heavy chain CDR 2 of SEQ ID NO: 31, heavy chain CDR 3 of SEQ ID NO: 45, light chain CDR 1 of SEQ ID NO: 64, light chain CDR 2 of SEQ ID NO: 69, and light chain CDR 3 of SEQ ID NO: 74;
(a2) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 18, heavy chain CDR 2 of SEQ ID NO: 32, heavy chain CDR 3 of SEQ ID NO: 46, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a3) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, heavy chain CDR 2 of SEQ ID NO: 33, heavy chain CDR 3 of SEQ ID NO: 47, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a4) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, heavy chain CDR 2 of SEQ ID NO: 33, heavy chain CDR 3 of SEQ ID NO: 47, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75;
(a5) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, heavy chain CDR 2 of SEQ ID NO: 34, heavy chain CDR 3 of SEQ ID NO: 48, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a6) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 22, heavy chain CDR 2 of SEQ ID NO: 36, heavy chain CDR 3 of SEQ ID NO: 50, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a7) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a8) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76;
(a9) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 24, heavy chain CDR 2 of SEQ ID NO: 38, heavy chain CDR 3 of SEQ ID NO: 52, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a10) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 25, heavy chain CDR 2 of SEQ ID NO: 39, heavy chain CDR 3 of SEQ ID NO: 53, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76;
(a11) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, heavy chain CDR 2 of SEQ ID NO: 40, heavy chain CDR 3 of SEQ ID NO: 54, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76;
(a12) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 26, heavy chain CDR 2 of SEQ ID NO: 40, heavy chain CDR 3 of SEQ ID NO: 54, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a13) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 27, heavy chain CDR 2 of SEQ ID NO: 41, heavy chain CDR 3 of SEQ ID NO: 55, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a14) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, heavy chain CDR 2 of SEQ ID NO: 42, heavy chain CDR 3 of SEQ ID NO: 56, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a15) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 82, heavy chain CDR 2 of SEQ ID NO: 83, heavy chain CDR 3 of SEQ ID NO: 84, light chain CDR 1 of SEQ ID NO: 65, light chain CDR 2 of SEQ ID NO: 70, and light chain CDR 3 of SEQ ID NO: 75;
(a16) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a17) an antibody variable region that comprises an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15). [28] A multispecific antigen-binding molecule comprising the antibody variable fragment (a16) that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15).
[30] The first antigen-binding portion is any one of the following (a1) to (a18):
(a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59;
(a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60;
(a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a8) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61;
(a9) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a10) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61;
(a11) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61;
(a12) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a13) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a14) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a15) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60;
(a16) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6, 8 to 14, and 81, and/or a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 61;
(a17) An antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a15); and (a18) an antibody variable region that comprises an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a15). Any one of the multispecific antigen-binding molecules of [28] to [29].

In yet another aspect of the present invention, there is provided a method for treating a pulmonary artery disease comprising:
[31] A combination selected from (a1) to (a15) below:
(a1) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 208, and two polypeptide chains (chain 4 and chain 5), each comprising the amino acid sequence of SEQ ID NO: 214;
(a2) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a3) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a4) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 205, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 209, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a5) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 216, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 229, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a6) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 217, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 210, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a7) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 219, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a8) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 220, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a9) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 221, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 211, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a10) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 222, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 230, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 214;
(a11) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 223, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 212, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215;
(a12) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 225, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215;
(a13) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 226, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 215;
(a14) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 227, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 213, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215; and (a15) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 228, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 231, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 215.
wherein the five polypeptide chains (chain 1 to chain 5) are linked and/or associated with each other according to the orientation shown in FIG. 1(a).
[31A] Combination of:
A polypeptide chain (chain 1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-205 and 216-228; a polypeptide chain (chain 2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 206-207; a polypeptide chain (chain 3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 208-213 and 229-231; and two polypeptide chains (chain 4 & chain 5) each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 214 and 215.
A multispecific antigen-binding molecule comprising five polypeptide chains.
[31B] The multispecific antigen-binding molecule of [31] or [31A], wherein each of polypeptide chain 2 and chain 5 is bound to polypeptide chain 1; polypeptide chain 4 is bound to polypeptide chain 3; and polypeptide chain 1 is bound to polypeptide chain 3.

Yet another aspect of the present invention relates to:
[32] Any one of (a1) to (a5) below:
(a1) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 233, heavy chain CDR 2 of SEQ ID NO: 234, heavy chain CDR 3 of SEQ ID NO: 235, light chain CDR 1 of SEQ ID NO: 237, light chain CDR 2 of SEQ ID NO: 238, and light chain CDR 3 of SEQ ID NO: 239;
(a2) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 276, heavy chain CDR 2 of SEQ ID NO: 277, heavy chain CDR 3 of SEQ ID NO: 278, light chain CDR 1 of SEQ ID NO: 279, light chain CDR 2 of SEQ ID NO: 280, and light chain CDR 3 of SEQ ID NO: 281;
(a3) heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 285, heavy chain CDR 2 of SEQ ID NO: 286, heavy chain CDR 3 of SEQ ID NO: 287, light chain CDR 1 of SEQ ID NO: 288, light chain CDR 2 of SEQ ID NO: 289, and light chain CDR 3 of SEQ ID NO: 290;
(a4) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a3); and (a5) an antigen-binding molecule capable of binding to DLL3, comprising an antibody variable region that comprises an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a3).
[33] Any one of (a1) to (a6) below:
(a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 232, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 236;
(a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 264, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 265;
(a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 266, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 267;
(a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 268, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 269;
(a5) an antibody variable region that binds to the same epitope as any one of the antibody variable regions selected from (a1) to (a4); and (a6) an antigen-binding molecule capable of binding to DLL3, comprising an antibody variable region that comprises an antibody variable fragment that competes with the binding of any one of the antibody variable fragments selected from (a1) to (a4).

Yet another aspect of the present invention relates to:
[33-1] (a) a first antigen-binding portion and a second antigen-binding portion, each of which comprises an antibody variable region that binds to human CD137, which may be the same or different, and wherein the antibody variable region is selected from the group consisting of:
(a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 17, a heavy chain CDR 2 of SEQ ID NO: 31, a heavy chain CDR 3 of SEQ ID NO: 45, a light chain CDR 1 of SEQ ID NO: 64, a light chain CDR 2 of SEQ ID NO: 69, and a light chain CDR 3 of SEQ ID NO: 74;
(a2) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 18, a heavy chain CDR 2 of SEQ ID NO: 32, a heavy chain CDR 3 of SEQ ID NO: 46, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a3) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, a heavy chain CDR 2 of SEQ ID NO: 33, a heavy chain CDR 3 of SEQ ID NO: 47, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a4) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 19, a heavy chain CDR 2 of SEQ ID NO: 33, a heavy chain CDR 3 of SEQ ID NO: 47, a light chain CDR 1 of SEQ ID NO: 65, a light chain CDR 2 of SEQ ID NO: 70, and a light chain CDR 3 of SEQ ID NO: 75;
(a5) an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, heavy chain CDR 2 of SEQ ID NO: 34, heavy chain CDR 3 of SEQ ID NO: 48, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a6) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 22, a heavy chain CDR 2 of SEQ ID NO: 36, a heavy chain CDR 3 of SEQ ID NO: 50, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a7) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, a heavy chain CDR 2 of SEQ ID NO: 37, a heavy chain CDR 3 of SEQ ID NO: 51, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a8) an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 23, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 51, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76;
(a9) an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 24, heavy chain CDR 2 of SEQ ID NO: 38, heavy chain CDR 3 of SEQ ID NO: 52, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a10) an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 25, heavy chain CDR 2 of SEQ ID NO: 39, heavy chain CDR 3 of SEQ ID NO: 53, light chain CDR 1 of SEQ ID NO: 66, light chain CDR 2 of SEQ ID NO: 71, and light chain CDR 3 of SEQ ID NO: 76;
(a11) an 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;
(a12) an 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;
(a13) an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 27, heavy chain CDR 2 of SEQ ID NO: 41, heavy chain CDR 3 of SEQ ID NO: 55, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73;
(a14) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, a heavy chain CDR 2 of SEQ ID NO: 42, a heavy chain CDR 3 of SEQ ID NO: 56, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73; and (a15) a first antigen-binding portion and a second antigen-binding portion independently selected from an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 82, a heavy chain CDR 2 of SEQ ID NO: 83, a heavy chain CDR 3 of SEQ ID NO: 84, a light chain CDR 1 of SEQ ID NO: 65, a light chain CDR 2 of SEQ ID NO: 70, and a light chain CDR 3 of SEQ ID NO: 75; and (b) a heavy chain CDR 1 that binds human delta-like 3 (DLL3) and comprises SEQ ID NO: 233, a heavy chain CDR 1 that comprises SEQ ID NO: 234, and a heavy chain CDR 1 that comprises SEQ ID NO: 235. 2. a third antigen-binding portion comprising an antibody variable region comprising a heavy chain CDR 3 comprising SEQ ID NO: 235, a light chain CDR 1 comprising SEQ ID NO: 237, a light chain CDR 2 comprising SEQ ID NO: 238, and a light chain CDR 3 comprising SEQ ID NO: 239.
[33-2] Each of the first and second antigen-binding moieties comprises an antibody variable region, which may be the same or different, and the antibody variable region is selected from the group consisting of:
(a1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59;
(a2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60;
(a5) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a6) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a7) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a8) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61;
(a9) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a10) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61;
(a11) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61;
(a12) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a13) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(a14) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58;
(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; and (a16) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6, 8 to 14, and 81, and/or a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 61. A multispecific antigen-binding molecule of [33-1].
[33-2A] The first antigen-binding portion and the second antigen-binding portion each comprise:
Any one of the multispecific antigen-binding molecules of [33-1] to [33-2], comprising an antibody variable region comprising heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, heavy chain CDR 2 of SEQ ID NO: 34, heavy chain CDR 3 of SEQ ID NO: 48, light chain CDR 1 of SEQ ID NO: 63, light chain CDR 2 of SEQ ID NO: 68, and light chain CDR 3 of SEQ ID NO: 73.
[33-2B] The first antigen-binding portion and the second antigen-binding portion each comprise:
A multispecific antigen-binding molecule of [33-2A] comprising an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58.
[33-2C] A multispecific antigen-binding molecule according to any one of [33-1] to [33-2B], wherein the third antigen-binding portion comprises an antibody variable region comprising a VH comprising sequence number: 232 and a VL comprising sequence number: 236.
[33-3] A multispecific antigen-binding molecule of any one of [33-1] to [33-2C], wherein each of the first and second antigen-binding portions is a Fab having a cysteine residue at position 191 (EU numbering) and a disulfide bond is present connecting the two cysteine residues.
[33-4] A multispecific antigen-binding molecule according to [33-3], wherein each of the first, second, and third antigen-binding moieties is a Fab comprising a heavy chain comprising a VH and CH1 domain and a light chain comprising a VL and a light chain constant (CL) domain, and the C-terminus of the heavy chain of the third antigen-binding moiety is fused, directly or via a peptide linker, to the N-terminus of the Fab heavy chain of either the first antigen-binding moiety or the second antigen-binding moiety.
[33-5A] The multispecific antigen-binding molecule of [33-4], wherein the C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion via a peptide linker, and the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259.
[33-5B] The multispecific antigen-binding molecule of [33-5A], wherein the third antigen-binding portion is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and each of the first antigen-binding portion and the second antigen-binding portion is a conventional Fab molecule.
[33-6] A multispecific antigen-binding molecule according to [33-5A], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding moieties are arginine and lysine, respectively, and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding moieties are glutamic acid.
[33-7] A multispecific antigen-binding molecule of [33-6], further comprising an Fc domain.
[33-8] the Fc domain comprises a first and a second Fc region subunit;
The first Fc region subunit comprises:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
and a second Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 354, and a tryptophan at position 366, and the second Fc region subunit is selected from the group comprising:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 349, and a serine at position 366, an alanine at position 368, and a valine at position 407;
All positions are according to EU numbering,
[33-7] Multispecific antigen-binding molecule.

Yet another aspect of the present invention relates to:
[34-1] (a) a first antigen-binding portion and a second antigen-binding portion, each of which comprises an antibody variable region that binds to human CD3, which may be the same or different, and which is selected from the group consisting of the following (a1) to (a15):
(a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 comprising SEQ ID NO: 17, a heavy chain CDR 2 comprising SEQ ID NO: 31, a heavy chain CDR 3 comprising SEQ ID NO: 45, a light chain CDR 1 comprising SEQ ID NO: 64, a light chain CDR 2 comprising SEQ ID NO: 69, and a light chain CDR 3 comprising SEQ ID NO: 74;
(a2) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 18, a heavy chain CDR2 comprising SEQ ID NO: 32, a heavy chain CDR3 comprising SEQ ID NO: 46, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73;
(a3) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 19, a heavy chain CDR2 comprising SEQ ID NO: 33, a heavy chain CDR3 comprising SEQ ID NO: 47, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73;
(a4) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 19, a heavy chain CDR2 comprising SEQ ID NO: 33, a heavy chain CDR3 comprising SEQ ID NO: 47, a light chain CDR1 comprising SEQ ID NO: 65, a light chain CDR2 comprising SEQ ID NO: 70, and a light chain CDR3 comprising SEQ ID NO: 75;
(a5) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 20, a heavy chain CDR2 comprising SEQ ID NO: 34, a heavy chain CDR3 comprising SEQ ID NO: 48, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73;
(a6) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 22, a heavy chain CDR2 comprising SEQ ID NO: 36, a heavy chain CDR3 comprising SEQ ID NO: 50, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73;
(a7) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 23, a heavy chain CDR2 comprising SEQ ID NO: 37, a heavy chain CDR3 comprising SEQ ID NO: 51, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73;
(a8) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 23, a heavy chain CDR2 comprising SEQ ID NO: 37, a heavy chain CDR3 comprising SEQ ID NO: 51, a light chain CDR1 comprising SEQ ID NO: 66, a light chain CDR2 comprising SEQ ID NO: 71, and a light chain CDR3 comprising SEQ ID NO: 76;
(a9) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 24, a heavy chain CDR2 comprising SEQ ID NO: 38, a heavy chain CDR3 comprising SEQ ID NO: 52, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73;
(a10) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 25, a heavy chain CDR2 comprising SEQ ID NO: 39, a heavy chain CDR3 comprising SEQ ID NO: 53, a light chain CDR1 comprising SEQ ID NO: 66, a light chain CDR2 comprising SEQ ID NO: 71, and a light chain CDR3 comprising SEQ ID NO: 76;
(a11) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 26, a heavy chain CDR2 comprising SEQ ID NO: 40, a heavy chain CDR3 comprising SEQ ID NO: 54, a light chain CDR1 comprising SEQ ID NO: 66, a light chain CDR2 comprising SEQ ID NO: 71, and a light chain CDR3 comprising SEQ ID NO: 76;
(a12) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 26, a heavy chain CDR2 comprising SEQ ID NO: 40, a heavy chain CDR3 comprising SEQ ID NO: 54, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73;
(a13) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO: 27, a heavy chain CDR2 comprising SEQ ID NO: 41, a heavy chain CDR3 comprising SEQ ID NO: 55, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 68, and a light chain CDR3 comprising SEQ ID NO: 73;
(a14) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO:28, a heavy chain CDR2 comprising SEQ ID NO:42, a heavy chain CDR3 comprising SEQ ID NO:56, a light chain CDR1 comprising SEQ ID NO:63, a light chain CDR2 comprising SEQ ID NO:68, and a light chain CDR3 comprising SEQ ID NO:73; and (a15) an antibody variable region comprising a heavy chain CDR1 comprising SEQ ID NO:82, a heavy chain CDR2 comprising SEQ ID NO:83, a heavy chain CDR3 comprising SEQ ID NO:84, a light chain CDR1 comprising SEQ ID NO:65, a light chain CDR2 comprising SEQ ID NO:70, and a light chain CDR3 comprising SEQ ID NO:75;
and (b) a third antigen-binding portion that binds to human delta-like 3 (DLL3) and comprises an antibody variable region comprising a heavy chain CDR 1 comprising SEQ ID NO: 233, a heavy chain CDR 2 comprising SEQ ID NO: 234, a heavy chain CDR 3 comprising SEQ ID NO: 235, a light chain CDR 1 comprising SEQ ID NO: 237, a light chain CDR 2 comprising SEQ ID NO: 238, and a light chain CDR 3 comprising SEQ ID NO: 239.
[34-2] (a) a first antigen-binding portion and a second antigen-binding portion, each of which comprises an antibody variable region, which may be the same or different, wherein the antibody variable region is selected from the group consisting of:
(a1) an antibody variable region comprising a heavy chain variable region (VH) comprising SEQ ID NO: 3 and a light chain variable region (VL) comprising SEQ ID NO: 59;
(a2) an antibody variable region comprising a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 58;
(a3) an antibody variable region comprising a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 58;
(a4) an antibody variable region comprising a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 60;
(a5) an antibody variable region comprising a VH comprising SEQ ID NO: 6 and a VL comprising SEQ ID NO: 58;
(a6) an antibody variable region comprising a VH comprising SEQ ID NO: 8 and a VL comprising SEQ ID NO: 58;
(a7) an antibody variable region comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 58;
(a8) an antibody variable region comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 61;
(a9) an antibody variable region comprising a VH comprising SEQ ID NO: 10 and a VL comprising SEQ ID NO: 58;
(a10) an antibody variable region comprising a VH comprising SEQ ID NO: 11 and a VL comprising SEQ ID NO: 61;
(a11) an antibody variable region comprising a VH comprising SEQ ID NO: 12 and a VL comprising SEQ ID NO: 61;
(a12) an antibody variable region comprising a VH comprising SEQ ID NO: 12 and a VL comprising SEQ ID NO: 58;
(a13) an antibody variable region comprising a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 58;
(a14) an antibody variable region comprising a VH comprising SEQ ID NO: 14 and a VL comprising SEQ ID NO: 58;
(a15) an antibody variable region comprising a VH comprising SEQ ID NO: 81 and a VL comprising SEQ ID NO: 60; and (a16) a first antigen-binding portion and a second antigen-binding portion independently selected from a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-6, 8-14, and 81, and/or a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58-61; and (b) a third antigen-binding portion comprising an antibody variable region comprising a VH comprising SEQ ID NO: 232 and a VL comprising SEQ ID NO: 236.
[34-3] The multispecific antigen-binding molecule of [34-1], wherein the antibody variable regions of the first and second antigen-binding portions are the same.
[34-4] The multispecific antigen-binding molecule of [34-2], wherein the antibody variable regions of the first and second antigen-binding portions are the same.
[34-5] A multispecific antigen-binding molecule of [34-3], wherein each of the first and second antigen-binding portions is a Fab having a cysteine residue at position 191 (EU numbering), and these two cysteine residues are linked by a disulfide bond.
[34-6] A multispecific antigen-binding molecule of [34-4], wherein each of the first and second antigen-binding portions is a Fab having a cysteine residue at position 191 (EU numbering), and these two cysteine residues are linked by a disulfide bond.
[34-7] A multispecific antigen-binding molecule of [34-5], wherein each of the first, second, and third antigen-binding moieties is in a Fab format comprising a VH, a VL, a CH1 domain, and a light chain constant (CL) domain, and the C-terminus of the CH1 domain of the third antigen-binding moiety is fused, directly or via a peptide linker, to the N-terminus of the VH of either the first antigen-binding moiety or the second antigen-binding moiety.
[34-8] A multispecific antigen-binding molecule according to [34-6], wherein each of the first, second and third antigen-binding moieties is in a Fab format comprising a VH, a VL, a CH1 domain and a light chain constant (CL) domain, and the C-terminus of the CH1 domain of the third antigen-binding moiety is fused, directly or via a peptide linker, to the N-terminus of the VH of either the first antigen-binding moiety or the second antigen-binding moiety.
[34-9] The multispecific antigen-binding molecule of [34-7], wherein the fusion is via a peptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259.
[34-10] The multispecific antigen-binding molecule of [34-8], wherein the fusion is via a peptide linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259.
[34-11] The multispecific antigen-binding molecule of [34-9], wherein the third antigen-binding portion is a crossover Fab in which the VH is linked to a CL domain and the VL is linked to a CH1 domain, and each of the first and second antigen-binding portions is a conventional Fab in which the VH is linked to a CH1 domain and the VL is linked to a CL domain.
[34-12] The multispecific antigen-binding molecule of [34-10], wherein the third antigen-binding portion is a crossover Fab in which the VH is linked to a CL domain and the VL is linked to a CH1 domain, and each of the first and second antigen-binding portions is a conventional Fab in which the VH is linked to a CH1 domain and the VL is linked to a CL domain.
[34-13] The multispecific antigen-binding molecule of [34-11], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding moieties are arginine and lysine, respectively, and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding moieties are glutamic acid.
[34-14] The multispecific antigen-binding molecule of [34-12], wherein the amino acids at positions 123 and 124 (Kabat numbering) in the CL domain of each of the first and second antigen-binding moieties are arginine and lysine, respectively, and the amino acids at positions 147 and 213 (EU numbering) in the CH1 domain of each of the first and second antigen-binding moieties are glutamic acid.
[34-15] A multispecific antigen-binding molecule of [34-13], further comprising an Fc domain.
[34-16] A multispecific antigen-binding molecule of [34-14], further comprising an Fc domain.
[34-17] the Fc domain comprises a first and a second Fc region subunit;
The first Fc region subunit comprises:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
and a second Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 354, and a tryptophan at position 366, and the second Fc region subunit is selected from the group comprising:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 349, and a serine at position 366, an alanine at position 368, and a valine at position 407;
All positions are according to EU numbering,
[34-15] Multispecific antigen-binding molecule.
[34-18] the Fc domain comprises a first and a second Fc region subunit;
The first Fc region subunit comprises:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
and a second Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 354, and a tryptophan at position 366, and the second Fc region subunit is selected from the group comprising:
an Fc region polypeptide comprising an alanine at each of positions 234 and 235;
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and
an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine at position 349, and a serine at position 366, an alanine at position 368, and a valine at position 407;
All positions are according to EU numbering,
[34-16] Multispecific antigen-binding molecules.

FIG. 1 shows the design and nomenclature rules for a) trivalent antibodies in the DUAL/LINC (1+2) format, b) bispecific antibodies with a conventional antibody format, and c) bispecific antibodies with a BiTE format. Figure 1 shows TDCC activity of antibodies against the SK-MEL30 cell line. a) Comparison of TDCC between DUAL/LINC and CD3 bispecific formats. b) Effect of linker length on cytotoxic activity. 1 shows the in vivo efficacy of antibodies against NCI-H1436 xenografts in the huNOG mouse model. Y-axis represents tumor volume (mm ³ ) and X-axis represents days after tumor implantation. Figure 1 shows the results of analyzing CD8 T cell infiltration. Tumors were harvested at the indicated time points after antibody injection, and T cell infiltration was analyzed using a flow cytometer. The results of analyzing exhaustion markers on CD8 T cells are shown. Tumors were harvested 7 days after antibody injection, and the expression of exhaustion markers was analyzed using a flow cytometer. Schematic diagram showing the structure of full-length human DLL3 and human DLL3 ECD fragment protein. The epitopes recognized by each of the anti-DLL3 antibodies are also shown. The EGF domain has six regions, EGF1 to EGF6, from the N-terminus to the C-terminus. The figure shows the T cell-dependent cytotoxicity (TDCC) activity of anti-DLL3-Dual antibodies (DLL3-DualAE05/DualAE05-FF110) against the NB-1 cell line (neuroblastoma) and the QGP-1 cell line (pancreatic islet cell carcinoma). Figure 1 shows the in vivo efficacy of anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF114) in reducing tumor volume as monotherapy or in combination with cisplatin (CDDP) or carboplatin (CBDCA) (Combo) in DMS53 small cell lung cancer model. CBDCA means CBDCA monotherapy, CDDP means CDDP monotherapy. Y-axis means tumor volume ( ^mm3 ), X-axis means days after tumor implantation. ^* P<0.05, ^** P<0.01. Figure 1 shows the in vivo efficacy of anti-DLL3-Dual antibodies (DLL3-DualAE05/DualAE05-FF114) or DLL3CD3BiTE administered once in a single dose or on a multiple weekly (QW) schedule to NCI-H1436 xenografts in a humanized NOG mouse model in reducing tumor volume. Y-axis represents tumor volume ( ^mm3 ) and X-axis represents days after tumor implantation. Figure 1 shows the in vivo efficacy of anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF114) in combination with steroids or tocilizumab. a) In steroid pretreatment experiments, dexamethasone was administered intraperitoneally to DLL3-expressing PC-10-bearing huNOG mice 1 and 24 hours or 1 hour prior to antibody injection. a) Top panels show antibody-induced tumor suppression with or without steroids. a) Middle panels show steroid-mediated inhibition of antibody-induced IFN-γ release (left panel) or TNFα release (right panel). a) Bottom panels show steroid-mediated inhibition of antibody-induced IL-6 release. b) In tocilizumab experiments, tocilizumab was administered to DLL3-expressing PC-10-bearing huNOG mice 1 day prior to or 6 hours after injection of anti-DLL3-Dual-LINC antibodies. The graph shows tumor inhibition by the antibody with or without prior or subsequent tocilizumab administration.

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

The following definitions and detailed descriptions are provided to facilitate understanding of the present disclosure described herein.

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

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

In this specification, the meaning of the term "and/or" when describing the site of amino acid modification includes any combination of "and" and "or" appropriately combined. Specifically, for example, "amino acids at positions 33, 55, and/or 96 are substituted" includes the following variations of amino acid modification: (a) amino acids at positions 33, (b) 55, (c) 96, (d) 33 and 55, (e) 33 and 96, (f) 55 and 96, and (g) 33, 55, and 96.

Furthermore, in the present specification, as an expression indicating an amino acid modification, an expression indicating the one-letter code or three-letter code of the amino acid before and after the modification, respectively, before and after the number indicating a specific position may be appropriately used. For example, the modification N100bL or Asn100bLeu used in replacing an amino acid contained in an antibody variable region represents the replacement of Asn at position 100b (according to Kabat numbering) with Leu. That is, the number indicates the position of the amino acid according to Kabat numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before the replacement, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after the replacement. Similarly, the modification P238D or Pro238Asp used in replacing an amino acid in the Fc region contained in an antibody constant region represents the replacement of Pro at position 238 (according to EU numbering) with Asp. That is, the number indicates the position of the amino acid according to the EU numbering system, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution.

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

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

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

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

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

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

Host cells derived from multicellular organisms (invertebrate and vertebrate) are also suitable for expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

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

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

Recombinant production of the antigen-binding molecules described herein can be performed in a manner similar to that described above, by using a host cell that contains (e.g., is transformed by) one or more vectors that contain a nucleic acid encoding an amino acid sequence that comprises the entire antigen-binding molecule or a portion of the antigen-binding molecule.

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

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

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

The first antigen-binding moiety and the second antigen-binding moiety may be "dual antigen-binding moieties" capable of binding to CD3 and CD137, but not simultaneously, as described in more detail below. The third antigen-binding moiety may be a "DLL3 antigen-binding moiety," as described in more detail below.

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

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

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

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

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

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

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

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

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

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

Antigen-binding moiety The term "antigen-binding moiety" as used herein refers to a polypeptide molecule that specifically binds to an antigen. In one embodiment, an antigen-binding moiety can direct the entity to which it is bound to a target site, for example, a specific type of tumor cell expressing a cancer antigen (DLL3). In another embodiment, an antigen-binding moiety can activate signaling through its target antigen, for example, a T-cell receptor complex antigen (particularly CD3) and/or a costimulatory receptor (CD137). Antigen-binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen-binding moieties include the antigen-binding domain of an antibody or antibody variable region, including an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding moiety may include an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: alpha, delta, epsilon, gamma, or mu. Useful light chain constant regions include any of the two isotypes: kappa and lambda.

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

In another aspect, the antigen-binding portions of the invention disclosed herein can be used in novel chimeric antigen receptors (CARs) comprising one or more of the antigen-binding portions disclosed herein. In certain embodiments, the CARs of the invention comprise scFv constructs, which in preferred embodiments comprise heavy and light chain variable regions as disclosed herein. In preferred embodiments, the disclosed chimeric antigen receptors are useful for treating or preventing proliferative diseases and any recurrence or metastasis thereof.

Antigen-binding moiety that can bind to CD3 and CD137 but not simultaneously The multispecific antigen-binding molecule described herein comprises at least one antigen-binding moiety that can bind to CD3 and CD137 but not simultaneously (also referred to herein as "Dual antigen-binding moiety" or "first antigen-binding moiety" or "Dual-Fab" or "Dual-Ig"). In one embodiment, the antigen-binding moiety that can bind to CD3 and CD137 but not simultaneously is an antigen-binding moiety that binds to human CD3. In another embodiment, the antigen-binding moiety that can bind to CD3 and CD137 but not simultaneously is an antigen-binding moiety that binds to human CD137. In another embodiment, the antigen-binding moiety capable of binding to CD3 and CD137, but not simultaneously, is an antigen-binding moiety capable of binding to human CD3 and human CD137, and the antigen-binding moiety binds to either human CD3 or human CD137. In certain embodiments, the multispecific antigen-binding molecule comprises two Dual antigen-binding moieties (a "first antigen-binding moiety" and a "second antigen-binding moiety," each of which may be referred to as a "Dual-Fab"). In some embodiments, each of the two Dual antigen-binding moieties (the "first antigen-binding moiety" or the "second antigen-binding moiety" or the "Dual-Fab") provides monovalent binding to CD3 or CD137, but does not simultaneously bind to CD3 and CD137. In certain embodiments, the multispecific antigen-binding molecule comprises no more than two Dual antigen-binding moieties (the "first antigen-binding moiety" or the "second antigen-binding moiety" or the "Dual-Fab").

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

In certain embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") specifically binds to the whole of CD3 or a portion of a peptide thereof. In certain embodiments, the CD3 is human CD3 or cynomolgus CD3, particularly human CD3. In certain embodiments, the first antigen-binding portion is cross-reactive with (i.e., specifically binds to) human and cynomolgus CD3. In some embodiments, the first antigen-binding portion can specifically bind to the ε subunit of CD3, particularly the human CD3ε subunit of CD3 as set forth in SEQ ID NO:7 (NP_000724.1) (RefSeq accession number in parentheses). In some embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") can specifically bind to the CD3ε chain expressed on the surface of a eukaryotic cell. In some embodiments, the Dual antigen-binding moiety ("first antigen-binding moiety" or "second antigen-binding moiety" or "Dual-Fab") binds to the CD3ε chain expressed on the surface of a T cell.

In certain embodiments, CD137 is human CD137. In some embodiments, preferred examples of the antigen-binding molecules of the present invention include the following:
an antibody recognizing a region including the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 21);
An antibody recognizing a region including the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 35);
and an antibody recognizing a region including the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 49), and an antibody recognizing a region including the sequence LQDPCSNCPAGTFCDNNRNQIC (SEQ ID NO: 105) in the human CD137 protein.

In certain embodiments, the Dual antigen-binding portion ("First antigen-binding portion" or "Second antigen-binding portion" or "Dual-Fab") comprises any one of the antibody variable region sequences shown in Table 1 below. In certain embodiments, the Dual antigen-binding portion ("First antigen-binding portion" or "Second antigen-binding portion" or "Dual-Fab") comprises any one of the combinations of heavy chain variable region and light chain variable region shown in Table 1.

Table 1: SEQ ID NOs of the variable regions of the dual antigen-binding portion (the "first antigen-binding portion" or the "second antigen-binding portion" or the "Dual-Fab").

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

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

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

In certain embodiments, the Dual antigen-binding portion ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab") comprises any one of the combinations of HVR sequences shown in Table 2 below.

Table 2: SEQ ID NOs of HVR (CDR) sequences of dual antigen-binding portions ("first antigen-binding portion" or "second antigen-binding portion" or "Dual-Fab")

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

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

Antigen-binding moiety capable of binding to DLL3 The multispecific antigen-binding molecules described herein comprise at least one antigen-binding moiety capable of binding to Delta-like 3 (DLL3) (also referred to herein as a "DLL3 antigen-binding moiety" or a "third antigen-binding moiety" or an "antigen-binding moiety that binds DLL3").

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

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

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

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

In certain embodiments, the multispecific antigen binding molecule or DLL3 antigen binding portion comprises any one of the antibody variable region sequences shown in Table 3 below. In certain embodiments, the multispecific antigen binding molecule or DLL3 antigen binding portion comprises any one of the combinations of heavy chain variable region and light chain variable region shown in Table 3. In some embodiments, the multispecific antigen binding molecule or DLL3 antigen binding portion comprises a domain comprising an antibody variable fragment that competes for binding to DLL3 with any one of the antibody variable regions shown in Table 3.

Table 3. SEQ ID NOs for the variable regions of exemplary DLL3 antigen binding moieties

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

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

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

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

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

In certain embodiments, the DLL3 antigen binding portion comprises any one of the combinations of HVR sequences shown in Table 4 below. In some embodiments, the multispecific antigen binding molecule or DLL3 antigen binding portion comprises a domain that comprises an antibody variable fragment that competes for binding to DLL3 with any one of the antibody variable regions shown in Table 4 or that competes for binding to DLL3 with any antibody variable fragment that comprises an HVR sequence that is identical to an HVR region of an antibody variable region shown in Table 4.

Table 4. SEQ ID NOs for HVR (CDR) sequences of exemplary DLL3 antigen binding portions

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

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

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

In certain 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 result in DLL3-sensitive lymphocytes that can be used to target and kill DLL3-positive tumor cells. As broadly discussed herein, the CARs of the invention typically include an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and include a DLL3-binding domain that activates certain lymphocytes and generates an immune response of DLL3-positive tumor cells. Selected embodiments of the invention include immunologically active host cells that exhibit the disclosed CARs, as well as various polynucleotide sequences and vectors that encode the DLL3 CARs of the invention. Other aspects include methods of enhancing the activity of T lymphocytes or natural killer (NK) cells in an individual by introducing host cells expressing a DLL3 CAR molecule into an individual suffering from cancer and treating the individual. Such aspects include, inter alia, lung cancer (e.g., small cell lung cancer) and melanoma.

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

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

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

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

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

The human DLL3 protein contains a transmembrane (TM) region and an intracellular domain at the C-terminus, and a DSL (Notch) domain at the N-terminus (see, for example, FIG. 6). In addition, DLL3 has an EGF domain, which includes six regions EGF1 to EGF6 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 an epitope within the extracellular domain (ECD), i.e., a domain from the N-terminus to just before the TM region, but not to the TM region or the C-terminal intracellular domain. The multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention may bind to an epitope within any of the above-mentioned 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 an epitope within the region from EGF6 to just before the TM region. More specifically, the multispecific antigen-binding molecule or DLL3 antigen-binding portion of the present invention may bind to an epitope within the region defined by SEQ ID NO: 89 in human DLL3. In some embodiments, the molecules/antibodies of the invention bind to an epitope within the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region, or the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region.

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

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

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

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

Antigen-binding domain The term "antigen-binding domain" refers to a portion of an antibody that comprises an area that specifically binds to and is complementary to a portion or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, an 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".

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

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

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

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

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

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

Specifically, for example, a region of a biotin-labeled test antigen-binding molecule capable of binding to CD3 and CD137, such as a low molecular weight antibody composed of the Fab region, or a monovalent antibody thereof (an antibody lacking one of the two Fab regions that a normal antibody has) is mixed with CD3 or CD137 labeled with a sulfo-tag (Ru complex), and the mixture is added to a streptavidin-immobilized plate. In this operation, the biotin-labeled test antigen-binding molecule binds to the streptavidin on the plate. Light is generated from the sulfo-tag, and the luminescence signal is detected using a Sector Imager 600 or 2400 (MSD K.K.), etc., thereby allowing the binding of the above-mentioned region of the test antigen-binding molecule to CD3 or CD137 to be confirmed.

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

Specifically, the assay can be performed using, for example, Biacore (GE Healthcare Japan Corp.), an interaction analysis instrument based on the surface plasmon resonance (SPR) phenomenon. Biacore analysis instruments include any model such as 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 chip, can be used as the sensor chip. Proteins for capturing the antigen-binding molecules of the present invention, such as protein A, protein G, protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human L chain antibody, anti-human Fc antibody, antigen protein, or antigen peptide, are immobilized on the sensor chip by a coupling method such as amine coupling, disulfide coupling, or aldehyde coupling. CD3 or CD137 is injected onto the sensor chip as an analyte, and the interaction is measured to obtain a sensorgram. In this procedure, the concentration of CD3 or CD137 can be selected within the range of several μM to several pM according to the strength of the interaction (e.g., KD) of the assay sample.

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

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

ALPHAScreen is implemented by ALPHA technology using two types of beads (donor and acceptor) based on the following principle: A luminescence signal is detected only when a biological interaction between a molecule bound to a donor bead and a molecule bound to an acceptor bead brings these two beads into close proximity. A photosensitizer in the donor bead excited by a laser converts the surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the acceptor bead located close to it, thereby triggering a chemiluminescence reaction in the bead, which ultimately emits light. In the absence of an interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead. Therefore, no chemiluminescence reaction occurs.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Alternatively, a method using fluorescence resonance energy transfer (FRET) may be used. FRET is a phenomenon in which excitation energy is transferred directly between two fluorescent molecules located in close proximity to each other through electronic resonance. When FRET occurs, the excitation energy of the donor (a fluorescent molecule having an excited state) is transferred to the acceptor (another fluorescent molecule located near the donor), so that the fluorescence emitted from the donor disappears (more precisely, the fluorescence lifetime is shortened) and instead, fluorescence is emitted from the acceptor. By using this phenomenon, it is possible to analyze whether or not an antibody binds to CD3 and CD137 simultaneously. For example, when CD3 having a fluorescent donor and CD137 having a fluorescent acceptor simultaneously bind to a test antigen-binding molecule, the fluorescence of the donor disappears, while fluorescence is emitted from the acceptor. Therefore, a change in the fluorescence wavelength is observed. Such an antibody is confirmed to bind to CD3 and CD137 simultaneously. On the other hand, if mixing CD3, CD137, and the test antigen-binding molecule does not change the fluorescence wavelength of the fluorescent donor bound to CD3, the test antigen-binding molecule can be considered as an antigen-binding domain that can bind to CD3 and CD137, but does not bind to CD3 and CD137 simultaneously.

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

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

Also, when CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or when both are present on the same cell, the variable region can bind to CD3 and CD137 simultaneously, but cannot bind to CD3 and CD137 each expressed on a different cell, and this can also be assayed by methods known in the art.

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

Alternatively, this assay may be performed by the ALPHAScreen method. A test antigen-binding molecule is mixed with cells expressing CD3 bound to donor beads and cells expressing CD137 bound to acceptor beads. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a signal is observed. If the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed.

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

Instead of these methods based on binding activity, an assay based on biological activity may be performed. For example, cells expressing CD3 and cells expressing CD137 are mixed and cultured with a test antigen-binding molecule. Two antigens expressed on the two cells, respectively, are mutually activated via the test antigen-binding molecule when the antigen-binding molecule simultaneously binds to these two antigens. Therefore, a change in activation signal, such as an increase in phosphorylation level downstream of each of the antigens, can be detected. Alternatively, the production of cytokines is induced as a result of activation. Therefore, the amount of cytokines produced can be measured, and thereby it can be confirmed whether or not they bind to the two cells simultaneously. Alternatively, cytotoxic activity against cells expressing CD137 is induced as a result of activation. Alternatively, the expression of a reporter gene is induced by a promoter that is activated downstream of the signaling pathway of CD137 or CD3 as a result of activation. Therefore, it can be confirmed whether or not they bind to the two cells simultaneously by measuring the cytotoxic activity or the amount of reporter protein produced.

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

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

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, each of 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) does not bind to antigens expressed on different cells, resulting in cross-linking of different cells. In the present application, such an antigen-binding mode of the antigen-binding molecule of the present invention can be referred to as "cis-binding", whereas the antigen-binding mode of the antigen-binding molecule in which each of the two antigen-binding portions of the antigen-binding molecule binds to antigens expressed on different cells, resulting in cross-linking of different cells, can be referred to as "trans-binding". In some embodiments, the antigen-binding molecule of the present invention mainly binds to antigens expressed on the same single cell in a "cis-binding" manner.

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

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

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

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

Fused "Fused" means that the components (e.g., a Fab molecule and an Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.

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

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

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

Methods for Determining Affinity In certain embodiments, an antigen-binding molecule or antibody provided herein has a dissociation constant (KD) for its antigen of 1 μM or less, 120 nM or less, 100 nM or less, 80 nM or less, 70 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 ⁻⁸ M or less, 10 ⁻⁸ M to 10 ⁻¹³ M, 10 ⁻⁹ M to 10 ⁻¹³ M). In certain embodiments, the KD value of the antibody/antigen binding molecule for CD3, CD137, or DLL3 falls within 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.

In one embodiment, KD is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab to the antigen is measured by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of an increasing series of unlabeled antigens, and then capturing the bound antigen with a plate coated with an anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish the assay conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM of [ ¹²⁵ I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although the incubation can be continued for longer (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation (e.g., 1 hour) at room temperature. The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl/well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

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

Following the above-described methods for measuring the affinity of an antigen-binding molecule or antibody, one skilled in the art can perform affinity measurements of other antigen-binding molecules or antibodies for various antigens.

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

Antibody Fragment An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994); in addition, WO93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab') ₂ fragments that contain salvage receptor binding epitope residues and have increased half-life in vivo. Diabodies are antibody fragments with two antigen binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies, production by recombinant host cells (e.g., E. coli or phage), as described herein.

Antibody ClassesAn antibody "class" refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

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

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

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

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

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

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

Polynucleotides (nucleic acids)
"Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can include modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.) and unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present on the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to generate additional linkages to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by: P(O)S ("thioate"), P(S)S ("dithioate"), (O) _NR2 ("amidate"), P(O)R, P(O)OR', CO, or _CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

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

Vector The term "vector" as used herein refers to a nucleic acid molecule that can propagate another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell to which it is introduced. A vector can cause the expression of a nucleic acid to which it is operatively linked. Such vectors are also referred to herein as "expression vectors." A vector can be introduced into a host cell using a virus or electroporation. However, the introduction of a vector is not limited to in vitro methods. For example, a vector can also be directly introduced into a subject using in vivo methods.

In another aspect of the invention, a vector containing a nucleic acid molecule encoding an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure capable of binding to CD3 and CD137 but not simultaneously, an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure may be introduced into a subject to directly express the antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure in the subject. An example of a vector that may be used is, but is not limited to, an adenovirus. It is also possible to directly administer a nucleic acid molecule encoding an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure to a subject, or to transfer a nucleic acid molecule encoding an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure to a subject via electroporation, or to administer a cell containing a nucleic acid molecule encoding an antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure to be expressed and secreted to a subject, thereby continuously expressing and secreting the antigen-binding portion, antigen-binding molecule, or antibody of the present disclosure in the subject.

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

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

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

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

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

scFv, single chain antibodies, and sc(Fv) ₂
As used herein, the terms "scFv", "single-chain antibody" and "sc(Fv) ₂ " all refer to a single polypeptide chain antibody fragment that contains variable regions derived from heavy and light chains but does not contain a constant region. Generally, single-chain antibodies further contain a polypeptide linker between the VH and VL domains that allows the formation of a desired structure that may allow antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in "The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994)". See also International Publication WO1988/001649, U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies can be bispecific and/or humanized.

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. U.S.A. (1988) 85 (16), 5879-5883). The peptide linker can hold the VH and VL in close proximity.

sc(Fv) ₂ is a single-chain antibody in which four variable regions, two VL and two VH, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). The two VH and two VL may be derived from different monoclonal antibodies. Suitable examples of such sc(Fv) ₂ include 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 produced by linking scFvs with a linker such as a peptide linker.

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

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

Furthermore, a carrier polymer such as PEG or an organic compound such as an anticancer drug may be conjugated to the antigen-binding molecule or antibody of the present disclosure. Alternatively, a glycosylation sequence is suitably inserted into the antigen-binding molecule or antibody so that the glycosylation provides the desired effect.

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

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

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

Three linkers are usually required to link four antibody variable regions. The linkers used may be of the same type or different types.

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

"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 digesting the immunoglobulin (monoclonal antibody) near the disulfide bond between the hinge regions of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond between the hinge regions of the two H chains, producing two homologous antibody fragments in which the L chain containing VL (light chain variable region) and CL (light chain constant region) is linked to the H chain fragment containing VH (light chain variable region) and CHγ1 (γ1 region in the heavy chain constant region) by disulfide bonds at their C-terminal regions. Each of these two homologous antibody fragments is called Fab'.

"F(ab') ₂ " consists of two light chains and two heavy chains containing constant regions of the CH1 domain and a part of the CH2 domain such that a disulfide bond is formed between the two heavy chains. The F(ab') ₂ disclosed herein can be suitably produced as follows. A monoclonal whole antibody or the like containing a desired antigen-binding site is partially digested with a protease such as pepsin, and the Fc fragment is removed by adsorption onto a protein A column. The protease is not particularly limited as long as it can cleave the whole antibody so as to selectively produce F(ab') ₂ under appropriately set enzyme reaction conditions such as pH. For example, such proteases include pepsin and ficin.

As used herein, the term "single domain antibody" is not limited by its structure, as long as the domain can exhibit antigen-binding activity by itself. It is known that while a general antibody, for example an IgG antibody, exhibits antigen-binding activity when the variable region is formed by pairing VH and VL, the domain structure of a single domain antibody can exhibit antigen-binding activity by itself without pairing with another domain. Usually, a single domain antibody has a relatively low molecular weight and exists in the form of a monomer.

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

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

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

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

In some embodiments, the Fc domain of a multispecific antigen-binding molecule consists of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the 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 molecules described herein comprise no more than one Fc domain.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Preferred antigen-binding molecules or antibodies include, for example, those that contain an Fc domain in which the amino acid at position 265 (EU numbering) in the amino acids that form the Fc domain of an IgG1 antibody is substituted with another amino acid. The type of amino acid after substitution is not particularly limited; however, antigen-binding molecules or antibodies that contain an Fc domain in which the amino acid at position 265 is substituted with alanine are particularly preferred.

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

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

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

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

On the other hand, "Fc ligand" or "effector ligand" refers to a molecule, and preferably a polypeptide, that binds to an antibody Fc domain to form an Fc/Fc ligand complex. The molecule may be from any organism. 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, C1q, and C3, mannan-binding lectin, mannose receptor, Staphylococcus protein A, Staphylococcus protein G, and viral Fcγ receptors. Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors that are homologous to Fcγ receptors (Davis et al., (2002) Immunological Reviews 190, 123-136). Fc ligands also include unidentified molecules that bind to Fc.

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

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

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

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

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

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

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

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

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

The protrusions and gaps can be created by modifying the nucleic acid encoding the polypeptide, for example by site-directed mutagenesis, or by peptide synthesis.

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

In yet a further 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 results in the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In other embodiments, other techniques can be applied to the multispecific antigen-binding molecules of the present invention to promote association between H chains and L chains having the desired combinations.

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

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

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

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

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

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

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

In a preferred embodiment, the first H chain CH3 region and the second H chain CH3 region of the antibody may be cross-linked by a disulfide bond.

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

In addition, other known techniques can also be used to form the multispecific antibodies of the present invention. Using a strand-exchange engineered domain CH3 generated by changing a portion of the CH3 of one of the antibody's H chains to a sequence derived from the corresponding IgA and introducing the corresponding IgA sequence into the complementary portion of the CH3 of the other H chain, association of polypeptides having different sequences can be efficiently induced by complementary association of CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). Using this known technique, the desired multispecific antibodies can also be efficiently formed.

In addition, for the formation of multispecific antigen-binding molecules, there are various techniques, such as those described in WO2011/028952, WO2014/018572 and Nat Biotechnol. 2014 Feb;32(2):191-8 for producing antibodies using the association of CH1 and CL and the association of VH and VL, those described in WO2008/119353 and WO2011/131746 for producing bispecific antibodies by combining monoclonal antibodies prepared separately (Fab Arm Exchange), those described in WO2012/058768 and WO2013/063702 for controlling the association between antibody heavy chain CH3, those described in WO2012/023053 for producing multispecific antibodies composed of two types of light chains and one type of heavy chain, those described in Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)) may be used to produce multispecific antibodies using two bacterial cell lines each expressing one half of an antibody chain, including one heavy chain and one light chain.

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

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

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

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

Complement-dependent cytotoxicity "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to the corresponding antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased C1q binding capacity are described, for example, in U.S. Patent No. 6,194,551 B1 and WO1999/51642. See also, for example, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

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

Measurement of T-cell-dependent cytotoxicity In an embodiment in which an antigen-binding molecule binds to both DLL3 and CD3/CD137, the following method is preferably used to evaluate or determine T-cell-dependent cytotoxicity (TDCC) caused by contacting an antigen-binding molecule of the present disclosure with a DLL3-expressing cell to which the antigen-binding site in the antigen-binding molecule of the present disclosure binds. Methods for evaluating or determining cytotoxic activity in vitro include methods for determining the activity of cytotoxic T cells, etc. Whether an antigen-binding molecule of the present 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 the cytotoxicity assay, an antigen-binding molecule capable of binding to an antigen different from DLL3 and not expressed in cells and to CD3/CD137 is used as a control antigen-binding molecule. The control antigen-binding molecule is assayed in the same manner. Activity is then evaluated by testing whether an antigen-binding molecule of the present disclosure exhibits stronger cytotoxic activity than that of a control antigen-binding molecule.

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

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

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

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

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

Pharmaceutical Compositions
In one aspect, the present disclosure provides a pharmaceutical composition or pharmaceutical preparation comprising the antigen-binding molecule or antibody of the present disclosure. In certain embodiments, the pharmaceutical composition or pharmaceutical preparation of the present disclosure induces T cell-dependent cytotoxicity, in other words, the pharmaceutical composition or pharmaceutical preparation of the present disclosure is a therapeutic agent for inducing cytotoxicity. In certain embodiments, the pharmaceutical composition or pharmaceutical preparation of the present disclosure is a pharmaceutical composition or pharmaceutical preparation for treating and/or preventing cancer. In certain embodiments, the pharmaceutical composition or pharmaceutical preparation of the present disclosure is a pharmaceutical composition or pharmaceutical preparation for treating and/or preventing DLL3-positive cancer or DLL3-expressing cancer, including lung cancer (including small cell lung cancer) and melanoma, or including DLL3-expressing neuroendocrine neoplasms (NEN), neuroendocrine tumors (NET) or neuroendocrine carcinomas (NEC), or other solid tumors of non-neuroendocrine origin that express DLL3. In some preferred embodiments, examples of cancer include pancreatic neuroendocrine tumors (PNET), small cell NEC (SCNEC) or large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine cancer, medullary thyroid cancer (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC); or other solid tumors, such as neuroblastoma, glioma or glioblastoma (GBM), melanoma, medullary thyroid cancer. In a preferred embodiment, the tumor or cancer disease is lung cancer, preferably SCLC. In another preferred embodiment, the tumor or cancer disease is glioma or glioblastoma (GBM). In another preferred embodiment, the tumor or cancer disease is neuroendocrine prostate cancer. In a particular embodiment, the pharmaceutical composition or pharmaceutical preparation of the present disclosure is a cytostatic agent. In a particular embodiment, the pharmaceutical composition or pharmaceutical preparation of the present disclosure is an anti-cancer agent.

The pharmaceutical composition or pharmaceutical preparation of the present disclosure, the therapeutic agent for inducing cytotoxicity of the present disclosure, the cell proliferation inhibitor, or the anticancer agent can be formulated with various types of antigen-binding molecules or antibodies as necessary. For example, a cocktail of multiple antigen-binding molecules or antibodies of the present disclosure can enhance the cytotoxic effect against cells expressing an antigen.

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

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

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

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

The pharmaceutical composition, cell proliferation inhibitor, or anticancer agent of the present disclosure may be administered to a patient either orally or parenterally. Parenteral administration is preferred. Specifically, such administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition, therapeutic agent for inducing cell damage, cell proliferation inhibitor, or anticancer agent of the present disclosure may be administered locally or systemically by injection. Furthermore, an appropriate administration method may be selected according to the age and symptoms of the patient. The administration dose may be selected, for example, from the range of 0.0001 mg to 1,000 mg per kg of body weight for each administration. Alternatively, the dose may be selected, for example, from the range of 0.001 mg to 100,000 mg per patient. However, the dose of the pharmaceutical composition of the present disclosure is not limited to these doses.

Preferably, the pharmaceutical composition of the present disclosure comprises an antigen-binding molecule or antibody as described herein. In one aspect, the composition is a pharmaceutical composition for use in inducing cytotoxicity. In another aspect, the composition is a pharmaceutical composition for use in treating or preventing cancer. Preferably, the cancer is lung cancer (including small cell lung cancer) and melanoma, or includes neuroendocrine neoplasms (NENs), neuroendocrine tumors (NETs) or neuroendocrine carcinomas (NECs) that express DLL3, or other solid tumors of non-neuroendocrine origin that express DLL3. In some preferred embodiments, examples of cancer include pancreatic neuroendocrine tumors (PNETs), small cell NEC (SCNEC) or large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine cancer, medullary thyroid cancer (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC); or other solid tumors, such as neuroblastoma, glioma or glioblastoma (GBM), melanoma, medullary thyroid cancer. In a preferred embodiment, the tumor or cancer disease is lung cancer, preferably SCLC. In another preferred embodiment, the tumor or cancer disease is glioma or glioblastoma (GBM). In another preferred embodiment, the tumor or cancer disease is neuroendocrine prostate cancer. The pharmaceutical composition of the present disclosure can be used to treat or prevent cancer. Thus, the present disclosure provides a method for treating or preventing cancer, in which an antigen binding molecule or antibody described herein is administered to a patient in need thereof.

The present disclosure also provides a method for damaging cells expressing DLL3 or inhibiting cell proliferation by contacting the cells expressing DLL3 with an antigen binding molecule of the present disclosure that binds to DLL3. The cells to which the antigen binding molecule of the present disclosure binds are not particularly limited, as long as they express DLL3. Specifically, in the present disclosure, preferred DLL3-expressing cells include lung cancer (including small cell lung cancer) and melanoma, or include neuroendocrine neoplasms (NEN), neuroendocrine tumors (NET) or neuroendocrine carcinomas (NEC) that express DLL3, or other solid tumors of non-neuroendocrine origin that express DLL3. In some preferred embodiments, examples of cancer include pancreatic neuroendocrine tumors (PNETs), small cell NEC (SCNEC) or large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine cancer, medullary thyroid cancer (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC); or other solid tumors, such as neuroblastoma, glioma or glioblastoma (GBM), melanoma, medullary thyroid cancer. In a preferred embodiment, the tumor or cancer disease is lung cancer, preferably SCLC. In another preferred embodiment, the tumor or cancer disease is glioma or glioblastoma (GBM). In another preferred embodiment, the tumor or cancer disease is neuroendocrine prostate cancer.

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

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

The present disclosure also provides a kit for use in the method of the present disclosure, containing an antigen-binding molecule of the present disclosure or an antigen-binding molecule produced by the method of the present disclosure. The kit may be packaged with additional pharma- ceutically acceptable carriers or vehicles, or instructions for use of the kit, etc.

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

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

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

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

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

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. In certain embodiments, the cancer is a DLL3-expressing or DLL3-positive cancer, including neuroendocrine neoplasms (NENs), neuroendocrine tumors (NETs) or neuroendocrine carcinomas (NECs) that express DLL3, or other solid tumors of non-neuroendocrine origin that express DLL3. In some embodiments, examples of cancer include pancreatic neuroendocrine tumors (PNETs), small cell NECs (SCNECs) or large cell NECs (LCNECs), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine carcinoma, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC); or other solid tumors, such as neuroblastoma, glioma or glioblastoma (GBM), melanoma, medullary thyroid carcinoma. In a preferred embodiment of the present invention, the tumor or cancer disease is lung cancer, preferably SCLC.In another preferred embodiment of the present invention, the tumor or cancer disease is glioma or glioblastoma (GBM).In another preferred embodiment of the present invention, the tumor or cancer disease is neuroendocrine prostate cancer.

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

Other Agents and Treatment The multispecific antigen-binding molecules described herein may be administered in combination with one or more other agents in treatment. For example, the multispecific antigen-binding molecules described herein may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredient that is suitable for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunotherapeutic agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis inducers.

In certain of the above embodiments, the additional therapeutic agent is an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an agent that modulates an immune response. In some embodiments, the immunotherapeutic agent is an agent that enhances an anti-tumor immune response. In some embodiments, the immunotherapeutic agent is an agent that increases cell-mediated immunity. In some embodiments, the immunotherapeutic agent is an agent that increases T cell activity. In some embodiments, the immunotherapeutic agent is an agent that increases cytolytic T cell (CTL) activity. In some embodiments, the immunotherapeutic agent is an agent that increases NK cell activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits suppression of an immune response. In some embodiments, the immunotherapeutic agent is an agent that inhibits suppressor cells or inhibits the activity of cells. In some embodiments, the immunotherapeutic agent is an agent that inhibits Treg activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits MDSC activity. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of an inhibitory immune checkpoint receptor. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of PD-1. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of PD-L1 and/or PD-L2. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of CTLA-4. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of CD80 and/or CD86. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of TIGIT. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of KIR. In some embodiments, the immunotherapeutic agent is an agent that inhibits the activity of IDO 1. In some embodiments, the immunotherapeutic agent is an agent that enhances or stimulates the activity of activating immune checkpoint receptors. In some embodiments, the immunotherapeutic agent is an agent that enhances or stimulates the activity of GITR. In some embodiments, the immunotherapeutic agent is an agent that enhances or stimulates the activity of OX 40. In some embodiments, the immunotherapeutic agent is an agent that enhances or stimulates the activity of CD40.

In some embodiments described herein, the immunotherapeutic agent is a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a TIGIT antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a CD96 antagonist, a CD20 antagonist, or an IDO1 antagonist. In some embodiments, the PD-1 antagonist is an antibody that specifically binds to PD-1. In some embodiments, the antibody that binds to PD-1 is pembrolizumab, pidilizumab (CT-011), or nimotuzumab (nivolumab). In some embodiments, the PD-L1 antagonist is an antibody that specifically binds to PD-L1. In some embodiments, the antibody that binds to PD-L1 is atezolizumab, durvalumab (MEDI4736), or BMS-936559. In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds to CTLA-4. In some embodiments, the antibody that binds to CTLA-4 is ipilimumab or tremelimumab (tremelimumab) (CP-675, 206). In some embodiments, the KIR antagonist is an antibody that specifically binds to KIR. In some embodiments, the antibody that binds to KIR is rituximab (lirilumab). In some embodiments of the methods described herein, the immunotherapeutic agent is a CD28 agonist, a 4-1BB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, or a GITR agonist. The multispecific antigen-binding molecules described herein may be administered, where appropriate or necessary, in combination with steroids or other antibody drugs, such as tocilizumab, to control cytokine release syndrome (CRS), a common adverse event of T cell-related therapeutics such as T cell engagers and CAR-T cells.

Chemotherapeutic Agents In certain of the above embodiments, the additional therapeutic agent is a chemotherapeutic agent, e.g., a microtubule disrupting agent, antimetabolite, topoisomerase inhibitor, DNA intercalator, alkylating agent, hormonal therapy, kinase inhibitor, receptor antagonist, activator of tumor cell apoptosis, or antiangiogenic agent. "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide (CYTOXAN®); alkylsulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines, e.g., benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine. ethylenimines and methylolmelamines, including; acetogenins (especially bullatasin and bullatasinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including the synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); elatherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride. hydrochloride), melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., enediyne antibiotics, e.g., the calicheamicins, particularly calicheamicin γ1I and calicheamicin ω11 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicins, including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (including MYOCET®), pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicinone), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), (registered trademark), epothilones, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements acid replenishers), such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; rosoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A) A), Roridin A, and Anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");thiotepa; taxoids such as paclitaxel (TAXOL®), albumin modified nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cytosine; vincas, which block tubulin polymerization so that it does not form microtubules, including suplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those targeting genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, those that inhibit expression of Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOOPE® vaccine and gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); sorafenib, ABT510; Bcl-2 inhibitors, e.g., oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, e.g., rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors, e.g., lonafarnib (SCH 6636, SARASAR™); lurbinectedin or amrubicin; and pharmacologic acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, e.g., CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is an abbreviation for a treatment regimen with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

Chemotherapeutic agents as defined herein may also include "anti-hormonal agents" or "endocrine therapeutic agents" that function to control, reduce, block, or inhibit the action of hormones that can promote cancer growth. They may be hormones themselves, including, but not limited to, antiestrogens with a mixed agonist/antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxyfene, ketoxifene, and selective estrogen receptor modulators (SERMs), e.g., SERM3; pure antiestrogens without agonist properties, e.g., fulvestrant (FASLODEX®), and EM800 ( Such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors, such as anastrozole (ARIMIDEX®), letrozole (FEMARA®), and aminoglutethimide, as well as vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and aromatase inhibitors, including other aromatase inhibitors including 4(5)-imidazoles; luteinizing hormone releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and triptorelin; sex steroids, including progestins, e.g., megestrol acetate and medroxyprogesterone acetate, estrogens, e.g., diethylstilbestrol and premarin, and androgens/retinoids, e.g., fluoxymesterone, all-trans retinoic acid, and fenretinide; onapristone; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens, e.g., flutamide, nilutamide, and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

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

Such combination therapy includes combined administration (wherein two or more therapeutic agents are included in the same or separate compositions) and separate administration, where administration of the multispecific antigen-binding molecules described herein may precede, be concurrent with, and/or follow administration of additional therapeutic agents and/or adjuvants. The multispecific antigen-binding molecules described herein may also be used in combination with radiation therapy.

All references cited herein are hereby incorporated by reference.

Below are examples of the methods and compositions of the present disclosure. It will be understood that various other embodiments may be practiced in light of the general description above.

[Example 1]

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

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

1.2.2 Affinity measurements for human CD3 and CD137 The binding affinity of Dual-Fab antibodies (Dual-Ig) for human CD3 was evaluated at 25 °C using a Biacore 8K instrument (GE Healthcare). Anti-human Fc (GE Healthcare) was immobilized onto all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies were captured onto the anti-Fc sensor surface, and then recombinant human CD3 or CD137 was injected onto the flow cells. All antibodies and analytes were prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, 0.005% _NaN3 . The sensor surface was regenerated every cycle with 3 M _MgCl2 . Binding affinity was determined by processing the data and fitting to a 1:1 binding model using Biacore Insight Evaluation software, version 2.0 (GE Healthcare). CD137 binding affinity assay was performed under the same conditions, except that the assay temperature was set at 37° C. The binding affinities of Dual-Fab antibodies to recombinant human CD3 and CD137 are shown in Tables 9-1 and 9-2 (the term E used to represent K _on , K _off and K D values in the tables means "10 to the power of __", e.g., 3.54E+04=3.54 ^* 10 ⁴ ). As shown in Tables 9-1 and 9-2, the Dual Fab variants showed different binding kinetics to CD3 and CD137 compared to H183/L072.

Table 5: Annotation of the SEQ ID NOs in Table 7 (SEQ ID NOs for human CD3 and CD137 antigens used in affinity measurements in Table 9)

Table 6: Annotation of SEQ ID NOs in Tables 8-1 to 8-6 (antibody name and SEQ ID NOs of variable regions including CDRs 1, 2, and 3)

Table 7. Full-length amino acid sequences of antigens

Table 8-1: Full length amino acid sequences of the variable regions of the antibodies annotated in Table 6, followed by the amino acid sequences of the CDR 1, 2, and 3 regions.

(Table 8-2) Continued from Table 8-1

(Table 8-3) Continued from Table 8-2

(Table 8-4) Continued from Table 8-3

(Table 8-5) Continued from Table 8-4

(Table 8-6) Continued from Table 8-5

(Table 9-1) Binding kinetics of dual variants to human CD3 and human CD137

(Table 9-2) Binding kinetics of dual variants to human CD3 and human CD137

[Example 2]
Sequence and preparation of bispecific and trispecific antibodies
2.1. Design and preparation of bispecific or trispecific antibody (1+1) formats
To evaluate the effect of the Dual-Ig variants (DualAE05, etc.) prepared in Example 1, bispecific and trispecific antibodies were generated with one arm recognizing the tumor antigen DLL3 (derived from anti-DLL3 antibody D30841AE13) and another arm recognizing effector cells, mainly T cells. DLL3/CD3ε (1+1) and DLL3/DualAE05 (1+1) antibodies were generated by using Fab-arm exchange (FAE) according to the method described in Proc Natl Acad Sci US A. 2013 Mar 26; 110(13):5145-5150. The (1+1) format is shown in Figure 1(b). The naming rules of the target antigens and each binding domain of each Fv region in the bispecific and trispecific antibodies in the (1+1) format are shown in Table 10-2, and the sequence numbers are shown in Table 12.

2.2. Design and Preparation of Trivalent Antibody (1+2) Format Containing One Monovalent DLL3 Binding Arm and Two Dual-Fabs Expression of target antigens in solid tumors is likely to be highly heterogeneous, and areas of tumors with low antigen expression may not result in sufficient cross-linking of CD3 or CD137. In particular, CD137 receptor clustering is critical for effective agonist activity (Trends Biohem Sci. 2002 Jan;27(1)19-26). To improve the cytotoxic activity mediated by CD3 binding and activation, a new trivalent trispecific antibody format, i.e., DLL3-DUAL/LINC, 1+2), was designed to increase the number of binding domains for the CD137 molecule. Specifically, the new antibody format is a trivalent trispecific antibody with a "1+2" format, comprising two monovalent Dual-Fabs (FvB and FvC in FIG. 1a), each of which can bind to one CD3 or CD137 but not simultaneously, and one monovalent DLL3-binding arm (FvA in FIG. 1a), in which one disulfide bond ("LINC") is introduced/engineered between the two Dual-Fabs, for example, by introducing a cysteine substitution (S191C in Kabat numbering) at position 191 in the CH1 domain of each of the two Dual-Fabs (FIG. 1(a) and Table 10-1). Without being bound by theory, the inventors contemplated that the disulfide bond ("LINC") designed in this way would restrict the antigen (CD3 or CD137) binding orientation of the two Dual-Fabs to cis antigen binding (i.e., binding to two antigens on the same cell) as a result of steric hindrance or short distance between the two Dual-Fabs, thereby improving the safety profile of the trispecific antibody by preventing undesired cross-linking of two CD3/CD137 expressing immune cells mediated by the two Dual-Fabs in a DLL3-independent manner. Specifically, a DLL3-Dual/Dual antibody variant (DLL3-Dual/LINC, 1+2) was prepared that has monovalent tumor antigen binding ability to DLL3, bivalent CD3 and bivalent CD137 binding properties due to the two Dual-Fabs (Figure 1(a) and Table 10-1). CrossMab technology (WO2017055539) was also utilized. The Fc region was FcγR silent and deglycosylated. The naming rules of the target antigens and each binding domain of each Fv region in the triabody are shown in Table 10-1, and the SEQ ID NOs are shown in Tables 11-1 and 12. For negative controls, Ctrl (VHR IC17HdK and VLR IC17L), which does not bind DLL3, was used instead of the monovalent DLL3 binding arm (Table 12). All antibodies were expressed in trivalent form by transient expression in Expi293 cells (Invitrogen) and purified according to Example 1.1. To improve purity, the antibodies were further purified by passing them through an affinity column that specifically binds to the UnLINC format (i.e., trivalent 1+2 antibodies without engineered disulfide bonds) but not to the LINC format (i.e., trivalent 1+2 antibodies with engineered disulfide bonds).

2.3. Construction of Bispecific Antibodies (BiTEs) To compare the effect of the trivalent antibody (DLL3-DUAL/LINC, 2+1) with other DLL3/CD3 bispecific format antibodies, an antibody with a half-life extended BiTE format was constructed and named DLL3CD3BiTE. DLL3CD3BiTE was composed of two single-chain variable fragments (ScFvs) in which a single-chain variable fragment directed against the tumor-associated antigen DLL3 was fused to one directed against CD3 (Figure 1(c)). The target antigens of each Fv region in the BiTE format bispecific antibody are shown in Table 10-3, and the sequence numbers are shown in Tables 11-2 and 14.

Table 10-1: Antibody name, target arm, and Fc information

Table 10-2: Bispecific and trispecific antibodies (1+1)

(Table 10-3) Bispecific antibody (BiTE)

Table 11-1: Antibody chain numbers and sequence IDs

Table 11-2: Antibody chain numbers and sequence IDs

Table 12: Variable regions and their CDR1-3 sequence IDs

Table 13: Sequence ID numbers of the Fc regions of Table 14

(Table 14-1)

(Table 14-2)

(Table 14-3)

(Table 14-4)

(Table 14-5)

(Table 14-6)

(Table 14-7)

(Table 14-8)

(Table 14-9)

(Table 14-10)

(Table 14-11)

[Example 3]
In vitro efficacy of DLL3-Dual-LINC antibodies against DLL3-expressing cell lines
3.1. Measurement of TDCC activity using anti-DLL3/Dual-Linc(1+2) antibody, Dual(1+1) antibody, DLL3CD3BiTE, and DLL3/CD3ε(1+1) antibody
Cytotoxic activity was evaluated by observing the tumor cell proliferation inhibition rate using xCELLigence Real-Time Cell Analyzer (Roche Diagnostics) in the presence of PBMC. Figure 2 shows the TDCC activity of anti-DLL3/Dual-Linc antibody (DLL3-DualAE05/DualAE05, 1+2), Dual (1+1) antibody (DLL3/DualAE05), DLL3CD3BiTE, and DLL3/CD3ε (1+1) antibody prepared according to Table 10-13. SK-MEL30 cell line was used as target cells. The target cells were detached from the dish, and the cells were plated on E-plate 96 (Roche Diagnostics) in an aliquot of 100 μL/well by adjusting the cells to 5×10 ³ cells/well, and the measurement of cell proliferation was started using xCELLigence Real-Time Cell Analyzer. After 24 hours, the plate was removed and 50 μL of each antibody prepared at each concentration (5-fold serial dilution starting from 10 nM, i.e., 0.25, 1.25, 2.5, 5, and 10 nM) was added to the plate. After 15 minutes of reaction at room temperature, 50 μL of fresh human PBMC solution was added at an effector:target ratio of 2 (i.e., 1× ¹⁰⁴ cells/well), and cell proliferation measurement was resumed using the xCELLigence Real-Time Cell Analyzer. The reaction was carried out at 37°C under 5% carbon dioxide gas. Because CD137 signaling enhances T cell survival and prevents activation-induced cell death, the TDCC assay was performed at a low E:T ratio. To observe the superior cytotoxic activity contributed by CD137 activation, an extended period may be required. Therefore, approximately 68 hours after the addition of PBMC, the cytostatic inhibition (CGI) rate (%) was determined using the following formula: The Cell Index value obtained from the xCELLigence Real-Time Cell Analyzer used in the calculation was a normalized value defined as 1 for the Cell Index value immediately before antibody addition.
Cell proliferation inhibition rate (%) = (A-B) x 100/(A-1)
A represents the average Cell Index value in wells without antibody (containing only target cells and human PBMCs), and B represents the average Cell Index value in target wells. The test was performed in duplicate.

As shown in Figure 2a), anti-DLL3/Dual-Linc antibody (DLL3-DualAE05/DualAE05, 1+2) showed stronger TDCC activity than Dual (1+1) antibody (DLL3/DualAE05) and DLL3/CD3ε (1+1) antibody, suggesting that the Dual-Linc molecular format contributes to improved cytotoxic activity. In addition, at these antibody concentrations, the TDCC activity of anti-DLL3/Dual-Linc antibody is comparable to that of DLL3/CD3BiTE.

3.2. Evaluation of the effect of linker length on cytotoxic activity To improve cytotoxic activity against tumor cells, closer and tighter binding between tumor cells and effector cells is crucial. This means that a shorter distance may help bring T cells and tumor cells closer together, which may be beneficial for TDCC. Therefore, we generated three anti-DLL3/Dual-Linc antibody variants with different linker lengths. The linker lengths of DLL3-DualAE05/DualAE05-FF110 (medium linker) and DLL3-DualAE05/DualAE05-FF111 (short linker) are shorter than that of DLL3-DualAE05/DualAE05-FF091 (long linker) (hereinafter these variants may be abbreviated as DLL3-AE05/AE05-FF110, DLL3-AE05/AE05-FF111, and DLL3-AE05/AE05-FF091, respectively). To evaluate the effect of linker length on cytotoxic activity, TDCC measurements were performed as described in Example 3.1 using 0.2, 1, and 5 nM of antibody.

As shown in Figure 2 b), DLL3-AE05/AE05-FF110 (medium linker) and DLL3-AE05/AE05-FF111 (short linker) showed stronger TDCC activity than that of DLL3-AE05/AE05-FF091 (long linker) at 0.2 nM. DLL3-AE05/AE05-FF110 (medium linker) showed the strongest TDCC activity. These results suggest that the linker length is important for TDCC activity, and that the medium-length linker is the most suitable for TDCC activity in vitro.

[Example 4]
Evaluation of the in vivo efficacy of DLL3-DualAE05/DualAE05-FF091 trispecific antibody compared to DLL3CD3BiTE
The antitumor activity of DLL3-DualAE05/DualAE05-FF091 antibody and DLL3CD3BiTE prepared in Example 2 was tested in a human small cell lung cancer NCI-H1436 cancer model. NCI-H1436 cells were subcutaneously implanted into NOG humanized mice. NOG female mice were purchased from In-Vivo Science. For humanization, mice were sublethally irradiated, followed by injection of 100,000 human umbilical cord blood cells (ALLCELLS) one day later. After humanization, NCI-H1436 (5× ¹⁰⁶ cells) were mixed with Matrigel Basement Membrane Matrix (Corning) and implanted into the right flank of humanized NOG mice. The day of implantation was defined as day 0. On day 11, mice were randomized based on tumor volume and body weight. The next day, mice were injected intravenously with either vehicle (PBS containing 0.05% Tween), 6.5 mg/kg DLL3-DualAE05/DualAE05-FF091, or 3.5 mg/kg DLL3CD3BiTE.

For analysis of T cell infiltration, xenografted tumors from mice were harvested at the indicated time points after antibody administration. Tumors were weighed and disaggregated by using a Tumor Dissociation Kit for Humans (Miltenyi) according to the manufacturer's protocol, and CountBright Absolute Counting Beads (ThermoFisher scientific) were added after disaggregation. The number of CD8+ T cells in the tumor was assessed using an LSRFortessa X-20 (BD).

For analysis of T cell exhaustion, mouse-borne xenografted tumors were harvested 7 days after antibody administration. Tumors were disaggregated using a human Tumor Dissociation Kit (Miltenyi) according to the manufacturer's protocol, and intratumoral T cells were evaluated for expression of T cell exhaustion markers, Tim-3, PD-1, and Lag-3.

As a result, DLL3-DualAE05/DualAE05-FF091 showed stronger efficacy and higher T cell infiltration than DLL3CD3BiTE (Figures 3 and 4). Furthermore, T cells in the DLL3-DualAE05/DualAE05-FF091-treated group had lower expression of PD-1, Tim-3, and LAG3 (Figure 5), suggesting that DLL3-DualAE05/DualAE05-FF091 induced lower T cell exhaustion.

[Example 5]
In vitro efficacy of anti-DLL3-Dual-LINC antibodies against non-SCLC cell lines
The in vitro cytotoxicity of anti-DLL3-Dual antibody (DLL3-DualAE05/DualAE05-FF110) and untargeted control Dual-LINC antibody (Ctrl-DualAE05/DualAE05-FF110) against non-SCLC cell lines was evaluated using the LDH cytotoxicity detection kit (Takara Bio). NB-1 cell line (neuroblastoma, JCRB Cell Bank IFO50295) and QGP-1 cell line (pancreatic islet cell carcinoma, JCRB Cell Bank JCRB0183) were used as target cells. Target cells were seeded at 1 × ¹⁰⁴ cells/well in a 96-well culture plate, and antibodies were added at various concentrations (10 nM to 0.00064 nM, 5-fold serial dilutions). Then, 2 × ¹⁰⁵ human PBMCs were added and incubated at 37°C. After 24 hours, the high control samples were lysed by Triton-X 100, and 50 μL of supernatant was collected from all wells. The cytotoxic activity (%) was calculated according to the manufacturer's protocol. As shown in Figure 7, anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF110) exhibited in vitro cell killing activity against non-SCLC cell lines, including NB-1 and QGP-1 cell lines.

[Example 6]
In vivo efficacy of anti-DLL3-Dual-LINC antibodies in combination with chemotherapy agents
The antitumor efficacy of combination treatment of anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF114) and platinum reagents was evaluated in the DMS53 small cell lung cancer model. NOG female mice were purchased from In-Vivo Science. For humanization, mice were sublethally irradiated and then injected with 100,000 human umbilical cord blood cells (LONZA) one day later. DMS53 cells (ATCC CRL-2062) were mixed with Matrigel Basement Membrane Matrix (Corning) and inoculated into the right flank of humanized NOG mice. On day 9, when the tumor volume was approximately 150 ^mm3 , mice were randomized based on tumor volume and body weight and intravenously (iv) injected with vehicle (PBS containing 0.05% Tween), 1.3 mg/kg anti-DLL3-Dual-LINC antibody (DLL3-DualAE05/DualAE05-FF114), 7.5 mg/kg cisplatin (CDDP), and 40 mg/kg carboplatin (CBDCA) as monotherapy or in combination. As a result, the combination of anti-DLL3-Dual-LINC antibody DLL3-DualAE05/DualAE05-FF114 with platinum reagents showed significantly higher efficacy than the monotherapy of CDDP, CBDCA, or anti-DLL3-Dual-LINC antibody alone (Figure 8).

[Example 7]
In vivo efficacy of anti-DLL3-Dual-LINC antibodies against a humanized model with NCI-H1436
The antitumor efficacy of anti-DLL3-Dual-LINC antibodies DLL3-DualAE05/DualAE05-FF114 was evaluated in the NCI-H1436 small cell lung cancer model. NOG female mice were purchased from In-Vivo Science. For humanization, mice were sublethally irradiated and then injected with 100,000 human umbilical cord blood cells (LONZA) one day later. NCI-H1436 cells (ATCC CRL-5871) were mixed with Matrigel Basement Membrane Matrix (Corning) and inoculated into the right flank of humanized NOG mice. On day 16, when the tumor volume was approximately 150 ^mm3 , mice were randomized based on tumor volume and body weight and injected iv with vehicle (PBS containing 0.05% Tween), 6.5 mg/kg anti-DLL3-Dual-LINC antibody (DLL3-DualAE05/DualAE05-FF114), and 3.5 mg/kg DLL3CD3BiTE. Anti-DLL3-Dual-LINC antibody DLL3-DualAE05/DualAE05-FF114 and DLL3CD3BiTE were administered in a single dose or weekly (QW). As a result, anti-DLL3-Dual-LINC antibody DLL3-DualAE05/DualAE05-FF114 showed stronger efficacy than DLL3CD3BiTE in both treatment regimens (Figure 9).

[Example 8]
In vitro efficacy of anti-DLL3-Dual-LINC antibodies against other non-SCLC cell lines
The in vitro cytotoxic activity of anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF110 or DLL3-DualAE05/DualAE05-FF114) and non-targeting control Dual LINC antibodies against other non-SCLC cell lines is evaluated as described in Example 5. Examples of non-SCLC cell lines evaluated are pancreatic neuroendocrine tumor (PNET), small cell NEC (SCNEC), large cell NEC (LCNEC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine carcinoma, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma (GBM), melanoma, and medullary thyroid carcinoma cell lines.

[Example 9]
In vitro and in vivo efficacy of anti-DLL3-Dual-LINC antibodies in combination with other chemotherapeutic agents or checkpoint inhibitors
The in vitro cytotoxic activity of anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF110 or DLL3-DualAE05/DualAE05-FF114) in combination with other chemotherapeutic agents or checkpoint inhibitors (anti-PD-L1 antibodies or anti-PD1 antibodies) against SCLC cell lines or other non-SCLC cell lines is evaluated using methods such as those described in Example 5.
The in vivo anti-tumor efficacy of anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF110 or DLL3-DualAE05/DualAE05-FF114) in combination with other chemotherapy reagents or checkpoint inhibitors (anti-PD-L1 antibodies or anti-PD1 antibodies) is evaluated in the DMS53 small cell lung cancer model and other non-SCLC cancer models as described in Example 6.
In this example, the non-SCLC cell line (or non-SCLC cancer model) evaluated is a cell line (or cancer model) of a cancer selected from the group consisting of pancreatic neuroendocrine tumors (PNETs), small cell NEC (SCNEC), large cell NEC (LCNEC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine carcinoma, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma (GBM), melanoma, and medullary thyroid carcinoma.
In this example, the checkpoint inhibitor evaluated for combination therapy with anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF110 or DLL3-DualAE05/DualAE05-FF114) is selected from the group consisting of pembrolizumab, pidilizumab (pidilizumab), nimotuzumab (nivolumab), atezolizumab, and durvalumab, in particular atezolizumab.

[Example 10]
In vivo efficacy of anti-DLL3-Dual-LINC antibodies in combination with steroids or tocilizumab
Cytokine release syndrome (CRS) is a common adverse event of T cell-related therapeutic agents such as T cell engagers and CAR-T cells. In clinical practice, treatment with steroids and/or tocilizumab is widely used to control CRS. Therefore, the in vivo efficacy of anti-DLL3-Dual-LINC antibodies (DLL3-DualAE05/DualAE05-FF114) in combination with steroids or tocilizumab was evaluated. Immunized humanized NOG mice were generated as described in Example 7. PC-10 cells (Immuno-Biological Laboratories) expressing human DLL3 were implanted into the right flank of humanized NOG mice. Mice were randomized based on tumor volume and body weight when the tumor volume was approximately 200 ^mm3 . In steroid pretreatment experiments, 33 mg/kg dexamethasone was administered intraperitoneally 1 and 24 hours or 1 hour before antibody injection. In the tocilizumab experiment, 10 mg/kg tocilizumab was administered 1 day before or 6 hours after injection of anti-DLL3-Dual-LINC antibody. Plasma samples were collected before and 6, 24, and 96 hours after injection of anti-DLL3-Dual-LINC antibody, and plasma concentrations of cytokines were measured using Human Cytokine/Chemokine Magnetic Bead Panel (HCYTMAG-60K-PX38, Millipore) according to the manufacturer's instructions. As a result, pretreatment with dexamethasone induced a significant reduction in cytokine production without affecting tumor growth inhibition (Fig. 10a). Similarly, pre- and post-treatment with tocilizumab did not have any effect on the anti-tumor efficacy of anti-DLL3-Dual-LINC antibody (Fig. 10b).

The present invention provides a multispecific antigen-binding molecule capable of binding to CD3 and CD137 (4-1BB), but not simultaneously to CD3 and CD137, and capable of binding to DLL3. The antigen-binding molecule of the present invention exhibits enhanced T cell-dependent cytotoxicity in a DLL3-dependent manner through binding to CD3/CD37 and DLL3. The antigen-binding molecule and pharmaceutical compositions thereof can be used to target cells expressing DLL3 for use in immunotherapy to treat various cancers, particularly cancers associated with DLL3, such as DLL3-positive cancers.

Claims (19)

1. A pharmaceutical formulation comprising an antibody and a pharma- ceutical acceptable carrier for use in the treatment of cancer, wherein the antibody is
(a) a first antigen-binding portion and a second antigen-binding portion that are Fab molecules comprising any one of the following (a1) or (a2):
(a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 20, a heavy chain CDR 2 of SEQ ID NO: 34, a heavy chain CDR 3 of SEQ ID NO: 48, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
(a2) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 28, a heavy chain CDR 2 of SEQ ID NO: 42, a heavy chain CDR 3 of SEQ ID NO: 56, a light chain CDR 1 of SEQ ID NO: 63, a light chain CDR 2 of SEQ ID NO: 68, and a light chain CDR 3 of SEQ ID NO: 73;
and (b) a third antigen-binding portion that binds human Delta-like 3 (DLL3) and comprises an antibody variable region comprising a heavy chain CDR 1 comprising SEQ ID NO: 233, a heavy chain CDR 2 comprising SEQ ID NO: 234, a heavy chain CDR 3 comprising SEQ ID NO: 235, a light chain CDR 1 comprising SEQ ID NO: 237, a light chain CDR 2 comprising SEQ ID NO: 238, and a light chain CDR 3 comprising SEQ ID NO: 239 ;
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 ; and
Each of the first antigen-binding moiety and the second antigen-binding moiety has the following properties:
(i) binds to human CD3;
(ii) binds to human CD137; or
(iii) capable of binding to human CD3 and human CD137, and capable of binding to either human CD3 or human CD137
having one, two or more of the following :
The pharmaceutical preparation. Each of the first antigen-binding moiety and the second antigen-binding moiety is any one of the following (a1) or (a2):
(a1) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 58;
(a2) VH comprising the amino acid sequence of SEQ ID NO: 14, and VL comprising the amino acid sequence of SEQ ID NO: 58;
Including,
2. The pharmaceutical formulation of claim 1. The pharmaceutical formulation according to claim 1 or 2, wherein the third antigen-binding portion comprises an antibody variable region comprising a VH comprising SEQ ID NO: 232 and a VL comprising SEQ ID NO: 236. The pharmaceutical formulation according to claim 1 or 2, wherein the at least one disulfide bond is formed with a cysteine residue at position 191 (EU numbering) of the CH1 region of each of the first antigen-binding portion and the second antigen-binding portion. The pharmaceutical formulation according to claim 1 or claim 2, wherein each of the first, second, and third antigen-binding moieties is a Fab comprising a heavy chain comprising a VH and CH1 domain and a light chain comprising a VL and a light chain constant (CL) domain, and the C-terminus of the heavy chain of the third antigen-binding moiety is fused, directly or via a peptide linker, to the N-terminus of the Fab heavy chain of either the first antigen-binding moiety or the second antigen-binding moiety. 6. The pharmaceutical formulation of claim 5, wherein the C-terminus of the heavy chain of the third antigen-binding portion is fused to the N-terminus of the Fab heavy chain of either the first antigen-binding portion or the second antigen-binding portion via a peptide linker, and the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO: 259 . 7. The pharmaceutical formulation of claim 6, wherein the third antigen-binding portion is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain have been exchanged, and each of the first antigen-binding portion and the second antigen-binding portion is a conventional Fab molecule. 8. The pharmaceutical formulation of claim 7, wherein in the CL domain of each of the first and second antigen-binding moieties, the amino acids at positions 123 and 124 (Kabat numbering) are arginine and lysine, respectively; and in the CH1 domain of each of the first and second antigen-binding moieties, the amino acids at positions 147 and 213 (EU numbering) are glutamic acid. 3. The pharmaceutical formulation of claim 1 or claim 2, wherein the antibody further comprises an Fc domain. The group consisting of the following (A) to (C):
(A) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:201, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:208, and two polypeptide chains (chains 4 and 5), each comprising the amino acid sequence of SEQ ID NO:214;
(B) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:203, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214; and (C) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO:204, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO:206, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO:209, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO:214.
23. A pharmaceutical formulation for use in the treatment of cancer comprising an antibody comprising five polypeptide chains in a combination selected from the following: 11. The pharmaceutical formulation of claim 1, 2 or 10 , wherein the cancer is a DLL3-expressing or DLL3-positive cancer. 12. The pharmaceutical formulation of claim 11, wherein the cancer is a neuroendocrine neoplasm (NEN), neuroendocrine tumor (NET), neuroendocrine carcinoma (NEC), or other solid tumor of non-neuroendocrine origin that expresses or is DLL3 positive. 11. The pharmaceutical formulation of claim 1, 2 or 10, wherein the cancer is selected from the group consisting of pancreatic neuroendocrine tumor (PNET), small cell NEC (SCNEC), large cell NEC (LCNEC), small cell lung cancer (SCLC), large cell neuroendocrine lung cancer, neuroendocrine prostate cancer (NEPC), Merkel cell carcinoma, small cell bladder cancer, GI neuroendocrine cancer, medullary thyroid carcinoma (MTC), gastrointestinal pancreatic neuroendocrine carcinoma (GEP NEC), neuroblastoma, glioma or glioblastoma ( GBM ), melanoma, and medullary thyroid cancer. 13. The pharmaceutical formulation of claim 1, 2 or 10 for use in combination with an additional therapeutic agent. 15. The pharmaceutical formulation of claim 14 , wherein the additional therapeutic agent is a chemotherapeutic agent or an immune checkpoint inhibitor. 16. The pharmaceutical formulation of claim 15 , wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist. 17. The pharmaceutical formulation of claim 16 , wherein the PD-1 axis binding antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody. 16. The pharmaceutical formulation of claim 15, wherein the chemotherapeutic agent is a microtubule disrupting agent, antimetabolite, topoisomerase inhibitor, DNA intercalating agent, alkylating agent, hormonal therapy, kinase inhibitor, receptor antagonist, activator of tumor cell apoptosis, antiangiogenic agent, etoposide, irinotecan, lurbinectedin, amrubicin, or a platinum agent. 19. The pharmaceutical preparation of claim 18 , wherein the platinum agent is cisplatin or carboplatin.

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