Antigen-binding molecule containing a modified antibody variable region

Abstract

To provide antigen-binding molecules which exhibit effective target-specific cell killing efficacy mediated by immune cells (e.g. T cells) while having reduced or minimal side effects, and to provide a pharmaceutical composition comprising the antigen binding molecule, and a method for producing the antigen-binding molecule.SOLUTION: An antigen-binding molecule capable of binding to multiple different antigens (e.g., CD3 on T cells), but does not nonspecifically crosslink two or more immune cells such as T cells is provided. Such multispecific antigen-binding molecule is capable of modulating and / or activating an immune response while circumventing the cross-linking between different cells resulting from the binding of the conventional multispecific antigen-binding molecule to antigens expressed on the different cells, which is considered to be responsible for adverse reactions when the multispecific antigen-binding molecule is used as a drug.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides antigen-binding molecules that can modulate and / or activate an immune response; pharmaceutical compositions comprising any of the antigen-binding molecules; and methods for producing the antigen-binding molecules. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and rarely cause adverse reactions (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-Patent Literature 1) and Eur J Pharm Biopharm. (2005) 59 (3), 389-396 (Non-Patent Literature 2)). Antibodies not only have antigen-binding activity and agonist or antagonist activity, but also induce effector cell-mediated cytotoxic activity (also called effector function), such as ADCC (antibody-dependent cytotoxicity), ADCP (antibody-dependent cytophagocytosis), or CDC (complement-dependent cytotoxicity). In particular, antibodies of the IgG1 subclass exhibit effector function against cancer cells, and numerous antibody drugs are being developed in the field of oncology.

[0003] For an antibody to exert ADCC, ADCP, or CDC effects, its Fc region must bind to the antibody receptor (FcγR) and various complement components present on effector cells (such as NK cells or macrophages). In humans, the FcγR protein family has been reported to include isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and their allotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (Non-Patent Literature 3)). Of these isoforms, FcγRIa, FcγRIIa, and FcγRIIIa have a domain called ITAM (immune receptor activating tyrosine motif) in their intracellular domain, which transmits activation signals. In contrast, only FcγRIIb has a domain called ITIM (immune receptor suppressing tyrosine motif) in its intracellular domain, which transmits suppression signals. All of these isoforms of FcγR are known to transmit signals through crosslinking by immune complexes (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-Patent Literature 4)). In fact, when an antibody exerts effector function against cancer cells, FcγR molecules on the effector cell membrane cluster together with the Fc regions of multiple antibodies bound to the cancer cell membrane, thereby transmitting an activation signal through the effector cell. As a result, a cytotoxic effect is exerted. In this respect, crosslinking of FcγR is limited to effector cells located near cancer cells, which indicates that immune activation is localized to cancer cells (Ann. Rev. Immunol. (1988). 6. 251-81 (Non-Patent Literature 5)).

[0004] Native immunoglobulins bind to antigens via their variable region and to receptors or complements such as FcγR, FcRn, FcαR, and FcεR via their constant region. Each FcRn molecule (a binding molecule that interacts with the Fc region of IgG) binds to each heavy chain of the antibody, one molecule at a time. Therefore, it has been reported that two FcRn molecules bind to one IgG antibody molecule. Unlike FcRn, FcγR interacts with the hinge region and CH2 domain of the antibody, and only one FcγR molecule binds to one IgG antibody molecule (J. Bio. Chem., (20001) 276, 16469-16477). It has been found that the binding between FcγR and the Fc region of the antibody is important for the hinge region of the antibody, several amino acid residues in the CH2 domain, and the glycan attached to Asn 297 (EU numbering) of the CH2 domain (Chem. Immunol. (1997), 65, 88-110 (Non-Patent Literature 6), Eur. J. Immunol. (1993) 23, 1098-1104 (Non-Patent Literature 7), and Immunol. (1995) 86, 319-324 (Non-Patent Literature 8)). Various Fc region variants with different FcγR binding characteristics have been studied, focusing on this binding site, and Fc region variants with higher binding activity to activated FcγR have been obtained (WO2000 / 042072 (Patent Literature 1) and WO2006 / 019447 (Patent Literature 2)). For example, Lazar et al. succeeded in increasing the binding activity of human IgG1 to human FcγRIIIa (V158) by approximately 370 times by substituting Ser 239, Ala 330, and Ile 332 (EU numbering) of human IgG1 with Asn, Leu, and Glu, respectively (Proc. Natl. Acad. Sci. USA (2006) 103, 4005-4010 (Non-Patent Literature 9) and WO2006 / 019447 (Patent Literature 2)). This modified form shows approximately 9 times greater binding activity compared to the wild type in terms of the ratio of FcγRIIIa to FcγIIb (A / I ratio).Alternatively, Shinkawa et al. succeeded in increasing the binding activity to FcγRIIIa by approximately 100 times by deleting the fucose in the sugar chain attached to Asn 297 (EU numbering) (J. Biol. Chem. (2003) 278, 3466-3473 (Non-Patent Literature 10)). These methods can significantly improve the ADCC activity of human IgG1 compared to natural human IgG1.

[0005] Natural IgG antibodies typically recognize and bind to only one epitope through their variable region (Fab), and therefore can only bind to one antigen. On the other hand, in cancer or inflammation, multiple proteins are known to be involved, and these proteins can crosstalk with each other. For example, in immune diseases, several inflammatory cytokines (TNF, IL1, and IL6) are known to be involved (Nat. Biotech., (2011) 28, 502-10 (Non-Patent Literature 11)). Furthermore, activation of other receptors is known to be one mechanism underlying the acquisition of drug resistance in cancer (Endocr Relat Cancer (2006) 13, 45-51 (Non-Patent Literature 12)). In such cases, a normal antibody that recognizes only one epitope cannot inhibit multiple proteins.

[0006] Antibodies that bind to two or more antigens with a single molecule (these antibodies are called bispecific antibodies) are being studied as molecules that inhibit multiple targets. By modifying naturally occurring IgG antibodies, binding activity to two different antigens (a first antigen and a second antigen) can be conferred (mAbs. (2012) Mar 1, 4(2)). Therefore, such antibodies not only neutralize these two or more antigens with a single molecule, but also enhance antitumor activity by cross-linking cytotoxic cells to cancer cells. Previously reported molecular forms of bispecific antibodies include molecules with an antigen-binding site added to the N-terminus or C-terminus of the antibody (DVD-Ig, TCB, and scFv-IgG), molecules in which the two Fab regions of the antibody have different sequences (common light chain bispecific antibodies and hybrid hybridomas), molecules in which one Fab region recognizes two antigens (Two-in-one IgG and DutaMab), and molecules with a CH3 domain loop as another antigen-binding site (Fcab) (Nat. Rev. (2010), 10, 301-316 (Non-Patent Literature 13) and Peds (2010), 23(4), 289-297 (Non-Patent Literature 14)). In all of these bispecific antibodies, the effector function of the antibody is conserved because it interacts with FcγR in its Fc region.

[0007] If all the antigens recognized by a bispecific antibody are antigens specifically expressed in cancer, then the bispecific antibody that binds to any of the antigens will exhibit cytotoxic activity against cancer cells, and thus a more efficient anticancer effect can be expected compared to conventional antibody drugs that recognize only one antigen. However, if any one of the antigens recognized by the bispecific antibody is expressed in normal tissue or on immune cells, cross-linking with FcγR can cause damage to normal tissue or the release of cytokines (J. Immunol. (1999) Aug 1, 163(3), 1246-52 (Non-patent Literature 15)). As a result, a strong adverse reaction can be induced.

[0008] For example, catumaxomab is known as a bispecific antibody that recognizes proteins expressed on T cells and proteins expressed on cancer cells (cancer antigens). Catumaxomab has two Fabs that bind to the cancer antigen (EpCAM) and the CD3ε chain expressed on T cells, respectively. By simultaneously binding to the cancer antigen and CD3ε, catumaxomab induces T cell-mediated cytotoxic activity, and by simultaneously binding to the cancer antigen and FcγR, it induces NK cell or antigen-presenting cell (e.g., macrophages)-mediated cytotoxic activity. By utilizing these two cytotoxic activities, catumaxomab has shown high therapeutic efficacy against malignant ascites when administered intraperitoneally, and is therefore approved in Europe (Cancer Treat Rev. (2010) Oct 36(6), 458-67 (Non-Patent Literature 16)). Furthermore, there have been reports of cases where catumakisomab administration resulted in the emergence of antibodies that react with cancer cells, demonstrating that adaptive immunity is induced (Future Oncol. (2012) Jan 8(1), 73-85 (Non-Patent Literature 17)). Based on these results, antibodies that possess both T cell-mediated cytotoxic activity and effects mediated by cells such as NK cells or macrophages via FcγR (these antibodies are specifically called trifunctional antibodies) are attracting attention because they are expected to have strong antitumor effects and induce adaptive immunity.

[0009] However, because trifunctional antibodies simultaneously bind to CD3ε and FcγR even in the absence of cancer antigens, they cross-link T cells expressing CD3ε to cells expressing FcγR, even in environments without cancer cells, causing the production of large amounts of various cytokines. Due to this induction of cancer antigen-independent cytokine production, the administration of trifunctional antibodies is currently limited to the intraperitoneal route (Cancer Treat Rev. 2010 Oct 36(6), 458-67 (Non-Patent Literature 16)). Systemic administration of trifunctional antibodies is extremely difficult due to severe cytokine storm-like adverse reactions (Cancer Immunol Immunother. 2007 Sep; 56(9): 1397-406 (Non-Patent Literature 18)). Conventional bispecific antibodies can bind to both antigens, namely the first antigen, the cancer antigen (EpCAM), and the second antigen, CD3ε, simultaneously with binding to FcγR. Therefore, such adverse reactions caused by simultaneous binding to FcγR and the second antigen, CD3ε, cannot be avoided due to their molecular structure. In recent years, improved antibodies have been provided that utilize an Fc region with reduced binding activity to FcγR, thereby avoiding adverse reactions while inducing T cell-mediated cytotoxicity (WO2012 / 073985). However, even with such antibodies, given their molecular structure, they cannot bind to cancer antigens while acting on two immune receptors, namely CD3ε and FcγR, and can only utilize one immune receptor, thus proving to be insufficiently effective (WO2014 / 116846 (Patent Document 4)). Furthermore, it is known that a very serious adverse event caused by cytokine release, known as cytokine release syndrome (CRS) or cytokine storm, can be caused by such bispecific antibodies that act only on CD3ε, and it has been reported that induction of IL-6 may be one of the main causes of CRS (Ferran, 1990, Eur J Immunol. Mar;20(3):509-15 (Non-Patent Document 26), Frey, 2016, Hematology Am Soc Hematol Educ Program. 2;2016(1):567-572 (Non-Patent Document 27)).

[0010] T cells play a crucial role in tumor immunity and are known to be activated by two signaling mechanisms: 1) binding of the T cell receptor (TCR) to antigen peptides presented by major histocompatibility complex (MHC) class I molecules and activation of the TCR; and 2) binding of costimulatory molecules on the surface of T cells to ligands on antigen-presenting cells and activation of these costimulatory molecules. Furthermore, the activation of molecules belonging to the tumor necrosis factor (TNF) superfamily and the TNF receptor superfamily, such as CD137(4-1BB) on the surface of T cells, has been described as important for T cell activation (Vinay, 2011, Cellular & Molecular Immunology, 8, 281-284 (Non-Patent Literature 19)).

[0011] CD137 agonist antibodies have already been demonstrated to exhibit antitumor effects, which have been experimentally shown to be mainly due to the activation of CD8-positive T cells and NK cells (Houot, 2009, Blood, 114, 3431-8 (Non-Patent Document 20)). T cells engineered to have a chimeric antigen receptor molecule consisting of a tumor antigen-binding domain as an extracellular domain and CD3 and CD137 signaling domains as intracellular domains (CAR-T cells) can enhance the durability of efficacy (Porter, N ENGL J MED, 2011, 365;725-733 (Non-Patent Document 21)). However, the side effects due to the non-specific hepatotoxicity of such CD137 agonist antibodies are clinical and preclinical problems, and drug development has not advanced (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22 (Non-Patent Document 22)). It has been suggested that the main cause of the side effects involves the binding of the antibody to the Fcγ receptor via the antibody constant region (Schabowsky, Vaccine, 2009, 28, 512-22 (Non-Patent Document 23)).

[0012] Furthermore, it has been reported that for an agonist antibody targeting a receptor belonging to the TNF receptor superfamily to exhibit agonist activity in vivo, antibody bridging by Fcγ receptor-expressing cells (FcγRII-expressing cells) is necessary (Li, Proc Natl Acad Sci USA. 2013, 110(48), 19501-6 (Non-Patent Document 24)). WO2015 / 156268 (Patent Document 3) describes that a bispecific antibody having a binding domain with CD137 agonist activity and a binding domain for a tumor-specific antigen exhibits CD137 agonist activity only in the presence of cells expressing the tumor-specific antigen and can activate immune cells, whereby the hepatotoxic adverse events of the CD137 agonist antibody can be avoided while retaining the anti-tumor activity of the antibody. WO2015 / 156268 further describes that by using this bispecific antibody in combination with another bispecific antibody having a binding domain with CD3 agonist activity and a binding domain for a tumor-specific antigen, the anti-tumor activity can be further enhanced and these adverse events can be avoided. A trispecific antibody having three binding domains for CD137, CD3, and a tumor-specific antigen (EGFR) has also been reported (WO2014 / 116846 (Patent Document 4)).

Prior Art Documents

Patent Documents

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Non-Patent Documents

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[0015] Antibodies that exhibit both cytotoxic activity mediated by immune cells (e.g., T cells) and activating activity of T cells and / or other immune cells via a co-stimulatory molecule (e.g., CD137), in a target antigen-specific manner, while avoiding adverse reactions, are not yet known. An object of the present invention is to provide an antigen-binding molecule that exhibits an effective target-specific cell death effect mediated by immune cells (e.g., T cells) while having reduced or minimal side effects. Another object of the present invention is to provide a pharmaceutical composition comprising the antigen-binding molecule and a method for producing the antigen-binding molecule. [Means for solving the problem]

[0016] An antigen-binding molecule is provided that can bind to multiple different antigens (e.g., CD3 on T cells, and CD137 on T cells, NK cells, and / or DC cells, etc.) but does not nonspecifically crosslink two or more immune cells, such as T cells. Such a multispecific antigen-binding molecule can modulate and / or activate the immune response while avoiding crosslinking between different cells (e.g., different T cells) resulting from the binding of conventional multispecific antigen-binding molecules to antigens expressed on different cells, which is thought to be the cause of adverse reactions when multispecific antigen-binding molecules are used as drugs.

[0017] In one aspect, the antigen-binding molecule of the present invention provides a novel antigen-binding molecule having a highly unique structural form that improves or enhances the effectiveness of multispecific antigen-binding molecules. The novel antigen-binding molecule having a unique structural form provides an increase in the number of antigen-binding domains, thereby giving an increase in binding titer and / or specificity to each antigen on effector cells and target cells, accompanied by a reduction in undesirable adverse reactions. In a further aspect, one of the antigen-binding molecules having such a novel and unique structural form of the present invention comprises at least two first and second antigen-binding domains (e.g., Fab domains) linked together (e.g., via Fc, disulfide bonds, or linkers, etc.), each binding to a first and / or second antigen on an effector cell (e.g., an immune cell such as a T cell, NK cell, or DC cell), and further comprises a third (and optionally fourth) antigen-binding domain linked to either the first or second antigen-binding domain, which binds to a third antigen on a target cell (e.g., a tumor cell).

[0018] In a further aspect, one of the antigen-binding molecules having such a novel and unique structural form of the present invention comprises at least two first and second antigen-binding domains (e.g., Fab domains) linked together (e.g., via Fc, disulfide bonds, or linkers, etc.) each binding to a first and / or second antigen on an effector cell (e.g., an immune cell such as a T cell, NK cell, or DC cell), and further comprises a third (and optionally fourth) antigen-binding domain linked to either the first or second antigen-binding domain, each of the first and second antigen-binding domains (e.g., Fab domains) capable of binding to the first and / or second antigen, wherein each contains at least one amino acid mutation, the amino acid mutation creating a linkage between the first and second antigen-binding domains, keeping them close to each other, and promoting, for example, cis-antigen binding to the same single effector cell. The antigen-binding molecule has such a unique structural form that it has been surprisingly found by the inventors to exhibit excellent efficacy while reducing or minimizing off-target side effects caused by undesirable cross-linking between different cells (e.g., effector cells such as T cells).

[0019] More specifically, the present invention relates to the following: [1] (i) a first antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) A second antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region. An antigen-binding molecule comprising at least two antigen-binding domains, The first antigen-binding domain and the second antigen-binding domain are linked via an Fc region, a disulfide bond, or a linker. The first antigen-binding domain and the second antigen-binding domain can each bind to the first antigen and to a second antigen different from the first antigen, respectively, but they cannot bind to both the first and second antigens simultaneously. The aforementioned antigen-binding molecule. [2] The antigen-binding molecule of [1] further comprising a third antigen-binding domain having a heavy chain variable (VH) region and a light chain variable (VL) region, which is capable of binding to a third antigen different from the first and second antigens, wherein the third antigen-binding domain is linked to either the first antigen-binding domain or the second antigen-binding domain or to an Fc region. [3] (i) a first antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) A second antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region. An antigen-binding molecule comprising at least two antigen-binding domains, The first antigen-binding domain and the second antigen-binding domain are linked via an Fc region, a disulfide bond, or a linker. The first antigen-binding domain can bind to the first antigen and to a second antigen different from the first antigen, but it cannot bind to both the first and second antigens simultaneously; The second antigen-binding domain can bind to either the first antigen or the second antigen, or only one of them. The aforementioned antigen-binding molecule. [4] The antigen-binding molecule of [3], further comprising a third antigen-binding domain having a heavy chain variable (VH) region and a light chain variable (VL) region, which is capable of binding to a third antigen different from the first and second antigens, wherein the third antigen-binding domain is linked to either the first antigen-binding domain or the second antigen-binding domain or to an Fc region. [5] (i) A first antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) A third antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region. An antigen-binding molecule comprising at least two antigen-binding domains, The third antigen-binding domain is linked to the first antigen-binding domain. The first antigen-binding domain can bind to the first antigen and to a second antigen different from the first antigen, but it cannot bind to both the first and second antigens simultaneously. The third antigen-binding domain is capable of binding to a third antigen that is different from the first and second antigens. The aforementioned antigen-binding molecule. [6] (i) A first antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) A second antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region. An antigen-binding molecule comprising at least two antigen-binding domains, The first antigen-binding domain and the second antigen-binding domain are linked via an Fc region, a disulfide bond, or a linker. The first antigen-binding domain and the second antigen-binding domain are each capable of binding to either the first antigen or the second antigen, respectively. The aforementioned antigen-binding molecule. [7] The antigen-binding molecule of [6], further comprising a third antigen-binding domain having a heavy chain variable (VH) region and a light chain variable (VL) region, which is capable of binding to a third antigen different from a first antigen and a second antigen, wherein the third antigen-binding domain is linked to either the first antigen-binding domain or the second antigen-binding domain or to an Fc region. [7A] Antigen-binding molecules represented by the following formula: TIFF0007871319000001.tif28170In formula, C is an Fc region; O is an integer, either 1 or 0; B 1 and B 2 Each of the following: (i) A first antigen-binding domain and a second antigen-binding domain, each capable of binding to a first antigen and a second antigen distinct from the first antigen, but not to both antigens simultaneously; (ii) A first antigen-binding domain and a second antigen-binding domain, wherein one antigen-binding domain can bind to a first antigen and a second antigen different from the first antigen, but not to both antigens simultaneously, and the other antigen-binding domain can bind to either the first antigen or the second antigen, but not both; (iii) a first antigen-binding domain and a second antigen-binding domain, each capable of binding to the first antigen; or (iv) The first antigen-binding domain and the second antigen-binding domain, each capable of binding to either the first antigen or the second antigen, respectively. and; Each B 1 and B 2 m is an integer of 1 or 0, provided that both m are not 0 at the same time; A 1and A 2 each of which is as follows: (i) The same antigen-binding domain that can bind to a third antigen, different from the first antigen and the second antigen; (ii) One antigen-binding domain that can bind to a third antigen, different from the first antigen and the second antigen, and the other antigen-binding domain that can bind to a fourth antigen, different from the first antigen, the second antigen, and the third antigen, different antigen-binding domains where; n of each A1 and A2 is an integer of 1 or 0, provided that when m is 0, n is 0; and B 1 each of the wavy lines between and B 2 and C is a covalent bond or a linker; B 1 between and A 1 and each of the wavy lines between B 2 and A 2 is a covalent bond or a linker; and the wavy line between B1 and B2 is one or more bonds that hold B 1 and B 2 close to each other, provided that: when B1 and B2 each contain an antibody heavy chain hinge region and B1 and B2 are linked to each other by one or more native disulfide bonds in their respective hinge regions, the bond is a bond existing between any other parts outside the hinge region, or an additional bond existing between the hinge regions. [8] An antigen-binding molecule of any one of [1] to [5], in which one or more of the first antigen-binding domain and the second antigen-binding domain, which can bind to a first antigen and a second antigen different from the first antigen but do not bind to both the first and second antigens simultaneously, have at least one amino acid modification. [9] The antigen-binding molecule of [8], wherein the modification is at least one amino acid substitution, insertion, or deletion.

[10] The antigen-binding molecule of [9], wherein the modification is the substitution of a portion of the amino acid sequence of the VH and / or VL region that binds to the first antigen by the amino acid sequence of the VH and / or VL region that binds to the second antigen, or the insertion of the amino acid sequence of the VH and / or VL region that binds to the second antigen into the amino acid sequence of the VH and / or VL region that binds to the first antigen.

[11] An antigen-binding molecule having 1 to 25 amino acids inserted or substituted, one of either [9] or

[10] .

[12] Any one antigen-binding molecule from [8] to

[11] , wherein the modified amino acid is an amino acid in one or more of the CDR1, CDR2, CDR3, and FR3 regions of the heavy chain variable (VH) region and / or light chain variable (VL) region.

[13] Any one antigen-binding molecule from [8] to

[12] , wherein the amino acid to be modified is an amino acid in one or more loops of the hypervariable region (HVR).

[14] Any one antigen-binding molecule from [8] to

[13] , wherein the modified amino acid is at least one amino acid selected from Kabat numbering positions 31–35, 50–65, 71–74, and 95–102 in the antibody heavy chain variable (VH) region, and Kabat numbering positions 24–34, 50–56, and 89–97 in the light chain variable (VL) region.

[15] An antigen-binding molecule in which the first antigen-binding domain and the second antigen-binding domain are linked via an Fc region, one of the antigen-binding molecules from [1] to

[14] .

[16] An antigen-binding molecule of

[15] in which the Fc region has reduced binding activity to FcγR compared to the Fc region of the wild-type human IgG1 antibody.

[17] An antigen-binding molecule, one of the [1] to

[14] , wherein each of the first antigen-binding domain and the second antigen-binding domain includes a hinge region and is linked by one or more disulfide bonds in the hinge region.

[18] An antigen-binding molecule, one of the [1] to

[14] , in which a first antigen-binding domain and a second antigen-binding domain are linked via a linker.

[19] One antigen-binding molecule from [1] to

[14] , each of which antigen-binding domains has a Fab, Fab', scFab, Fv, scFv, or VHH structure.

[20] One antigen-binding molecule from [1] to

[14] , each having a Fab in its antigen-binding domain.

[21] An antigen-binding molecule comprising a Fab and a hinge region, each of which has a first antigen-binding domain and a second antigen-binding domain that together form an F(ab')2 structure, one of the antigen-binding molecules from [1] to

[20] .

[22] The third antigen-binding domain is as follows: (i) Between the C-terminus of a polypeptide containing a heavy chain variable (VH) region of a third antigen-binding domain and the N-terminus of a polypeptide containing a heavy chain variable (VH) region of either the first antigen-binding domain or the second antigen-binding domain, (ii) Between the C-terminus of a polypeptide containing the heavy chain variable (VH) region of the third antigen-binding domain and the N-terminus of a polypeptide containing the light chain variable (VL) region of either the first antigen-binding domain or the second antigen-binding domain, (iii) Between the C-terminus of a polypeptide containing the light chain variable (VL) region of the third antigen-binding domain and the N-terminus of a polypeptide containing the heavy chain variable (VH) region of either the first antigen-binding domain or the second antigen-binding domain, (iv) Between the C-terminus of a polypeptide containing the light chain variable (VL) region of the third antigen-binding domain and the N-terminus of a polypeptide containing the light chain variable (VL) region of either the first or second antigen-binding domain One antigen-binding molecule from [2], [4], [5] and [7]-

[21] is linked to either the first antigen-binding domain or the second antigen-binding domain via one of the following linkages.

[23] The antigen-binding molecule of [1]-

[22] , wherein the first antigen-binding domain and the second antigen-binding domain are linked to each other via at least one bond that keeps the first antigen-binding domain and the second antigen-binding domain close to each other, provided that the first antigen-binding domain includes a heavy chain hinge region and the second antigen-binding domain includes a heavy chain hinge region, and the first antigen-binding domain and the second antigen-binding domain are linked to each other by one or more native disulfide bonds in their respective hinge regions, the bond being a bond present between any other parts other than the hinge regions, or a further bond present between the hinge regions. [23A] An antigen-binding molecule of [1]-

[23] wherein at least one binding that keeps a first antigen-binding domain and a second antigen-binding domain close to each other limits the antigen-binding of the first antigen-binding domain and the second antigen-binding domain to cis-antigen binding (i.e., binding to an antigen on the same cell).

[24] An antigen-binding molecule of

[23] in which at least one bond is covalent.

[25] The antigen-binding molecule of

[24] in which a covalent bond is formed by direct crosslinking between an amino acid residue in the first antigen-binding domain and an amino acid residue in the second antigen-binding domain.

[26] An antigen-binding molecule of

[25] in which the crosslinked amino acid residue is cysteine.

[27] Antigen-binding molecules of

[26] in which the covalent bond formed is a disulfide bond.

[28] The antigen-binding molecule of

[24] wherein the covalent bond is formed by crosslinking between an amino acid residue in the first antigen-binding domain and an amino acid residue in the second antigen-binding domain via a crosslinking agent.

[29] The antigen-binding molecule of

[28] , wherein the crosslinking agent is an amine-reactive crosslinking agent.

[30] The antigen-binding molecule of

[29] in which the crosslinked amino acid residue is lysine.

[31] An antigen-binding molecule of

[23] in which at least one bond is non-covalent.

[32] Antigen-binding molecules of

[31] , wherein the non-covalent bond is an ionic bond, a hydrogen bond, or a hydrophobic bond.

[33] The first antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, as well as a light chain variable (VL) region and a light chain constant region (CL), and the second antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, as well as a light chain variable (VL) region and a light chain constant region (CL), and at least one binding is located between an amino acid residue in the CH1 region of the first antigen-binding domain and an amino acid residue in the CH1 region of the second antigen-binding domain. One antigen-binding molecule from

[23] to

[32] .

[34] The antigen-binding molecule of

[33] wherein the amino acid residue is located at a position selected from the group consisting of positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 165, 167, 174, 176, 177, 178, 190, 191, 192, 194, 195, 197, 213, and 214 in the CH1 region according to EU numbering.

[35] The antigen-binding molecule of

[34] , wherein the amino acid residue is located at position 191 in the CH1 region according to EU numbering.

[36] The antigen-binding molecule of

[35] , in which the amino acid residue at position 191 according to EU numbering in the CH1 region of the first antigen-binding domain and the second antigen-binding domain are linked to each other to form a bond.

[37] An antigen-binding molecule comprising a first antigen-binding domain comprising a heavy chain variable (VH) region, a CH1 region, and a hinge region, as well as a light chain variable (VL) region and a light chain constant region, and a second antigen-binding domain comprising a heavy chain variable (VH) region, a CH1 region, and a hinge region, as well as a light chain variable (VL) region and a light chain constant region, wherein at least one binding is located between an amino acid residue in the hinge region of the first antigen-binding domain and an amino acid residue in the hinge region of the second antigen-binding domain, and any one of the antigen-binding molecules of

[23] to

[32] .

[38] The antigen-binding molecule of

[37] wherein the amino acid residue is located at a position selected from the group consisting of positions 216, 218, and 219 in the hinge region according to EU numbering.

[39] An antigen-binding molecule comprising any one of

[23] to

[32] , wherein the first antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, as well as a light chain variable (VL) region and a light chain constant region (CL), and the second antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, as well as a light chain variable (VL) region and a light chain constant region (CL), and at least one binding is located between an amino acid residue in the CL region of the first antigen-binding domain and an amino acid residue in the CL region of the second antigen-binding domain.

[40] An antigen-binding molecule of

[39] in which the amino acid residue is located at a position selected from the group consisting of positions 109, 112, 121, 126, 128, 151, 152, 153, 156, 184, 186, 188, 190, 200, 201, 202, 203, 208, 210, 211, 212, and 213 in the CL region according to EU numbering.

[41] The antigen-binding molecule of

[40] , wherein the amino acid residue is located at position 126 in the CL region according to EU numbering.

[42] The antigen-binding molecule of

[42] in which the amino acid residue at position 126 according to EU numbering in the CL region of the first antigen-binding domain and the second antigen-binding domain are linked to each other to form a bond.

[43] An antigen-binding molecule, any one of

[23] to

[32] , wherein the first antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, and a light chain variable (VL) region and a light chain constant region (CL), and the second antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, and a light chain variable (VL) region and a light chain constant region (CL), and at least one bond exists between an amino acid residue in the CH1 region of the first antigen-binding domain and an amino acid residue in the CL region of the second antigen-binding domain, and is linked to form a bond.

[44] An antigen-binding molecule from any of

[23] to

[32] , wherein the first antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, and a light chain variable (VL) region and a light chain constant region (CL), and the second antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, and a light chain variable (VL) region and a light chain constant region (CL), and at least one bond exists between an amino acid residue in the CH1 region of the second antigen-binding domain and an amino acid residue in the CL region of the first antigen-binding domain, and is linked to form a bond.

[45] The antigen-binding molecule of

[43] , in which the amino acid residue at position 191 according to EU numbering in the CH1 region of the first antigen-binding domain and the amino acid residue at position 126 according to EU numbering in the CL region of the second antigen-binding domain are linked to form a bond.

[46] The antigen-binding molecule of

[44] , in which the amino acid residue at position 191 according to EU numbering in the CH1 region of the second antigen-binding domain and the amino acid residue at position 126 according to EU numbering in the CL region of the first antigen-binding domain are linked to form a bond.

[47] An antigen-binding molecule whose CH1 and / or light chain constant region (CL) are derived from human, one of the

[33] -

[46] .

[48] ​​An antigen-binding molecule of any one of the following

[33] -

[46] , wherein the CH1 region subclass is γ1, γ2, γ3, γ4, α1, α2, μ, δ, or ε.

[49] An antigen-binding molecule from any one of

[33] to

[46] whose CL region subclass is κ or λ.

[50] Any one antigen-binding molecule of

[23] -

[32] wherein at least one bond exists between an amino acid residue in the heavy chain variable (VH) region or light chain variable (VL) region of the first antigen-binding domain and an amino acid residue in the heavy chain variable (VH) region or light chain variable (VL) region of the second antigen-binding domain.

[51] An antigen-binding molecule having at least one bond between an amino acid residue in the VH region of a first antigen-binding domain and an amino acid residue in the VH region of a second antigen-binding domain,

[50]

[52] An antigen-binding molecule of

[51] in which an amino acid residue is located at a position selected from the group consisting of positions 8, 16, 28, 74, and 82b in the VH region according to Kabat numbering.

[53] An antigen-binding molecule having at least one bond between an amino acid residue in the VL region of a first antigen-binding domain and an amino acid residue in the VH region of a second antigen-binding domain,

[50]

[54] The antigen-binding molecule of

[53] wherein the amino acid residue is located at a position selected from the group consisting of positions 100, 105, and 107 in the VL region according to Kabat numbering.

[55] The first antigen is an antigen-binding molecule, one of the [1] to

[54] molecules, which is specifically expressed on T cells.

[56] One antigen-binding molecule from [1] to

[55] , wherein the first antigen is a T cell receptor complex molecule.

[57] An antigen-binding molecule from any one of [1] to

[56] , wherein the first antigen is CD3, preferably human CD3.

[58] The second antigen is one antigen-binding molecule, [1] to

[57] which is expressed on a T cell or any other immune cell.

[59] The second antigen is one antigen-binding molecule from [1] to

[58] , which is a costimulatory molecule expressed on a T cell or any other immune cell.

[60] A single antigen-binding molecule from [1] to

[59] whose second antigen is a TNFR superfamily molecule.

[61] One antigen-binding molecule from [1] to

[60] , wherein the second antigen is CD137(4-1BB).

[62] An antigen-binding molecule from any of [1] to

[61] , wherein the first antigen is CD3 and the second antigen is CD137.

[63] A third antigen distinct from the first and second antigens, which is one of the antigen-binding molecules [1] to

[62] that is specifically expressed on cancer cells.

[64] A third antigen distinct from the first and second antigens, which is one of the antigen-binding molecules [1] to

[63] , glypican-3 (GPC3).

[65] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) Sequence ID: VH region containing a sequence having at least 95% sequence identity to any one of the amino acid sequences 1-11 and 61; and (b) Sequence ID: VL region containing a sequence having at least 95% sequence identity to any one of the amino acid sequences 45-48 One antigen-binding molecule from [1] to

[64] , including [1]. [65A] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65B] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 46 One antigen-binding molecule from [1] to

[64] , including [1]. [65C] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65D] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65E] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65F] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65G] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65H] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65H] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 9; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65I] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 10; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 45 One antigen-binding molecule from [1] to

[64] , including [1]. [65J] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 11; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 48 One antigen-binding molecule from [1] to

[64] , including [1]. [65K] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) A VH region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 61; and (b) VL region containing a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 48 One antigen-binding molecule from [1] to

[64] , including [1].

[66] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) Sequence ID: VH region containing a sequence having one of the amino acid sequences 1-11 and 61; and (b) VL region containing one of the amino acid sequences from 45 to 48. One antigen-binding molecule from [1] to

[64] that competes for binding with the antibody containing [the specified substance].

[67] One or more of the first antigen-binding domain or the second antigen-binding domain, (a) Sequence ID: VH region containing a sequence having one of the amino acid sequences 1-11 and 61; and (b) VL region containing one of the amino acid sequences from 45 to 48. A single antigen-binding molecule from [1] to

[64] that binds to the same epitope as the antibody containing [the specified substance].

[68] One or more of the first antigen-binding domain or the second antigen-binding domain, (i) VH region including the following: (a) Sequence ID: An HCDR1 sequence having at least 95% sequence identity to any one of the amino acid sequences 12-22 and 62; (b) Sequence ID: An HCDR2 sequence having at least 95% sequence identity to any one of the amino acid sequences 23-33 and 63; and / or (c) SEQ ID NO: An HCDR3 sequence having at least 95% sequence identity to any one of the amino acid sequences 34-44 and 64; and / or (ii) VL area including the following: (d) Sequence ID: LCDR1 sequence having at least 95% sequence identity to any one of the amino acid sequences 49-52; (e) Sequence ID: An LCDR2 sequence having at least 95% sequence identity to any one of the amino acid sequences 53-54 and 56; and / or (f) Sequence ID: LCDR3 sequence having at least 95% sequence identity to any one of the amino acid sequences 57-58 and 60 One antigen-binding molecule from [1] to

[64] , including [1]. [68A] One antigen-binding molecule from [1] to

[64] in which one or more of the first or second antigen-binding domains include a VH region containing HCDR1-3 and a VL region containing LCDR1-3 sequences, as shown in Table 1.1.

[69] Below: (a) A polypeptide chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 71, 73, 75, 78, 80, and 83; (b) A polypeptide chain containing an amino acid sequence selected from the group consisting of Sequence IDs 68 and 72; (c) A polypeptide chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 74, 76, 79, 81, and 84; and (d) Polypeptide chain containing an amino acid sequence selected from the group consisting of Sequence IDs 70, 77, and 82. One antigen-binding molecule from [1] to

[64] , including one or more of the above. [69A] One antigen-binding molecule from [1] to

[64] , containing a polypeptide chain as listed in Table 2.2. A pharmaceutical composition comprising one antigen-binding molecule from

[70] [1] to

[69] and a pharmaceutically acceptable carrier.

[71] One or more polynucleotides encoding one or more polypeptides of any one of the antigen-binding molecules of [1] to

[69] . One or more vectors containing the polynucleotides of

[72]

[71] . Cells containing the vectors

[73] and

[72] . A method for producing antigen-binding molecules, comprising the steps of culturing the cells of

[74]

[73] and isolating antigen-binding molecules from the culture supernatant.

[75] (a) A step of providing one or more nucleic acids encoding one or more polypeptides that form a first antigen-binding domain and a second antigen-binding domain, (i) The first antigen-binding domain and the second antigen-binding domain can bind to the first antigen and to a second antigen different from the first antigen, but cannot bind to both the first and second antigens simultaneously, or (ii) The first antigen-binding domain can bind to the first antigen and a second antigen different from the first antigen, but not to both the first and second antigens simultaneously; and the second antigen-binding domain can bind to either the first or the second antigen, or (iii) The first antigen-binding domain and the second antigen-binding domain can each bind to either the first antigen or the second antigen, but not both. The process to be provided; (b) The process of introducing nucleic acids into host cells; (c) A step of culturing host cells so that two or more polypeptides are produced; and (d) Step to obtain antigen-binding molecule A method for producing antigen-binding molecules, including [the specified element].

[76] As defined in steps (i) and (ii), providing an antigen-binding domain that does not bind to the first antigen and the second antigen simultaneously, - A step of preparing a library of antigen-binding domains, each of which binds to a first or second antigen, wherein at least one amino acid is modified in its heavy chain variable (VH) region and light chain variable (VL) region, wherein the modified variable regions are different from each other by at least one amino acid; and - A step of selecting antigen-binding domains from the prepared library that have binding activity to the first and second antigens, but do not bind to the first and second antigens simultaneously, and include heavy chain variable (VH) and light chain variable (VL) regions. The method of

[75] , including the method of

[75] . [76A] The method of

[76] , wherein the modification is a modification of at least one amino acid selected from Kabat numbering positions 31–35, 50–65, 71–74, and 95–102 in the heavy chain variable (VH) region, and Kabat numbering positions 24–34, 50–56, and 89–97 in the light chain variable (VL) region. [76B] Any one of the methods

[75] -[76A] wherein the antigen-binding domain, which does not simultaneously bind to the first antigen and the second antigen as defined in (i) and (ii), is itself an antigen-binding domain that does not simultaneously bind to the first antigen and the second antigen expressed on different cells, respectively.

[77] A method of any one of the

[75] to [76B], further comprising step (a) providing one or more nucleic acids encoding one or more polypeptides having a third antigen-binding domain that binds to a third antigen different from the first and second antigens. [77A] The host cells cultured in step (c) further comprise nucleic acids encoding the antibody Fc region, one of the methods

[75] to [76B]. [77B] The method of [77A] wherein the Fc region is an Fc region in which binding activity to FcγR is reduced compared to the Fc region of a natural human IgG1 antibody.

[78] The first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain are encoded by a single nucleotide in any one of the following ways:

[75] -[77B].

[79] A method of any one of the

[75] -

[78] , further comprising the step of introducing one or more mutations into the nucleic acid sequences encoding each of the first and second antigen-binding domains, wherein step (a) introduces one or more bindings that, when translated, link the first and second antigen-binding domains close together.

[80] The method of

[79] wherein a first antigen-binding domain and a second antigen-binding domain are linked to each other via at least one bond that keeps the first antigen-binding domain and the second antigen-binding domain close to each other, provided that the first antigen-binding domain includes a heavy chain hinge region and the second antigen-binding domain includes a heavy chain hinge region and the first antigen-binding domain and the second antigen-binding domain are linked to each other by one or more native disulfide bonds in their respective hinge regions, the bond is a bond present between any other parts other than the hinge regions or a further bond present between the hinge regions.

[81] The first antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, as well as a light chain variable (VL) region and a light chain constant region (CL), and the second antigen-binding domain comprises a heavy chain variable (VH) region and a CH1 region, as well as a light chain variable (VL) region and a light chain constant region (CL), and one or more mutations are present. (i) within the CH1 region of the first antigen-binding domain and within the CH1 region of the second antigen-binding domain; (ii) In the CH1 region of the first antigen-binding domain and in the CL region of the second antigen-binding domain; (iii) In the CL region of the first antigen-binding domain and in the CH1 region of the second antigen-binding domain; (iv) within the CL region of the first antigen-binding domain and within the CL region of the second antigen-binding domain; or (v) In the VH or VL region of the first antigen-binding domain, and in the VH or VL region of the second antigen-binding domain The methods found in

[79] or

[80] .

[82] One or more mutations are cysteine ​​substitutions or insertions in any one of the following ways:

[79] –

[81] .

[83] One of the following methods

[79] -

[81] is used, in which a cysteine ​​amino acid residue is introduced at EU numbering position 191 in the CH1 region of the first antigen-binding domain and the second antigen-binding domain, respectively.

[84] Any one of the methods of

[79] to

[83] further comprising the step of performing an assay to determine whether the first antigen-binding domain and the second antigen domain do not simultaneously bind to the first and second antigens expressed on different cells, respectively.

[85] The first antigen is a molecule specifically expressed on T cells, one of the methods described in

[75] to

[84] .

[86] The first antigen is a T cell receptor complex molecule, in any one of the following ways,

[75] to

[84] .

[87] Any one of the methods

[75] -

[86] , wherein the first antigen is CD3, preferably human CD3.

[88] The second antigen is a molecule expressed on a T cell or any other immune cell, in any one of the ways of

[75] -

[87] .

[89] The second antigen is a costimulatory molecule expressed on a T cell or any other immune cell, in any one of the ways of

[75] -

[88] .

[90] The second antigen is a TNFR superfamily molecule, one of the methods described in

[75] -

[89] .

[91] Any one of the methods

[75] -

[90] , wherein the second antigen is CD137(4-1BB).

[92] The first antigen is CD3 and the second antigen is CD137, one of the methods

[75] to

[91] .

[93] Any one of the following

[75] -

[92] is a molecule that is specifically expressed on cancer cells, wherein the third antigen is different from the first and second antigens.

[94] Any one of the following methods,

[75] -

[93] , wherein the third antigen, distinct from the first and second antigens, is glypican-3 (GPC3). [Brief explanation of the drawing]

[0020] [Figure 1.1]The results of measuring the CD137 agonist activity of affinity-matured GPC3 / Dual-Ig variant tripspecific antibodies are shown. (a) Mean luminescence units + / - standard deviation (sd) detected by the selected antibody group in SK-pca60 cell line co-cultured with CD137-overexpressing Jurkat NFκB receptor cells. (b) Similarly to (a), mean luminescence units + / - standard deviation (sd) detected by the other antibody group in SK-pca60 cell line co-cultured with CD137-overexpressing Jurkat NFκB receptor cells were analyzed in a second plate. [Figure 1.2] This shows the mean cytotoxic activity (inhibition of cell proliferation (%) + / - sd) of GPC3 / Dual-Ig variants. SK-pca60 was co-cultured with PBMCs in the presence of selected GPC3 / Dual-Ig trispecific molecules at 5 nM and 10 nM concentrations and E:T 0.5, and analyzed using a real-time xCELLigence system. The mean cell proliferation inhibition (%) + / - sd obtained at 120 hours is plotted on the graph. [Figure 2.1] Various antibody forms of the present invention are illustrated. The annotations for each Fv region correspond to those shown in Table 2.1. Figure (a) illustrates the 1+2 trivalent antibody, (b) illustrates the 1+2 trivalent antibody to which linc technology has been applied, (c) illustrates the 2Fab bivalent antibody form, and (d) illustrates the conventional IgG-based bivalent antibody form. [Figure 2.2.1] Tables 2.2 and 2.3 illustrate the antibody format and naming conventions for the sequence IDs listed. [Figure 2.2.2] Tables 2.2 and 2.3 illustrate the antibody format and naming conventions for the sequence IDs listed. [Figure 2.3]This paper presents the results of evaluating the cytotoxic activity of different antibody forms in GPC3-low-expressing cancer cells. (a) Histograms from flow cytometry analysis of GPC3 expression (solid black line) in SK-pca60 cell lines (left panel), Huh7 cell lines (center panel), and NCI-H446 cell lines (right panel). Anti-KLH antibody was used as a control (gray histogram). (b) shows a comparison of the cytotoxic activity of GPC3 / CD3 and GPC3 / Dual in 1+1 form, and (c) shows a comparison of the cytotoxic activity of 1+2 trivalent antibody and 2Fab antibody compared to 1+1 form antibody in low-GPC3-expressing Huh7 cell lines (left panel) and NCI-H446 cell lines (right panel). Tumor cell lines were co-cultured with PBMCs at an E:T ratio of 1. Data was acquired using the xCELLigence system, and the values ​​were displayed as the average + / - sd of cell proliferation inhibition (%) over 72 hours. [Figure 3.1] This diagram illustrates how introducing crosslinking in the 1+2 form, such as with GPC3-Dual / Dual antibodies, can reduce toxicity. Linc-Ig can primarily limit binding to immune cells in the cis form. In contrast, the 1+2 trivalent form could result in trans binding between two immune cells independently of tumor antigen binding. This can cause crosslinking between two immune cells independently of tumor antigen binding, potentially increasing toxicity. [Figure 3.2] This shows the antigen-independent cytotoxic activity against GPC3-negative cells in the presence of each antibody. CD137-overexpressing CHO cells were co-cultured with purified in vitro activated T cells in E:T 5 for 48 hours and analyzed using an LDH assay. The graph illustrates the mean cell lysis (%) + / - sd of different antibody formulations incubated at 1.25, 5, and 20 nM. [Figure 3.3]This graph shows the results of evaluating the cytotoxic activity (cell proliferation inhibition) of different antibody formulations in the NCI-H446 cell line. The 1+2 trivalent formulation showed stronger cytotoxic activity than the 1+1 formulation, with and without linc technology. NCI-H446 was co-cultured with PBMCs at an E:T ratio of 0.5 along with various antibody formulations at 1, 3, and 10 nM. Data were acquired using the xCELLigence system, and the values ​​are displayed as the average + / - sd of cell proliferation inhibition (%). [Figure 3.4] Figure 3.3 shows the results of evaluating cytokine release using different antibody formulations in the NCI-H446 cell line. The graphs show the mean concentrations + / - sd of the cytokines IFNγ (upper left), IL-2 (upper right), and TNFα (lower left). The supernatant of the co-culture in Figure 3.3, co-cultured with PBMCs at E:T 1.0, was analyzed at 40 hours. Antibodies were added at 0.6, 2.5, and 10 nM. [Figure 4] The design of the C3NP1-27 and CD3ε peptide antigens, which are biotin-labeled by a disulfide linker, is shown. [Figure 5] This graph shows the results of phage ELISA for clones obtained using phage display for CD3 and CD137. The Y-axis represents the specificity for CD137-Fc, and the X-axis represents the specificity for each clone for CD3. [Figure 6] This graph shows the results of phage ELISA for clones obtained using phage display for CD3 and CD137. The Y-axis represents the specificity for CD137-Fc in the bead ELISA for each clone, and the X-axis represents the specificity for CD3 in the same plate ELISA as in Figure 5. [Figure 7] This shows comparative data between the human CD137 amino acid sequence and the cynomolgus monkey CD137 amino acid sequence. [Figure 8] This graph shows the ELISA results of IgG obtained by phage display for CD3 and CD137. The Y-axis represents the specificity of each clone to cynomolgus monkey CD137-Fc, and the X-axis represents the specificity to human CD137. [Figure 9] This graph shows the ELISA results of IgG obtained using phage display for CD3 and CD137. The Y-axis represents the specificity for CD3e. [Figure 10] This graph shows the results of competitive ELISA for IgG obtained by phage display against CD3 and CD137. The Y-axis represents the ELISA response against biotin-human CD137-Fc or biotin-human Fc. Excess amounts of human CD3 or human Fc were used as competitors. [Figure 11A] This graph shows the results of phage ELISA for the phage display panning output pool for CD3 and CD137. The Y-axis represents specificity for human CD137. The X-axis represents the panning output pool, with R1 to R6 representing the pool before phage display panning, and R1 to R6 representing the panning output pool after rounds 1 to 6 of phage display panning, respectively. [Figure 11B] This graph shows the results of phage ELISA for the phage display panning output pool for CD3 and CD137. The Y-axis represents the specificity for cynomolgus monkey CD137. The X-axis represents the panning output pool, with R1 to R6 representing the panning output pool after rounds 1 to 6 of phage display panning, respectively. [Figure 11C] This graph shows the results of phage ELISA for the phage display panning output pool for CD3 and CD137. The Y-axis represents specificity for CD3. The X-axis represents the panning output pool, with R1 to R6 representing the pool before phage display panning, and R1 to R6 representing the panning output pool after rounds 1 to 6 of phage display panning, respectively. [Figure 12.1]This is a series of graphs showing the ELISA results of IgG obtained with phage display for CD3 and CD137. The Y-axis represents the specificity of each clone for human CD137-Fc, and the X-axis represents the specificity for human CD137 or CD3. [Figure 12.2] This is a series of graphs showing the ELISA results of IgG obtained with phage display for CD3 and CD137. The Y-axis represents the specificity of each clone for human CD137-Fc, and the X-axis represents the specificity for human CD137 or CD3. [Figure 12.3] This is a series of graphs showing the ELISA results of IgG obtained with phage display for CD3 and CD137. The Y-axis represents the specificity of each clone for human CD137-Fc, and the X-axis represents the specificity for human CD137 or CD3. [Figure 13] This is a series of graphs showing the ELISA results of IgG obtained with phage display for CD3 and CD137. The Y-axis represents the specificity of each clone for human CD137-Fc, and the X-axis represents the specificity for human CD137 or CD3. [Figure 14] This graph shows the results of competitive ELISA for IgG obtained by phage display against CD3 and CD137. The Y-axis represents the ELISA response against biotin-human CD137-Fc or biotin-human Fc. An excess amount of human CD3 was used as the competitor. [Figure 15] This graph shows the results of ELISA of IgG obtained by phage display against CD3 and CD137 to identify the epitope domains of each clone. The Y-axis represents the ELISA response against each domain of human CD137. [Figure 16] This is a series of graphs showing the ELISA results of IgG obtained by phage display affinity maturation for CD3 and CD137. The Y-axis represents the specificity of each clone to human CD137-Fc, and the X-axis represents the specificity to human CD137 or CD3. [Figure 17.1] This is a series of graphs showing the results of competitive ELISA of IgG obtained with phage display against CD3 and CD137. The Y-axis represents the ELISA response against biotin-human CD137-Fc or biotin-human Fc. An excess amount of human CD3 was used as the competitor. [Figure 17.2] This is a series of graphs showing the results of competitive ELISA of IgG obtained with phage display against CD3 and CD137. The Y-axis represents the ELISA response against biotin-human CD137-Fc or biotin-human Fc. An excess amount of human CD3 was used as the competitor. [Figure 17.3] This is a series of graphs showing the results of competitive ELISA of IgG obtained with phage display against CD3 and CD137. The Y-axis represents the ELISA response against biotin-human CD137-Fc or biotin-human Fc. An excess amount of human CD3 was used as the competitor. [Figure 17.4] This is a series of graphs showing the results of competitive ELISA of IgG obtained with phage display against CD3 and CD137. The Y-axis represents the ELISA response against biotin-human CD137-Fc or biotin-human Fc. An excess amount of human CD3 was used as the competitor. [Figure 17.5] This is a series of graphs showing the results of competitive ELISA of IgG obtained with phage display against CD3 and CD137. The Y-axis represents the ELISA response against biotin-human CD137-Fc or biotin-human Fc. An excess amount of human CD3 was used as the competitor. [Figure 18A] This diagram schematically illustrates the mechanism of IL-6 secretion from activated B cells mediated by anti-human GPC3 / Dual-Fab antibodies. [Figure 18B] This graph shows the results of evaluating the CD137-mediated agonist activity of various anti-human GPC3 / Dual-Fab antibodies based on the level of IL-6 production secreted from activated B cells. Ctrl represents the negative control human IgG1 antibody. [Figure 19A]This diagram schematically illustrates the mechanism of luciferase expression in activated Jurkat T cells mediated by anti-human GPC3 / Dual-Fab antibodies. [Figure 19B] This series of graphs shows the results of evaluating the CD3-mediated agonist activity of various anti-human GPC3 / Dual-Fab antibodies based on the level of luciferase production expressed in activated Jurkat T cells. Ctrl represents the negative control human IgG1 antibody. [Figure 20] This series of graphs shows the results of evaluating cytokine (IL-2, IFN-γ, and TNF-α) release from human PBMC-derived T cells in the presence of each immobilized antibody. The Y-axis represents the concentration of each secreted cytokine, and the X-axis represents the concentration of the immobilized antibody. The control anti-CD137 antibody (B), control anti-CD3 antibody (CE115), negative control antibody (Ctrl), and one of the dual antibodies (L183L072) were used for the assay. [Figure 21] This is a series of graphs showing the results of evaluating the T cell-dependent cytotoxic activity (TDCC) against GPC3-positive target cells (SK-pca60 and SK-pca13a) using each bispecific antibody. The Y-axis represents the ratio of cell proliferation inhibition (CGI), and the X-axis represents the concentration of each bispecific antibody. Anti-GPC3 / Dual bispecific antibody (GC33 / H183L072), negative control / Dual bispecific antibody (Ctrl / H183L072), anti-GPC3 / anti-CD137 bispecific antibody (GC33 / B), and negative control / anti-CD137 bispecific antibody (Ctrl / B) were used for this assay. Five-fold effector (E) cells were added to tumor (T) cells (ET5). [Figure 22] This graph shows the results of cell ELISA for CE115 against CD3e. [Figure 23] This is a diagram showing the molecular morphology of EGFR_ERY22_CE115. [Figure 24] This graph shows the results of TDCC (SK-pca13a) for EGFR_ERY22_CE115. [Figure 25]This is an exemplary sensorgram of an antibody with a binding ratio of less than 0.8. The vertical axis shows the RU value (response), and the horizontal axis shows time. [Figure 26] The figure shows examples of modified antibodies in which Fabs are crosslinked with each other. The figure schematically illustrates the structural differences between wild-type antibody (WT), modified antibody in which the CH1 regions of the antibody H chain are crosslinked with each other (HH type), modified antibody in which the CL regions of the antibody L chain are crosslinked with each other (LL type), and modified antibody in which the CH1 region of the antibody H chain is crosslinked with the CL region of the antibody L chain (HL type or LH type). [Figure 27] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody (as described in Reference Example 15), and the modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 28] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody (as described in Reference Example 15), and the modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 29] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody (as described in Reference Example 15), and the modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 30]The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody (as described in Reference Example 15), and the modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 31] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody (as described in Reference Example 15), and the modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 32] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody (as described in Reference Example 15), and the modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 33] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody (as described in Reference Example 15), and the modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 34]The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAH.xxx-G1T4) produced by introducing a cysteine ​​substitution into the heavy chain variable region of the anti-IL6R antibody (as described in Reference Example 15), and the modified antibody (MRAH-G1T4.xxx) produced by introducing a cysteine ​​substitution into the heavy chain constant region of the anti-IL6R antibody are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 35] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 36] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 37] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 38]The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 39] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 40] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 41] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 42]The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 43] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 44] The results of protease treatment of the anti-IL6R antibody (MRA), the modified antibody (MRAL.xxx-k0) produced by introducing a cysteine ​​substitution into the light chain variable region of the anti-IL6R antibody (as described in Reference Example 16), and the modified antibody (MRAL-k0.xxx) produced by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody (as described in Reference Example 16) are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody. [Figure 45] The results of protease treatment of the anti-IL6R antibody (MRA) and the modified antibody (MRAL-k0.K126C) prepared by introducing a cysteine ​​substitution into the light chain constant region of the anti-IL6R antibody, as described in Reference Example 17, are shown. Each protease-treated antibody was subjected to non-reducing capillary electrophoresis, followed by band detection with an anti-κ chain antibody or an anti-human Fc antibody. [Figure 46]This shows the correspondence between the molecular weight of each band obtained by protease treatment of the antibody sample described in Reference Example 17 and its estimated structure. The structure of each molecule and whether the molecule can react with anti-κ chain antibody or anti-Fc antibody (whether the band is detected by electrophoresis in Figure 45) are also described. [Modes for carrying out the invention]

[0021] Description of the manner In the present invention, the "antigen-binding domain" means a domain that includes at least a portion of the heavy chain variable (VH) region and / or a portion of the light chain variable (VL) region of an antibody, insofar as it has the activity to bind to part or all of an antigen, each comprising four framework regions (FRs) and three adjacent complementarity-determining regions (CDRs). Specifically, in the present invention, an "antigen-binding domain" including a light chain variable (VL) region or a heavy chain variable (VH) region is preferred. More specifically, in the present invention, an "antigen-binding domain" including both a light chain variable (VL) region and a heavy chain variable (VH) region is preferred.

[0022] In the present invention, the "antigen-binding domain" is also defined as follows: (i) CH1 region of the variable heavy chain (VH) region and the constant antibody heavy chain region; (ii) The variable (VH) region of the heavy chain, the CH1 region of the constant region of the antibody heavy chain, and the hinge region of the antibody heavy chain; (iii) Light chain variable (VL) region and light chain steady (CL) region; (iv) The CH1 region of the heavy chain variable (VH) region and the constant region of the antibody heavy chain, as well as the light chain variable (VL) region; (v) The CH1 region of the heavy chain variable (VH) region and the antibody heavy chain constant region, as well as the light chain variable (VL) region and the light chain constant (CL) region; (vi) The variable heavy chain (VH) region, the CH1 region of the constant antibody heavy chain region, and the hinge region of the antibody heavy chain, as well as the variable light chain (VL) region; (vii) the variable heavy chain (VH) region, the CH1 region of the antibody heavy chain constant region, and the hinge region of the antibody heavy chain, as well as the variable light chain (VL) region and the light chain constant (CL) region; or (viii) Heavy chain variable (VH) region, as well as light chain variable (VL) region and light chain steady (CL) region This also refers to domains that include [the specified domain].

[0023] The antigen-binding domain of the present invention may have any sequence and may be derived from any antibody, such as a mouse antibody, rat antibody, rabbit antibody, goat antibody, camel antibody, a humanized antibody obtained by humanizing any of these non-human antibodies, and a human antibody. A "humanized antibody," also called a reshaped human antibody, is obtained by transplanting the complementarity-determining region (CDR) of a non-human mammalian antibody, such as a mouse antibody, into the CDR of a human antibody. Methods for identifying CDRs are known in the art (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; and Chothia et al., Nature (1989) 342: 877). General recombination techniques for this purpose are also known in the art (see European Patent Application Publication EP 125023 and WO 96 / 02576).

[0024] In the present invention, the "antigen-binding molecule" is not particularly limited as long as the molecule contains the "antigen-binding domain" of the present invention. The antigen-binding molecule may further include a peptide or protein having a length of approximately 5 amino acids or more. The peptide or protein is not limited to peptides or proteins derived from living organisms, and may, for example, be a polypeptide consisting of an artificially designed sequence. Natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc., may also be used.

[0025] In some embodiments, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising an antibody Fc region. The "Fc region" in the present invention is defined as follows.

[0026] In some embodiments, the “antigen-binding molecule” of the present invention may be an antigen-binding molecule that includes an antigen-binding domain as defined above, which comprises a heavy chain variable (VH) region and a light chain variable (VL) region in a single polypeptide chain linked by one or more linkers, such as diabody(Db), a single-chain antibody, or sc(Fab')2, but lacks an Fc region.

[0027] As used in this application, the term "antibody fragment" may refer to a molecule other than an intact antibody that binds to an antigen to which an intact antibody binds, and which contains a portion of the intact antibody. Examples of antibody fragments, but not limited to, include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); single-chain Fab (scFab); monodomain antibodies; and multispecific antibodies formed from antibody fragments.

[0028] When used in this application, the term "variable fragment (Fv)" may refer to the smallest unit of antibody-derived portion that binds to an antigen, consisting of a pair of antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In 1988, Skerra and Pluckthun found that homogeneous and active antibodies could be prepared from the periplasmic fraction of Escherichia coli by inserting an antibody gene downstream of a bacterial signaling sequence and inducing the expression of the gene in Escherichia coli (Science (1988) 240(4855), 1038-1041). In Fv prepared from the periplasmic fraction, VH associates with VL to bind to the antigen.

[0029] When used in this application, the terms “scFv,” “single-chain antibody,” and “sc(Fv)2” refer to antibody fragments of a single polypeptide chain that include variable regions derived from the heavy and light chains but do not include a constant region. Generally, single-chain antibodies also include a polypeptide linker between the VH and VL domains that can form a desirable structure thought to enable antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in “The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994).” See also International Patent Publication WO 1988 / 001649; U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may be bispecific and / or humanized.

[0030] When used in this application, the term "scFv" may refer to a single-chain polypeptide in which the VH and VL forming Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be held in close proximity by the peptide linker.

[0031] When used in this application, the term "sc(Fv)2" may refer to a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231(1-2), 177-189). The two VHs and two VLs may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes a bispecific sc(Fv)2 that recognizes two epitopes present on a single antigen, for example, as described in the Journal of Immunology (1994) 152(11), 5368-5374. sc(Fv)2 can be prepared by methods known to those skilled in the art. For example, sc(Fv)2 can be prepared by linking scFv with a linker such as a peptide linker. In this specification, sc(Fv)2 takes the form in which the two VH units and two VL units of the antibody are arranged in the order VH, VL, VH, and VL starting from the N-terminus of the single-chain polypeptide ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). The order of the two VH units and two VL units is not limited to the above form and may be arranged in any order. Exemplary orders of the form 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]

[0032] When used in this application, the terms "Fab," "F(ab')2," and "Fab'" may mean the following: "Fab" consists of one light chain and a CH1 region and variable region derived from one heavy chain. The heavy chain of the wild-type Fab molecule cannot form disulfide bonds with other heavy-chain molecules. Depending on the purpose, Fab variants are also included in which amino acid residues in the wild-type Fab molecule may be modified by substitution, addition, or deletion. In certain embodiments, the mutant amino acid residues contained in the Fab variant (e.g., cysteine ​​or lysine residues after substitution, addition, or insertion) can form disulfide bonds with other heavy-chain molecules or parts thereof (e.g., the Fab molecule).

[0033] scFab is an antigen-binding domain in which the CH1 region and variable region derived from one light chain and one heavy chain forming the Fab are linked together by a peptide linker. The CH1 region and variable region derived from the light chain and heavy chain can be kept in close proximity by the peptide linker.

[0034] "F(ab')2" or "Fab" refers to an antibody fragment produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, by digesting the immunoglobulin (monoclonal antibody) near the disulfide bond present between the hinge regions of each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond present between the hinge regions of each of the two H chains, producing two homologous antibody fragments in which the L chain, containing VL (variable L chain region) and CL (constant L chain region), is linked via a disulfide bond at its C-terminal region to an H chain fragment containing VH (variable H chain region) and CHγ1 (γ1 region in the constant H chain region). These two homologous antibody fragments are each called Fab'.

[0035] "F(ab')2" consists of two light chains and two heavy chains containing the constant regions of the CH1 and CH2 domains, such that a disulfide bond is formed between the two heavy chains. For example, the F(ab')2 described herein can be prepared as follows: A full-length monoclonal antibody or similar containing the desired antigen-binding domain is partially digested by a protease such as pepsin; the Fc fragment is removed by adsorption onto a protein A column. The protease is not particularly limited as long as it can selectively cleave the full-length antibody to produce F(ab')2 under appropriately set enzymatic reaction conditions such as pH. Such proteases include, for example, pepsin and ficin.

[0036] When used in this application, the term "monodomain antibody" is not particularly limited in its structure, as long as the domain itself can exert antigen-binding activity. Conventional antibodies, exemplified by IgG antibodies, exert antigen-binding activity when the variable region is formed by pairing of VH and VL domains. In contrast, monodomain antibodies are known to exert antigen-binding activity through their own domain structure alone, without pairing with another domain. Monodomain antibodies usually have a relatively low molecular weight and exist in monomeric form. Examples of monodomain antibodies include, but are not limited to, antigen-binding molecules that naturally lack a light chain, such as VHH from camelid animals and VNAR from sharks, as well as antibody fragments containing all or part of an antibody VH domain or all or part of an antibody VL domain. Examples of monodomain antibodies that are antibody fragments containing all or part of an antibody VH / VL domain include, but are not limited to, artificially prepared monodomain antibodies originating from human antibody VH or human antibody VL, such as those described in U.S. Patent No. 6,248,516 B1. In some aspects of the present invention, a single monodomain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0037] Monodomain antibodies can be obtained from animals capable of producing monodomain antibodies, or by immunizing animals capable of producing monodomain antibodies. Examples of animals capable of producing monodomain antibodies include, but are not limited to, camels, and transgenic animals into which genes for the ability to produce monodomain antibodies have been introduced. Camels include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which genes for the ability to produce monodomain antibodies have been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 or U.S. Patent Publication US2011 / 0123527 A1. Humanized single-chain antibodies can also be obtained by substituting the framework sequence of a monodomain antibody obtained from an animal having a human germline sequence or a similar sequence. A humanized monodomain antibody (e.g., humanized VHH) is one embodiment of the monodomain antibody of the present invention.

[0038] Alternatively, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by methods such as ELISA and panning. 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 immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), and synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), and AIDS 2016 30:11). (1691-1701) is one example.

[0039] When used in this application, the term "Db" may refer to a dimer composed of two polypeptide chains (e.g., Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; and W093 / 11161). These polypeptide chains are linked, for example, through a short linker of approximately 5 residues, such that the light chain variable domain (VL) and heavy chain variable domain (VH) on the same polypeptide chain cannot pair with each other. Because of this short linker, VL and VH encoded on the same polypeptide chain cannot form a single-stranded Fv, but instead dimerize with VH and VL, respectively, on different polypeptide chains to form two antigen-binding sites.

[0040] In the present invention, the "Fc region" refers to a region in an antibody molecule that includes the hinge or a portion thereof, as well as a fragment consisting of CH2 and CH3 domains. The Fc region of an IgG class, but not limited to, means, for example, the region from cysteine ​​226 (EU numbering (also referred to herein as the EU index)) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus. The Fc region can preferably be 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 onto a protein A column or protein G column. Such proteases are not particularly limited, as long as they can digest the full-length antibody to restrictively form Fab or F(ab')2 under appropriately set enzyme reaction conditions (e.g., pH). Examples may include pepsin and papain.

[0041] The "antigen-binding domain" of the present invention, which "can bind to a first antigen and a second antigen different from the first antigen, but does not bind to both the first and second antigens simultaneously," means that when the antigen-binding domain of the present invention is bound to the first antigen, it cannot bind to the second antigen, and conversely, when the variable region is bound to the second antigen, it cannot bind to the first antigen. In this situation, the phrase "does not bind to both the first and second antigens simultaneously" also means that the "antigen-binding domain," as a single antigen-binding domain, does not crosslink cells expressing the first antigen (e.g., effector cells such as T cells, NK cells, or DC cells) with cells expressing the second antigen (e.g., effector cells such as T cells, NK cells, or DC cells), or does not simultaneously bind to the first and second antigens expressed on different cells. This clause further includes cases where the antigen-binding domain can bind to both the first and second antigens simultaneously, but not simultaneously, when the first and second antigens are expressed on different cells, such as when they are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell. Such antigen-binding domains are not particularly limited, as long as they possess these functions. An example of such a domain may be an antigen-binding domain derived from an IgG antibody, in which some of its amino acids have been modified to bind to a desired antigen. The amino acids to be modified are selected, for example, from amino acids in the antigen-binding domain that bind to the first or second antigen, such that the modification does not result in a loss of binding to the antigen. In this context, the phrase "expressed on different cells" simply means that the antigen is expressed on separate cells. Such cell combinations may be of the same type, such as a T cell and another T cell, or they may be of different types, such as a T cell and an NK cell.

[0042] In this application, the "antigen-binding domain" of the present invention, as defined above, which "can bind to a first antigen and a second antigen different from the first antigen," may be described by the abbreviation "Dual" or "dual." In some embodiments, when both the first antigen-binding domain and the second binding domain of the antigen-binding molecule of the present invention are "Dual," it may be expressed as "Dual / Dual" or "dual / dual." In some embodiments, when one of the first antigen-binding domain and the second binding domain of the antigen-binding molecule of the present invention is "Dual," and the other antigen-binding domain binds to only one antigen, for example, CD3 or CD137 (i.e., binds to only one of the first or second antigen), it may be expressed as "Dual / CD3," "CD3 / Dual," "Dual / CD137," or "CD137 / Dual," etc. In some further embodiments, in which either the first antigen-binding domain or the second binding domain of the antigen-binding molecule of the present invention is linked to a third antigen-binding domain that can bind to a third antigen (as defined below; e.g., GPC3) different from the first and second antigens, the molecule may be represented as, for example, "GPC3-Dual / Dual", "GPC3-Dual / CD3", "GPC3-CD3 / Dual", "GPC3-Dual / CD137", or "GPC3-CD137 / Dual". In some further embodiments, the above embodiments may be represented as follows: (as defined below) "the first antigen-binding domain and the second antigen-binding domain are linked to each other via at least one binding that keeps the first antigen-binding domain and the second antigen-binding domain close to each other," for example, "Dual / CD3(linc)", "CD3 / Dual(linc)", "Dual / CD137(linc)", "CD137 / Dual(linc)", "GPC3-Dual / Dual(linc)", "GPC3-Dual / CD3(linc)", "GPC3-CD3 / Dual(linc)", "GPC3-Dual / CD137(linc)", or "GPC3-CD137 / Dual(linc)", etc.

[0043] In the present invention, the term "capable of binding to either the first antigen or the second antigen" means: (i) the antigen-binding domain of the present invention has binding activity to either the first antigen or the second antigen which is different from the first antigen, and does not have binding activity to the other antigen of the first or second antigen; (ii) the antigen-binding domain of the present invention has preferential binding activity to either the first antigen or the second antigen which is different from the first antigen; (iii) the antigen-binding domain of the present invention has significant binding activity to either the first antigen or the second antigen which is different from the first antigen (for example, KD is 1 × 10⁻¹⁶). -5 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, or 1 × 10 -9 While possessing a KD of less than 1, it has weak binding activity (e.g., KD is 1 × 10⁻¹⁰) to the other antigen among the first and second antigens. -3 Super M, 1×10 -4 M or 1 × 10 -5 (iv) The antigen-binding domain of the present invention has binding activity to either the first antigen or a second antigen different from the first antigen, while the binding activity to the other of the first or second antigen is undetectable, as determined by methods known in the art, such as electrochemiluminescence (ECL) or surface plasmon resonance (SPR); (v) The antigen-binding domain of the present invention has binding activity that is 1, 5, 10, 50, 100, 1000, 10000, 100000 or more higher with respect to the first antigen (second antigen) compared to binding to the second antigen (first antigen) different from the first antigen.

[0044] In some embodiments, the binding activity or affinity of the antigen-binding domain of the present invention to a first or second antigen (e.g., CD3, CD137) is evaluated at 25°C or 37°C using, for example, a Biacore T200 instrument (GE Healthcare). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). The antigen-binding domain is captured on the anti-Fc sensor surface, and then the antigen (e.g., recombinant human CD3 or CD137) is injected onto the flow cell. All antigen-binding domains and analytes are prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The sensor surface is regenerated with 3 M MgCl2 in each cycle. Binding affinity is determined by processing the data and fitting it to a 1:1 binding model, for example, using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). In some embodiments, the CD3 binding affinity assay was performed under the above conditions with the assay temperature set to 25°C, and the CD137 binding affinity assay was performed under the same conditions except that the assay temperature was set to 37°C.

[0045] In some embodiments of the present invention, "the first antigen-binding domain and the second antigen-binding domain are linked to each other via at least one binding." The at least one binding for linking the first antigen-binding domain and the second antigen-binding domain can be introduced into one or more of the following: (i) Between the constant CH1 region of the antibody heavy chain of the first antigen-binding domain and the constant CH1 region of the antibody heavy chain of the second antigen-binding domain; (ii) Between the hinge region of the antibody heavy chain of the first antigen-binding domain and the hinge region of the antibody heavy chain of the second antigen-binding domain; (iii) Between the light chain constant (CL) region of the first antigen-binding domain and the light chain constant (CL) region of the second antigen-binding domain; (iv) Between the CH1 region of the antibody heavy chain constant state of the first antigen-binding domain and the light chain constant state (CL) region of the second antigen-binding domain; (v) Between the light chain constant (CL) region of the first antigen-binding domain and the CH1 region of the antibody heavy chain constant of the second antigen-binding domain; and / or (vi) Between the heavy chain variable (VH) region of the first antigen-binding domain and the heavy chain variable (VH) region of the second antigen-binding domain.

[0046] In this specification, in the case of (ii) above, “at least one bond” introduced between the two hinge regions is one or more additional bonds other than the one or more native disulfide bonds between cysteine ​​residues that wild-type antibodies typically have between the hinge regions of their respective heavy chains. For example, IgG1 antibody has two native disulfide bonds between the hinge regions of its respective heavy chains, while IgG2 and IgG3 have more disulfide bonds between the hinge regions of their respective heavy chains. Examples of such cysteine ​​residues include the cysteine ​​residues at positions 226 and 229 according to EU numbering. In the present invention, in the case of (ii) above, “at least one bond” introduced between the hinge regions is one or more additional bonds in the hinge regions of IgG1, IgG2, or IgG3, other than such originally present disulfide bonds. In the present invention, in any of the cases (i) to (vi) above, “at least one bond” can be introduced at any amino acid position in each of the two CH1 regions; at any amino acid position in each of the two hinge regions; or at any amino acid position in each of the two CL regions, to the extent that the antigen-binding molecule of the present invention exhibits, achieves, and / or maintains the desired properties.

[0047] In the embodiments described above, in at least one of the first and second antigen-binding domains, one or more (e.g., multiple) amino acid residues that result in binding between the antigen-binding domains are located at positions of seven amino acids or more apart from each other in the primary structure. This means that between any two amino acids of the above-mentioned multiple amino acid residues, there are six or more amino acid residues other than those residues. In certain embodiments, the combination of multiple amino acid residues that results in binding between the antigen-binding domains includes a pair of amino acid residues located at positions of less than seven amino acids apart in the primary structure. In certain embodiments, if the first and second antigen-binding domains are linked to each other via three or more bindings, the binding between the antigen-binding domains may result from three or more amino acid residues, including a pair of amino acid residues located at positions of seven amino acids or more apart in the primary structure. In a particular embodiment, amino acid residues located at the same position in the first antigen-binding domain and the second antigen-binding domain are linked to each other, forming a bond. In a particular embodiment, amino acid residues located at different positions in the first antigen-binding domain and the second antigen-binding domain are linked to each other, forming a bond.

[0048] The positions of amino acid residues within antigen-binding domains can be indicated by the Kabat numbering system or 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. For example, if the amino acid residues that cause binding between the first and second antigen-binding domains are located at the same corresponding position in the antigen-binding domains, these amino acid residues can be represented by the same number in the Kabat numbering system or the EU numbering system. Alternatively, if the amino acid residues that cause binding between the first and second antigen-binding domains are located at different, uncorresponding positions in the antigen-binding domains, these amino acid residues can be represented by different numbers in the Kabat numbering system or the EU numbering system.

[0049] As described above, in the above-described aspect, at least one amino acid residue that causes binding between antigen-binding domains is located within the constant region. According to a particular theory, the amino acid residues are located within the CH1 region of the antibody heavy chain constant region, and are situated at positions selected from the group consisting of positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 165, 167, 174, 176, 177, 178, 190, 191, 192, 194, 195, 197, 213, and 214 in the CH1 region according to EU numbering. In an exemplary embodiment, the amino acid residue is located at position 191 in the CH1 region according to EU numbering, and the amino acid residues at position 191 in the CH1 region of the two antigen-binding domains are linked to each other, forming a bond.

[0050] In a particular embodiment, at least one amino acid residue that causes binding between antigen-binding domains is located within the hinge region, for example, at a position selected from the group consisting of positions 216, 218, and 219 in the hinge region according to EU numbering. In a particular embodiment, at least one amino acid residue that causes binding between antigen-binding domains is located within the light chain constant (CL) region, for example, at a position selected from the group consisting of EU numbering positions 109, 112, 121, 126, 128, 151, 152, 153, 156, 184, 186, 188, 190, 200, 201, 202, 203, 208, 210, 211, 212, and 213 in the CL region. In an exemplary embodiment, the amino acid residue is located at EU numbering position 126 in the CL region, and the amino acid residues at EU numbering position 126 in the CL region of the two antigen-binding domains are linked to each other to form a bond.

[0051] As described above, in a particular embodiment, an amino acid residue in the CH1 region of the first antigen-binding domain and an amino acid residue in the CL region of the second antigen-binding domain are linked to form a bond. In an exemplary embodiment, an amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain and an amino acid residue at EU numbering position 126 in the CL region of the second antigen-binding domain are linked to form a bond.

[0052] As described above, in the embodiments described above, at least one amino acid residue that causes binding between antigen-binding domains is located within the heavy chain (VH) variable region and / or the light chain variable (VL) region. In certain embodiments, the amino acid residue is located within the VH region, for example, at a position selected from the group consisting of positions 8, 16, 28, 74, and 82b according to Kabat numbering within the VH region. In certain embodiments, the amino acid residue is located within the VL region, for example, at a position selected from the group consisting of positions 100, 105, and 107 according to Kabat numbering within the VL region.

[0053] In the present invention, the “at least one binding” introduced to link the first antigen-binding domain and the second antigen-binding domain as described above may be any type of binding selected from the following, but is not limited to these: (i) covalent bonds (e.g., covalent bonds formed by direct crosslinking between amino acids, such as disulfide bonds between cysteine ​​residues; or covalent bonds formed by crosslinking between amino acids via a crosslinking agent, such as covalent bonds between lysine residues via an amine-reactive crosslinking agent); and / or (ii) Non-covalent bonds (e.g., ionic bonds, hydrogen bonds, or hydrophobic bonds).

[0054] In the present invention, “at least one binding” introduced to link the first antigen-binding domain and the second antigen-binding domain as described above can keep the first antigen-binding domain and the second antigen-binding domain close to each other. In this specification, the term “keeping the first antigen-binding domain and the second antigen-binding domain close to each other” is described below, but is not limited to the following.

[0055] In the embodiments described above, “at least one binding” introduced to link the first antigen-binding domain and the second antigen-binding domain as described above can hold the two binding domains (i.e., the first antigen-binding domain and the second antigen-binding domain as described above) in a spatially close position. The linkage between the first antigen-binding domain and the second antigen-binding domain via the binding allows the antigen-binding molecule of the present invention to hold the two antigen-binding domains in a closer position than a control antigen-binding molecule, which differs from the antigen-binding molecule of the present invention only in that it does not have any further binding introduced between the two antigen-binding domains. In some embodiments, the terms “spatially close position” or “closer position” include the meaning that the first antigen-binding domain and the second antigen-binding domain as described above are held at a shorter distance and / or with reduced flexibility.

[0056] As a result, the two antigen-binding domains of the antigen-binding molecule of the present invention (i.e., the first antigen-binding domain and the second antigen-binding domain described above) bind to antigens expressed on the same single cell. In other words, each of the two antigen-binding domains of the antigen-binding molecule of the present invention (i.e., the first antigen-binding domain and the second antigen-binding domain described above) does not bind to antigens expressed on different cells and does not cause cross-linking between different cells. In this application, such a mode of antigen-binding of the antigen-binding molecule of the present invention may be called "cis binding," while a mode of antigen-binding of the antigen-binding molecule in which each of the two antigen-binding domains of the antigen-binding molecule binds to antigens expressed on different cells in such a way that it causes cross-linking between different cells may be called "trans binding." In some embodiments, the antigen-binding molecule of the present invention preferentially binds to antigens expressed on the same single cell in a "cis binding" manner.

[0057] In the embodiments described above, the antigen-binding molecule of the present invention can reduce and / or suppress undesirable crosslinking and activation of immune cells (e.g., T cells, NK cells, or DC cells) by linking between the first antigen-binding domain and the second antigen-binding domain via the binding described above. That is, in some embodiments of the present invention, the first antigen-binding domain of the antigen-binding molecule of the present invention binds to any signaling molecule expressed on immune cells such as T cells (e.g., a first antigen), and the second antigen-binding domain of the antigen-binding molecule of the present invention also binds to any signaling molecule expressed on immune cells such as T cells (e.g., a first antigen, or a second antigen different from the first antigen). Thus, the first and second antigen-binding domains of the antigen-binding molecule of the present invention can bind to either the first or second signaling molecule expressed on the same single immune cell, such as a T cell (i.e., cis-binding mode), or on different immune cells, such as T cells (i.e., trans-binding mode). When the first and second antigen-binding domains bind in a trans-binding manner to signaling molecules expressed on different immune cells, such as T cells, these different immune cells, such as T cells, become cross-linked, and under certain circumstances, such cross-linking of immune cells, such as T cells, can lead to undesirable activation of these immune cells.

[0058] On the other hand, in another embodiment of the antigen-binding molecule of the present invention, i.e., an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain linked to each other via at least one bond that keeps the two antigen-binding domains close together, both the first antigen-binding domain and the second antigen-binding domain can bind in a "cis-binding" manner to a signaling molecule expressed on the same single immune cell, such as a T cell, thereby reducing cross-linking of different immune cells, such as T cells, via the antigen-binding molecule and avoiding undesirable activation of immune cells. In this application, the above feature, namely, "the first antigen-binding domain and the second antigen-binding domain are linked to each other via at least one bond that keeps the first antigen-binding domain and the second antigen-binding domain close to each other," may be abbreviated as "linc." Using this abbreviation, in some embodiments, the antigen-binding molecule of the present invention may be represented as, for example, "Dual / CD3(linc)", "CD3 / Dual(linc)", "Dual / CD137(linc)", "CD137 / Dual(linc)", "GPC3-Dual / Dual(linc)", "GPC3-Dual / CD3(linc)", "GPC3-CD3 / Dual(linc)", "GPC3-Dual / CD137(linc)", or "GPC3-CD137 / Dual(linc)", etc.

[0059] In some embodiments, the antigen-binding molecule of the present invention may include modifications of one or more amino acids in any one or more portions of the antigen-binding domain, the heavy chain variable (VH) region, the light chain variable (VL) region, the CH1 region of the heavy chain constant region, the light chain constant (CL) region, the hinge region of the antibody heavy chain, and the Fc region (as described below). One amino acid modification may be used alone, or multiple amino acid modifications may be used in combination. When multiple amino acid modifications are used in combination, the number of modifications to be combined is not particularly limited and can be set as appropriate within a range that can achieve the objective of the invention. For example, the number of modifications to be combined may be 2 to 30, preferably 2 to 25, 2 to 22, 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 2 to 3.

[0060] The combination of multiple amino acid modifications may be applied only to the heavy chain variable domain or the light chain variable domain of the antibody, or may be appropriately distributed to both the heavy chain and light chain variable domains. One or more amino acid residues in the variable region are acceptable as modified amino acid residues, as long as antigen-binding activity is maintained. When amino acids in the variable region are modified, the resulting variable region preferably maintains the binding activity of the corresponding unmodified antibody, and preferably has a binding activity that is, for example, 50% or more, more preferably 80% or more, and even more preferably 100% or more higher than before modification, but the variable regions according to the present invention are not limited to these. Binding activity may be increased by the amino acid modification, for example, to 2 times, 5 times, or 10 times the binding activity before modification.

[0061] Examples of regions preferred for amino acid modification include solvent-exposed regions and loops within the variable domain. Of these, CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, Kabat numbering positions 31-35, 50-65, 71-74, and 95-102 in the heavy (H) chain variable domain, and Kabat numbering positions 24-34, 50-56, and 89-97 in the light (L) chain variable domain are preferred. Kabat numbering positions 31, 52a-61, 71-74, and 97-101 in the heavy (H) chain variable domain, and Kabat numbering positions 24-34, 51-56, and 89-96 in the light (L) chain variable domain are more preferred. In addition, amino acids that increase antigen-binding activity may be introduced during amino acid modification.

[0062] In the present invention, the terms “hypervariable region” or “HVR” as used herein refer to each region of an antibody variable domain that has a hypervariable sequence (“complementarity-determining region” or “CDR”) and / or forms a structurally defined loop (“hypervariable loop”) and / or contains residues that come into contact with an antigen (“antigen contact region”). Generally, antibodies include six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs as used herein include: (a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact sites located at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c) containing 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 specified, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al. above.

[0063] In the present invention, "loop" means a region containing residues that are not involved in maintaining the β-barrel structure of immunoglobulin. In this invention, amino acid modification means substitution, deletion, addition, insertion, modification, or a combination thereof. In this invention, amino acid modification is used interchangeably with amino acid mutation and may be used in the same sense.

[0064] Amino acid residue substitutions are carried out, for example, by replacing one amino acid residue with another, for the purpose of modifying any of the following (a) to (c): (a) the polypeptide backbone structure of a region having a sheet or helical structure; (b) the charge or hydrophobicity of the target site; and (c) the size of the side chain. Amino acid residues are classified into the following groups based on the general properties of the side chain: (1) hydrophobic residues: norleucine, Met, Ala, Val, Leu, and Ile; (2) neutral hydrophilic residues: Cys, Ser, Thr, Asn, and Gln; (3) acidic residues: Asp and Glu; (4) basic residues: His, Lys, and Arg; (5) residues that affect chain orientation: Gly and Pro; and (6) aromatic residues: Trp, Tyr, and Phe.

[0065] Substitutions of amino acid residues within each of these groups are called conservative substitutions, while substitutions of an amino acid residue in one of these groups with an amino acid residue in another group are called non-conservative substitutions. The substitutions according to the present invention may be conservative substitutions or non-conservative substitutions. Alternatively, a combination of conservative and non-conservative substitutions may be used.

[0066] Modifications of amino acid residues also include selecting a variable region in the antibody variable region that binds to a first or second antigen, from those obtained by random modification of amino acids in which the modification does not result in loss of binding to the antigen, that can bind to both the first and second antigens but cannot bind to both antigens simultaneously; and modifications that involve inserting a peptide previously known to have binding activity to a desired antigen into the aforementioned region. Examples of peptides previously known to possess binding activity to a desired antigen include those listed in the table below.

[0067] [Table A]

[0068] Several antibodies that bind to different epitopes of human CD3ε are known in the art, for example, antibody OKT3 (e.g., Kung, P. et al, Science 206 (1979) 347-349; Salmeron, A. et al, J Immunol 147 (1991) 3047-3052; see US9226962B2), antibody UCHT1 (e.g., Callard, RE et al, Clin Exp Immunol 43 (1981) 497-505; see Arnett et al. PNAS 2004), or antibody SP34 (human-cynomolgus monkey CD3 cross-reactivity; e.g., Pessano, S. et al, EMBO J 4 (1985) 337-344, Conrad ML, et al, Cytometry A 71 (2007)). (See 925-933). WO2015181098A1 also describes a human-cynomolgus monkey cross-reactive antibody that specifically binds to human and cynomolgus monkey T cells, activates human T cells, and does not bind to the same epitopes as antibody OKT3, antibody UCHT1, and / or antibody SP34.

[0069] WO2015068847A1 (incorporated herein by reference) describes a method for preparing Dual-Fab and examples of peptides known to be able to bind to different target proteins, where such peptides can function as a second antigen-binding site when inserted into the variable region of an antibody that binds to a first antigen, such as human CD3. Specifically, WO2015068847A1 describes the following: Example 3 - An anti-CD3 antibody that binds to integrin and CD3, but not simultaneously. Example 4 - An anti-CD3 antibody that binds to TLR2 and CD3 but not simultaneously. Example 8 - An anti-CD3 antibody that binds to IgA and CD3, but not simultaneously. Example 9 - An anti-CD3 antibody that binds to CD154 and CD3, but not simultaneously. In addition, WO2015068847A1 describes numerous sites within the heavy chain variable region and light chain variable region that allow for the placement of the antigen-binding site without losing the ability of the first antigen-binding site to bind to CD3. See the examples described above, as well as the experiment described in Example 6, in which GGS peptides of various lengths (3, 6, or 9 residues) were inserted into three different VH sites (CDR2, FR3, or CDR3).

[0070] In the present invention, modifications to the heavy chain variable (VH) region and / or light chain variable (VL) region described above may be combined with modifications known in the art. For example, modification of the N-terminal glutamine in the variable region to pyroglutamic acid by pyroglutamylation is a modification well known to those skilled in the art. Therefore, the antigen-binding molecule of the present invention having glutamine at the N-terminus of its heavy chain variable (VH) region may contain a variable region in which this N-terminal glutamine is modified to pyroglutamic acid.

[0071] In the present invention, the heavy chain variable (VH) region and / or light chain variable (VL) region of the antigen-binding domain of the antigen-binding molecule may further have amino acid modifications to improve, for example, antigen binding, pharmacokinetics, stability, or antigenicity. In the present invention, the heavy chain variable (VH) region and / or light chain variable (VL) region of the antigen-binding domain of the antigen-binding molecule may be modified to have pH-dependent binding activity to the antigen, thereby enabling repeated binding to the antigen (WO2009 / 125825).

[0072] Furthermore, in the present invention, an amino acid modification (WO2013 / 180200) that alters antigen-binding activity depending on the concentration of a target tissue-specific compound may be applied, for example, to the heavy chain variable (VH) region and / or light chain variable (VL) region of the third antigen-binding domain of an antigen-binding molecule that binds to a third antigen (e.g., a tumor antigen).

[0073] In the present invention, the heavy chain variable (VH) region and / or light chain variable (VL) region in the antigen-binding domain of the antigen-binding molecule may be further modified for purposes such as enhancing binding activity, improving specificity, reducing pI, conferring pH-dependent antigen-binding properties, improving the thermal stability of binding, improving solubility, improving stability to chemical modifications, improving heterogeneity derived from glycans, avoiding T cell epitopes identified by in silico prediction or in vitro T cell-based assays for reduced immunogenicity, or introducing T cell epitopes for activation of regulatory T cells (mAbs 3:243-247, 2011).

[0074] In the present invention, whether the antigen-binding domain and / or antigen-binding molecule of the present invention can bind to an antigen, and whether it "can bind to an antigen but does not bind to any other antigen," can be determined by methods known in the art. This can be determined, for example, by electrochemiluminescence (ECL) (BMC Research Notes 2011, 4:281).

[0075] Specifically, for example, with respect to the low molecular weight antigen-binding molecule of the present invention, a biotin-labeled test antigen-binding molecule is mixed with an antigen labeled with a sulfo-tag (Ru complex) (e.g., each of the first, second, or third antigens), 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 emission signal is detected using a Sector Imager 600 or 2400 (MSD KK), thereby confirming the binding of the above-mentioned test antigen-binding molecule to the antigen (e.g., each of the first, second, or third antigens).

[0076] Alternatively, this assay may be performed by methods such as ELISA, FACS (fluorescence-activated cell sorting), ALPHAScreen (amplified luminescence proximity homogeneous assay screen), or the BIACORE method based on surface plasmon resonance (SPR) phenomena (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0077] Specifically, the assay can be performed using, for example, Biacore (GE Healthcare Japan Corp.), an interaction analysis instrument based on surface plasmon resonance (SPR) phenomena. Biacore analyzers include any model such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any Biacore sensor chip such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chip can be used as the sensor chip. Proteins for capturing the antigen-binding molecule 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. An antigen (e.g., the first antigen, the second antigen, or the third antigen) is injected as an analyte, and the interaction is measured to obtain a sensorgram. In this procedure, the concentration of the antigen (e.g., the first antigen, the second antigen, or the third antigen) can be selected within a range of several μM to several pM depending on the strength of the interaction of the assay sample (e.g., KD).

[0078] Alternatively, an antigen (e.g., a first antigen, a second antigen, or a third antigen) may be immobilized on the sensor chip instead of the antigen-binding molecule, and then the antigen may be interacted with the antigen-binding molecule sample to be evaluated. Whether the antigen-binding domain and / or antigen-binding molecule of the present invention has binding activity to the antigen (e.g., a first antigen, a second antigen, or a third antigen) 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 relative to the level of the antigen-binding molecule sample before the action.

[0079] In some embodiments, the binding affinity of the antigen-binding molecules (antibodies) of the present invention to antigens (e.g., CD3, CD137) is evaluated at 25°C or 37°C, for example, using a Biacore T200 instrument (GE Healthcare). Anti-human Fc (e.g., GE Healthcare) is immobilized on all flow cells of the CM4 sensor chip using an amine coupling kit (e.g., GE Healthcare). The antigen-binding molecules (antibodies) are captured on the anti-Fc sensor surface, and then the antigen (e.g., recombinant human CD3 or CD137) is injected onto the flow cell. All antigen-binding molecules (antibodies) and analytes are prepared in ACES pH 7.4 containing 20 mM ACES, 150 mM NaCl, 0.05% Tween 20, and 0.005% NaN3. The sensor surface is regenerated with 3 M MgCl2 after each cycle. Binding affinity is determined by processing the data and fitting it to a 1:1 binding model, for example, using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). In some embodiments, the CD3 binding affinity assay is performed under the same conditions, except that the assay temperature is set to 25°C, and the CD137 binding affinity assay is performed under the same conditions, except that the assay temperature is set to 37°C.

[0080] ALPHAScreen operates using ALPHA technology, which employs two types of beads (donor and acceptor), based on the following principle: A light emission signal is detected only when the two beads are located in close proximity due to a biological interaction between molecules bound to the donor bead and molecules bound to the acceptor bead. A photosensitiver in the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, upon reaching the nearby acceptor bead, triggers a chemiluminescent reaction in the bead, ultimately emitting light. If there is no interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead. Therefore, no chemiluminescent reaction occurs.

[0081] One of the substances whose interaction is to be observed (ligand) is immobilized on a thin gold film of the sensor chip. Light is shone from the back of the sensor chip so that total internal reflection occurs at the interface between the gold film and the glass. As a result, a region with reduced reflectivity (SPR signal) is formed in a portion of the reflected light. The other substance whose interaction is to be observed (analyte) 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 a y-axis 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 the response on the sensorgram before and after interaction with the analyte) can be determined from the sensorgram. However, since the amount of binding also depends on the amount of ligand, comparisons must be made under conditions using substantially the same amount of ligand. Kinetics, namely the binding 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 suitably used in the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0082] Whether the antigen-binding molecule of the present invention "does not bind to the first antigen and the second antigen simultaneously" can be confirmed by confirming that the antigen-binding molecule has binding activity to both the first antigen and the second antigen; then pre-binding either the first antigen or the second antigen to the antigen-binding molecule containing the variable region having this binding activity; and then determining whether or not it has binding activity to the other antigen by the method described above. Alternatively, this can also be confirmed by determining whether the binding of the antigen-binding molecule to either the first antigen or the second antigen immobilized on an ELISA plate or sensor chip is inhibited by the addition of the other antigen to a solution. In some embodiments, the binding of the antigen-binding molecule of the present invention to either the first antigen or the second antigen is inhibited by the binding of the antigen-binding molecule to the other antigen 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.

[0083] In one aspect, while immobilizing one antigen (e.g., a first antigen), the inhibition of binding of the antigen-binding molecule to the first antigen can be determined in the presence of another antigen (e.g., a second antigen) by a method known in the prior art (i.e., ELISA, BIACORE, etc.). In another aspect, while immobilizing the second antigen, the inhibition of binding of the antigen-binding molecule to the second antigen can also be determined in the presence of the first antigen. If, when either of the two aspects described above 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, then it is determined that the antigen-binding molecule of the present invention does not bind to the first antigen and the second antigen simultaneously. In some embodiments, the concentration of the antigen injected as an analyte is at least 1, 2, 5, 10, 30, 50, or 100 times higher than the concentration of the other antigen being immobilized. In a preferred configuration, the concentration of the antigen injected as an analyte is 100 times higher than the concentration of other antigens immobilized, and binding is inhibited by at least 80%.

[0084] In one embodiment, the ratio of the KD value of the antigen-binding molecule for the first antigen (analyte) binding activity to the second antigen (immobilized) binding activity of the antigen-binding molecule (KD(first antigen) / KD(second antigen)) is calculated, and a first antigen (analyte) concentration that is 10, 50, 100, or 200 times higher than the second antigen (immobilized) concentration by the KD ratio (KD(first antigen) / KD(second antigen)) can be used for the competitive measurement described above. (For example, if the KD ratio is 0.1, concentrations 1, 5, 10, or 20 times higher can be selected. Furthermore, if the KD ratio is 10, concentrations 100, 500, 1000, or 2000 times higher can be selected.)

[0085] In one aspect, while immobilizing one antigen (e.g., a first antigen), the attenuation of the binding signal of the antigen-binding molecule to the first antigen can be determined in the presence of another antigen (e.g., a second antigen) by a method known in the prior art (i.e., ELISA, ECL, etc.). In another aspect, while immobilizing the second antigen, the attenuation of the binding signal of the antigen-binding molecule to the second antigen can also be determined in the presence of the first antigen. If, when either of the two aspects described above is performed, the binding signal is attenuated by at least 50%, preferably 60%, preferably 70%, more preferably 80%, more preferably 90%, or even more preferably 95%, then it is determined that the antigen-binding molecule of the present invention does not bind to the first antigen and the second antigen simultaneously (see Reference Examples 2-5, 3-9, and 4-4). In some embodiments, the concentration of the antigen injected as an analyte is at least 1, 2, 5, 10, 30, 50, or 100 times higher than the concentration of the other antigen being immobilized. In a preferred configuration, the concentration of the antigen injected as an analyte is 100 times higher than the concentration of other antigens immobilized, and binding is inhibited by at least 80%.

[0086] In one embodiment, the ratio of the KD value of the antigen-binding molecule for the first antigen (analyte) binding activity to the second antigen (immobilized) binding activity of the antigen-binding molecule (KD(first antigen) / KD(second antigen)) is calculated, and a first antigen (analyte) concentration that is 10, 50, 100, or 200 times higher than the second antigen (immobilized) concentration can be used for the above measurement. (For example, if the KD ratio is 0.1, concentrations 1, 5, 10, or 20 times higher can be selected. Furthermore, if the KD ratio is 10, concentrations 100, 500, 1000, or 2000 times higher can be selected.)

[0087] Specifically, for example, when using the ECL method, a biotin-labeled test antigen-binding molecule, a first antigen labeled with a sulfo-tag (Ru complex), and an unlabeled second antigen are prepared. If the test antigen-binding molecule can bind to both the first and second antigens, but not simultaneously, the sulfo-tag luminescence signal can be detected in the absence of the unlabeled second antigen by adding a mixture of the test antigen-binding molecule and the labeled first antigen onto a streptavidin-immobilized plate and observing the subsequent luminescence. In contrast, the luminescence signal decreases in the presence of the unlabeled second antigen. The relative binding activity can be determined by quantifying this decrease in the luminescence signal. This analysis can be similarly performed using a labeled second antigen and an unlabeled first antigen.

[0088] In the case of ALPHAScreen, the test antigen-binding molecule interacts with the first antigen in the absence of a competing second antigen, generating a signal in the 520-620 nm range. The untagged second antigen competes with the first antigen for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence can be quantified to determine the relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin, etc., is known in the art. For example, the first antigen can be tagged with GST by a method appropriately adopted, which includes in-frame fusion of a polynucleotide encoding the first antigen with a polynucleotide encoding GST; and expression of the resulting fusion gene in cells possessing a vector capable of expressing it, followed by purification using a glutathione column. The resulting signal is preferably analyzed using software such as GRAPHPAD PRISM (GraphPad Software, Inc., San Diego), which is adapted to a one-site competition model based on nonlinear regression analysis. This analysis can be similarly performed using a tagged second antigen and an untagged first antigen.

[0089] Alternatively, a method using fluorescence resonance energy transfer (FRET) may be used. FRET is a phenomenon in which excitation energy is directly transferred between two closely located fluorescent molecules via electron resonance. When FRET occurs, the excitation energy of the donor (the fluorescent molecule in the excited state) is transferred to the acceptor (another fluorescent molecule located near the donor), causing the fluorescence emitted from the donor to disappear (more precisely, its fluorescence lifetime to be shortened), and instead, fluorescence is emitted from the acceptor. This phenomenon can be used to analyze whether an antibody binds simultaneously to both the first and second antigens. For example, when a first antigen with a fluorescent donor and a second antigen with a fluorescent acceptor bind simultaneously to a test antigen-binding molecule, the fluorescence of the donor disappears, while fluorescence is emitted from the acceptor. Therefore, a change in fluorescence wavelength is observed. Such an antibody is confirmed to bind simultaneously to both the first and second antigens. On the other hand, if the mixture of the first antigen, the second antigen, and the test antigen-binding molecule does not change the fluorescence wavelength of the fluorescent donor bound to the first antigen, then this test antigen-binding molecule can be considered an antigen-binding domain that can bind to both the first and second antigens, but not to both simultaneously.

[0090] For example, a biotin-labeled test antigen-binding molecule is bound to streptavidin on donor beads, while a first antigen tagged with glutathione S-transferase (GST) is bound to acceptor beads. The test antigen-binding molecule interacts with the first antigen in the absence of a competing second antigen, generating a signal in the 520-620 nm range. The untagged second antigen competes with the first antigen for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence can be quantified to determine the relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin, etc., is known in the art. For example, the first antigen can be tagged with GST by a method appropriately adopted, which includes in-frame fusion of a polynucleotide encoding the first antigen and a polynucleotide encoding GST; and expression of the resulting fusion gene in cells possessing a vector capable of expressing it, followed by purification using a glutathione column. The obtained signals are preferably analyzed using software such as GRAPHPAD PRISM (GraphPad Software, Inc., San Diego), which is fitted to a one-site competition model based on nonlinear regression analysis.

[0091] Tagging is not limited to GST tagging and may be performed with any tag, including but not limited to histidine tags, MBP, CBP, Flag tags, HA tags, V5 tags, c-myc tags, etc. Binding of the test antigen-binding molecule to the donor beads is not limited to binding using a biotin-streptavidin reaction. In particular, if 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.

[0092] Furthermore, when the first and second antigens are not expressed on the cell membrane, like soluble proteins, or when both are present on the same cell, the variable region can bind to both the first and second antigens simultaneously. However, when the first and second antigens are expressed on different cells, they cannot bind to each other simultaneously. In such cases, assays can also be performed using methods known in the art. Specifically, a test antigen-binding molecule, which has been confirmed to be positive in ECL-ELISA for the simultaneous binding of a first and second antigen, is mixed with cells expressing the first antigen and cells expressing the second antigen. It can be shown that the test antigen-binding molecule cannot simultaneously bind to the first and second antigens expressed on different cells unless the antigen-binding molecule and these cells bind to each other simultaneously. This assay can be performed, for example, by cell-based ECL-ELISA. Cells expressing the first antigen are immobilized on a plate beforehand. After binding the test antigen-binding molecule to it, cells expressing the second antigen are added to the plate. Different antigens expressed only on cells expressing the second antigen are detected using an antibody labeled with a sulfo-tag against this antigen. If the antigen-binding molecule simultaneously binds to the two antigens expressed on two different cells, a signal is observed. If the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed.

[0093] Alternatively, this assay may be performed using the ALPHAScreen method. The test antigen-binding molecule is mixed with cells expressing a first antigen bound to donor beads and cells expressing a second antigen bound to acceptor beads. A signal is observed if the antigen-binding molecule simultaneously binds to the two antigens expressed on the two cells, respectively. If the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed. Alternatively, this assay may be performed using the Octet interaction analysis method. First, cells expressing a first antigen tagged with a peptide tag are bound to a biosensor that recognizes the peptide tag. Cells expressing a second antigen and the test antigen-binding molecule are placed in wells and their interactions are analyzed. If the antigen-binding molecule binds simultaneously to two antigens expressed on two different cells, a large wavelength shift is observed due to the binding of the test antigen-binding molecule and the cells expressing the second antigen to the biosensor. If the antigen-binding molecule does not bind to these antigens simultaneously, a small wavelength shift is observed due to the binding of only the test antigen-binding molecule to the biosensor.

[0094] Instead of these methods based on binding activity, assays based on biological activity may be performed. For example, cells expressing a first antigen and cells expressing a second antigen are cultured together with a test antigen-binding molecule. The two antigens expressed on each of the two cells are mutually activated via the test antigen-binding molecule if the antigen-binding molecule binds to both antigens simultaneously. Therefore, changes in activation signals, such as an increase in the phosphorylation levels downstream of each antigen, can be detected. Alternatively, cytokine production is induced as a result of activation. Therefore, the amount of cytokine produced can be measured to determine whether it binds to the two cells simultaneously. Alternatively, cytotoxic activity against cells expressing the second antigen is induced as a result of activation. Alternatively, the expression of a reporter gene is induced as a result of activation by a promoter that is activated downstream of the signaling pathway of the second or first antigen. Therefore, cytotoxic activity or the amount of reporter protein produced can be measured to determine whether it binds to the two cells simultaneously.

[0095] 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 belonging to a class of antibodies that contain the same amino acid sequence as naturally occurring IgG and are substantially encoded by the immunoglobulin γ gene. Natural human IgG means, for example, natural human IgG1, natural human IgG2, natural human IgG3, or natural human IgG4. Natural IgG also includes variants that arise spontaneously. Multiple allotype sequences based on genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242 as 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 EU numbering positions 356-358.

[0096] The antibody Fc region can be found, for example, as an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM type Fc region. For example, an Fc region derived from a naturally occurring human IgG antibody can be used as the antibody Fc region of the present invention. For example, an Fc region derived from the constant region of naturally occurring IgG, specifically the constant region originating from naturally occurring human IgG1 (SEQ ID NO: 498), the constant region originating from naturally occurring human IgG2 (SEQ ID NO: 499), the constant region originating from naturally occurring human IgG3 (SEQ ID NO: 500), or the constant region originating from naturally occurring human IgG4 (SEQ ID NO: 501), can be used as the Fc region of the present invention. The constant region of naturally occurring IgG also includes variants that arise spontaneously therefrom.

[0097] The Fc region of the present invention is particularly preferably an Fc region in which binding activity to the Fcγ receptor is reduced. Here, the Fcγ receptor (also referred to herein as FcγR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4, and means any member of the protein family substantially encoded by the Fcγ receptor gene. In humans, this family includes FcγRI(CD64), which contains isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which contains isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), which contains isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); as well as any undiscovered human FcγR or FcγR isoform or allotype. FcγR includes those derived from humans, mice, rats, rabbits, and monkeys. FcγR is not limited to these molecules and may originate from any organism. Mouse FcγR includes, but is not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype. Preferred examples of such Fcγ receptors include human FcγRI(CD64), FcγRIIa(CD32), FcγRIIb(CD32), FcγRIIIa(CD16), and / or FcγRIIIb(CD16).

[0098] FcγR is found in the form of active receptors possessing ITAM (immune receptor activating tyrosine motif) and suppressive receptors possessing ITIM (immune receptor suppressive tyrosine motif). FcγR is classified into active FcγR (FcγRI, FcγRIIa R, FcγRIIa H, FcγRIIIa, and FcγRIIIb) and suppressive FcγR (FcγRIIb). The polynucleotide and amino acid sequences of FcγRI are described in NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIa are described in BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIb are described in BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIa are described in BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are described in BC128562.1 and AAI28563.1, respectively (RefSeq registration numbers). FcγRIIa has two genetic polymorphisms in which the 131st amino acid of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990). FcγRIIb has two genetic polymorphisms in which the 232nd amino acid of FcγRIIb is substituted with isoleucine (I type) or threonine (T type) (Arthritis. Rheum. 46: 1242-1254 (2002)). FcγRIIIa has two genetic polymorphisms in which the 158th amino acid of FcγRIIIa is substituted with valine (V type) or phenylalanine (F type) (J. Clin. Invest. 100(5): 1059-1070 (1997)). FcγRIIIb has two genetic polymorphisms (NA1 and NA2) (J. Clin. Invest. 85: 1287-1295 (1990)).

[0099] Reduced binding activity to the Fcγ receptor can be confirmed by well-known methods such as FACS, ELISA, ALPHAScreen (amplified luminescence proximity homogeneous assay screen), or BIACORE method based on surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHAScreen method is performed using ALPHA technology, which employs two types of beads (donor and acceptor), based on the following principle: A light emission signal is detected only when the two beads are located in close proximity due to a biological interaction between molecules bound to the donor bead and molecules bound to the acceptor bead. A photosensitiver 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, upon reaching the nearby acceptor bead, triggers a chemiluminescent reaction in the bead, ultimately emitting light. If there is no interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead. Therefore, no chemiluminescent reaction occurs.

[0100] For example, a biotin-labeled test antigen-binding molecule is bound to donor beads, while a glutathione S-transferase (GST)-tagged Fcγ receptor is bound to acceptor beads. In the absence of antigen-binding molecules with competing mutant Fc regions, antigen-binding molecules with wild-type Fc regions interact with the Fcγ receptor, generating a signal in the 520-620 nm range. Antigen-binding molecules with untagged mutant Fc regions compete with antigen-binding molecules with wild-type Fc regions for interaction with the Fcγ receptor. The resulting decrease in fluorescence can be quantified to determine the relative binding affinity. Biotinylation of antigen-binding molecules (e.g., antibodies) using sulfo-NHS-biotin is known in the art. For example, the Fcγ receptor can be tagged with GST by a method appropriately adopted, which includes in-frame fusion of a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST; and expression of the resulting fusion gene in cells possessing a vector capable of expressing it, followed by purification using a glutathione column. The obtained signal is preferably analyzed using software such as GRAPHPAD PRISM (GraphPad Software, Inc., San Diego), which is adapted to a one-site competition model based on nonlinear regression analysis.

[0101] One of the substances whose interaction is to be observed (ligand) is immobilized on a gold thin film of the sensor chip. Light is shone from the back of the sensor chip so that total internal reflection occurs at the interface between the gold thin film and the glass. As a result, a region of reduced reflectivity (SPR signal) is formed in a portion of the reflected light. The other substance whose interaction is to be observed (analyte) 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 a y-axis and displays the time-dependent change in mass as assay data (sensorgram). Kinetics, i.e., the binding 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 suitably used in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0102] In this specification, reduced binding activity to the Fcγ receptor means that the test antigen-binding molecule exhibits a binding activity of, for example, 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, 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 binding activity of a control antigen-binding molecule containing the Fc region, based on the analysis method described above. Antigen-binding molecules containing the Fc region of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used as appropriate control antigen-binding molecules. The structure of the Fc region is described in SEQ ID NO: 502 (A added to the N-terminus of RefSeq registration number AAC82527.1), SEQ ID NO: 503 (A added to the N-terminus of RefSeq registration number AAB59393.1), SEQ ID NO: 504 (A added to the N-terminus of RefSeq registration number CAA27268.1), or SEQ ID NO: 505 (A added to the N-terminus of RefSeq registration number AAB59394.1). When using an antigen-binding molecule containing a variant of the Fc region of a particular isotype of antibody as a test substance, the effect of the variant mutation on the binding activity to the Fcγ receptor is tested using this antigen-binding molecule containing the Fc region of that particular isotype of antibody as a control. Antigen-binding molecules containing Fc region variants that have been confirmed to have reduced binding activity to the Fcγ receptor are prepared as appropriate.

[0103] For example, variants such as 231A-238S deletion (WO 2009 / 011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54), C226S, C229S, E233P, L234V, or L235A (Blood (2007) 109, 1185-1192) (these amino acids are defined according to EU numbering) are known in the art as such variants.

[0104] A preferred example of this is an antigen-binding molecule having an Fc region derived from the Fc region of a particular isotype of antibody, by substitution of any of the following constituent amino acids: positions 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, and 332, as defined according to EU numbering. The antibody isotype from which the Fc region originates is not particularly limited, and Fc regions originating from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies may be used as appropriate. An Fc region originating from a natural human IgG1 antibody is preferably used. For example, the following group of substitutions of constituent amino acids, as defined according to EU numbering (the numbers indicate the position of the amino acid residue as defined according to EU numbering; the single-letter amino acid notation before the number indicates the amino acid residue before substitution; and the single-letter amino acid notation after the number indicates the amino acid residue before substitution): (a) L234F, L235E, and P331S, (b) C226S, C229S, and P238S, (c) C226S and C229S, and (d) C226S, C229S, E233P, L234V, and L235A Antigen-binding molecules having an Fc region derived from the IgG1 antibody Fc region, either due to one of the above or due to a deletion in the amino acid sequence at positions 231-238, may also be used as appropriate.

[0105] The following groups of amino acid substitutions, as defined according to EU numbering (the numbers indicate the position of the amino acid residue as defined according to EU numbering; the single-letter amino acid notation before the number indicates the amino acid residue before substitution; and the single-letter amino acid notation after the number indicates the amino acid residue before substitution): (e) H268Q, V309L, A330S, and P331S, (f) V234A, (g) G237A, (h) V234A and G237A, (i) A235E and G237A, and (j) V234A, A235E, and G237A Antigen-binding molecules having an Fc region derived from the Fc region of an IgG2 antibody, by any of the above, may also be used as appropriate.

[0106] The following substitution groups of constituent amino acids, defined according to EU numbering (the numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid notation preceding the number represents the amino acid residue before substitution; and the single-letter amino acid notation following the number represents the amino acid residue before substitution): (k) F241A, (l) D265A, and (m) V264A Antigen-binding molecules having an Fc region derived from the Fc region of an IgG3 antibody, by any of the above, may also be used as appropriate.

[0107] The following substitution groups of constituent amino acids, defined according to EU numbering (the numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid notation preceding the number represents the amino acid residue before substitution; and the single-letter amino acid notation following the number represents the amino acid residue before substitution): (n) L235A, G237A, and E318A, (o) L235E, and (p) F234A and L235A Antigen-binding molecules having an Fc region derived from the Fc region of an IgG4 antibody, by any of the above, may also be used as appropriate.

[0108] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of a native human IgG1 antibody by substitution of the amino acid at the corresponding EU numbering position in the Fc region of the counter-part IgG2 or IgG4 with any of the following constituent amino acids: the amino acids at positions 233, 234, 235, 236, 237, 327, 330, and 331 as defined according to EU numbering.

[0109] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of a native human IgG1 antibody by substitution of any one or more of the following constituent amino acids: the amino acids at positions 234, 235, and 297 as defined according to EU numbering with different amino acids. The type of amino acid present after substitution is not particularly limited. Antigen-binding molecules having an Fc region in which any one or more of the amino acids at positions 234, 235, and 297 are substituted with alanine are particularly preferred.

[0110] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of an IgG1 antibody by substitution of the constituent amino acid at position 265 as defined according to EU numbering with a different amino acid. The type of amino acid present after substitution is not particularly limited. Antigen-binding molecules having an Fc region in which the amino acid at position 265 is substituted with alanine are particularly preferred.

[0111] In some embodiments, the antigen-binding molecule may have an increased half-life and increased binding to the fetal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) (as described in US2005 / 0014934A1 (Hinton et al.)). These antigen-binding molecules include an Fc region having one or more substitutions therein that increase the binding of the Fc region to FcRn. Such Fc variants include substitutions at one or more positions of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, those having a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan, Nature 322:738-40 (1988); U.S. Patents No. 5,648,260 and 5,624,821; and WO 1994 / 29351 for other examples of Fc region variants.

[0112] In another embodiment, the active ingredient may be encapsulated in microcapsules prepared by coacervation techniques in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). In yet another embodiment, the antigen-binding molecule of the present invention may be conjugated with a “heteropolymer,” for example, to improve half-life or stability, or to otherwise improve the antibody. For example, the antibody may be linked to one of various non-proteinogenic polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. An antibody fragment linked to one or more PEG molecules, e.g., Fab', is an exemplary embodiment of the present invention. In yet another embodiment, the antigen-binding molecule of the present invention may have improved pharmacokinetics by fusing with a domain capable of binding to an embryonic Fc receptor, such as an albumin protein, preferably human serum albumin; see, for example, Muller, Dafne, et al. Journal of Biological Chemistry 282.17 (2007): 12650-12660; and Biotechnol Lett (2010) 32:609-622.

[0113] In some embodiments, the "antigen-binding molecule" of the present invention may be a multispecific antigen-binding molecule comprising, for example, (i) a first antigen-binding domain linked to an Fc region and a second antigen-binding domain distinct from the first antigen-binding domain; (ii) a third antigen-binding domain linked to an Fc region and having its C-terminus linked to the N-terminus of the first antigen-binding domain and a second antigen-binding domain distinct from the first antigen-binding domain; and (iii) a third antigen-binding domain linked to an Fc region and having its C-terminus linked to the N-terminus of the second antigen-binding domain and a first antigen-binding domain distinct from the second antigen-binding domain.

[0114] To suppress unintended association between the heavy (H) chains of the first and second antigen-binding domains, a technique can be applied to the association of multispecific antigen-binding molecules by introducing charge repulsion at the interface between the second constant domain (CH2) or the third constant domain (CH3) of the Fc region (WO2006 / 106905). In a technique for suppressing unintended association between the heavy (H) chains of a first antigen-binding domain and a second antigen-binding domain by introducing charge repulsion at the interface of CH2 or CH3, examples of amino acid residues that come into contact with each other at the interface between the constant heavy (H) chain domains may include the residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in one CH3 domain, as well as their partner residues in the other CH3 domain.

[0115] More specifically, for example, an antigen-binding molecule containing two heavy (H) chain CH3 domains can be prepared, in which one to three pairs of amino acid residues selected from the following pairs of amino acid residues (1) to (3) in the first H chain CH3 domain have the same charge: (1) amino acid residues at EU numbering positions 356 and 439 contained in the H chain CH3 domain; (2) amino acid residues at EU numbering positions 357 and 370 contained in the H chain CH3 domain; and (3) amino acid residues at EU numbering positions 399 and 409 contained in the H chain CH3 domain.

[0116] The antigen-binding molecule can further be prepared as an antigen-binding molecule in which one to three pairs of amino acid residues are selected from pairs of amino acid residues (1) to (3) in a second H chain CH3 domain different from the first H chain CH3 domain, such that each pair of amino acid residues corresponds to a pair of homogeneous amino acid residues (1) to (3) in the first H chain CH3 domain, and has a charge opposite to that of the corresponding amino acid residues in the first H chain CH3 domain.

[0117] Each amino acid residue described in pairs (1) to (3) is located near its partner in the associated heavy chain. A person skilled in the art can find the positions corresponding to each of the amino acid residues described in pairs (1) to (3) in a desired heavy chain CH3 domain or heavy chain constant domain by following homology modeling using commercially available software, and can modify the amino acid residues at those positions as appropriate.

[0118] In the antigen-binding molecule described above, each of the "charged amino acid residues" is preferably selected from, for example, amino acid residues belonging to either of the following groups (a) and (b): (a) Glutamic acid (E) and aspartic acid (D); and (b) Lysine (K), arginine (R), and histidine (H).

[0119] In the antigen-binding molecule described above, the phrase "having the same charge" means, for example, that all of the two or more amino acid residues belong to either group (a) or (b). The phrase "having opposite charges" means, for example, that at least one of the two or more amino acid residues may belong to either group (a) or (b), while the remaining amino acid residues belong to the other group.

[0120] In a preferred embodiment, the antigen-binding molecule may have a first H chain CH3 domain and a second H chain CH3 domain crosslinked by a disulfide bond. As described above, the amino acid residues modified in accordance with the present invention are not limited to amino acid residues in the antibody variable region or antibody constant region as described above. Those skilled in the art can identify amino acid residues constituting the interface of polypeptide variants or heterologous polymers by homology modeling using commercially available software, and can modify the amino acid residues at those positions to control association.

[0121] The association of the multispecific antigen-binding molecules of the present invention can also be carried out by alternative techniques known in the art. An amino acid side chain in a heavy chain variable (VH) region is substituted with a larger side chain (knob), and the amino acid side chain of its partner in another heavy chain variable (VH) region is substituted with a smaller side chain (hole). Knobs can be placed in holes to efficiently associate polypeptides of different Fc domains (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; and Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0122] In addition to this technique, further alternative techniques known in the art may be used to form the multispecific antigen-binding molecule of the present invention. A portion of the CH3 of one heavy (H) chain is converted to its corresponding IgA-derived sequence, and the complementary portion of the CH3 of the other heavy (H) chain is converted to its corresponding IgA-derived sequence. By using the resulting chain exchange domain CH3, efficient association between polypeptides with different sequences can be induced by complementary CH3 association (Protein Engineering Design & Selection, 23; 195-202, 2010). The multispecific antigen-binding molecule of interest can also be efficiently formed by using this technique known in the art.

[0123] Alternatively, multispecific antigen-binding molecules may be formed by, for example, antibody preparation techniques using CH1-CL and VH-VL association of antibodies as described in WO2011 / 028952, techniques for preparing bispecific antibodies using separately prepared monoclonal antibodies as described in WO2008 / 119353 and WO2011 / 131746 (Fab arm exchange), techniques for controlling the association between antibody heavy chain CH3 domains as described in WO2012 / 058768 and WO2013 / 063702, techniques for preparing bispecific antibodies composed of two light chains and one heavy chain as described in WO2012 / 023053, or techniques for preparing bispecific antibodies using two bacterial cell lines, each expressing a half-unit of an antibody consisting of one H chain and one L chain, as described in Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)). In addition to these association techniques, the CrossMab technology (Scaefer et al., Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192), a known heterogeneous light chain association technique in which a light chain forming a variable region that binds to a first epitope and a light chain forming a variable region that binds to a second epitope are associated with a heavy chain forming a variable region that binds to a first epitope and a heavy chain forming a variable region that binds to a second epitope, respectively, can also be used to prepare the multispecific or multiparatopic antigen-binding molecules provided by the present invention.

[0124] Examples of techniques for preparing bispecific antibodies using separately prepared monoclonal antibodies may include a method that promotes heterodimerization of an antibody by placing a monoclonal antibody in which specific amino acids in the heavy chain CH3 domain have been substituted under reducing conditions to obtain a desired bispecific antibody. Examples of preferred amino acid substitution sites for this method may include the residues at EU numbering positions 392 and 397 in the CH3 domain. Furthermore, bispecific antigen-binding molecules can also be prepared by using an antibody in which one to three pairs of amino acid residues selected from the following pairs (1) to (3) in the first H chain CH3 domain have the same charge: (1) the amino acid residues at EU numbering positions 356 and 439 contained in the H chain CH3 domain; (2) the amino acid residues at EU numbering positions 357 and 370 contained in the H chain CH3 domain; and (3) the amino acid residues at EU numbering positions 399 and 409 contained in the H chain CH3 domain. Bispecific antigen-binding molecules can also be prepared by using antibodies in which pairs of amino acid residues (1) to (3) in a second H chain CH3 domain, distinct from the first H chain CH3 domain, are selected such that one to three pairs of amino acid residues correspond to pairs of homogeneous amino acid residues (1) to (3) in the first H chain CH3 domain, and have opposite charges to the corresponding amino acid residues in the first H chain CH3 domain.

[0125] Even if the desired multispecific antigen-binding molecule cannot be efficiently formed, the multispecific antigen-binding molecule of the present invention can be obtained by separating and purifying the desired multispecific antigen-binding molecule from the produced antigen-binding molecules. For example, a previously reported method involves introducing amino acid substitutions into the variable domains of two types of H chains to confer an isoelectric point difference so that two types of homodimers and the desired heterodimerized antibody can be purified separately by ion-exchange chromatography (WO2007114325). A method using protein A to purify a heterodimerized antibody consisting of a mouse IgG2a H chain that can bind to protein A and a rat IgG2b H chain that cannot bind to protein A has been previously reported as a method for purifying heterodimers (WO98050431 and WO95033844). Alternatively, the amino acid residues at EU numbering positions 435 and 436, which constitute the protein A binding site of IgG, may be substituted with amino acids such as Tyr and His, which provide different protein A binding strengths, and the resulting heavy chains are used to alter the interaction between each heavy chain and protein A. As a result, only heterodimerized antibodies can be efficiently purified using a protein A column.

[0126] Multiple of these techniques, for example, two or more, may be used in combination. Furthermore, these techniques can be applied separately to the two heavy (H) chains to be associated, as appropriate. The antigen-binding molecule of the present invention is based on these modified forms, but may also be prepared as an antigen-binding molecule having the same amino acid sequence.

[0127] Modification of amino acid sequences can be carried out by various methods known in the art. Examples of these methods that may be performed include site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz) Methods may include, but are not limited to, HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; and Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82, 488-492), PCR mutagenesis, and cassette mutagenesis.

[0128] In addition to the above-described amino acid modifications, the antigen-binding molecule of the present invention can further contain additional modifications. The additional modifications can be selected, for example, from amino acid substitutions, deletions, and modifications, and combinations thereof. For example, the antigen-binding molecule of the present invention can be further optionally modified without substantially changing the intended function of the molecule. Such mutations can be made, for example, by conservative substitution of amino acid residues. Alternatively, even modifications that change the intended function of the antigen-binding molecule of the present invention may be carried out as long as the function changed by such modifications is within the scope of the object of the present invention.

[0129] The modification of the amino acid sequence according to the present invention also includes post-translational modification. Specifically, post-translational modification can refer to the addition or deletion of sugar chains. For example, the antigen-binding molecule of the present invention having a constant region of IgG1 type can have an amino acid residue modified with a sugar chain at position 297 of EU numbering. The sugar chain structure for use in modification is not limited. Generally, antibodies expressed by eukaryotic cells contain sugar chain modifications in the constant region. Therefore, antibodies expressed by the following cells are usually modified with several sugar chains: Antibody-producing cells of mammals; and Eukaryotic cells transformed with an expression vector containing DNA encoding an antibody. Here, eukaryotic cells include yeast and animal cells. For example, CHO cells or HEK293H cells are typical animal cells for transformation with an expression vector containing DNA encoding an antibody. On the other hand, the antibodies of the present invention also include antibodies without sugar chain modification at that position. Antibodies having a constant region not modified with a sugar chain can be obtained by expression of genes encoding these antibodies in prokaryotic cells such as Escherichia coli.

[0130] The additional modification according to the present invention may more specifically be, for example, the addition of sialic acid to a sugar chain in the Fc region (mAbs. 2010 Sep-Oct;2(5):519-27).

[0131] If the antigen-binding molecule of the present invention has an Fc region, for example, amino acid substitutions to improve binding activity to FcRn (J Immunol. 2006 Jan 1;176(1):346-56;J Biol Chem. 2006 Aug 18;281(33):23514-24;Int Immunol. 2006 Dec;18(12):1759-69;Nat Biotechnol. 2010 Feb;28(2):157-9;WO2006 / 019447;WO2006 / 053301;and WO2009 / 086320) or amino acid substitutions to improve antibody heterogeneity or stability ((WO2009 / 041613)) may be added.

[0132] When the term "antibody" is used in this application, it is interpreted in its broadest sense and includes any antibody, such as monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, and humanized antibodies, as long as they exhibit the desired biological activity.

[0133] When the term "antibody" is used in this application, it may refer to any antibody, without being limited by the type of antigen or its origin. Examples of antibody origins may include, but are not limited to, human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

[0134] Antibodies can be prepared by methods well known to those skilled in the art. For example, monoclonal antibodies may be produced by hybridoma (Kohler and Milstein, Nature 256:495 (1975)) or by recombinant methods (U.S. Patent No. 4,816,567). Alternatively, monoclonal antibodies may be isolated from phage display antibody libraries (Clackson et al., Nature 352:624-628 (1991); and Marks et al., J.Mol.Biol. 222:581-597 (1991)). Furthermore, monoclonal antibodies may be isolated from a single B cell clone (N. Biotechnol. 28(5): 253-457 (2011)).

[0135] Humanized antibodies are also called reconstituted human antibodies. Specifically, for example, humanized antibodies consisting of human antibodies transplanted with CDRs of non-human animal (e.g., mouse) antibodies are known in the art. General genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known in the art as a method for transplanting mouse antibody CDRs into human FRs.

[0136] A vector for humanized antibody expression can be prepared by inserting DNA encoding an antibody variable domain, each containing three linked CDRs and four FRs, and DNA encoding a human antibody constant domain into an expression vector such that the variable domain DNA fuses in-frame with the constant domain DNA. These vectors containing the inserts are then transferred into a host to establish recombinant cells. The recombinant cells are then cultured for the expression of the DNA encoding the humanized antibody to produce the humanized antibody in the cell culture (see European Patent Publication No. EP 239400 and International Publication No. WO1996 / 002576).

[0137] If necessary, amino acid residues in the FR may be substituted so that the reconstituted human antibody CDR forms an appropriate antigen-binding site. For example, the amino acid sequence of the FR can be mutated by applying the PCR method used in transplantation of mouse CDRs into human FRs.

[0138] The desired human antibody can be obtained by DNA immunization using transgenic animals (see International Publication Numbers WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) that possess the entire repertoire of human antibody genes as immunized animals.

[0139] In addition, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage using phage display. A phage expressing antigen-binding scFv can be selected. The genes of the selected phage can be analyzed to determine the DNA sequence encoding the V region of the antigen-binding human antibody. After determining the DNA sequence of the antigen-binding scFv, the V region sequence can be fused in-frame with the sequence of the desired human antibody C region, and then inserted into a suitable expression vector to prepare an expression vector. The expression vector is then transferred into preferred expression cells listed above for the expression of the gene encoding the human antibody to obtain the human antibody. These methods are known in the art (see International Publication Numbers WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0140] In addition to phage display technology, other known technologies for obtaining human antibodies by panning using human antibody libraries include, for example, technologies using cell-free translation systems, technologies for presenting antigen-binding molecules on the surface of cells or viruses, and technologies using emulsions. For example, ribosome display methods, which involve forming a complex between mRNA and translated protein via ribosomes by removing stop codons, cDNA or mRNA display methods, which involve covalently binding translated proteins to gene sequences using compounds such as puromycin, or CIS display methods, which involve forming a complex between genes and translated proteins using nucleic acid-binding proteins, can be used as technologies using cell-free translation systems. Phage display methods, as well as E. coli display methods, Gram-positive bacterial display methods, yeast display methods, mammalian cell display methods, and virus display methods, can be used as technologies for presenting antigen-binding molecules on the surface of cells or viruses. For example, in vitro virus display methods using genes and translation-related molecules encapsulated in emulsions can be used as technologies using emulsions. These methods are known in the art (Nat Biotechnol. 2000 Dec; 18 (12): 1287-92; Nucleic Acids Res. 2006; 34 (19): e127; Proc Natl Acad Sci US A. 2004 Mar 2; 101 (9): 2806-10; Proc Natl Acad Sci US A. 2004 Jun 22; 101 (25): 9193-8; Protein Eng Des Sel. 2008 Apr; 21 (4): 247-55; Proc Natl Acad Sci US A. 2000 Sep 26; 97 (20): 10701-5; MAbs. 2010 Sep-Oct; 2 (5): 508-18; and Methods Mol Biol. 2012; 911: 183-98).

[0141] One antibody variable region contained in each antigen-binding domain of the antigen-binding molecule of the present invention can bind to two different antigens, but cannot bind to these antigens simultaneously. In some embodiments, one antibody variable region contained in each antigen-binding domain of the antigen-binding molecule of the present invention can bind to a first antigen but not to a second antigen. The "first antigen" or "second antigen" to which the first antigen-binding domain and / or the second antigen-binding domain bind is preferably, for example, an immune cell surface molecule (e.g., T cell surface molecule, NK cell surface molecule, dendritic cell surface molecule, B cell surface molecule, NK T cell surface molecule, MDSC cell surface molecule, and macrophage surface molecule), or an antigen expressed not only in tumor cells, tumor blood vessels, and stromal cells, but also in normal tissues (e.g., integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF, etc.). With respect to the combination of the "first antigen" or the "second antigen," preferably, one of the first antigen and the second antigen is, for example, a molecule specifically expressed on T cells, and the other antigen is a molecule expressed on the surface of T cells or any other immune cells. In another embodiment of the combination of the "first antigen" and the "second antigen," preferably, one of the first antigen and the second antigen is, for example, a molecule specifically expressed on T cells, and the other antigen is a molecule expressed on immune cells that is different from a pre-selected antigen.

[0142] Specific examples of molecules specifically expressed on T cells include CD3 and T cell receptors. CD3 is particularly preferred. For example, in the case of human CD3, the site in CD3 to which the antigen molecule of the present invention binds may be any epitope present in the sequence of the γ, δ, or ε chains constituting human CD3. Particularly preferred is an epitope present in the extracellular region of the ε chain in the human CD3 complex. The polynucleotide sequences of the structures of the γ, δ, and ε chains constituting CD3 are NM_000073.2, NM_000732.4, and NM_000733.3, and their polypeptide sequences are NP_000064.1, NP_000723.1, and NP_000724.1 (RefSeq registry numbers). Examples of other antigens include Fcγ receptors, TLRs, lectins, IgA, immune checkpoint molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules.

[0143] In one embodiment, the first antigen is a T cell receptor complex molecule, preferably a molecule specifically expressed on T cells, such as CD3, and more preferably a molecule such as human CD3. In another embodiment, the second antigen is a molecule expressed on T cells or any other immune cell, preferably a cell surface regulator on an immune cell, more preferably a costimulatory molecule expressed on T cells, and even more preferably, but not limited to, proteins of the “TNF superfamily” or “TNF receptor superfamily,” including human CD137(4-1BB), CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL. In one preferred embodiment, the first antigen is CD3 and the second antigen is CD137. In this specification, the first antigen and the second antigen are defined interchangeably.

[0144] In this specification, the term "CD137" is also known as 4-1BB and is a member of the tumor necrosis factor (TNF) receptor family. Examples of factors belonging to the TNF superfamily or TNF receptor superfamily include CD137, CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL.

[0145] In some embodiments of the present invention, the antigen-binding molecule of the present invention further comprises a third antigen-binding domain that binds to a "third antigen" different from the "first antigen" and "second antigen" described above. The third antigen-binding domain that binds to the third antigen of the present invention may be an antigen-binding domain that recognizes any antigen. The third antigen-binding domain that binds to the third antigen of the present invention may be an antigen-binding domain that recognizes a molecule specifically expressed in cancer tissue.

[0146] In this specification, the “third antigen” is not particularly limited and may be any antigen. Examples of antigens include 17-IA, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Adresin, Adiponectin, ADP-ribosylcyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, Allergen, α1-Antichemotrypsin, α1-Antitrypsin, α-Synuclein, α-V / β-1 Antagonist, aminin, amyloid-beta, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, ApoA1, Apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthrax anthracis) protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, β-2-microglobulin, β-lactamase, bFGF, BID, Bik, BIM, BLC, BL -CAM, BLK, B lymphocyte stimulating factor (BlyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR,BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, bombesin, bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B lymphocyte cell adhesion molecule, C10, C1 inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, cadherin-3, calcitonin, cAMP, carbonic anhydrase-IX, carcinoembryonic antigen (CEA), cancer-associated antigen, cardiotrophin-1, cathepsin A, cathepsin B, cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1 / I-309, CCL11 / Eotaxin, CCL12 / MCP-5, CCL13 / MCP-4, CCL14 / HCC-1, CCL15 / HCC-2, CCL16 / HCC-4, CCL17 / TARC, CCL18 / PARC, CCL19 / ELC, CCL2 / MCP-1, CCL20 / MIP-3-α, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF-1, CCL24 / eotaxin-2, CCL25 / TECK, CCL26 / eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CCL3 / M1P-1-α, CCL3Ll / LD-78-β, CCL4 / MIP-l-β, CCL5 / RANTES, C CL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / 10 / MTP-1-γ, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD1 1b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD 26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51,CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80(B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, claudin 18, CLC, Clostridium botulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D, corticosteroid-binding globulin, colony-stimulating factor-1 receptor, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1 / fractalkine, CX3CR1, CXCL, CXCL1 / Gro-α, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF -l-α / β, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15 / Lungkine, CXCL16, CXCL16, CXCL2 / Gro-β, CXCL3 / G ro-γ, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCLlO / IP- 10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cystatin C, Cytokeratin Tumor-Associated Antigen, DAN, DCC, DcR3, DC-SIGN, Disintegration Promoter, Delta-like Protein Ligand 4, des(1-3)-IGF-1 (Brain IGF-1), Dhh, DHICA Oxidase, Dickkopf-1, Digoxin, Dipeptidyl Peptidase IV, DKl, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EGF-like Domain-containing Protein 7, Elastase, Elastin, EMA, EMMPRIN, ENA, ENA-78, Endosialin, Endothelin Receptor, Endotoxin, Enkephalinase, eNOS,Eot, eotaxin, eotaxin-2, eotaxini, EpCAM, ephrin B2 / EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO), erythropoietin receptor, E-selectin, ET-1, Exodus-2, F protein of RSV, F10, F11, F12, F13, F5, F9, factor Ia, factor IX, factor Xa, factor VII, factor VIII, factor VIIIc, Fas, FcαR, FcεRI, FcγIIb, FcγRI, FcγR IIa, FcγRIIIa, FcγRIIIb, FcRn, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acid, FGF-basic, fibrin, fibroblast-activating protein (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, folate receptor, follicle-stimulating hormone (FSH), fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-1), GDF-3 (Vgr-2), GDF-5 (BMP-14 / CDMP-1), GDF-6 (BMP-13 / CDMP-2), GDF-7 (BMP-12 / CDMP-3), GDF-8 (myostatin), GDF-9, GDNF, Gelsolin, GFAP, GF-CSF, GFR-α1, GFR-α2, GFR-α3, GF-β1, gH envelope glycoprotein, GITR, glucagon, glucagon receptor, glucagon-like peptide 1 receptor, Glut 4, glutamate carboxypeptidase II, glycoprotein hormone receptor, glycoprotein IIb / IIIa (GP IIb / IIIa), glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO / MGSA, growth hormone-releasing factor, GRO-β, GRO-γ, H. pylori,Haptens (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, heparin cofactor II, hepatic growth factor, anthrax protective antigen, hepatitis C virus E2 glycoprotein, hepatitis E, hepcidin, Her1, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV envelope protein, e.g., GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue plasminogen activator (t-PA), huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-α, IFN-β, IFN-γ, IgA, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptor, IL-11, IL-11 receptor, IL-12, IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-1 6, IL-16 receptor, IL-17, IL-17 receptor, IL-18(IGIF), IL-18 receptor, IL-1α, IL-1β, IL-1 receptor, IL-2, IL-2 receptor, IL-20, IL-20 receptor, IL-21, IL-21 receptor, IL-23, IL-23 receptor, IL-2 receptor, IL-3, IL-3 receptor, IL-31, IL-31 receptor, IL-3 receptor, IL-4, IL-4 receptor, IL-5, IL-5 receptor, IL-6, IL-6 receptor, IL-7 , IL-7 receptor, IL-8, IL-8 receptor, IL-9, IL-9 receptor, immunoglobulin immune complex, immunoglobulin, INF-α, INF-α receptor, INF-β, INF-β receptor, INF-γ, INF-γ receptor, type I IFN,Type I IFN receptor, influenza, inhibin, inhibin α, inhibin β, iNOS, insulin, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein, integrin, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α-V / β-3, integrin α-V / β-6, integrin α4 / β7, integrin α5 / β1, integrin α5 / β3, integrin α5 / β6, integrin ασ(αV), integrin αθ, integrin β1, integrin β2, integrin β3 (GPIIb-IIIa), IP-10, I-TA C, JE, kalliklein, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, calistatin, KC, KDR, keratinocyte growth factor (KGF), keratinocyte growth factor 2 (KGF-2), KGF, killer immunoglobulin-like receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (amylin, pancreatic islet amyloid polypeptide), LAP (TGF-1), latency-related peptide, latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, leptin, luteinizing hormone (LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptor, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotactin, lymphotoxin β receptor, lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, Maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC(67 aa), MDC(69 aa), megsin, Mer,MET tyrosine kinase receptor family, metalloproteinases, membrane glycoprotein OX2, mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial proteins, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14 MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, Monocyte attracting protein, Monocyte colony suppressor, Mouse gonadotropin-related peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, Mucin (Mud), Müllerian duct inhibitor, Mug, MuSK, Myelin-related glycoprotein, Bone marrow progenitor cell inhibitor-1 (MPIF-I), NAIP, Nanobody, NAP, NAP-2, NCA 90, NCAD, N-cadherin, NCAM, neprilysin, nerve cell adhesion molecule, neuroserpin, nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin-1, neuroturin, NGF-β, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural protein type 3 (NS3) derived from hepatitis C virus, NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM, OSM receptor, bone induction factor, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet growth factor, plgR, PLP, polyglycol chains of various sizes (e.g., PEG-20, PEG-30, PEG-40), PP14, prekallikreinPrion protein, procalcito, Renin, programmed cell death protein 1, proinsulin, prolactin, proprotein convertase PC9, prorelaxin, prostate-specific membrane antigen (PSMA), protein A, protein C, protein D, protein S, protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, rheumatoid factor, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF / KL, SCGF, sclerostin, SDF-1, SDF1α, SDF1β, SERINE, serum amyloid P, serum albumin, sFRP-3, Shh, Shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine-1-phosphate receptor 1, Staphylococcus lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor) Cerebral ulcer-associated glycoprotein (72), TARC, TB, TCA-3, T-cell receptor α / β, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, Pan-specific TGF-β, TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-βRl (ALK-5), TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thymic stromal lymphoproteinlymphoprotein) and activator Ck-1 activates thyroid hormone (TSH). Commercial Tie, TIMP, TIQ. Built-in bathrooms, bathrooms, snowflakes Liquids TMEFF2, Tmpo, TMPRSS2, TNF complex I, TNF complex II, TNF-α TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1 Apo-2 / DR4)、TNFRSF10B(TRAIL R2 DR5 / KILLER / TRICK-2A / TRICK-B)、TNFRSF10C(TRAIL R3 DcR1 / LIT / TRID)、TNFRSF10D(TRAIL R4 DcR2 / TRUNDD)、TNFRSF11A(RANK ODF R / TRANCE R)、TNFRSF11B(OPG OCIF / TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF12A、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHO ATAR / HveA / LIGHT R / TR2、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY CROWN / TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF Rl CD120a / p55-60, TNFRSF1B (TNF RII CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3). Apo-3 / LARD / TR-3 / TRAMP / WSL-1); Apo-1 / APT1 / CD95), TNFRSF6B(DcR3 M68 / TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9(4-1 BB CD137 / ILA), TNFRST23(DcTRAIL R1 TNFRH1), TNFSF10 (TRAILTNFSF11 (TRANCE / RANK ligand ODF / OPG ligand), TNFSF12 (TWEAK Apo-3 ligand / DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT HVEM ligand / LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand / TL6), TNFSF1A (TNF-α connectin / DIF / TNFSF2), TNFSF1B (TNF-β LTa / TNFSF1), TNFSF3 (LTb TNFC / p33), TNFSF4 (OX40 ligand gp34 / TXGP1), TNFSF5 (CD40 ligand CD154 / gp39 / HIGM1 / IMD3 / TRAP), TNFSF6 (Fas ligand Apo-1 ligand / APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1 BB ligand CD137 ligand), TNF-α, TNF-β, TNIL-I, toxic metabolites, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factor (TGF), e.g., TGF-α and TGF-β, transmembrane glycoprotein NMB, transthyretin, TRF, Trk, TROP-2, trophotrophic glycoprotein, TSG, TSLP, tumor necrosis factor (TNF), tumor-associated antigen CA125, Tumor-associated antigens expressing Lewis Y-related glucose, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VAP-1, Vascular endothelial growth factor (VEGF), Vaspin, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viral antigen, VitB12 receptor, Vitronectin receptor, VLA, VLA-1 This includes VLA-4, VNR integrin, von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2 / SCM-l-β, XCLl / lymphotactin, XCR1, XEDAR, XIAP, XPD, and glypican-3 (GPC3).

[0147] In the present invention, the third antigen-binding domain in the antigen-binding molecule of the present invention binds to a "third antigen" that is different from the "first antigen" and "second antigen" described above. In some embodiments, the third antigen is derived from a human, mouse, rat, monkey, rabbit, or dog. In some embodiments, the third antigen is a molecule that is specifically expressed on cells or organs derived from a human, mouse, rat, monkey, rabbit, or dog. Preferably, the third antigen is a molecule that is not systemically expressed on cells or organs. Preferably, the third antigen is, for example, a tumor cell-specific antigen, and also includes antigens that are expressed in conjunction with the malignant transformation of cells, and abnormal glycans that appear on the cell surface or on protein molecules during malignant transformation of cells. Specific examples include ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate-specific antigen (PSA), melanoma-related antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-related antigens (e.g., CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1, and LEA), and Burkitt lymphoma antigen 38.13, CD19, human B lymphoma antigens CD20, CD33, melanoma-specific antigens (e.g., ganglioside GD2, ganglioside GD3, ganglioside GM2, and ganglioside GM3), tumor-specific transplantation antigens (TSTA), T antigens, virus-induced tumor antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), colon CEA, oncoemetic antigens α-fetoprotein (e.g., oncoemetic trophoblast glycoprotein 5T4 and oncoemetic bladder tumor antigen), differentiation antigens (e.g., human Lung cancer antigens (L6 and L20), fibrosarcoma antigen, human T-cell leukemia-associated antigen Gp37, neoglycoprotein, sphingolipids, breast cancer antigen (e.g., EGFR (epidermal growth factor receptor)), NY-BR-16, NY-BR-16 and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen APO-1, differentiation antigens such as antigen I found in fetal erythrocytes, early endoderm I antigen found in adult erythrocytes, I(Ma) found in pre-transplant embryos or gastric cancer, and found in mammary epithelium. M18, M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5 found in bone marrow cells, D156-22, TRA-1-85 (blood type H) found in colorectal cancer, SCP-1 found in testicular and ovarian cancer, C14 found in colon cancer, F3 found in lung cancer, AH6, Y hapten found in gastric cancer, Ley, TL5 (blood type A) found in embryonic cancer cells, EGF receptor found in A431 cells, E1 series found in pancreatic cancer. Blood type B), FC10.2 found in embryonic cancer cells, gastric cancer antigen, CO-514 (blood type Lea) found in adenocarcinoma, NS-10 found in adenocarcinoma, CO-43 (blood type Leb), G49 found in the EGF receptor of A431 cells, MH2 (blood type ALeb / Ley) found in colon cancer, 19.9 found in colon cancer, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2, GD3, D1 found in embryonic cancer cells.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8, SSEA-3 and SSEA-4 found in 4-cell to 8-cell stage embryos, cutaneous T-cell lymphoma-associated antigen, MART-1 antigen, sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, paraneoplastic brain-testicular cancer antigen (tumor neuronal antigen MA2 and paraneoplastic neuronal antigen), neuro-oncological abdominal antigen 2 (NOVA2), hematological cell carcinoma antigen gene 520, tumor-associated antigen CO-029, tumor This includes the ulcer-associated antigens MAGE-C1 (cancer / testicular antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b, MAGE-X2, cancer-testicular antigen (NY-EOS-1), YKL-40, and any fragments of these polypeptides, as well as their modified structures (such as the modified phosphate groups and glycans mentioned above), EpCAM, EREG, CA19-9, CA15-3, sialyl SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A.

[0148] In one preferred embodiment, the third antigen is a molecule specifically expressed in cancer tissue, preferably glypican-3 (GPC3).

[0149] In one aspect, the antigen-binding molecule of the present invention has at least one feature selected from the group consisting of (1) to (4) below. (1) At least one of the first antigen-binding domain or the second antigen-binding domain binds to the extracellular domain of CD3ε (epsilon) containing the amino acid sequence of SEQ ID NO: 159. (2) The antigen-binding molecule of the present invention has agonist activity against CD137. (3) The antigen-binding molecule of the present invention induces T cell activation through binding to CD3 and exhibits cytotoxic activity against cells expressing a third antigen (e.g., a tumor antigen on cancer cells), but does not induce T cell activation via CD3 signaling or activation of immune cells expressing CD137 independently of the presence of cells expressing the third antigen (i.e., in the absence of cells expressing the third antigen), and (4) The antigen-binding molecule of the present invention does not induce cytokine release from PBMCs in the absence of cells expressing the third antigen molecule.

[0150] Where the terms “CD137 agonist antibody” or “antigen-binding molecule having agonist activity against CD137” are used in this application, they refer to an antibody or antigen-binding molecule that, when added to cells, tissues, or organisms expressing CD137, activates at least about 5%, specifically at least about 10%, or more specifically at least about 15% of CD137-expressing cells, where 0% activation is the background level of CD137-expressing inactive cells (e.g., IL-6 secretion). In various specific examples, the "CD137 agonist antibody" or "antigen-binding molecule having agonist activity against CD137" for use as a pharmaceutical composition in this application can activate cells by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, or 1000%.

[0151] Where the terms “CD137 agonist antibody” or “antigen-binding molecule having agonist activity against CD137” are used in this application, they also refer to an antibody or antigen-binding molecule that, when added to cells, tissues, or organisms expressing CD137, activates at least about 5%, more specifically at least about 10%, or more specifically at least about 15% of the cells expressing CD137, where 100% activation is the level of activation achieved by equimolar amounts of binding partners under physiological conditions. In various specific examples, the "CD137 agonist antibody" or "antigen-binding molecule having agonist activity against CD137" for use as a pharmaceutical composition in this application can activate cells by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, or 1000%.

[0152] In some embodiments, the term “binding partner” means a molecule known to bind to CD137 and induce activation of CD137-expressing cells. In further embodiments, examples of binding partners include urelumab (CAS registry number 934823-49-1) and its variants, as described in WO2005 / 035584A1, utomilumab (CAS registry number 1417318-27-4) and its variants, as described in WO2012 / 032433A1, and various known CD137 agonist antibodies. In certain embodiments, examples of binding partners include CD137 ligands. In further embodiments, activation of CD137-expressing cells by an anti-CD137 agonist antibody or “antigen-binding molecule having agonist activity against CD137” can be determined using an ELISA characterizing IL-6 secretion (see, for example, Reference Example 5-2 herein). The anti-CD137 antibody or "antigen-binding molecule having agonist activity against CD137" used as the binding partner, and the antibody concentration for measurement, may refer to Reference Example 5-2, where 100% activation is the level of activation achieved by the antibody or antigen-binding molecule. In a further embodiment, an antibody containing the heavy-chain amino acid sequence of SEQ ID NO: 142 and the light-chain amino acid sequence of SEQ ID NO: 144 may be used as the binding partner at 30 μg / mL for measurement (see, for example, Reference Example 5-2 herein).

[0153] In a non-limiting embodiment, the present invention provides a "CD137 agonist antibody" or an "antigen-binding molecule having agonist activity against CD137" comprising an Fc region, wherein the Fc region has enhanced binding activity to the inhibitory Fcγ receptor.

[0154] In a non-limiting manner, CD137 agonist activity can be confirmed using B cells that are known to express CD137 on their surface. In a non-limiting manner, the HDLM-2 B cell line can be used as the B cell line. Since IL-6 expression is induced as a result of CD137 activation, CD137 agonist activity can be evaluated by the amount of human interleukin-6 (IL-6) produced. In this evaluation, by using the amount of IL-6, it is possible to determine what percentage of CD137 agonist activity the molecule being evaluated possesses by evaluating the increase in IL-6 expression from inactivated B cells as a 0% background level.

[0155] In some embodiments, the antigen-binding molecules of the present invention induce T cell activation through binding to CD3 and exhibit cytotoxic activity against cells expressing a third antigen (e.g., a tumor antigen on cancer cells), but do not induce T cell activation or CD137-expressing immune cells independently of the presence of cells expressing the third antigen (i.e., in the absence of cells expressing the third antigen). Whether the antigen-binding molecules induce T cell activation through binding to CD3 and exhibit cytotoxic activity against cells expressing the third antigen can be determined, for example, by co-culturing cells expressing the third antigen with T cells in the presence of the antigen-binding molecules and assaying T cell activation via CD3 signaling. T cell activation can be assayed, for example, by using recombinant T cells that express a reporter gene (e.g., luciferase) in response to CD3 signaling, and by detecting the expression of the reporter gene or the activity of the reporter gene product as an indicator of T cell activation. When recombinant T cells expressing a reporter gene in response to CD3 signaling are co-cultured with cells expressing a third antigen in the presence of an antigen-binding molecule, the expression of the reporter gene or the detection of the activity of the reporter gene product in a dose-dependent manner of the antigen-binding molecule indicates that the antigen-binding molecule induces T cell activation against cells expressing the third antigen.

[0156] Similarly, whether an antigen-binding molecule induces T cell activation via CD3 signaling in cells expressing CD137, independently of the presence of cells expressing a third antigen (i.e., in the absence of cells expressing the third antigen molecule), can be determined, for example, by co-culturing CD137-expressing cells and T cells in the presence of the antigen-binding molecule and assaying T cell CD3 activation as described above. When recombinant T cells expressing a reporter gene in response to CD3 signaling are co-culturned with CD137-expressing cells in the presence of the antigen-binding molecule, if there is no expression of the reporter gene or activity of the reporter gene product, or if it is below the detection limit or below that of the negative control, then it is determined that the antigen-binding molecule does not induce T cell activation in cells expressing CD137. In one scenario, when recombinant T cells expressing a reporter gene in response to CD3 signaling are co-cultured with cells expressing CD137 in the presence of an antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is at most about 50%, 30%, 20%, 10%, 5%, or 1%, then the antigen-binding molecule is determined not to induce T cell activation in the CD137-expressing cells, where 100% activation is the level of activation achieved by an antigen-binding molecule that binds simultaneously to both CD3 and CD137. In one scenario, when recombinant T cells expressing a reporter gene in response to CD3 signaling are co-cultured with cells expressing CD137 in the presence of an antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is at most about 50%, 30%, 20%, 10%, 5%, or 1%, then the antigen-binding molecule is determined not to induce T cell activation in the CD137-expressing cells, where 100% activation is the level of activation achieved by the same antigen-binding molecule in cells expressing a third antigen molecule.

[0157] In some embodiments, the antigen-binding molecule of the present invention does not induce cytokine release from PBMCs in the absence of cells expressing the third antigen molecule. Whether the antigen-binding molecule does not induce cytokine release in the absence of cells expressing the third antigen can be determined, for example, by incubating PBMCs and the antigen-binding molecule in the absence of cells expressing the third antigen, and by measuring cytokines such as IL-2, IFNγ, and TNFα released from PBMCs into the culture supernatant using methods known in the art. If no significant levels of cytokines are detected or significant cytokine expression is not induced in the culture supernatant of PBMCs incubated with the antigen-binding molecule in the absence of cells expressing the third antigen, then it is determined that the antigen-binding molecule does not induce cytokine release from PBMCs in the absence of cells expressing the third antigen.

[0158] In one scenario, "no significant cytokine levels detected" also means that the cytokine concentration level is at most approximately 50%, 30%, 20%, 10%, 5%, or 1%, where 100% is the cytokine concentration achieved by antigen-binding molecules simultaneously binding to the first antigen (CD3) and the second antigen (CD137). In one scenario, "no significant cytokine levels detected" also means that the cytokine concentration level is at most approximately 50%, 30%, 20%, 10%, 5%, or 1%, where 100% is the cytokine concentration achieved in the presence of cells expressing a molecule of the third antigen. In one scenario, "no significant induction of cytokine expression" also means that the level of cytokine concentration increase is at most 5 times, 2 times, or 1 time the concentration of each cytokine before the addition of the antigen-binding molecule.

[0159] In some embodiments, with respect to binding to CD137, the antigen-binding molecules of the present invention compete for binding to CD137 with antibodies selected from the group consisting of: (a) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 104 and a VL region having the amino acid sequence of SEQ ID NO: 124, (b) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 119 and a VL region having the amino acid sequence of SEQ ID NO: 126, (c) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 114 and a VL region having the amino acid sequence of SEQ ID NO: 129, (d) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 104 and a VL region having the amino acid sequence of SEQ ID NO: 131, (e) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 114 and a VL region having the amino acid sequence of SEQ ID NO: 134, (f) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 1 and a VL region having the amino acid sequence of SEQ ID NO: 45, (g) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 2 and a VL region having the amino acid sequence of SEQ ID NO: 46, (h) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 3 and a VL region having the amino acid sequence of SEQ ID NO: 45, (i) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 4 and a VL region having the amino acid sequence of SEQ ID NO: 45, (j) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 5 and a VL region having the amino acid sequence of SEQ ID NO: 45, (k) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 6 and a VL region having the amino acid sequence of SEQ ID NO: 45, (l) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 7 and a VL region having the amino acid sequence of SEQ ID NO: 45, (m)An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 8 and a VL region having the amino acid sequence of SEQ ID NO: 45, (n) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 9 and a VL region having the amino acid sequence of SEQ ID NO: 45, (o) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 10 and a VL region having the amino acid sequence of SEQ ID NO: 46. (p) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 11 and a VL region having the amino acid sequence of SEQ ID NO: 48, and (q) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 61 and a VL region having the amino acid sequence of SEQ ID NO: 48.

[0160] In some embodiments, with respect to binding to CD137, the antigen-binding molecule of the present invention binds to the same CD137 molecule epitope as an antibody selected from the group consisting of: (a) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 104 and a VL region having the amino acid sequence of SEQ ID NO: 124, (b) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 119 and a VL region having the amino acid sequence of SEQ ID NO: 126, (c) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 114 and a VL region having the amino acid sequence of SEQ ID NO: 129, (d) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 104 and a VL region having the amino acid sequence of SEQ ID NO: 131, (e) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 114 and a VL region having the amino acid sequence of SEQ ID NO: 134, (f) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 1 and a VL region having the amino acid sequence of SEQ ID NO: 45, (g) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 2 and a VL region having the amino acid sequence of SEQ ID NO: 46, (h) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 3 and a VL region having the amino acid sequence of SEQ ID NO: 45, (i) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 4 and a VL region having the amino acid sequence of SEQ ID NO: 45, (j) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 5 and a VL region having the amino acid sequence of SEQ ID NO: 45, (k) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 6 and a VL region having the amino acid sequence of SEQ ID NO: 45, (l) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 7 and a VL region having the amino acid sequence of SEQ ID NO: 45, (m)An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 8 and a VL region having the amino acid sequence of SEQ ID NO: 45, (n) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 9 and a VL region having the amino acid sequence of SEQ ID NO: 45, (o) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 10 and a VL region having the amino acid sequence of SEQ ID NO: 46. (p) An antibody comprising a VH region having the amino acid sequence of SEQ ID NO: 11 and a VL region having the amino acid sequence of SEQ ID NO: 48, and (q) An antibody containing a VH region having the amino acid sequence of SEQ ID NO: 61 and a VL region having the amino acid sequence of SEQ ID NO: 48.

[0161] In some embodiments, with respect to binding to CD137, the antigen-binding molecules of the present invention may have activity equivalent to any one of (a) to (q) above. In this specification, “equivalent activity” means CD137 agonist activity that is 70% or more, preferably 80% or more, and more preferably 90% or more, of the binding activity of any one of (a) to (q) above.

[0162] Whether the test antigen-binding molecule of the present invention shares a common epitope with a particular antibody, as described above, can be evaluated based on the competition between the two for the same epitope. Competition between the two can be detected by a cross-blocking assay, for example. A competitive ELISA assay is a preferred cross-blocking assay. Specifically, in a cross-blocking assay, the CD137 protein used to coat the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antibody, and then the antigen-binding molecule of the present invention is added thereto. The amount of the antigen-binding molecule of the present invention bound to the CD137 protein in the wells is indirectly correlated with the binding ability of the candidate competing antibody (test antibody) that competes for binding to the same epitope. That is, the higher the affinity of the test antibody for the same epitope, the less the antigen-binding molecule of the present invention bound to the CD137 protein-coated wells, and the greater the amount of test antibody bound to the CD137 protein-coated wells.

[0163] The amount of antigen-binding molecules of the present invention bound to the wells can be easily determined by pre-labeling the antigen-binding molecules. For example, biotin-labeled antigen-binding molecules can be measured using an avidin / peroxidase conjugate and a suitable substrate. In particular, cross-blocking assays using enzymatic labeling such as peroxidase are called "competitive ELISA assays." The antigen-binding molecules of the present invention can be labeled with other labeling substances that enable detection or measurement. Specifically, radiolabeling, fluorescent labeling, and the like are known.

[0164] Furthermore, if the test antibody has a constant region derived from a different species than the antigen-binding molecule of the present invention, the amount of the antigen-binding molecule of the present invention bound to the well can be measured by using a labeled antibody that recognizes the constant region of the antigen-binding molecule. Alternatively, if the test antibody and the antigen-binding molecule of the present invention belong to different classes derived from the same species, the amounts of the two bound to the well can be measured using an antibody that distinguishes between the individual classes.

[0165] If the candidate antigen-binding molecule of the present invention can block the binding of the anti-CD137 antibody by at least 20%, preferably at least 20% to 50%, and more preferably at least 50%, compared to the binding activity obtained in a control experiment performed in the absence of the candidate competing antigen-binding molecule of the present invention, then the candidate competing antigen-binding molecule of the present invention is either an antigen-binding molecule that substantially binds to the same epitope as the anti-CD137 antibody, or an antigen-binding molecule that competes for binding to the same epitope.

[0166] In another embodiment, the ability of a test antibody or antigen-binding molecule to bind competitively or cross-competitively with another antibody or antigen-binding molecule can be appropriately determined by those skilled in the art using standard binding assays known in the art, such as BIAcore analysis or flow cytometry.

[0167] Methods for determining the spatial structure of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance (see Epitope Mapping Protocols in Methods in Molecular Biology, GE Morris (ed.), Vol. 66 (1996)).

[0168] Whether a test antibody or antigen-binding molecule shares a common epitope with the CD137 ligand can also be evaluated based on competition between the test antibody or antigen-binding molecule and the CD137 ligand for the same epitope. Competition between the antibody or antigen-binding molecule and the CD137 ligand can be detected by cross-blocking assays, etc., as described above. In another embodiment, the ability of a test antibody or antigen-binding molecule to bind competitively or cross-competitively to the CD137 ligand can be appropriately determined by those skilled in the art using standard binding assays known in the art, such as BIAcore analysis or flow cytometry.

[0169] In some embodiments, with respect to binding to CD137, favorable examples of antigen-binding molecules of the present invention include antigen-binding molecules that bind to the same epitope as the human CD137 epitope to which an antibody selected from the group consisting of the following binds: In the human CD137 protein An antibody that recognizes a region containing the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (Sequence ID: 154). An antibody that recognizes a region containing the DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC sequence (SEQ ID NO: 149). An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (Sequence ID: 152), and An antibody that recognizes the region containing the LQDPCSNCPAGTFCDNNRNQIC sequence (Sequence ID: 147).

[0170] Depending on the target cancer antigen, a person skilled in the art can appropriately select heavy chain variable region sequences and light chain variable region sequences that bind to the cancer antigen from the cancer-specific antigen-binding domain. If the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope.

[0171] An "epitope" refers to an antigenic determinant in an antigen, and specifically to the antigenic site to which various binding domains in the antigen-binding molecules disclosed herein bind. Therefore, for example, an epitope can be defined according to its structure. Alternatively, an epitope may be defined according to the antigen-binding activity of the antigen-binding molecule that recognizes the epitope. If the antigen is a peptide or polypeptide, the epitope can be identified by the amino acid residues that form the epitope. Alternatively, if the epitope is a glycan, the epitope can be identified by its specific glycan structure.

[0172] A linear epitope is an epitope whose primary amino acid sequence contains other recognizable epitopes. Such linear epitopes typically contain at least three, and most commonly at least five, amino acids in their specific sequence, for example, about 8-10 or 6-20 amino acids.

[0173] In contrast to linear epitopes, "structural epitopes" are epitopes in which the primary amino acid sequence containing the epitope is not the sole determinant of the recognized epitope (for example, the primary amino acid sequence of a structural epitope is not necessarily recognized by the antibody that defines the epitope). Structural epitopes may contain a greater number of amino acids compared to linear epitopes. Antibodies or antigen-binding molecules that recognize structural epitopes recognize the three-dimensional structure of the peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, the amino acids and / or polypeptide backbone that form the structural epitope align, making the epitope recognizable by the antibody. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-specific spin labeling, and electron paramagnetic resonance spectroscopy. For example, see Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0174] An example of a method for evaluating the binding of epitopes in cancer-specific antigens by a test antigen-binding molecule is shown below. Methods for evaluating the binding of epitopes in target antigens by other binding domains can also be appropriately implemented according to the following examples.

[0175] For example, whether a test antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen recognizes a linear epitope in the antigen molecule can be confirmed, for example, as described below. For example, a linear peptide containing an amino acid sequence that forms the extracellular domain of a cancer-specific antigen is synthesized for the above purpose. The peptide can be chemically synthesized or obtained by genetic engineering techniques using a region in the cDNA of a cancer-specific antigen that encodes the amino acid sequence corresponding to the extracellular domain. Next, the test antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen is evaluated for its binding activity to the linear peptide containing the amino acid sequence that constitutes the extracellular domain. For example, the binding activity of the antigen-binding molecule to the peptide can be evaluated by ELISA using an immobilized linear peptide as the antigen. Alternatively, the binding activity to the linear peptide can be evaluated based on the level to which the linear peptide inhibits the binding of the antigen-binding molecule to cancer-specific antigen-expressing cells. The binding activity of the antigen-binding molecule to the linear peptide can be demonstrated by these tests.

[0176] Whether a test antigen molecule containing an antigen-binding domain for the aforementioned antigen recognizes a structural epitope can be confirmed as follows. For example, an antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen strongly binds to cancer-specific antigen-expressing cells upon contact, but does not substantially bind to an immobilized linear peptide containing an amino acid sequence that forms the extracellular domain of the cancer-specific antigen. In this specification, "substantially not binding" means that the binding activity is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less, compared to the binding activity to antigen-expressing cells in ELISA or fluorescence-activated cell sorting (FACS) using antigen-expressing cells as the antigen.

[0177] In ELISA, the binding activity of a test antigen-binding molecule containing an antigen-binding domain to antigen-expressing cells can be quantitatively evaluated by comparing the signal levels produced by the enzymatic reaction. Specifically, the test antigen-binding molecule is added to an ELISA plate immobilized with antigen-expressing cells. Then, the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, when using FACS, a dilution series of the test antigen-binding molecule can be prepared, and the antigen-binding titer to antigen-expressing cells can be determined to compare the binding activity of the test antigen-binding molecule to antigen-expressing cells.

[0178] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices: FACSCanto(TM) II FACSAria(trademark) FACSArray (trademark) FACSVantage(TM) SE FACSCalibur (trademark) (all are product names of BD Biosciences) EPICS ALTRA HyPerSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (all are Beckman Coulter product names).

[0179] Suitable methods for assaying the binding activity of a test antigen-binding molecule containing an antigen-binding domain to the aforementioned antigen include, for example, the following: First, antigen-expressing cells are reacted with the test antigen-binding molecule, then stained with a FITC-labeled secondary antibody, and FACSCalibur (BD) is used. The fluorescence intensity, i.e., the geometric mean, obtained by analysis using CELL QUEST Software (BD) reflects the amount of antibody bound to the cells. That is, the binding activity of the test antigen-binding molecule, expressed by the amount of bound test antigen-binding molecule, can be measured by determining the geometric mean.

[0180] Whether a test antigen-binding molecule containing the antigen-binding domain of the present invention shares a common epitope with another antigen-binding molecule can be evaluated based on competition between the two molecules for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. A competitive ELISA assay is a preferred cross-blocking assay.

[0181] Specifically, in a cross-blocking assay, the antigen coating the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antigen-binding molecule, and then the test antigen-binding molecule is added. The amount of test antigen-binding molecule bound to the antigen in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule that competes for binding to the same epitope. That is, the higher the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to the antigen-coated well.

[0182] The amount of test antigen-binding molecules bound to the wells via the antigen can be easily determined by pre-labeling the antigen-binding molecules. For example, biotin-labeled antigen-binding molecules can be measured using an avidin / peroxidase conjugate and a suitable substrate. In particular, cross-blocking assays using enzymatic labeling such as peroxidase are called "competitive ELISA assays." Antigen-binding molecules can also be labeled with other labeling substances that enable detection or measurement. Specifically, radiolabeling and fluorescent labeling are well known. If a candidate competing antigen-binding molecule can block the binding of a test antigen-binding molecule containing an antigen-binding domain by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity in a control experiment performed in the absence of the competing antigen-binding molecule, then the test antigen-binding molecule is determined to substantially bind to the same epitope to which the competing antigen-binding molecule binds, or to compete for binding to the same epitope.

[0183] If the structure of the epitope to which the test antigen-binding molecule containing the antigen-binding domain of the present invention binds has already been identified, whether the test antigen-binding molecule and the control antigen-binding molecule share a common epitope can be evaluated by comparing the binding activity of the two antigen-binding molecules to a peptide prepared by introducing amino acid mutations into the peptide that forms the epitope.

[0184] As a method for measuring such binding activity, for example, the binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide into which a mutation has been introduced can be measured by comparison in the ELISA format described above. In addition to the ELISA method, the binding activity to the mutant peptide bound to the column can also be determined by passing the test antigen-binding molecule and the control antigen-binding molecule through a column, and then quantifying the eluted antigen-binding molecule in the eluate. For example, a method for adsorbing the mutant peptide onto a column in the form of a GST fusion peptide is known.

[0185] Alternatively, if the identified epitope is a structural epitope, whether the test antigen-binding molecule and the control antigen-binding molecule share a common epitope can be evaluated by the following method. First, prepare cells expressing the antigen targeted by the antigen-binding domain and cells expressing the antigen with the mutated epitope. Add the test antigen-binding molecule and the control antigen-binding molecule to the cell suspension prepared by suspending these cells in a suitable buffer such as PBS. Next, wash the cell suspension with buffer as appropriate and add FITC-labeled antibodies that can recognize the test antigen-binding molecule and the control antigen-binding molecule. Determine the fluorescence intensity and number of cells stained with the labeled antibody using FACSCalibur (BD). The test antigen-binding molecule and the control antigen-binding molecule are diluted as appropriate with a suitable buffer and used at the desired concentration. For example, they may be used at concentrations in the range of 10 μg / ml to 10 ng / ml. The fluorescence intensity, i.e., the geometric mean, determined by analysis using CELL QUEST Software (BD) reflects the amount of labeled antibody bound to the cells. In other words, the binding activity of test antigen-binding molecules and control antigen-binding molecules, which is expressed by the amount of bound labeled antibody, can be measured by determining the geometric mean.

[0186] In some embodiments, the antigen-binding molecule of the present invention comprises an amino acid sequence obtained by introducing one or more amino acid modifications into a template sequence consisting of the heavy chain variable region sequence described in SEQ ID NO: 160 and / or the light chain variable region sequence described in SEQ ID NO: 161, wherein the one or more amino acids to be modified are located at the following positions: H chains: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, and 100g (Kabat numbering); and L chains: 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52, 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, and 96 (Kabat numbering) Selected from, The modified heavy chain variable region sequence of HVR-H3 is The amino acids at position 98 are Ala, Pro, Ser, Arg, His, or Thr; Ala, Ser, Thr, Gln, His, or Leu at amino acid position 99; The amino acid at position 100 is Tyr, Ala, Ser, Pro, or Phe; Tyr, Val, Ser, Leu, or Gly at amino acid position 100a; Asp, Ser, Thr, Leu, Gly, or Tyr at the 100b position of the amino acid; The amino acids Val, Leu, Phe, Gly, His, or Ala at position 100c; Leu, Phe, Ile, or Tyr at the 100d position of the amino acid; Gly, Pro, Tyr, Gln, Ser, or Phe at the 100e position of amino acids; Tyr, Ala, Gly, Ser, or Lys at the 100f position of the amino acid; Approximately 100g of amino acids (Kabat numbering) Gly, Tyr, Phe, or Val It contains at least one amino acid selected from the following.

[0187] In some embodiments, the antigen-binding molecule of the present invention comprises (a) a VH region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 115, 104, 119, or 114; (b) a VL region containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 124-130; or (c) a VH region containing the amino acid sequence of (a) and a VL region containing the amino acid sequence of (b).

[0188] The antigen-binding molecules of the present invention can be prepared by methods generally known to those skilled in the art. For example, the antigen-binding molecules of the present invention can be prepared according to, or by reference to, the antibody preparation methods described below, but the methods for preparing the antigen-binding molecules of the present invention are not limited thereto. Many combinations of host cells and expression vectors are known in the art for antibody preparation by transferring isolated genes encoding polypeptides into suitable hosts. All of these expression systems can be applied to the isolation of the antigen-binding molecules of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, examples of animal cells may include the following cells: (1) Mammalian cells, e.g., CHO (Chinese hamster ovary cell line), COS (monkey kidney cell line), myeloma cells (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), HEK293 (human fetal kidney cell line with sheared adenovirus (Ad)5 DNA), PER.C6 cells (human fetal retinal cell line transformed with adenovirus type 5 (Ad5) E1A and E1B genes), HeLa, and Vero (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1)); (2) Amphibian cells, such as the oocytes of the African clawed frog (Xenopus); and (3) Insect cells, e.g., sf9, sf21, and Tn5. The antigen-binding molecules of the present invention can also be prepared using Escherichia coli (mAbs 2012 Mar-Apr; 4(2): 217-225) or yeast (WO2000023579). Antibodies and antigen-binding molecules prepared using Escherichia coli are not sugar-chained. On the other hand, antibodies and antigen-binding molecules prepared using yeast are sugar-chained.

[0189] DNA encoding an antibody heavy chain and DNA encoding an antibody light chain are expressed, wherein one or more amino acid residues in the variable domain are substituted with different amino acids of interest. DNA encoding a heavy chain or light chain in which one or more amino acid residues in the variable domain are substituted with different amino acids of interest can be obtained, for example, by obtaining DNA encoding an antibody variable domain prepared by a method known in the art for a particular antigen, and by introducing substitutions as appropriate so that the codon encoding a specific amino acid in the domain encodes a different amino acid of interest.

[0190] Alternatively, DNA encoding a protein in which one or more amino acid residues in an antibody variable domain prepared by a method known in the art for a specific antigen are substituted with different amino acids of interest may be pre-designed and chemically synthesized to obtain DNA encoding a heavy chain in which one or more amino acid residues in the variable domain are substituted with different amino acids of interest. The amino acid substitution sites and types of substitutions are not particularly limited. Examples of regions preferred for amino acid modification include solvent-exposed regions and loops in the variable domain. Among these, CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, Kabat numbering positions 31-35, 50-65, 71-74, and 95-102 in the H chain variable domain, and Kabat numbering positions 24-34, 50-56, and 89-97 in the L chain variable domain are preferred. Kabat numbering positions 31, 52a-61, 71-74, and 97-101 in the H chain variable domain, and Kabat numbering positions 24-34, 51-56, and 89-96 in the L chain variable domain are more preferred. Amino acid modification is not limited to substitution, but may also include deletion, addition, insertion, modification, or a combination thereof.

[0191] DNA encoding a heavy chain in which one or more amino acid residues in the variable domain are substituted with different amino acids of interest can also be prepared as separate partial DNAs. Examples of partial DNA combinations include, but are not limited to, DNA encoding a variable domain and DNA encoding a constant domain; and DNA encoding a Fab domain and DNA encoding an Fc domain. Similarly, DNA encoding a light chain can also be prepared as separate partial DNAs.

[0192] These DNAs can be expressed by the following methods: for example, a heavy chain expression vector is constructed by incorporating the DNA encoding the heavy chain variable region together with the DNA encoding the heavy chain constant region into an expression vector. Similarly, a light chain expression vector is constructed by incorporating the DNA encoding the light chain variable region together with the DNA encoding the light chain constant region into an expression vector. These heavy and light chain genes may also be incorporated into a single vector.

[0193] The DNA encoding the target antibody is incorporated into an expression vector so that it is expressed under the control of expression regulatory regions, such as enhancers and promoters. Next, host cells are transformed with the resulting expression vector to express the antibody. In this case, a suitable host and expression vector can be used in combination.

[0194] Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. In addition to these vectors, for example, pGEM-T, pDIRECT, or pT7 can also be used for the purpose of cDNA subcloning and excision.

[0195] In particular, expression vectors are useful for using vectors to produce antibodies of the present invention. For example, when the host is Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue, it is essential that the expression vector has a promoter that enables efficient expression in Escherichia coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; and FASEB J. (1992) 6, 2422-2427, which is incorporated herein by reference in its entirety), the araB promoter (Better et al., Science (1988) 240, 1041-1043, which is incorporated herein by reference in its entirety), or the T7 promoter. Examples of such vectors include the vectors mentioned above, as well as pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen NV), pEGFP, and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase).

[0196] The vector may contain a signal sequence for polypeptide secretion. In the case of production in the periplasm of E. coli, the pelB signal sequence (the entire sequence of which is incorporated herein by reference Lei, SP et al., J. Bacteriol. (1987) 169, 4397) can be used as the signal sequence for polypeptide secretion. The vector can be transferred into host cells, for example, by the lipofectin method, the calcium phosphate method, or the DEAE-dextran method.

[0197] In addition to expression vectors for E. coli, examples of vectors for producing the antigen-binding molecules of the present invention include mammalian expression vectors (e.g., pcDNA3 (Invitrogen Corp.), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, which is incorporated herein by reference in its entirety), pEF, and pCDM8), insect cell expression vectors (e.g., "Bac-to-BAC Baculovirus Expression System" (GIBCO BRL), and pBacPAK8), plant expression vectors (e.g., pMH1 and pMH2), animal virus expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus expression vectors (e.g., pZIPneo), yeast expression vectors (e.g., "Pichia Expression Kit" (Invitrogen Corp.), pNV11, and SP-Q01), and Bacillus subtilis. This includes expression vectors derived from subtilis (e.g., pPL608 and pKTH50).

[0198] For expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, or HEK293 cells, the vector must have a promoter necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, which is incorporated herein by reference in its entirety), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, which is incorporated herein by reference in its entirety), the CAG promoter (Gene. (1991) 108, 193, which is incorporated herein by reference in its entirety), or the CMV promoter. More preferably, it must have a gene for screening transformed cells (e.g., a drug resistance gene that can act as a marker by a drug (such as neomycin, G418, etc.)). Examples of vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13. In addition, the EBNA1 protein may be co-expressed to increase the gene copy number. In this case, a vector containing the origin of replication OriP is used (Biotechnol Bioeng. 2001 Oct 20;75(2):197-203; and Biotechnol Bioeng. 2005 Sep 20;91(6):670-7).

[0199] Exemplary methods intended to stably express a gene and increase the gene copy number within a cell include transforming CHO cells lacking the nucleic acid synthesis pathway with a vector containing the DHFR gene that acts as a complement (e.g., pCHOI), and using methotrexate (MTX) in gene amplification. Exemplary methods intended to transiently express a gene include transforming COS cells, which have the SV40 T antigen gene on their chromosome, with a vector containing an origin of replication for SV40 (e.g., pcD). Origins of replication derived from polyomaviruses, adenoviruses, bovine papillomavirus (BPV), etc., can also be used. To increase the gene copy number in a host cell line, expression vectors may contain select markers such as aminoglycoside phosphotransferase (APH) genes, thymidine kinase (TK) genes, Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) genes, or dihydrofolate reductase (dhfr) genes.

[0200] The antigen-binding molecules of the present invention can be recovered, for example, by culturing transformed cells, and then separating antibodies from the molecularly transformed cells or the culture medium. The antigen-binding molecules of the present invention can be separated and purified by using a combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, C1q, FcRn, protein A and protein G columns, affinity chromatography, ion exchange chromatography, and gel filtration chromatography, as appropriate.

[0201] The aforementioned technologies, such as knob-into-hole technology (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; and Merchant AM et al., Nature Biotechnology (1998) 16, 677-681) or techniques that suppress unintended association between H chains by introducing charge repulsion (WO2006 / 106905), can be applied to methods for efficiently preparing multispecific antigen-binding molecules.

[0202] The inventors have also succeeded in developing a more efficient method for obtaining antigen-binding domains that bind to two or more different antigens. In some embodiments, a method for screening antigen-binding domains that bind to at least two or more different antigens for the purposes of the present invention is: (a) A step of providing a library containing multiple antigen-binding domains, (b) A step of contacting the library provided in step (a) with the target first antigen and collecting the antigen-binding domain bound to the first antigen. (c) A step of contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting the antigen-binding domains bound to the second antigen, and (d) A step of amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains. The method includes, and does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b). In the above method, the number of steps in which the antigen-binding domain is brought into contact with the antigen is not particularly limited. In some embodiments, the screening method of the present invention may include three or more contact steps if there are two or more target antigens. In further embodiments, the screening method of the present invention may include two or more steps of bringing the antigen-binding domain into contact with one or more of the target antigens. In this case, the antigen-binding domain may be brought into contact with each antigen in any order. For example, the antigen-binding domain may be brought into contact with each antigen two or more times consecutively, or it may be brought into contact with one antigen once or more times first, and then into contact with other antigens before bringing the same antigen into contact again. Even if the screening method of the present invention includes three or more steps of bringing the antigen-binding domain into contact with the antigen, the method does not include a step of amplifying the nucleic acid encoding the collected antigen-binding domain between any two consecutive contact steps.

[0203] In some embodiments, the antigen-binding domain of the present invention is a fusion polypeptide formed by fusing the antigen-binding domain with a scaffold and crosslinking the antigen-binding domain with the nucleic acid encoding the antigen-binding domain.

[0204] In some embodiments, the scaffold of the present invention is a bacteriophage. In some embodiments, the scaffold of the present invention is a ribosome, a RepA protein, or a DNA puromycin linker.

[0205] In some embodiments, elution is carried out in steps (b) and (c) above using an elution solution which is an acid solution, a base solution, DTT, or IdeS. In some embodiments, the elution solution used in steps (b) and (c) above of the present invention is EDTA or IdeS.

[0206] In some embodiments, a method for screening antigen-binding domains that bind to at least two or more different antigens for the purposes of the present invention is: (a) A step of providing a library containing multiple antigen-binding domains, (b) A step of contacting the library provided in step (a) with the target first antigen and collecting the antigen-binding domain bound to the first antigen. (b)' A step of translating the nucleic acid encoding the antigen-binding domain collected in step (b), (c) A step of contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting the antigen-binding domains bound to the second antigen, and (d) A step of amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains. The method includes, and does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b).

[0207] In some embodiments, a method for producing antigen-binding domains that bind to at least two or more different antigens for the purposes of the present invention is: (a) A step of providing a library containing multiple antigen-binding domains, (b) A step of contacting the library provided in step (a) with the target first antigen and collecting the antigen-binding domain bound to the first antigen. (c) A step of contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting the antigen-binding domains bound to the second antigen, and (d) A step of amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains. (e) A step of linking a polynucleotide encoding a candidate antigen-binding domain selected in step (d) with a polynucleotide encoding a polypeptide containing an Fc region. (f) A step of culturing cells into which a vector functionally linked to the polynucleotides obtained in step (d) above has been introduced, and (g) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (f) above. The method includes, and does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b).

[0208] In one embodiment, each antigen-binding domain in a library of antigen-binding domains has at least one amino acid modification in either or both of the heavy chain and light chain variable regions that bind to a first antigen (e.g., CD3 or CD137) or a second antigen (e.g., CD137 if the first antigen is CD3; or CD3 if the first antigen is CD137), where each antigen-binding domain in the library is different from the others in at least one amino acid that is thus modified from the others.

[0209] In the present invention, one amino acid modification may be used alone, or multiple amino acid modifications may be used in combination. When multiple amino acid modifications are used in combination, the number of modifications combined is not particularly limited, for example, 2 to 30, preferably 2 to 25, 2 to 22, 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 2 to 3. The combined amino acid modifications may be applied only to the heavy chain variable domain or the light chain variable domain of the antibody, or they may be appropriately distributed to both the heavy chain variable domain and the light chain variable domain.

[0210] As already mentioned above, examples of regions preferred for amino acid modification include solvent-exposed regions and loops within the variable region. Among these, CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, Kabat numbering positions 31-35, 50-65, 71-74, and 95-102 in the H chain variable region, and Kabat numbering positions 24-34, 50-56, and 89-97 in the L chain variable region are preferred. More preferred are Kabat numbering positions 31, 52a-61, 71-74, and 97-101 in the H chain variable region, and Kabat numbering positions 24-34, 51-56, and 89-96 in the L chain variable region.

[0211] Modification of amino acid residues also includes random modification of amino acids in the aforementioned region within the antibody variable region that binds to a first antigen (e.g., CD3 or CD137) or a second antigen (e.g., CD137 if the first antigen is CD3; or CD3 if the first antigen is CD137); and insertion of peptides previously known to have binding activity to a first antigen (e.g., CD3 or CD137) or a second antigen (e.g., CD137 if the first antigen is CD3; or CD3 if the first antigen is CD137) into the aforementioned region. The antigen-binding molecule of the present invention can be obtained by selecting from such modified antigen-binding molecules a variable region that can bind to a first antigen (e.g., CD3 or CD137) and a second antigen (e.g., CD137 if the first antigen is CD3; or CD3 if the first antigen is CD137), but cannot bind to these antigens simultaneously.

[0212] Whether the variable region can bind to a first antigen (e.g., CD3 or CD137) and a second antigen (e.g., CD137 if the first antigen is CD3; or CD3 if the first antigen is CD137), but cannot bind to these antigens simultaneously, and furthermore, whether the variable region can bind to both the first antigen (e.g., CD3 or CD137) and the second antigen (e.g., CD137 if the first antigen is CD3; or CD3 if the first antigen is CD137), when one of the antigens is present on a cell and the other antigen is present alone, when both antigens are present individually, or when both antigens are present on the same cell, but cannot bind to these antigens simultaneously when they are expressed on different cells, can also be confirmed according to the method described above.

[0213] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a first antigen-binding domain, which may optionally further include heavy chain constant regions (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a first antigen-binding domain, which may optionally further include a light chain constant (CL) region; (iii) a nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a second antigen-binding domain, which may optionally further include a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); and (iv) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a second antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; and (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes.

[0214] In some embodiments, the antigen-binding molecule thus produced comprises a first antigen-binding domain and a second antigen-binding domain linked to each other via at least one bond. The at least one bond for linking the first antigen-binding domain and the second antigen-binding domain is introduced into one or more of the following: (i) Between the constant CH1 region of the antibody heavy chain of the first antigen-binding domain and the constant CH1 region of the antibody heavy chain of the second antigen-binding domain; (ii) Between the hinge region of the antibody heavy chain of the first antigen-binding domain and the hinge region of the antibody heavy chain of the second antigen-binding domain; (iii) Between the light chain constant (CL) region of the first antigen-binding domain and the light chain constant (CL) region of the second antigen-binding domain; (iv) Between the CH1 region of the antibody heavy chain constant state of the first antigen-binding domain and the light chain constant state (CL) region of the second antigen-binding domain; (v) Between the light chain constant (CL) region of the first antigen-binding domain and the CH1 region of the antibody heavy chain constant of the second antigen-binding domain; and / or (vi) Between the heavy chain variable (VH) region of the first antigen-binding domain and the heavy chain variable (VH) region of the second antigen-binding domain. In some embodiments, the linkage between the first antigen-binding domain and the second antigen-binding domain can be created, for example, by introducing at least one amino acid modification (e.g., substitution with cysteine ​​or lysine) into each of the polypeptides (i) to (vi) above.

[0215] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a third antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1), and a heavy chain variable (VH) region of a first antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a third antigen-binding domain, which may optionally further include a light chain constant (CL) region; (iii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a first antigen-binding domain, which may optionally further include a light chain constant (CL) region; (iv) nucleic acids encoding a polypeptide, comprising a heavy chain variable (VH) region of a second antigen-binding domain, which may optionally further include heavy chain constant regions (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); and (v) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a second antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; and (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes.

[0216] In some embodiments, the antigen-binding molecule thus constructed comprises a first antigen-binding domain and a second antigen-binding domain linked to each other via at least one binding. The at least one binding for linking the first antigen-binding domain and the second antigen-binding domain is introduced into one or more of the following: (i) Between the constant CH1 region of the antibody heavy chain of the first antigen-binding domain and the constant CH1 region of the antibody heavy chain of the second antigen-binding domain; (ii) Between the hinge region of the antibody heavy chain of the first antigen-binding domain and the hinge region of the antibody heavy chain of the second antigen-binding domain; (iii) Between the light chain constant (CL) region of the first antigen-binding domain and the light chain constant (CL) region of the second antigen-binding domain; (iv) Between the CH1 region of the antibody heavy chain constant state of the first antigen-binding domain and the light chain constant state (CL) region of the second antigen-binding domain; (v) Between the light chain constant (CL) region of the first antigen-binding domain and the CH1 region of the antibody heavy chain constant of the second antigen-binding domain; and / or (vi) Between the heavy chain variable (VH) region of the first antigen-binding domain and the heavy chain variable (VH) region of the second antigen-binding domain. In some embodiments, the linkage between the first antigen-binding domain and the second antigen-binding domain can be created, for example, by introducing at least one amino acid modification (e.g., substitution with cysteine ​​or lysine) into each of the polypeptides (i) to (vi) above.

[0217] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a third antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1), and a heavy chain variable (VH) region of a second antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a third antigen-binding domain, which may optionally further include a light chain constant (CL) region; (iii) nucleic acids encoding polypeptides, comprising a light chain variable (VL) region of a second antigen-binding domain, which may optionally further include a light chain constant (CL) region; and (iv) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a first antigen-binding domain, which may optionally further include heavy chain constant regions (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (v) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a first antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; and (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes.

[0218] In some embodiments, the antigen-binding molecule thus produced comprises a first antigen-binding domain and a second antigen-binding domain linked to each other via at least one bond. The at least one bond for linking the first antigen-binding domain and the second antigen-binding domain is introduced into one or more of the following: (i) Between the constant CH1 region of the antibody heavy chain of the first antigen-binding domain and the constant CH1 region of the antibody heavy chain of the second antigen-binding domain; (ii) Between the hinge region of the antibody heavy chain of the first antigen-binding domain and the hinge region of the antibody heavy chain of the second antigen-binding domain; (iii) Between the light chain constant (CL) region of the first antigen-binding domain and the light chain constant (CL) region of the second antigen-binding domain; (iv) Between the CH1 region of the antibody heavy chain constant state of the first antigen-binding domain and the light chain constant state (CL) region of the second antigen-binding domain; (v) Between the light chain constant (CL) region of the first antigen-binding domain and the CH1 region of the antibody heavy chain constant of the second antigen-binding domain; and / or (vi) Between the heavy chain variable (VH) region of the first antigen-binding domain and the heavy chain variable (VH) region of the second antigen-binding domain. In some embodiments, the linkage between the first antigen-binding domain and the second antigen-binding domain can be created, for example, by introducing at least one amino acid modification (e.g., substitution with cysteine ​​or lysine) into each of the polypeptides (i) to (vi) above.

[0219] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a third antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1), and a heavy chain variable (VH) region of a first antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) nucleic acids encoding polypeptides, comprising a light chain variable (VL) region of a third antigen-binding domain, which may optionally further include a light chain constant (CL) region; and (iii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a first antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes.

[0220] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a third antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1), and a heavy chain variable (VH) region of a second antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) nucleic acids encoding polypeptides, comprising a light chain variable (VL) region of a third antigen-binding domain, which may optionally further include a light chain constant (CL) region; and (iii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a second antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes.

[0221] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a third antigen-binding domain, which may optionally further comprise a light chain constant (CL) region, and a heavy chain variable (VH) region of a first antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a third antigen-binding domain, which may optionally further include a heavy chain constant region (e.g., CH1;CH1 and hinge); (iii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a first antigen-binding domain, which may optionally further include a light chain constant (CL) region; (iv) nucleic acids encoding a polypeptide, comprising a heavy chain variable (VH) region of a second antigen-binding domain, which may optionally further include heavy chain constant regions (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); and (v) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a second antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; and (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes.

[0222] In some embodiments, the antigen-binding molecule thus produced comprises a first antigen-binding domain and a second antigen-binding domain linked to each other via at least one bond. The at least one bond for linking the first antigen-binding domain and the second antigen-binding domain is introduced into one or more of the following: (i) Between the constant CH1 region of the antibody heavy chain of the first antigen-binding domain and the constant CH1 region of the antibody heavy chain of the second antigen-binding domain; (ii) Between the hinge region of the antibody heavy chain of the first antigen-binding domain and the hinge region of the antibody heavy chain of the second antigen-binding domain; (iii) Between the light chain constant (CL) region of the first antigen-binding domain and the light chain constant (CL) region of the second antigen-binding domain; (iv) Between the CH1 region of the antibody heavy chain constant state of the first antigen-binding domain and the light chain constant state (CL) region of the second antigen-binding domain; (v) Between the light chain constant (CL) region of the first antigen-binding domain and the CH1 region of the antibody heavy chain constant of the second antigen-binding domain; and / or (vi) Between the heavy chain variable (VH) region of the first antigen-binding domain and the heavy chain variable (VH) region of the second antigen-binding domain. In some embodiments, the linkage between the first antigen-binding domain and the second antigen-binding domain can be created, for example, by introducing at least one amino acid modification (e.g., substitution with cysteine ​​or lysine) into each of the polypeptides (i) to (vi) above.

[0223] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a third antigen-binding domain, which may optionally further comprise a light chain constant (CL) region, and a heavy chain variable (VH) region of a second antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a third antigen-binding domain, which may optionally further include a heavy chain constant region (e.g., CH1;CH1 and hinge); (iii) nucleic acids encoding polypeptides, comprising a light chain variable (VL) region of a second antigen-binding domain, which may optionally further include a light chain constant (CL) region; and (iv) A nucleic acid encoding a polypeptide, comprising a heavy chain variable (VH) region of a first antigen-binding domain, which may optionally further include heavy chain constant regions (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (v) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a first antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; and (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes.

[0224] In some embodiments, the antigen-binding molecule thus constructed comprises a first antigen-binding domain and a second antigen-binding domain linked to each other via at least one binding. The at least one binding for linking the first antigen-binding domain and the second antigen-binding domain is introduced into one or more of the following: (i) Between the constant CH1 region of the antibody heavy chain of the first antigen-binding domain and the constant CH1 region of the antibody heavy chain of the second antigen-binding domain; (ii) Between the hinge region of the antibody heavy chain of the first antigen-binding domain and the hinge region of the antibody heavy chain of the second antigen-binding domain; (iii) Between the light chain constant (CL) region of the first antigen-binding domain and the light chain constant (CL) region of the second antigen-binding domain; (iv) Between the CH1 region of the antibody heavy chain constant of the first antigen-binding domain and the light chain constant (CL) region of the second antigen-binding domain; (v) Between the light chain constant (CL) region of the first antigen-binding domain and the CH1 region of the antibody heavy chain constant of the second antigen-binding domain; and / or (vi) Between the heavy chain variable (VH) region of the first antigen-binding domain and the heavy chain variable (VH) region of the second antigen-binding domain. In some embodiments, the linkage between the first antigen-binding domain and the second antigen-binding domain can be created, for example, by introducing at least one amino acid modification (e.g., substitution with cysteine ​​or lysine) into each of the polypeptides (i) to (vi) above.

[0225] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a third antigen-binding domain, which may optionally further comprise a light chain constant (CL) region, and a heavy chain variable (VH) region of a first antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) nucleic acids encoding a polypeptide, comprising a heavy chain variable (VH) region of a third antigen-binding domain, which may optionally further include a heavy chain constant region (e.g., CH1;CH1 and hinge); and (iii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a first antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes.

[0226] In one aspect, this application also provides a method for producing the antigen-binding molecule of the present invention. The method is, for example, as follows: (a) at least: (i) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a third antigen-binding domain, which may optionally further comprise a light chain constant (CL) region, and a heavy chain variable (VH) region of a second antigen-binding domain, which may optionally further comprise a heavy chain constant region (e.g., CH1;CH1 and hinge;CH1, hinge, and CH2;CH1, hinge, CH2, and CH3); (ii) nucleic acids encoding a polypeptide, comprising a heavy chain variable (VH) region of a third antigen-binding domain, which may optionally further include a heavy chain constant region (e.g., CH1;CH1 and hinge); and (iii) A nucleic acid encoding a polypeptide, comprising a light chain variable (VL) region of a second antigen-binding domain, which may optionally further include a light chain constant (CL) region. A process to provide; (b) The process of introducing the nucleic acid prepared in (a) into host cells; (c) A step of culturing host cells so that two polypeptides are expressed; (d) A step of collecting antigen-binding molecules from the culture medium of the cells cultured in step (c). Includes. In some embodiments, the antigen-binding molecule of the present invention is an antigen-binding molecule prepared by the method described above. In one aspect, the screening method of the present invention makes it possible to more efficiently obtain antigen-binding domains that bind to at least two or more different target antigens.

[0227] In this application, “library” means multiple antigen-binding molecules, multiple antigen-binding domains, multiple fusion polypeptides containing antigen-binding molecules, multiple fusion polypeptides containing antigen-binding domains, or multiple nucleic acids or polynucleotides encoding these. The multiple antigen-binding molecules, multiple antigen-binding domains, or multiple fusion polypeptides containing antigen-binding molecules, or multiple fusion polypeptides containing antigen-binding domains contained in the library are antigen-binding molecules, antigen-binding domains, or fusion polypeptides whose sequences are distinct from each other and do not have a single sequence. In some embodiments, the library of the present invention is a design library. In further embodiments, the design library is a design library as disclosed in WO2016 / 076345.

[0228] In one embodiment of the present invention, a fusion polypeptide of the antigen-binding molecule or antigen-binding domain of the present invention and a heterologous polypeptide can be prepared. In one embodiment, the fusion polypeptide may include, for example, an antigen-binding molecule or antigen-binding domain of the present invention fused with at least a portion of a viral coat protein selected from the group consisting of viral coat proteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, and their variants.

[0229] In one embodiment, the present invention provides a library essentially comprising a plurality of sequencely distinct fusion polypeptides, each comprising either of these antigen-binding molecules or antigen-binding domains and a heterologous polypeptide. Specifically, the present invention provides a library essentially comprising a plurality of sequencely distinct fusion polypeptides, each comprising either of these antigen-binding molecules or antigen-binding domains fused with at least a portion of viral coat proteins selected from the group consisting of, for example, viral coat proteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, and their variants. The antigen-binding molecules or antigen-binding domains of the present invention may further comprise a dimerization domain. In one embodiment, the dimerization domain may be located between the variable region of the heavy or light chain of an antibody and at least a portion of the viral coat protein. This dimerization domain may comprise at least one dimerization sequence and / or a sequence comprising one or more cysteine ​​residues. This dimerization domain may preferably be ligated to the C-terminus of the heavy chain variable or constant region. The dimerization domain can take on various structures depending on whether the antibody variable region is prepared as a fusion polypeptide component with a viral coat protein component (without an amber stop codon after the dimerization domain) or whether the antibody variable region is prepared primarily without a viral coat protein component (for example, with an amber stop codon after the dimerization domain). When the antibody variable region is prepared primarily as a fusion polypeptide with a viral coat protein component, bivalent presentation is brought about by one or more disulfide bonds and / or a single dimerization sequence.

[0230] In the context of multiple antigen-binding molecules or antigen-binding domains with distinct sequences as described herein, the term “different sequences” means that each antigen-binding molecule or antigen-binding domain in the library has a distinct sequence. Specifically, the number of distinct sequences in the library reflects the number of independent clones with different sequences in the library and may be referred to as the “library size.” The library size of a typical phage display library is 10 6 ~10 12 Therefore, by applying known techniques in this field, such as ribosome display methods, 10 14 It can be expanded up to 10,000 times. However, the actual number of phage particles used in panning selection of a phage library is usually 10 to 10,000 times larger than the library size. This excess multiplier is also called the "library equivalent number" and represents the fact that 10 to 10,000 individual clones may have the same amino acid sequence. Therefore, the term "sequences distinct from one another" as used in this invention means that the individual antigen-binding molecules in the library, excluding the library equivalent number, have distinct sequences, and more specifically, the library may have 10 6 ~10 14 Preferably 10 7 ~10 12 , more 10 8 ~10 11 , particularly preferably 10 8 ~10 10 This means that the sequences have different antigen-binding molecules or antigen-binding domains.

[0231] As described herein, “phage display” refers to a technique for presenting variant polypeptides as fusion proteins with at least a portion of the coat protein on the particle surface of a phage, such as a filamentous phage. Phage display is useful because it allows for rapid and efficient screening of large libraries of randomized protein variants for sequences that bind to target antigens with high affinity. Presentation of peptide and protein libraries on phages has been used to screen millions of polypeptides for those with specific binding properties. Multivalent phage display methods have been used to present small random peptides and small proteins through fusion with gene III or gene VIII of filamentous phages (Wells and Lowman, Curr. Opin. Struct. Biol. (1992) 3, 355-362; and references cited therein). Monovalent phage display involves fusioning a library of proteins or peptides with gene III or a portion thereof such that each phage particle presents one or zero copies of the fusion protein, and expressing the fusion protein at low levels in the presence of the wild-type gene III protein. Monovalent phages have lower avidity than multivalent phages, and therefore are screened based on endogenous ligand affinity using phagemide vectors, which simplifies DNA manipulation (Lowman and Wells, Methods: A Companion to Methods in Enzymology (1991) 3, 205-216).

[0232] A "phagemide" refers to a plasmid vector containing a bacterial origin of replication, such as ColE1, and a copy of the intergenetic region of a bacteriophage. Phagemids derived from any bacteriophage known in the art, such as filamentous bacteriophages or lambdoid bacteriophages, can be used as appropriate. Plasmids typically also contain selection markers for antibiotic resistance. DNA fragments cloned into these vectors can be grown as plasmids. When cells containing these vector...

Claims

1. (i) A first antigen-binding domain comprising a heavy chain variable (VH) region and a CH1 region, as well as a light chain variable (VL) region and a light chain constant region (CL); (ii) A second antigen-binding domain comprising the heavy chain variable (VH) region and the CH1 region, and the light chain variable (VL) region and the light chain constant region (CL). (iii) A third antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, which binds to a third antigen, An antigen-binding molecule comprising at least three antigen-binding domains, (a) The first antigen-binding domain and the second antigen-binding domain (i) Linked via the Fc region, forming the structural form of an IgG-type antibody, and (ii) linked to each other by at least one disulfide bond, Here, the at least one disulfide bond is located between the amino acid residue at position 191 according to EU numbering in the CH1 region of the first antigen-binding domain and the amino acid residue at position 191 according to EU numbering in the CH1 region of the second antigen-binding domain. (b) (i) The first antigen-binding domain and the second antigen-binding domain can each bind to the first antigen and to a second antigen different from the first antigen, but they cannot bind to both the first and second antigens at the same time, or (ii) The first antigen-binding domain can bind to the first antigen and to a second antigen different from the first antigen, but not to both the first and second antigens simultaneously, and the second antigen-binding domain can bind only to the second antigen. (c) The first antigen is CD3 and the second antigen is CD137, (d) The third antigen-binding domain is as follows: (i) Between the C-terminus of the polypeptide containing the heavy chain variable (VH) region of the third antigen-binding domain and the N-terminus of the polypeptide containing the heavy chain variable (VH) region of the first antigen-binding domain, (ii) Between the C-terminus of the polypeptide containing the heavy chain variable (VH) region of the third antigen-binding domain and the N-terminus of the polypeptide containing the light chain variable (VL) region of the first antigen-binding domain, (iii) Between the C-terminus of a polypeptide containing the light chain variable (VL) region of the third antigen-binding domain and the N-terminus of a polypeptide containing the heavy chain variable (VH) region of the first antigen-binding domain, (iv) Between the C-terminus of the polypeptide containing the light chain variable (VL) region of the third antigen-binding domain and the N-terminus of the polypeptide containing the light chain variable (VL) region of the first antigen-binding domain It is linked to the first antigen-binding domain through one of the following linkages: (e) The third antigen is a molecule that is different from the first and second antigens and is specifically expressed on cancer cells. A method for producing the aforementioned antigen-binding molecule, A step of culturing cells containing one or more vectors comprising one or more polynucleotides encoding one or more polypeptides of the antigen-binding molecule, and Steps to isolate antigen-binding molecules from the culture supernatant. A method for producing antigen-binding molecules, including [the specified element].

2. (i) A first antigen-binding domain comprising a heavy chain variable (VH) region and a CH1 region, and a light chain variable (VL) region and a light chain constant region (CL); (ii) A second antigen-binding domain comprising the heavy chain variable (VH) region and the CH1 region, and the light chain variable (VL) region and the light chain constant region (CL). (iii) A third antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, which binds to a third antigen, An antigen-binding molecule comprising at least three antigen-binding domains, (a) The first antigen-binding domain and the second antigen-binding domain (i) Linked via the Fc region, forming the structural form of an IgG-type antibody, and (ii) linked to each other by at least one disulfide bond, Here, the at least one disulfide bond is located between the amino acid residue at position 191 according to EU numbering in the CH1 region of the first antigen-binding domain and the amino acid residue at position 191 according to EU numbering in the CH1 region of the second antigen-binding domain. (b) (i) The first antigen-binding domain and the second antigen-binding domain can each bind to the first antigen and to a second antigen different from the first antigen, but they cannot bind to both the first and second antigens at the same time, or (ii) The first antigen-binding domain can bind to the first antigen and to a second antigen different from the first antigen, but not to both the first and second antigens simultaneously, and the second antigen-binding domain can bind only to the second antigen. (c) The first antigen is CD3 and the second antigen is CD137, (d) The third antigen-binding domain is as follows: (i) Between the C-terminus of the polypeptide containing the heavy chain variable (VH) region of the third antigen-binding domain and the N-terminus of the polypeptide containing the heavy chain variable (VH) region of the first antigen-binding domain, (ii) Between the C-terminus of the polypeptide containing the heavy chain variable (VH) region of the third antigen-binding domain and the N-terminus of the polypeptide containing the light chain variable (VL) region of the first antigen-binding domain, (iii) Between the C-terminus of a polypeptide containing the light chain variable (VL) region of the third antigen-binding domain and the N-terminus of a polypeptide containing the heavy chain variable (VH) region of the first antigen-binding domain, (iv) Between the C-terminus of the polypeptide containing the light chain variable (VL) region of the third antigen-binding domain and the N-terminus of the polypeptide containing the light chain variable (VL) region of the first antigen-binding domain It is linked to the first antigen-binding domain through one of the following linkages: (e) The third antigen is a molecule that is different from the first and second antigens and is specifically expressed on cancer cells. A method for producing the aforementioned antigen-binding molecule, comprising the following steps: (1) A step of providing one or more nucleic acids that encode one or more polypeptides that form a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain; (2) The process of introducing nucleic acids into host cells; (3) A step of culturing host cells so that two or more polypeptides are produced; and (4) A step to obtain an antigen-binding molecule.

3. To provide an antigen-binding domain that does not bind to the first antigen and the second antigen simultaneously, - A step of preparing a library of antigen-binding domains, each of which binds to a first or second antigen, wherein at least one amino acid is modified in its heavy chain variable (VH) region and light chain variable (VL) region, wherein the modified variable regions are different from each other by at least one amino acid; and - A step of selecting antigen-binding domains from the prepared library that have binding activity to the first antigen and the second antigen, but do not bind to the first antigen and the second antigen simultaneously, and include heavy chain variable (VH) and light chain variable (VL) regions. The method according to claim 2, including the method described in claim 2.

4. The method according to claim 3, wherein the modification is a modification of at least one amino acid selected from Kabat numbering positions 31-35, 50-65, 71-74, and 95-102 in the heavy chain variable (VH) region, using the amino acid sequence shown in SEQ ID NO: 184 as a template sequence, and from Kabat numbering positions 24-34, 50-56, and 89-97 in the light chain variable (VL) region, using the amino acid sequence shown in SEQ ID NO: 185 as a template sequence.

5. The method according to any one of claims 2 to 4, wherein the antigen-binding domain that does not simultaneously bind to the first antigen and the second antigen is itself an antigen-binding domain that does not simultaneously bind to the first antigen and the second antigen expressed on different cells, respectively.

6. The method according to claim 5, wherein the Fc region is an Fc region in which binding activity to FcγR is reduced compared to the Fc region of a natural human IgG1 antibody.

7. The method according to any one of claims 2 to 6, wherein the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain are encoded by a single nucleotide.

8. The method according to any one of claims 2 to 7, wherein step (a) further comprises introducing one or more mutations into the nucleic acid sequences encoding each of the first and second antigen-binding domains, which, when translated, introduce one or more disulfide bonds that link the first and second antigen-binding domains close together.

9. The method of claim 8, wherein the at least one disulfide bond is further present between any other parts other than the hinge regions, or between the hinge regions, if the first antigen-binding domain includes a heavy chain hinge region and the second antigen-binding domain includes a heavy chain hinge region, and the first antigen-binding domain and the second antigen-binding domain are linked to each other by one or more native disulfide bonds in their respective hinge regions.

10. One or more mutations, (i) in the CH1 region of the first antigen-binding domain and in the CH1 region of the second antigen-binding domain; (ii) In the CH1 region of the first antigen-binding domain and in the CL region of the second antigen-binding domain; (iii) In the CL region of the first antigen-binding domain and in the CH1 region of the second antigen-binding domain; (iv) in the CL region of the first antigen-binding domain and in the CL region of the second antigen-binding domain; or (v) In the VH or VL region of the first antigen-binding domain, and in the VH or VL region of the second antigen-binding domain The method according to claim 8 or 9, which exists in the present.

11. The method according to any one of claims 8 to 10, wherein one or more mutations are cysteine ​​substitutions or insertions.

12. The method according to any one of claims 8 to 11, further comprising the step of performing an assay to determine whether the first antigen-binding domain and the second antigen domain do not simultaneously bind to the first antigen and the second antigen expressed on different cells, respectively.

13. The method according to any one of claims 2 to 12, wherein the third antigen is glypican-3 (GPC3).

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