Antigen-binding molecule comprising altered antibody variable region binding cd3 and cd137

Abstract

This invention provides antigen-binding domains that can bind to CD3 and CD137, but not simultaneously to both CD3 and CD137, and methods for using them. It also provides methods for more efficiently obtaining antigen-binding domains that bind to two or more antigens.

Description

Technical Field

[0001] This invention relates to antigen-binding molecules that bind to CD3 and CD137 (4-1BB) and methods of using them. Prior Technology

[0002] Antibodies have attracted attention as drugs due to their high stability in plasma and low incidence of 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 possess antigen-binding and agonist / antagonist functions, but also induce cytotoxic activities mediated by effector cells (also known as effector functions), such as ADCC (antibody-dependent cytotoxicity), ADCP (antibody-dependent cell phagocytosis), or CDC (complement-dependent cytotoxicity). In particular, antibodies against the IgG1 subtype exhibit effector functions against cancer cells. Therefore, a large number of antibody drugs have been developed in the field of cancer.

[0003] In order for antibodies to exert their ADCC, ADCP, or CDC effects, their Fc region must bind to antibody receptors (FcγR) and various complement components present in effector cells (such as NK cells or macrophages). In humans, isoforms of FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb have been reported as a protein family of FcγR, and their respective allotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (Non-Patent Literature 3)). Among these isoforms, FcγRIa, FcγRIIa, and FcγRIIIa possess a domain called ITAM (immunoreceptor tyrosine-based activation motif) in their intracellular domain, which transduces activation signals. In contrast, only FcγRIIb possesses a domain called ITIM (immunoreceptor tyrosine-based inhibitory motif) within its intracellular domain, which transduces inhibitory signals. Isotypes of these FcγRs are known to transduce signals via cross-linking with immune complexes or analogues (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-Patent Literature 4)). Indeed, when antibodies exert their effector function against cancer cells, FcγR molecules on the effector cell membrane cluster by binding to the Fc regions of multiple antibodies on cancer cells, thereby transducing activation signals through the effector cells. This results in a cytotoxic effect. In this case, the cross-linking of FcγRs is limited to effector cells close to cancer cells, indicating that immune activation is localized within cancer cells (Ann. Rev. Immunol. (1988). 6.251-81 (Non-Patent Literature 5)).

[0004] Naturally occurring immunoglobulins bind to antigens via their variable regions and to receptors and complement such as FcγR, FcRn, FcαR, and FcεR via their constant regions. Each FcRn molecule (the binding molecule that interacts with the Fc region of IgG) binds to the heavy chains of an antibody in a one-to-one connection. Therefore, two FcRn molecules have been reported to bind to one IgG antibody molecule. Unlike FcRn, FcγR interacts with the antibody hinge region and CH2 domain, and only one FcγR molecule binds to one IgG antibody molecule (J. Biol. Chem. (2001) 276, 16469-16477). For the binding between FcγR and the Fc region of the antibody, certain amino acid residues in the hinge region and CH2 domain of the antibody, as well as the sugar chain in the CH2 domain that has been added to Asn297 (EU number), have been found to be important (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)). Fc region variants with various FcγR binding properties have previously been studied by focusing on this binding site to produce Fc region variants with higher binding activity against activated FcγR (WO2000 / 042072 (Patent Literature 1) and WO2006 / 019447 (Patent Literature 2)). For example, Lazar et al. successfully increased the binding activity of human IgG against human FcγRIIIa (V158) by approximately 370-fold by replacing Ser239, Ala330, and Ile332 (EU designations) of human IgG with Asn, Leu, and Glu, respectively (Proc. Natl. Acad. Sci. USA (2006) 103, 4005-4010 (Non-Patent Document 9) and WO2006 / 019447 (Patent Document 2)). In terms of the FcγRIIIa to FcγRIIb ratio (A / I ratio), this modified form exhibited approximately 9-fold activity compared to the wild type. Alternatively, Shinkawa et al. have successfully increased the binding activity against FcγRIIIa by approximately 100-fold by deleting trehalose (fucose) added to Asn297 (EU designation) (J. Biol. Chem. (2003) 278, 3466-3473 (Non-Patent Literature 10)). Compared to naturally occurring human IgG1, these methods can dramatically improve the ADCC activity of human IgG1.

[0005] Naturally occurring IgG antibodies typically recognize and bind to a single antigenic determinant via their variable region (Fab), thus binding to only one antigen. Meanwhile, many types of proteins are known to be involved in cancer or inflammation, and these proteins can crosstalk with each other. For example, some inflammatory interstitials (TNF, IL1, and IL6) are known to be involved in immune diseases (Nat. Biotech., (2011) 28, 502-10 (Non-Patent Literature 11)). Furthermore, the activation of other receptors is known to be a fundamental mechanism for cancer to acquire drug resistance (Endor Relat Vancer (2006) 13, 45-51 (Non-Patent Literature 12)). In this case, a typical antibody that recognizes a single antigenic determinant cannot inhibit multiple proteins.

[0006] Antibodies that bind two or more types of antigens in a single molecule (these antibodies are also known as bispecific antibodies) have been studied as molecules that inhibit multiple targets. Binding activity against two different antigens (a primary antigen and a secondary antigen) can be conferred by modification of naturally occurring IgG antibodies (mAbs. (2012) Mar 1, 4(2)). Therefore, these antibodies not only have the ability to neutralize two or more types of antigens in a single molecule, but also enhance antitumor activity through cross-linking with cells that are cytotoxic to cancer cells. Molecules with antigen-binding sites at the N or C terminals of the antibody (DVD-Ig, TCB, and scFv-IgG), molecules with different sequences of two Fab regions of the antibody (common L-chain bispecific antibody and heterozygous fusion adenomas), molecules with one Fab region recognizing two antigens (two-in-one IgG and DutaMab), and molecules with a CH3 domain loop as another antigen-binding site (Fcab) have previously been reported as bispecific antibody molecules (Nat. Rev. (2010), 10, 301-316 (Non-Patent Literature 13) and Peds (2010), 23(4), 289-297 (Non-Patent Literature 14)). Because any of these bispecific antibodies interacts with FcγR in its Fc region, antibody effector function is retained therein.

[0007] If all antigens recognized by bispecific antibodies are antigens specifically expressed in cancer cells, bispecific antibodies binding to any of these antigens may exhibit cytotoxic activity against cancer cells, thus potentially offering more efficient anticancer efficacy compared to conventional antibody drugs that recognize only one antigen. However, in cases where any antigen recognized by a bispecific antibody is expressed in normal tissues or in immune cells, cross-linking with FcγR can cause damage to the normal tissue or release of interferons (J. Immunol. (1999) Aug 1, 163(3), 1246-52 (Non-Patent Literature 15)). This can induce serious adverse reactions.

[0008] For example, catuxomab is known as a bispecific antibody that recognizes proteins expressed on T cells and proteins expressed on cancer cells (cancer antigens). Catuxomab binds to two Fab-binding cancer antigens (EpCAM) and the CH3ε chain expressed on T cells. Catuxomab induces T cell-mediated cytotoxic activity by simultaneously binding to the cancer antigen and the CH3ε chain, and induces NK cell or antigen-presenting cell (e.g., macrophage)-mediated cytotoxic activity by simultaneously binding to the cancer antigen and FcγR. Through the use of these two cytotoxic activities, catuxomab has shown high therapeutic efficacy against malignant ascites by intraperitoneal administration, and has therefore been approved in Europe (Cancer Treat Rev. (2010) Oct 36(6), 458-67 (Non-Patent Literature 16)). Furthermore, caputuzumab administration has been reported to produce cancer cell-reactive antibodies in some cases, demonstrating the induction of acquired immune systems (Future Oncol. (2012) Jan 8(1), 73-85 (Non-Patent Literature 17)). Based on these results, antibodies with T-cell-mediated cytotoxic activity and efficacy via FcγR through cells such as NK cells or macrophages (specifically referred to as trifunctional antibodies) have attracted attention due to their expected strong antitumor efficacy and induction of acquired immunity.

[0009] However, even in the absence of cancer antigens, this trifunctional antibody still binds to both CD3ε and FcγR, thus crosslinking CD3ε-expressing T cells to FcγR-expressing cells even in a cancer-free environment to produce large quantities of various intercellular substances. This cancer antigen-independent induction of various intercellular substance production limits the administration of current trifunctional antibodies to the intraperitoneal route (Cancer Treat Rev. 2010 Oct 36(6), 458-67 (Non-Patent Literature 16)). Due to severe adverse reactions such as intercellular cytokine storm, this trifunctional antibody is very difficult to administer systemically (Cancer Immunol Immunother. 2007 Sep;56(9):1397-406 (Non-Patent Literature 18)).

[0010] Traditional bispecific antibodies can bind to two antigens, namely the first antigen EpCAM and the second antigen CD3ε, and also bind to FcγR. Therefore, considering their molecular structure, it is impossible to avoid the adverse reaction caused by the simultaneous binding to FcγR and the second antigen CD3ε.

[0011] In recent years, modified antibodies that induce T cell-mediated cytotoxicity and circumvent adverse reactions have been developed by using Fc regions that have reduced binding activity against FcγR (WO2012 / 073985).

[0012] However, considering its molecular structure, even this antibody failed to act on two immune receptors, namely CD3ε and FcγR, when it bound to the cancer antigen.

[0013] Antibodies that can exert both T-cell-mediated and non-T-cell-mediated cytotoxic activities in a cancer antigen-specific manner while avoiding adverse reactions are currently unknown.

[0014] T cells play a crucial role in tumor immunity and are known to be activated through two signals: 1) the binding of the T cell receptor (TCR) to antigenic peptides presented by type I molecules of the major histocompatibility complex (MHC) and activation of the TCR; and 2) the binding of co-stimulatory molecules on the T cell surface to ligands of antigen-presenting cells and activation of the co-stimulatory 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 T cell surface, has been described as important for T cell activation (Vinay, 2011, Cellular & Molecular Immunology, 8, 281-284 (Non-Patent Literature 19)).

[0015] CD137 agonist antibodies have shown antitumor efficacy, experimentally demonstrated primarily due to activation of CD8-positive T and NK cells (Houot, 2009, Blood, 114, 3431-8 (Non-Patent Literature 20)). It is also understood that engineered T cells with chimeric antigen receptor molecules (CAR-T cells) can enhance drug efficacy persistence, the latter consisting of an extracellular tumor antigen-binding domain and intracellular CD3 and CD137 signaling domains (Porter, N ENGL J MED, 2011, 365; 725-733 (Non-Patent Literature 21)). However, the non-specific hepatotoxic side effects of these CD137 agonist antibodies have become a problem both clinically and non-clinically, and pharmaceutical development has not progressed (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22 (Non-Patent Literature 22)). The main cause of side effects is suggested to involve the binding of the antibody to the Fcγ receptor via the antibody constant domain (Schabowsky, Vaccine, 2009, 28, 512-22 (Non-Patent Literature 23)). Furthermore, it has been reported that in order for agonist antibodies targeting receptors belonging to the TNF receptor superfamily to exert in vivo agonist activity, antibody cross-linking with Fcγ receptor-expressing cells (FcγRII-expressing cells) is necessary (Li, Proc Natl Acad Sci USA. 2013, 110(48), 19501-6 (Non-Patent Literature 24)). WO2015 / 156268 (Patent Literature 3) describes that a bispecific antibody having a binding domain containing CD137 agonist activity and a binding domain for tumor-specific antigens can exert CD137 agonist activity and activate immune cells only in the presence of cells expressing the tumor-specific antigen, thereby avoiding the hepatotoxic adverse events of CD137 agonist antibodies and preserving the antitumor activity of the antibody. WO2015 / 156268 further describes that the antitumor activity can be further enhanced, and that such adverse events can be avoided by combining this bispecific antibody with another bispecific antibody having a binding domain containing CD3 activating activity and a binding domain to tumor-specific antigens. Trispecific antibodies having three binding domains to CD137, CD3, and tumor-specific antigen (EGFR) have also been reported (WO2014 / 116846 (Patent Document 4)). However, antibodies that exert both T-cell-mediated cytotoxic activity and T-cell and other immune cell activation activity via CD137 in a cancer antigen-specific manner while simultaneously avoiding adverse reactions are currently unknown.

[0016] The technique of using libraries to obtain binding domains for any antigen is well known (Clackson et al., Nature 352:624-628 (1991) (Non-Patent Literature 25); Marks et al., J.Mol.Biol.222:581-597 (1991) (Non-Patent Literature 26)). For example, phage display, ribosome display, mRNA display, CIS display, E. coli display, cell display, and yeast display are known techniques for obtaining binding domains using libraries (Nat Biotechnol. 1996 Mar; 14(3): 309-14 (Non-Patent Literature 27); Nat Biotechnol. 2000 Dec; 18(12): 1287-92 (Non-Patent Literature 28); Nucleic Acids Res. 2006; 34(19): e127 (Non-Patent Literature 29); Proc Natl Acad Sci USA. 2004 Mar 2; 101(9): 2806-10 (Non-Patent Literature 30); Proc Natl Acad Sci USA. 2004 June 22; 101(25): 9193-8 (Non-Patent Literature 31); Protein Eng Des Sel. 2008 Apr;21(4):247-55 (Non-Patent Literature 32);Proc Natl Acad Sci USA.2000 Sep 26;97(20):10701-5 (Non-Patent Literature 33);MAbs.2010 Sep-Oct;2(5):508-18 (Non-Patent Literature 34);andMethods Mol Biol.2012;911:183-98 (Non-Patent Literature 35)).

[0017] Binding domains to two different antigens have been obtained using library methods (Bostrom et al., Science 323:1610-4 (2009) (Non-Patent Literature 36)). Several techniques have been reported for obtaining these domains to two different antigens, such as alternating use of different antigens in different panning rounds, and methods for first obtaining the first binding domain to the first antigen from a library prepared by randomizing the binding domain of the first antigen, and then obtaining the binding domain to the second antigen. However, these strategies require a gene amplification step after recovering the first antigen binding domain to amplify the recovered polynucleotides.

[0018] A phage display library method that sequentially applies selection pressure to one antigen twice without an intermediate step of nucleic acid amplification has been reported and is known as double-round selection (Hawkins et al., J. Mol. Biol. 226: 889-96 (1992) (Non-Patent Literature 37)). However, no more efficient method is known for collecting binding domains to two or more different antigens by sequentially applying selection pressure to two or more different antigens twice or more.

[0019] List of documents

[0020] Patent documents

[0021] Patent Document 1: WO2000 / 042072

[0022] Patent Document 2: WO2006 / 019447

[0023] Patent Document 3: WO2015 / 156268

[0024] Patent Document 4: WO2014 / 116846

[0025] Non-patent literature

[0026] Non-patent literature 1: Nat. Biotechnol. (2005) 23, 1073-1078

[0027] Non-patent literature 2: Eur J Pharm Biopharm. (2005) 59(3), 389-396

[0028] Non-patent literature 3: Immunol. Lett. (2002) 82, 57-65

[0029] Non-patent literature 4: Nat. Rev. Immunol. (2008) 8, 34-47

[0030] Non-patent literature 5: Ann. Rev. Immunol. (1988). 6. 251-81

[0031] Non-patent literature 6: Chem. Immunol. (1997), 65, 88-110

[0032] Non-patent literature 7: Eur. J. Immunol. (1993) 23, 1098-1104

[0033] Non-patent literature 8: Immunol. (1995) 86, 319-324

[0034] Non-patent literature 9: Proc. Natl. Acad. Sci. USA (2006) 103, 4005-4010

[0035] Non-patent literature 10: J. Biol. Chem. (2003) 278, 3466-3473

[0036] Non-patent literature 11: Nat. Biotech., (2011) 28, 502-10

[0037] Non-patent literature 12: Endocr Relat Cancer (2006) 13, 45-51

[0038] Non-patent literature 13: Nat. Rev. (2010), 10, 301-316

[0039] Non-patent literature 14: Peds (2010), 23(4), 289-297

[0040] Non-patent literature 15: J. Immunol. (1999) Aug 1, 163(3), 1246-52

[0041] Non-patent literature 16: Cancer Treat Rev. (2010) Oct 36(6), 458-67

[0042] Non-patent literature 17: Future Oncol. (2012) Jan 8(1), 73-85

[0043] Non-patent literature 18: Cancer Immunol Immunother. 2007 Sep; 56(9): 1397-406

[0044] Non-patent literature 19: Vinary, 2011, Cellular & Molecular Immunology, 8, 281-284

[0045] Non-patent literature 20: Houot, 2009, Blood, 114, 3431-8

[0046] Non-patent literature 21: Porter, N ENGL J MED, 2011, 365; 725-733

[0047] Non-patent literature 22: Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22

[0048] Non-patent literature 23: Schabowsky, Vaccine, 2009, 28, 512-22

[0049] Non-patent literature 24: Li, Proc Natl Acad Sci USA. 2013, 110(48), 19501-6

[0050] Non-patent literature 25: Clackson et al., Nature 352: 624-628 (1991)

[0051] Non-patent literature 26: Marks et al., J. Mol. Biol. 222: 581-597 (1991)

[0052] Non-patent literature 27: Nat Biotechnol. 1996 Mar; 14(3): 309-14

[0053] Non-patent literature 28: Nat Biotechnol. 2000 Dec; 18(12): 1287-92

[0054] Non - Patent Document 29: Nucleic Acids Res. 2006; 34(9): e127

[0055] Non - Patent Document 30: Proc Natl Acad Sci USA. 2004 Mar 2; 101(9): 2806 - 10

[0056] Non - Patent Document 31: Proc Natl Acad Sci USA 2004 Jun 22; 101(25): 9193 - 8

[0057] Non - Patent Document 32: Protein Eng Des Sel. 2008 Apr; 21(4): 247 - 55

[0058] Non - Patent Document 33: Proc Natl Acad Sci USA. 2000 Sep 26; 97(20): 10701 - 5

[0059] Non - Patent Document 34: MAbs. 2010 Sep - Oct; 2(5): 508 - 18

[0060] Non - Patent Document 35: Methods Mol Biol. 2012; 911: 183 - 98

[0061] Non - Patent Document 36: Bpstrom et al., Science 323: 1610 - 4 (2009)

[0062] Non - Patent Document 37: Hawkins et al., J. Mol. Biol. 226: 889 - 9 (1992) Summary of the Invention Problems to be Solved by the Invention

[0063] Trispecific antibodies containing a tumor-specific antigen (EGFR) binding domain, a CD137 binding domain, and a CD3 binding domain have been reported (WO2014116846). However, since antibodies with this molecular format can bind to three different antigens simultaneously, the inventors hypothesize that such trispecific antibodies may cause cross-linking between CD3ε-expressing T cells and CD137-expressing cells (e.g., T cells, B cells, NK cells, DCs, etc.) by simultaneously binding to CD3 and CD137.

[0064] Furthermore, it has been reported that bispecific antibodies against CD8 and CD3ε induce cytotoxicity in CD8-positive T cells due to cross-linking with these antibodies (Wong, Clin. Immunol. Immunopathol. 1991, 58(2), 236-250). Therefore, the inventors hypothesize that bispecific antibodies against molecules expressed on T cells and CD3ε will also induce cytotoxicity in T cells because they will cross-link with cells expressing these molecules and CD3ε.

[0065] Several previously reported techniques for obtaining antigen-binding domains that bind to two different antigens have been employed, such as methods that alternately use different antigens in different panning rounds, and methods that first obtain a first binding domain for the first antigen from a library prepared by randomizing the binding domain of the first antigen, and then obtain a binding domain for the second antigen. However, these strategies require steps such as recovering the binding domain for the first antigen, amplifying the recovered nucleotides encoding the binding domain for the first antigen, and further recovering and amplifying the nucleic acid that can also bind to the second antigen. The inventors believe that, as a result of this process, each panning round will ultimately concentrate the binding domain, which exhibits a stronger binding to one of the different antigens used in the process compared to other antigens, and is more specific than binding domains that bind to each of the different antigens, thus preventing the desired molecule from being efficiently recovered.

[0066] It should be understood that in some methods, such as cell display, yeast display, or bacterial display that can use FACS (fluorescence activated cell sorting) for selection, two or more selection pressures on two or more different antigens may be applied simultaneously. However, the inventors believe that in methods such as phage display, ribosome display, mRNA display, or CIS display, where FACS cannot be used, it is difficult to apply two or more selection pressures on two or more different antigens simultaneously. The means to solve the problem

[0067] This invention provides antigen-binding domains that bind to CD3 and CD137, and methods for using them. This invention also provides methods for obtaining antigen-binding domains that bind to two or more antigens more efficiently.

[0068] In some embodiments, the antigen-binding molecule of the present invention comprises an antibody variable region capable of binding to CD3 and CD137 (4-1BB) but not simultaneously to CD3 and CD137, and an antigen-binding molecule comprising a variable region binding to a third antigen different from CD3 and CD137.

[0069] In some embodiments, the antigen-binding molecule of the present invention comprises an antibody variable region capable of binding to T cell receptor and CD137 (4-1BB) but not simultaneously to T cell receptor and CD137; and an antigen-binding molecule comprising a variable region binding to a third antigen different from T cell receptor and CD137.

[0070] In some embodiments, the antigen-binding molecule of the present invention comprises an antibody variable region capable of binding to CD3 and CD137 but not simultaneously binding to CD3 and CD137, and an antigen-binding molecule whose variable region binds to a molecule specifically expressed in cancer tissue.

[0071] In some embodiments, the antigen-binding domain of the present invention can bind to CD3 and CD137 but not simultaneously to the variable regions of CD3 and CD137. In some embodiments, the antibody variable region of the present invention can bind to CD3 and CD137 but not simultaneously to the variable regions of CD3 and CD137.

[0072] In some embodiments, the present invention also provides antigen-binding domains that do not bind to CD3 and CD137 simultaneously, which are variable regions that bind to CD3 and CD137 expressed on different cells at different times.

[0073] In some embodiments, the antigen-binding molecule of the present invention comprises an antibody Fc region. In other embodiments, the antigen-binding molecule of the present invention comprises an antibody Fc region having weakened binding activity against FcγR compared to the Fc region of naturally occurring human IgG1 antibodies.

[0074] In some embodiments, the antigen-binding molecule of the present invention has at least one feature selected from the group consisting of (1) to (4) below: (1) the variable region binds to the extracellular domain of CD3ε (epsilon) containing the amino acid sequence of sequence number: 91; (2) the antigen-binding molecule has activating activity against CD137; (3) the antigen-binding molecule induces CD3 activation of T cells against cells expressing the third antigen, but does not induce activation of T cells against cells expressing CD137; and (4) the antigen-binding molecule does not induce the release of cytokines from PBMCs in the absence of cells expressing the third antigen.

[0075] In some embodiments, the antigen-binding molecule of the present invention has at least one feature selected from the group consisting of (1) to (2) below: (1) the antigen-binding molecule does not compete with the CD137 ligand for binding to CD137, and (2) the antigen-binding molecule induces cytotoxicity of T cells against cells expressing a third antigen, but does not induce cytotoxicity of T cells against cells expressing CD137.

[0076] In some embodiments, the antigen-binding molecule of the present invention competes with antibodies selected from the group consisting of: (a) an antibody comprising a VH sequence having an amino acid sequence of sequence number 30 and a VL sequence having an amino acid sequence of sequence number 51; (b) an antibody comprising a VH sequence having an amino acid sequence of sequence number 46 and a VL sequence having an amino acid sequence of sequence number 53; (c) an antibody comprising a VH sequence having an amino acid sequence of sequence number 40 and a VL sequence having an amino acid sequence of sequence number 56; (d) an antibody comprising a VH sequence having an amino acid sequence of sequence number 30 and a VL sequence having an amino acid sequence of sequence number 58; and (e) an antibody comprising a VH sequence having an amino acid sequence of sequence number 40 and a VL sequence having an amino acid sequence of sequence number 61.

[0077] In some embodiments, the antigen-binding molecule of the present invention comprises an amino acid sequence resulting from the introduction of one or more amino acids into the template sequence consisting of the heavy chain variable domain sequence of sequence number 92 and / or the light chain variable domain sequence of sequence number 93, wherein the one or more amino acids comprise at least one amino acid selected from the following positions: H chain: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 10 0b, 100c, 100d, 100e, 100f, and 100g (Kabat numbers); 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 numbers), wherein the HVR-H3 of the modified heavy chain variable field sequence contains the sequence selected from the following. At least one amino acid: Ala, Pro, Ser, Arg, His, or Thr at amino acid position 98; Ala, Ser, Thr, Gln, His, or Leu at amino acid position 99; Tyr, Ala, Ser, Pro, or Phe at amino acid position 100; Tyr, Val, Ser, Leu, or Gly at amino acid position 100a; Asp, Ser, Thr, Leu, Gly, or Tyr at amino acid position 10. 0b; Val, Leu, Phe, Gly, His, or Ala at amino acid position 100c; Leu, Phe, Ile, or Tyr at amino acid position 100d; Gly, Pro, Tyr, Gln, Ser, or Phe at amino acid position 100e; Tyr, Ala, Gly, Ser, or Lys at amino acid position 100f; Gly, Tyr, Phe, or Val at amino acid position 100g (Kabat number).

[0078] In some embodiments, the antigen-binding molecule of the present invention comprises (a) a VH sequence having at least 95% sequence identity with an amino acid sequence of sequence number 41, 30, 46 or 40; (b) a VL sequence having at least 95% sequence identity with an amino acid sequence of sequence number 51, 52, 53, 54, 55, 56 or 57; or (c) the VH sequence of (a) and the VL sequence of (b).

[0079] In some embodiments, the antigen-binding molecule of the present invention is a monoclonal antibody. In some embodiments, the antigen-binding molecule of the present invention is a human antibody, a humanized antibody, or a chimeric antibody. In other embodiments, the antigen-binding molecule of the present invention is a full-length IgG1, IgG2, IgG3, or IgG4 antibody.

[0080] This invention also provides isolated nucleic acids numbered as antigen-binding molecules of this invention. This invention also provides host cells comprising the nucleic acids of this invention. This invention also provides a method for manufacturing antibodies, comprising culturing the host cells of this invention to manufacture the antibodies.

[0081] The present invention also provides pharmaceutical formulations comprising the antigen-binding molecule of the present invention and a pharmaceutically acceptable carrier.

[0082] The antigen-binding molecule of the present invention can be used as a medicine. The antigen-binding molecule of the present invention can be used to treat various types of cancer.

[0083] The antigen-binding molecule of the present invention can be used in the manufacture of pharmaceuticals. In some embodiments, the pharmaceuticals are used to treat various types of cancer. The present invention also provides methods for treating individuals with various types of cancer. In some embodiments, this method comprises administering an effective amount of the antigen-binding molecule of the present invention to the individual.

[0084] The inventors have successfully prepared an antigen-binding molecule comprising: an antigenic variable region having binding activity against two different antigens (CD3 and CD137) but not simultaneously binding to the two antigens, and a variable region binding to an antigen different from the two antigens (a third antigen), and have found that it causes enhanced activity induced by using an antigen-binding molecule with binding activity against the three different antigens. Furthermore, the inventors have successfully prepared an antigen-binding molecule that avoids cross-linking between different cells caused by the binding of conventional multispecific antigen-binding molecules to antigens expressed on different cells, and believe that when multispecific antigen-binding molecules are used as drugs, they are responsible for adverse reactions.

[0085] The inventors have successfully developed a more efficient method for obtaining antigen-binding domains that bind to two or more different antigens.

[0086] In some embodiments, the screening method of the present invention for antigen-binding domains bound to at least two or more different antigens of interest comprises: (a) providing a library containing a plurality of antigen-binding domains, (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains bound to the first antigen, (c) contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting antigen-binding domains bound to the second antigen, and (d) amplifying the gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains, wherein the method does not include amplifying the nucleic acid encoding the antigen-binding domains collected in step (b) between steps (b) and (c).

[0087] In some embodiments, the antigen-binding domain of the present invention is Fab, scFv, Fab'2, VHH, VH, or VL.

[0088] In some embodiments, the antigen-binding domain of the present invention is a fusion polypeptide, which is formed by fusing an antigen-binding domain with a scaffold to crosslink the antigen-binding domain with a nucleic acid encoding the antigen-binding domain.

[0089] In some embodiments, the scaffold of the present invention is a bacteriophage. In some embodiments, the scaffold of the present invention is a ribosome, RepA protein, or DNA puromycin linker.

[0090] In some embodiments, an extraction solution, such as an acidic solution, an alkaline solution, DTT, or IdeS, is used for extraction in steps (b) and (c) above.

[0091] In some embodiments, the extraction solution used in steps (b) and (c) of the present invention is EDTA or IdeS.

[0092] In some embodiments, the screening method of the present invention for antigen-binding domains bound to at least two or more different antigens of interest includes: (a) providing a library containing a plurality of antigen-binding domains; (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains bound to the first antigen; (c) translating nucleic acids encoding the antigen-binding domains collected in step (b); (d) contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting antigen-binding domains bound to the second antigen; and (e) amplifying the gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains, wherein the method does not include amplifying the nucleic acid encoding the antigen-binding domains collected in step (b) between steps (b) and (c).

[0093] In some embodiments, the method of manufacturing antigen-binding domains of the present invention, which bind to at least two or more different antigens of interest, comprises: (a) providing a library comprising a plurality of antigen-binding domains; (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains bound to the first antigen; (c) contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting antigen-binding domains bound to the second antigen; (d) amplifying a gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; (e) linking a polynucleotide encoding a candidate antigen-binding domain selected in step (d) with a polynucleotide encoding a polypeptide comprising an Fc region; (f) culturing cells introduced into a vector in which the polynucleotides obtained in step (d) are operatively linked; and (g) collecting antigen-binding molecules from the culture solution of the cells cultured in step (f), wherein the method does not include amplifying a nucleic acid encoding the antigen-binding domains collected in step (b) between steps (b) and (c).

[0094] In some embodiments, the library provided in step (a) of the present invention is a design library.

[0095] In some embodiments, the antigen-binding molecule of the present invention is an antibody prepared by the above method.

[0096] More specifically, the present invention relates to the following:

[0097] [1] An antigen-binding molecule comprising: an antibody variable region capable of binding to CD3 and CD137, but not simultaneously binding to CD3 and CD137; and a variable region capable of binding to a third antigen different from CD3 and CD137.

[0098] [2] Such as the antigen-binding molecule in [1], wherein the third antigen is a molecule specifically expressed in cancerous tissue.

[0099] [3] An antigen-binding molecule such as [1] or [2], wherein the variable region that binds CD3 and CD137 at different times binds to CD3 and CD137 at different times, respectively, which are expressed in different cells.

[0100] [4] Any of the antigen-binding molecules in [1] to [3] further includes the antibody Fc region.

[0101] [5] The antigen-binding molecule as in [4], wherein the Fc region is an Fc region with reduced binding activity against FcγR compared to the Fc region of naturally occurring human IgG1 antibodies.

[0102] [6] An antigen-binding molecule of any one of [1] to [5], wherein the antigen-binding molecule has at least one feature selected from the group consisting of (1) to (4) below: (1) the variable region binds to the extracellular domain of CD3ε (epsilon) containing the amino acid sequence of sequence number: 91; (2) the antigen-binding molecule has activating activity against CD137; (3) the antigen-binding molecule induces CD3 activation of T cells against cells expressing the third antigen, but does not induce CD3 activation of T cells against cells expressing CD137; and (4) the antigen-binding molecule does not induce the release of cytokines from PBMCs in the absence of cells expressing the third antigen.

[0103] [7] An antigen-binding molecule of any one of [1] to [6] competes with an antibody selected from the group consisting of: (a) an antibody comprising a VH sequence having an amino acid sequence of sequence number 30 and a VL sequence having an amino acid sequence of sequence number 51; (b) an antibody comprising a VH sequence having an amino acid sequence of sequence number 46 and a VL sequence having an amino acid sequence of sequence number 53; (c) an antibody comprising a VH sequence having an amino acid sequence of sequence number 40 and a VL sequence having an amino acid sequence of sequence number 56; (d) an antibody comprising a VH sequence having an amino acid sequence of sequence number 30 and a VL sequence having an amino acid sequence of sequence number 58; and (e) an antibody comprising a VH sequence having an amino acid sequence of sequence number 40 and a VL sequence having an amino acid sequence of sequence number 61.

[0104] [8] An antigen-binding molecule as described in any of [1] to [7], comprising an amino acid sequence resulting from the introduction of one or more amino acids into a template sequence consisting of the heavy chain variable domain sequence of sequence number 92 and / or the light chain variable domain sequence of sequence number 93, wherein the one or more amino acids comprise at least one amino acid selected from the following positions: H chain: 31, 52b, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 1 00b, 100c, 100d, 100e, 100f, and 100g (Kabat numbers); 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 numbers), wherein the HVR-H3 of the altered heavy chain domain sequence contains the sequence selected from the following. One less amino acid: Ala, Pro, Ser, Arg, His, or Thr at amino acid position 98; Ala, Ser, Thr, Gln, His, or Leu at amino acid position 99; Tyr, Ala, Ser, Pro, or Phe at amino acid position 100; Tyr, Val, Ser, Leu, or Gly at amino acid position 100a; Asp, Ser, Thr, Leu, Gly, or Tyr at amino acid position 100a. b; Val, Leu, Phe, Gly, His, or Ala at amino acid position 100c; Leu, Phe, Ile, or Tyr at amino acid position 100d; Gly, Pro, Tyr, Gln, Ser, or Phe at amino acid position 100e; Tyr, Ala, Gly, Ser, or Lys at amino acid position 100f; Gly, Tyr, Phe, or Val at amino acid position 100g (Kabat number).

[0105] [9] An antigen-binding molecule of any one of [1] to [8] comprises (a) a VH sequence having at least 95% sequence identity with an amino acid sequence of sequence number 41, 30, 46 or 40; (b) a VL sequence having at least 95% sequence identity with an amino acid sequence of sequence number 51, 52, 53, 54, 55, 56 or 57; or (c) the VH sequence of (a) and the VL sequence of (b).

[0106]

[10] A pharmaceutical composition comprising an antigen-binding molecule of any one of [1] to [9] and a pharmaceutically acceptable carrier.

[0107]

[11] A method for screening antigen-binding domains that bind at least two or more different antigens of interest, comprising: (a) providing a library comprising a plurality of antigen-binding domains, (b) contacting the library provided in step (a) with a first antigen of interest and collecting the antigen-binding domains that bind to the first antigen, (c) contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting the antigen-binding domains that bind to the second antigen, and (d) amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains, wherein the method does not include amplifying the nucleic acids encoding the antigen-binding domains collected in step (b) between step (b) and step (c).

[0108]

[12] The method of

[11] , wherein the antigen-binding domain is a fusion polypeptide formed by cross-linking an antigen-binding domain with a nucleic acid encoding the antigen-binding domain by fusing the antigen-binding domain with a scaffold.

[0109]

[13] The method of

[12] , wherein the scaffold is a phage.

[0110]

[14] The method of

[12] , further comprising, between step (b) and (c), a step of translating the nucleic acid encoding the antigen-binding domain collected in step (b).

[0111]

[15] The method of

[12] or

[14] , wherein the scaffold is a ribosome, a RepA protein, or a DNA puromycin linker. Advantages of the Invention

[0112] Brief Description of the Drawings

[0113] FIG. 1 is a conceptual diagram of an antibody that binds to CD3 and CD137 but does not bind to these antigens simultaneously.

[0114] FIG. 2 is a conceptual diagram of an antibody that does not cause cross-linking because the antibody does not bind to CD3 and CD137 simultaneously. In contrast, a trifunctional antibody against CD3, CD137, and a third antigen causes cross-linking of T cells and CD137-positive cells.

[0115] FIG. 3 is a conceptual diagram of an antibody that binds to CD3 and CD137 but does not connect two cells simultaneously.

[0116] Figure 4 is a conceptual schematic diagram of an antibody that crosslinks positive cells for the third antigen to T cells expressing CD3 and CD137.

[0117] Figure 5 is a conceptual schematic diagram of an antibody that crosslinks positive cells for the third antigen to cells expressing CD137.

[0118] Figure 6 is a schematic diagram of the design and construction process of the dual scFv VH ribosome display library.

[0119] Figure 7 shows the ELISA results of the pure strains obtained by ribosome display of CD3 and CD137. The Y-axis represents the specificity for CD137-Fc, and the X-axis represents the specificity of each pure strain for CD3. The black-stained pure strains were identified as positive scFvs showing binding to both CD137 and CD3.

[0120] Figure 8 shows the results of ECL analysis of IgG obtained by ribosome display of CD3 and CD137. The Y-axis represents the response to both CD137, CD3, and the disc itself.

[0121] Figure 9 shows the ELISA results of pure strains obtained from ribosome display of CD3 and CD137. The Y-axis represents the specificity of each pure strain for CD137-Fc, and the X-axis represents the specificity of each pure strain for CD3. Campaign 3 refers to the panning of the ribosome display library with dual rounds of selection.

[0122] Figure 10 shows the ELISA results of the pure strains obtained by displaying the ribosomes of CD3 and CD137. The Y-axis represents the specificity of each pure strain for CD137-Fc, and the X-axis represents the specificity of each pure strain for CD3.

[0123] Figure 11 shows the results of ELISA for IgG obtained by ribosome display of CD3 and CD137. The Y-axis represents the specificity of each pure strain for CD137-Fc, and the X-axis represents the specificity of each pure strain for CD3.

[0124] Figure 12 is a schematic diagram of the design of the dual scFv VL ribosome display library and the dual Fab VL ribosome display library.

[0125] Figure 13 shows the ELISA results of IgG obtained by maturation with ribosome affinity for CD3 and CD137. The Y-axis represents the specificity of each pure strain for CD137-Fc, and the X-axis represents the specificity of each pure strain for CD3.

[0126] Figure 14 shows the results of a competitive ELISA of IgG obtained by maturation with ribosome-displaying affinity for CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. Excess human CD3 or human Fc is used as a competitor.

[0127] Figure 15 shows the design of C3NP1-27, where the CD3ε peptide antigen is biotin-labeled via a disulfide linker.

[0128] Figure 16 shows the results of phage ELISA of pure strains obtained by displaying phages of CD3 and CD137. The Y-axis represents the specificity of each pure strain to CD137-Fc, and the X-axis represents the specificity of each pure strain to CD3.

[0129] Figure 17 shows the results of phage ELISA for pure strains obtained by displaying phages of CD3 and CD137. The Y-axis represents the specificity for CD137-Fc in bead ELISA and the X-axis represents the specificity for CD3 in disc ELISA, as shown in Figure 16 for each pure strain.

[0130] Figure 18 shows comparative data of human CD137 amino acid sequences and cynomolgus monkey CD137 amino acid sequences.

[0131] Figure 19 shows the ELISA results of IgG obtained by phage display of CD3 and CD137. The Y-axis represents the specificity of each pure strain to cynomolgus monkey CD137-Fc, and the X-axis represents the specificity of each pure strain to human CD137.

[0132] Figure 20 shows the ELISA results of IgG obtained by phage display of CD3 and CD137. The Y-axis indicates the specificity for CD3e.

[0133] Figure 21 shows the results of a competitive ELISA for IgG obtained by phage display of CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. Excess human CD3 or human Fc is used as a competitor.

[0134] Figure 22 shows the results of phage ELISA for phage display and panning output pools of CD3 and CD137. The Y-axis represents the specificity for human CD137 (Figure 22A), cynomolgus monkey CD137 (Figure 22B), and CD3 (Figure 22C). The X-axis represents the panning output pools. The primary pool is the pool before phage display and panning, and R1 to R6 represent the panning output pools after phage display and panning rounds 1 to 6, respectively.

[0135] Figure 23 shows the ELISA results of IgG obtained by phage display of CD3 and CD137. The Y-axis represents the specificity of each pure strain for human CD137-Fc, and the X-axis represents the specificity of each pure strain for human CD137 or CD3.

[0136] Figure 24 shows the ELISA results of IgG obtained by phage display of CD3 and CD137. The Y-axis represents the specificity of each pure strain for human CD137-Fc, and the X-axis represents the specificity of each pure strain for human CD137 or CD3.

[0137] Figure 25 shows the competitive ELISA results of IgG obtained by phage display of CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. Excess human CD3 or human Fc is used as a competitor.

[0138] Figure 26 shows the ELISA results of IgG obtained by phage display of CD3 and CD137 to identify the antigenic determinant domains of each pure strain. The Y-axis represents the ELISA response to each domain of human CD137.

[0139] Figure 27 shows the ELISA results of IgG obtained by maturing with phage display affinity for CD3 and CD137. The Y-axis represents the specificity of each pure strain for human CD137-Fc, and the X-axis represents the specificity of each pure strain for human CD137 or CD3.

[0140] Figure 28 shows a set of competitive ELISA results for IgG obtained by phage display of CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. Excess human CD3 is used as a competitor.

[0141] Figure 29B shows the results of evaluating the CD137-mediated agonist activity of various anti-human GPC3 / double Fab antibodies by assessing the level of IL-6 production secreted by self-activated B cells. Ctrl represents the negative control human IgG1 antibody. Figure 29A shows the IL-6 secretion mechanism of self-activated B cells via anti-human GPC3 / double Fab antibody.

[0142] Figure 30B shows the results of evaluating the CD3-mediated activating activity of various anti-human GPC3 / double Fab antibodies by assessing the level of luciferase production in activated Jurkat T cells. Ctrl represents the negative control human IgG1 antibody. Figure 30A shows the mechanism of luciferase expression in activated Jurkat T cells via anti-human GPC3 / double Fab antibodies.

[0143] Figure 31 shows the results of assessing the release of cytokines (IL-2, IFN-γ, and TNF-α) from human PBMC-derived T cells in the presence of various immobilized antibodies. The Y-axis represents the concentration of each secreted cytokine, and the X-axis represents the concentration of the immobilized antibody. Control anti-CD137 antibody (B), control anti-CD3 antibody (CE-115), negative control antibody (Ctrl), and one of the two antibodies (L183L072) were used for the assay.

[0144] Figure 32 shows the results of evaluating T-cell dependent cellular cytotoxicity (TDCC) against GPC3-positive target cells (SK-pca60 and SK-pca13a) using various bispecific antibodies. The Y-axis represents the proportion of cell growth 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 in this assay. Five-fold doses of effector (E) cell lines were added to tumor (T) cells (ET5).

[0145] Figure 33 shows the design and construction process of trispecific antibodies (mAb AB).

[0146] Figure 34 shows the naming principles of the prepared trispecific antibodies.

[0147] Figure 35 shows the results of Biacore analysis of simultaneous binding of GPC3 / CD137xCD3 trispecific antibodies and anti-GPC3 / bis-Fab antibodies. The Y-axis represents the binding response to each antigen. Initially, human CD3 (hCD3) was used as the analyte, and then hCD3 (shown as dashed lines) or a mixture of human CD137 (hCD137) and hCD3 (shown as solid lines) was also used as the analyte.

[0148] Figure 36 shows a sensor array illustrating the FACS analysis results of various antibodies on CD137-positive CHO cells or Jurkat cells. Figures 36(a) and 36(c) show the results of binding to human CD137-positive CHO cells, and Figures 36(b) and 36(d) show the results of binding to parental CHO cells. In Figures 36(a) and 36(b), solid lines show the results of anti-GPC-3 / double antibody (GC33 / H183L072), and filled-in lines show the results of the control antibody (Ctrl). In Figures 36(c) and 36(d), solid lines, dark gray filled-in lines, and light gray filled-in lines show the results of GPC3 / CD137xCtrl trispecific antibody, GPC3 / CD137xCD3 trispecific antibody, and Ctrl / CtrlxCD3 trispecific antibody, respectively. Figures 36(e) and 36(f) show the results of binding to Jurkat CD3-positive cells. In Figure 36(e), the solid line and filled area show the results of anti-GPC3 / double antibody (GC33 / H183L072) and control antibody (Ctrl), respectively. In Figure 36(f), the solid line, dark gray filled area, and light gray filled area show the results of GPC3 / CtrlxCD3 trispecific antibody, GPC3 / CD137xCD3 trispecific antibody, and Ctrl / CD137xCtrl trispecific antibody, respectively.

[0149] Figure 37 shows the results of evaluating the CD3-mediated activating activity of various antibodies on GPC3-positive target cells SK-pca60 by assessing the level of luciferase production in activated Jurkat T cells. Six tri-specific antibodies, anti-GPC3 / double Fab antibody (GPC3 / H183L072), and control / double Fab antibody (Ctrl / H183L072) were used in this assay. The X-axis represents the concentration of each antibody used.

[0150] Figure 38 shows the results of assessing the CD3-mediated activating activity of various antibodies on human CD137-positive CHO cells and parental CHO cells by evaluating the level of luciferase production expressed in activated Jurkat T cells. Six tri-specific antibodies, anti-GPC3 / double Fab antibody (GPC3 / H183L072), and control / double Fab antibody (Ctrl / H183L072) were used in this assay. The X-axis represents the concentration of each antibody used.

[0151] Figure 39 shows the results of assessing the release of intercytokines (IL-2, IFN-γ, and TFN-α) from human PBMCs in the presence of various soluble antibodies. The Y-axis represents the concentration of each secreted intercytokine, and the X-axis represents the concentration of the antibody used. Ctrl / CD137xCD3 trispecific antibody and control / double Fab antibody (Ctrl / H183L072) were used in this assay.

[0152] Figure 40 shows the cell ELISA results of CE115 against CD3e.

[0153] Figure 41 shows the molecular form of EGFR_ERY22_CE115.

[0154] Figure 42 shows the results of TDCC (SK-pca13a) for EGFR_ERY22_CE115.

[0155] Figure 43 is an illustrative sensor plot showing a proportion with binding amounts below 0.8. The vertical axis represents the RU value (response). The horizontal axis represents time. Implementation

[0156] In one embodiment, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising an antibody variable region that can bind to CD3 and CD137 (4-1BB) but not simultaneously to CD3 and CD137, and a variable region that binds to a third antigen different from CD3 and CD137.

[0157] In one embodiment, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising an antibody variable region that can bind to T cell receptor and CD137 (4-1BB) but not simultaneously to T cell receptor and CD137, and a variable region that binds to a third antigen different from T cell receptor and CD137.

[0158] In one embodiment, the antigen-binding molecule of the present invention comprises an antibody variable region that can bind to CD3 and CD137 but not simultaneously to CD3 and CD137, and a variable region that binds to molecules specifically expressed in cancer tissue.

[0159] In one state, the antigen-binding domain of the present invention is a variable region that can bind to CD3 and CD137 but not simultaneously to both CD3 and CD137. In one state, the antibody variable region of the present invention is a variable region that can bind to CD3 and CD137 but not simultaneously to both CD3 and CD137.

[0160] In some embodiments, the antigen-binding molecule of the present invention can activate T cells through its activating activity against CD3, and can induce cytotoxicity of T cells against target cells. Furthermore, it enhances T cell activation, survival, and differentiation into memory T cells through its co-stimulatory activating activity against CD137 and CD3. Simultaneously, because it does not bind to both CD3 and CD137 simultaneously, the antigen-binding molecule of the present invention can avoid adverse events caused by the cross-linking of CD137 and CD3.

[0161] In some embodiments, the antigen-binding molecule of the present invention can also activate CD137-expressing immune cells and enhance the immune response to target cells by activating CD137.

[0162] In this invention, "antibody variable region" generally refers to a region comprising a domain consisting of four framework regions (FRs) and three complementarity-determining regions (CDRs) flanked thereto, and also includes a portion of its sequence, provided that the portion of the sequence has the activity of binding to a partial or complete antigen. Preferably, it comprises a region containing both a light chain variable domain (VL) and a heavy chain variable domain (VH). The antibody variable region of this invention can have any sequence and can be a variable region derived from any antibody, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, and humanized antibodies obtained by humanizing such non-human antibodies, as well as human antibodies. "Humanized antibody," also known as a reshaped human antibody, is obtained by grafting the complementarity-determining region (CDR) of a non-human mammalian-derived antibody, such as a mouse antibody, onto the CDR of a human antibody. The method for identifying CDRs is known in the relevant technical field (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md; and Chothia et al., Nature (1989) 342:877). The aforementioned general gene recombination scheme is also known in the relevant technical field (see European Patent Publications EP 125023 and WO 96 / 02576).

[0163] The "antibody variable region" in the present invention's "not simultaneously binding to CD3 and CD137 (4-1BB)" means that the antibody variable region of the present invention cannot bind to CD137 in a state bound to CD3, and conversely, the variable region cannot bind to CD3 in a state bound to CD137. Throughout this text, the term "not simultaneously binding to CD3 and CD137" also includes cross-linking CD3-expressing cells to CD137-expressing cells, or not simultaneously binding to CD3 and CD137 expressed on different cells. This term further includes situations where CD3 and CD137 are not expressed on the cell membrane, like soluble proteins, or both are located on the same cell, and the variable region can simultaneously bind to both CD3 and CD137, but cannot simultaneously bind to CD3 and CD137 expressed on different cells. Such antibody variable regions are not particularly limited, as long as the antibody variable region has these functions. Examples may include variable regions derived from IgG-type antibody variable regions that bind to a desired antigen by altering some of their amino acids. The amino acid to be modified is selected from the variable region of an antibody that binds to CD3 or CD137, for example, an amino acid whose modification does not cancel the binding to the antigen.

[0164] Throughout this text, the phrase "expressed in different cells" means only that the antigen is expressed in separate cells. This combination of cells can be, for example, cells of the same type, such as T cells and another T cell, or cells of different types, such as T cells and NK cells.

[0165] In this invention, a single amino acid can be modified, or multiple amino acid modifications can be used in combination.

[0166] In cases where multiple amino acid changes are used in combination, the number of changes to be combined is not particularly limited and can be suitably set within a range that achieves the objectives of the present invention. The number of changes to be combined is, for example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, or 2 or more and 3 or less.

[0167] The multiple amino acid modifications to be combined can be added only to the antibody heavy chain variable domain or the light chain variable domain, or can be appropriately distributed in both the heavy chain variable domain and the light chain variable domain.

[0168] One or more amino acid residues in the variable region may be accepted as the amino acid residues to be modified, provided that antigen-binding activity is maintained. In the case of modifying the amino acids in the variable region, the resulting variable region preferably maintains the binding activity of the corresponding unmodified antibody, and preferably has, for example, 50% or more, more preferably 80% or more, and even more preferably 100% or more, the binding activity before modification, but the variable region according to the invention is not limited thereto. Binding activity can be increased by modifying the amino acids, and can be, for example, 2, 5, or 10 times, the binding activity before modification.

[0169] Examples of preferred regions for amino acid modification include solvent-exposed regions and rings within the variable region. Preferably, these include CDR1, CDR2, CDR3, FR3, and the ring. Specifically, preferred regions are Kabat positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the H-chain variable domain and Kabat positions 24 to 34, 50 to 56, and 89 to 97 in the L-chain variable domain. More preferably, these are Kabat positions 31, 52a to 61, 71 to 74, and 97 to 101 in the H-chain variable domain and Kabat positions 24 to 34, 51 to 56, and 89 to 96 in the L-chain variable domain. Furthermore, amino acids that increase antigen-binding activity can be introduced simultaneously with amino acid modification.

[0170] As used herein, the term "hypervariable region" or "HVR" refers to a region in the sequence that is highly variable ("complementarity determining region" or "CDR") and / or forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact"). Generally, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0171] The exemplary HVRs described in this article include: (a) highly variable rings occurring at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), 26–32 (H1), 53–55 (H2), and 96–101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901–917 (1987)); and (b) CDRs occurring at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., Sequence of Proteins of Immunological Interest, 5th Ed. Public Health Service, National of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J.Mol.Biol.262:732-745 (1996)); (d) combinations of (a), (b), and / or (c), including HVR amino acid residues 46 to 56 (L2), 47 to 56 (L2), 48 to 56 (L2), 49 to 56 (L2), 26 to 35 (H1), 26 to 35b (H1), 49 to 65 (H2), 93 to 102 (H3), and 94 to 102 (H3).

[0172] Unless otherwise specified, in this paper, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered according to Kabat et al., as above.

[0173] In this invention, "ring" refers to a region containing residues that are not involved in maintaining the barrel-shaped structure of immunoglobulin β.

[0174] In this invention, amino acid alteration refers to substitution, deletion, addition, insertion, or modification, or a combination thereof. In this invention, amino acid alteration can be used interchangeably with amino acid mutation and has the same meaning.

[0175] The substitution of amino acid residues is altered by replacing them with another amino acid residue, for example, as described in (a) to (c) below: (a) the polypeptide backbone structure of regions having a plate-like or helical structure; (b) the charge or hydrophobicity of the target site; and (c) the size of the side chain.

[0176] Based on the usual side chain properties, amino acid residues are classified into the following groups: (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.

[0177] The substitution of amino acid residues within each group is called conservative substitution, while the substitution of amino acid residues in one group by amino acid residues in another group is called non-conservative substitution.

[0178] The substitutions according to the present invention can be conservative substitutions or non-conservative substitutions. Alternatively, conservative substitutions and non-conservative substitutions can be combined.

[0179] The alteration of amino acid residues also includes: obtaining from random alterations of amino acids that do not cancel binding to antigens by means of such alterations; selecting variable regions in antibody variable regions that can bind to both CD3 and CD137 but cannot simultaneously bind to such antigens; and inserting peptides previously known to have binding activity against the desired antigen into the aforementioned regions.

[0180] In the variable region of the antibody of the present invention, the above-mentioned modifications can be combined with modifications known in the art. For example, the modification of the N-terminal glutamine in the variable region to pyroglutamylation is a modification known to those skilled in the art. Therefore, the antibody of the present invention having glutamine at the N-terminus of its heavy chain may contain a variable region having this N-terminal glutamine modified to pyroglutamylation.

[0181] This antibody variable region can be further modified, for example, by altering amino acids related to antigen binding, pharmacokinetics, stability, or antigenicity. The antibody variable region of the present invention can be modified to have pH-dependent binding activity against the antigen, thereby enabling repeated binding to the antigen (WO2009 / 125825).

[0182] Furthermore, amino acid alterations that change antigen-binding activity based on the concentration of target tissue-specific compounds can be added to, for example, the variable region of an antibody that binds to a third antigen (WO2013 / 180200).

[0183] The variable region can be further modified, for example, to enhance binding activity, improve specificity, reduce pI, impart pH-dependent antigen-binding properties, improve the thermal stability of binding, improve solubility, improve stability against chemical modifications, improve heterogeneity derived from glycans, avoid T cell antigenic determinants identified by computer (in silico) prediction or in vitro T cell-based assays to reduce immunogenicity, or introduce T cell antigenic determinants to activate regulatory T cells (mAbs 3:243-247, 2011).

[0184] Whether the antibody variable region of the present invention can "bind to CD3 and CD137" can be determined by methods known in the art.

[0185] This can be determined by, for example, the electrochemiluminescence method (ECL method) (BMC Research Notes 2011, 4:281).

[0186] Specifically, for example, a low-molecular-weight antibody consisting of the Fab region of a biotinylated antigen-binding molecule to be tested (e.g., an antibody lacking one of the two Fab regions typically found in general antibodies) is mixed with CD3 or CD137 labeled with a sulfur tag (ruthenium complex), and this mixture is added to a streptomycin-immobilization disk. In this operation, the biotinylated antigen-binding molecule to be tested binds to the streptomycin in the disk. Light is emitted from the sulfur tag, and the luminescence signal can be detected using a Sector Imager 600 or 2400 (MSD KK) to confirm the binding of the aforementioned antigen-binding molecule's region to CD3 or CD137.

[0187] Alternatively, this assay can be performed using ELISA, FACS (fluorescence activated cell sorting), ALPHAScreen (amplified luminescent proximity homogeneous assay screen), or the BIACORE method based on surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0188] Specifically, this measurement can be performed using an interaction analyzer such as Biacore (GE Health Japan Corp.) based on surface plasma resonance (SPR) phenomena. Biacore analyzers include any model such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any sensing chip used with Biacore, such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chips, can be used as the sensing chip. Proteins used to capture the antigen-binding molecules of the present invention, such as protein A, protein G, protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human L-chain antibody, anti-human Fc antibody, antigenic protein, or antigenic peptide, are immobilized onto the sensing chip by coupling methods such as amine coupling, disulfide coupling, or aldehyde coupling. CD3 or CD137 is injected thereon as an analyte, and the interaction is measured to obtain a sensing map. In this operation, the concentration of CD3 or CD137 can be selected in the range of several μM to several pM, depending on the interaction strength (e.g., KD) of the sample being measured.

[0189] Alternatively, CD3 or CD137 can be immobilized on the sensing chip in place of the antigen-binding molecule, allowing the antibody sample to be evaluated to interact with it. Whether the antibody variable region of the antigen-binding molecule of the present invention has binding activity against CD3 or CD137 can be confirmed based on the dissociation constant (KD) calculated from the sensing map of the interaction or based on the degree of increase in the sensing map of the layer after the antigen-binding molecule sample has been treated compared to before treatment.

[0190] The ALPHA technique, using two types of beads (donor and recipient), implements the ALPHAScreen method based on the following principle: luminescence is detected only when the two beads are brought close together through interactions between molecules bound to the donor bead and molecules bound to the recipient bead. The laser-excited photosensitizer in the donor bead converts ambient oxygen into excited singlet oxygen. This singlet oxygen diffuses around the donor bead and contacts the nearby recipient bead, thereby inducing chemiluminescence within the bead, which ultimately emits light. When interactions between molecules bound to the donor and recipient beads are absent, the singlet oxygen generated by the donor bead does not contact the recipient bead. Therefore, no chemiluminescence reaction occurs.

[0191] One of the substances whose interaction is to be observed (the ligand) is immobilized on a thin gold film of the sensing chip. The sensing chip is backlit, causing total internal reflection at the interface between the gold film and the glass. Therefore, a point of decrease in the reflected intensity (SPR signal) is formed on a portion of the reflected light. The other substance whose interaction is to be observed (the analyte) is injected onto the surface of the sensing chip. Once the analyte binds to the ligand, the mass of the immobilized ligand molecules increases, changing the refractive index of the solvent on the sensing chip surface. This change in refractive index shifts the position of the SPR (relative to which the dissociation of the bound molecules returns the signal to its original position). The Biacore system plots this shift on an axis representing the mass change on the sensing chip surface, showing the time-dependent mass change as measurement data (sensing map). The amount of analyte bound to the ligand and captured on the sensing chip surface (the change in the sensing map response between before and after the analyte interaction) can be determined by the sensing map. However, since the amount bound also depends on the amount of ligand, comparisons must be performed under conditions where the amount of ligand used is substantially the same. The kinetics, namely the association rate constant (ka) and dissociation rate constant (kd), can be determined from the sensing curve, and the affinity (KD) can be determined from the ratio between these constants. Inhibition assays are also preferred for the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0192] Whether the antigen-binding molecule of the present invention "does not bind to CD3 and CD137 simultaneously" can be verified by: verifying that the antigen-binding molecule has binding activity against both CD3 and CD137; then pre-binding CD3 or CD137 to the antigen-binding molecule containing a variable region having this binding activity; and then determining, by the above method, whether it has binding activity against the other. Alternatively, this can also be verified by determining whether the binding of the antigen-binding molecule to CD3 or CD137 immobilized on an ELISA disc or sensor chip is inhibited by the addition of the other to the solution. In some embodiments, the binding of the antigen-binding molecule of the present invention to CD3 or CD137 is inhibited 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, by the binding of the antigen-binding molecule to the other.

[0193] In one state, when an antigen (e.g., CD3) is immobilized, the inhibition of binding of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by methods known in the art (i.e., ELISA, BIACORE, etc.). In another state, when CD137 is immobilized, the inhibition of binding of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either of the above two states is performed, the antigen-binding molecule of the present invention is determined not to bind to both CD3 and CD137 simultaneously, and the binding is inhibited 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.

[0194] In some embodiments, the concentration of the injected antigen as an analyte is at least 1, 2, 5, 10, 30, 50, or 100 times higher than the concentration of another fixed antigen.

[0195] In a preferred manner, the concentration of the injected antigen as an analyte is 100 times higher than the concentration of the other antigen to be immobilized, and the binding is inhibited by at least 80%.

[0196] In one embodiment, the ratio of the KD value (KD(CD3) / KD(CD137)) of the CD3 (analyte) binding activity of the antigen-binding molecule to the CD137 (immobilized) binding activity of the antigen-binding molecule is calculated. Concentrations of CD3 (analyte) higher than the CD137 (immobilized) concentration that are 10, 50, 100, or 200 times the KD value (KD(CD3) / KD(CD137)) can be used for the above competitive measurement. (For example, when the KD ratio is 0.1, concentrations higher than 1, 10, or 20 times can be selected. Furthermore, when the KD ratio is 10, concentrations higher than 100, 500, 1000, or 2000 times can be selected.)

[0197] In one state, when an antigen (e.g., CD3) is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by methods known in the art (i.e., ELISA, ECL, etc.). In another state, when CD137 is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either of the above two states is performed, the antigen-binding molecule of the present invention is determined not to bind to CD3 and CD137 simultaneously, such that the binding signal is attenuated 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 (see Examples 5-5, 7-5, 8-9, 9-4).

[0198] In some embodiments, the concentration of the injected antigen as an analyte is at least 1, 2, 5, 10, 30, 50, or 100 times higher than the concentration of another fixed antigen.

[0199] In a preferred manner, the concentration of the injected antigen as an analyte is 100 times higher than the concentration of the other antigen to be immobilized, and the binding is inhibited by at least 80%.

[0200] In one embodiment, the ratio of the KD value (KD(CD3) / KD(CD137)) of the CD3 (analyte) binding activity of the antigen-binding molecule to the CD137 (immobilized) binding activity of the antigen-binding molecule is calculated. A CD3 (analyte) concentration higher than the CD137 (immobilized) concentration, which is 10, 50, 100, or 200 times the KD (CD3) / KD(CD137) ratio, can be used for the above competitive measurement. (For example, when the KD ratio is 0.1, concentrations higher than 1, 10, or 20 times can be selected. Furthermore, when the KD ratio is 10, concentrations higher than 100, 500, 1000, or 2000 times can be selected.)

[0201] Specifically, for example, in the case of using the ECL method, a biotin-labeled antigen-binding molecule to be tested, CD3 labeled with a sulfur-tag (ruthenium complex), and unlabeled CD137 are prepared. When the antigen-binding molecule to be tested can bind to both CD3 and CD137, but not simultaneously, a mixture of the antigen-binding molecule to be tested and the labeled CD3 is added to a streptomycin immobilization disk, followed by fluorescence detection. The luminescence signal of the sulfur-tag is detected in the absence of unlabeled CD137. Conversely, in the presence of unlabeled CD137, the luminescence signal decreases. This decrease in luminescence signal can be quantified to determine the relative binding activity. This analysis can be performed similarly using labeled CD137 and unlabeled CD3.

[0202] In the case of an ALPHAScreen, in the absence of competing CD137, the antigen-binding molecule to be tested interacts with CD3 to generate a signal at 520 to 620 nm. Untagged CD137 competes with CD3 for interaction with the antigen-binding molecule to be tested. The fluorescence reduction caused by the competition can be quantified to determine the relative binding activity. Biotinylation of peptides using thio-NHS-biotin, etc., is known in the art. CD3 can be tagged with GST by a suitable method, for example involving: fusing a peptide encoding CD3 in the framework with a peptide encoding GST; and expressing the resulting fusion gene in cells carrying a vector capable of expressing it, followed by purification using a glutathione column. Preferably, the resulting signal is analyzed using software such as GraphPad PRISM (GraphPad Software, Inc., San Diego), which is suitable for a site competition model based on nonlinear regression analysis. This analysis can be performed similarly using tagged CD137 and untagged CD3.

[0203] Alternatively, fluorescence resonance energy transfer (FRET) can be used. FRET is the phenomenon of direct transfer of excitation energy between two nearby fluorescent molecules via electronic resonance. When FRET occurs, the excitation energy of the donor (an excited-state fluorescent molecule) is transferred to the recipient (another fluorescent molecule located near the donor), causing the fluorescence emitted by the donor to disappear (more precisely, the fluorescence lifetime to shorten), and conversely, the recipient to emit fluorescence. By using this phenomenon, it is possible to analyze whether simultaneous binding to CD3 and CD137 is observed. For example, when CD3 carrying a fluorescent donor and CD137 carrying a fluorescent recipient simultaneously bind to the antigen-binding molecule to be tested, the fluorescence of the donor disappears and the fluorescence of the recipient is emitted. Therefore, a change in fluorescence wavelength is observed. This antibody has been shown to bind to both CD3 and CD137 simultaneously. On the other hand, if the mixture of CD3, CD137 and the antigen-binding molecule to be tested does not change the fluorescence wavelength of the fluorescent donor that binds to CD3, the antigen-binding molecule to be tested can be considered to be an antigen-binding domain that can bind to both CD3 and CD137, but not simultaneously to both CD3 and CD137.

[0204] For example, streptomycin allows the biotinylated antigen-binding molecule to be tested to bind to donor beads, while CD3 tagged with glutathione S-transferase (GST) allows binding to recipient beads. In the absence of a competing secondary antigen, the antigen-binding molecule to be tested interacts with CD3 to produce a signal at 520 to 620 nm. An untagged secondary antigen competes with CD3 for interaction with the antigen-binding molecule to be tested. The fluorescence reduction caused by the competition can be quantified to determine relative binding activity. Biotinylation of peptides using thio-NHS-biotin, etc., is known in the art. CD3 can be tagged with GST by appropriate methods, for example, involving: fusing a polynucleotide encoding CD3 in the framework with a polynucleotide encoding GST; and expressing the resulting fusion gene in cells carrying a vector capable of expressing it, followed by purification using a glutathione column. It is preferable to use, for example, software such as GraphPad PRISM (GraphPad Software, Inc., San Diego), which is suitable for one-site competition models based on nonlinear regression analysis, to analyze the obtained signals.

[0205] Tagging is not limited to GST tags and can be done with any tag, such as, but not limited to, histidine tags, MBP, CBP, Flag tags, HA tags, V5 tags, or c-myc tags. The binding of the antigen-binding molecule to be tested to the donor beads is not limited to binding using a biotin-streptomycin reaction. In particular, when the antigen-binding molecule to be tested contains an Fc, possible methods involve allowing the antigen-binding molecule to be tested to recognize proteins, such as protein A or protein G of the donor beads, via the Fc for binding.

[0206] Furthermore, when CD3 and CD137 are not expressed on the cell membrane but are soluble proteins, or when both are present in the same cell, the variable region can bind to both CD3 and CD137 simultaneously but not to CD3 and CD137 expressed in different cells. This situation can also be determined using methods known in the art.

[0207] Specifically, it has been confirmed that the antigen-binding molecule to be tested for a positive ECL-ELISA for simultaneous binding to CD3 and CD137 is mixed with cells expressing CD3 and cells expressing CD137. Unless the antigen-binding molecule and these cells bind to each other simultaneously, the antigen-binding molecule to be tested may show that it cannot simultaneously bind to CD3 and CD137 expressed on different cells. This test can be performed, for example, by a cell-based ECL-ELISA. Cells expressing CD3 are pre-immobilized onto a disc. After the antigen-binding molecule to be tested binds to it, cells expressing CD137 are added to the disc. Different antigens expressed only on cells expressing CD137 are detected using a sulfur-tagged antibody against that antigen. A signal is observed when the antigen-binding molecule binds simultaneously to two antigens individually expressed on two types of cells. No signal is observed when the antigen-binding molecule binds to the antigens separately.

[0208] Alternatively, this test can be performed using the ALPHAScreen method. The antigen-binding molecule to be tested is mixed with cells expressing CD3 that has bound to donor mitochondria and cells expressing CD137 that has bound to recipient mitochondria. A signal is observed when the antigen-binding molecule binds simultaneously to two antigens individually expressed on both types of cells. No signal is observed when the antigen-binding molecule binds to the antigens separately.

[0209] Alternatively, this test can be performed using the octet interaction method. First, a cell line expressing a peptide-tagged CD3 is allowed to bind to a biosensor that recognizes that peptide tag. Cells expressing CD137 and the antigen-binding molecule to be tested are placed in wells, and the interaction is analyzed. When the antigen-binding molecule binds simultaneously to two antibodies expressed on two different cells, a large wavelength shift is observed due to the binding of the antigen-binding molecule to be tested and the cell expressing CD137 to the biosensor. When the antigen-binding molecule does not bind simultaneously to the antigens, a small wavelength shift is observed only due to the binding of the antigen-binding molecule to be tested to the biosensor.

[0210] Methods not based on binding activity can be used for bioactivity-based assays. For example, cells expressing CD3 and cells expressing CD137 are mixed with the antigen-binding molecule to be tested and cultured. When the antigen-binding molecule to be tested binds to both antigens simultaneously, the two antigens individually expressed on the two cells are mutually activated by the antigen-binding molecule to be tested. Therefore, changes in activation signals, such as an increase in the degree of individual downstream phosphorylation of the antigen, can be detected. Alternatively, activation may induce the production of intercytokines. Therefore, the amount of intercytokines produced can be measured to confirm whether simultaneous binding occurs to both cells. Alternatively, activation may induce cytotoxicity against cells expressing CD137. Alternatively, activation may induce the expression of reporter genes by means of promoters downstream of the CD137 or CD3 signaling pathways. Therefore, the amount of cytotoxicity or reporter protein produced can be measured to confirm whether simultaneous binding occurs to both cells.

[0211] In this invention, the term "Fc region" refers to a region comprising a segment consisting of a hinge or portion thereof in an antibody molecule, and CH2 and CH3 domains. For IgG class, the Fc region refers to, but is not limited to, the region from cysteine ​​226 (EU number, also referred to herein as the EU indicator) to the C-terminus or proline 230 (EU number) to the C-terminus. Preferably, the Fc region can be obtained by partially breaking down monoclonal antibodies, such as IgG1, IgG2, IgG3, or IgG4, with a proteolytic enzyme such as pepsin, followed by re-washing of the fraction adsorbed onto a protein A column or protein G column. This proteolytic enzyme is not particularly limited, as long as, under appropriately set reaction conditions (e.g., pH), the enzyme can break down whole antibodies to form Fab or F(ab')2 in a restricted manner. Examples include pepsin and papain.

[0212] In some embodiments, the "antigen-binding molecule" is not particularly limited, as long as the molecule contains the "antibody variable region" of the present invention. The antigen-binding molecule may further comprise a peptide or protein having a length of about 5 or more amino acids. The peptide or protein is not particularly limited to those derived from an organism, and may be, for example, a polypeptide composed of an artificially designed sequence. Furthermore, natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc., may be used.

[0213] In some embodiments, the "antigen-binding molecule" of the present invention is not particularly limited to a molecule containing an "antibody variable region". In some embodiments, antigen-binding molecules that are not antibodies containing a variable region and can bind to two different antigens, such as affibody, can be obtained by methods known to those skilled in the art (PLoS One.2011;6(10):e25791;PLoS One.2012;7(8):e42288;J Mol Biol.2011 Aug 5;411(1):201-19;Proc Natl Acad Sci USA.2011 Aug 23;108(34):14067-72).

[0214] Preferred examples of antigen-binding molecules of the present invention may include antigen-binding molecules comprising the Fc region of an antibody.

[0215] The Fc region derived from, for example, naturally occurring IgG can be used as the "Fc region" of this invention. Herein, naturally occurring IgG refers to a polypeptide containing the same amino acid sequence as naturally occurring IgG and belonging to the antibody type substantially encoded by the immunoglobulin γ gene. Naturally occurring human IgG refers to, for example, naturally occurring human IgG1, naturally occurring human IgG2, naturally occurring human IgG3, or naturally occurring human IgG4. Naturally occurring IgG also includes variants derived spontaneously. In Sequences of proteins of immunological interest, NIH Publication No. 91-3242, multiple allotype sequences based on genotype polymorphism are described as constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, any of which can be used in this invention. In particular, the sequence of human IgG1 may have DEL or EEM as the amino acid sequence at EU number positions 356 to 358.

[0216] The antibody Fc region is found to be, for example, an Fc region of the type IgA1, IgA2, IgD1, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. For example, the Fc region derived from naturally occurring human IgG antibodies can be used as the antibody Fc region of the present invention. For example, the Fc region derived from the constant region of naturally occurring IgG, specifically, the constant region derived from naturally occurring human IgG1 (SEQ ID N: YY004), the constant region derived from naturally occurring human IgG2 (SEQ ID N: YY005), the constant region derived from naturally occurring human IgG3 (SEQ ID N: YY006), or the constant region derived from naturally occurring human IgG4 (SEQ ID N: YY007), can be used as the Fc region of the present invention. The constant region of naturally occurring IgG also includes variants spontaneously derived therefrom.

[0217] The Fc region of this invention is particularly preferably an Fc region with reduced binding activity against the Fcγ receptor. Hereinafter, the Fcγ receptor (also referred to herein as FcγR) means a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4, and implies any member of the protein family substantially encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to: FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; and isoforms FcγRIIa (including alloforms H131 (H type) and R131 (R type)). FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc of FcγRII (CD32); and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIII-NA2); and any undiscovered human FcγR or FcγR isoforms or allotypes. FcγR includes those derived from humans, mice, rats, rabbits, and monkeys. FcγR does not... The term is limited to such molecules and can be derived from any organism. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγR or FcγR isoforms or allotypes. Preferred examples of such FcγR receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).

[0218] FcγR was found to exist in two forms: an activating receptor with an ITAM (immunoreceptor tyrosine-based activation motif) and an inhibitory receptor with an ITIM (immunoreceptor tyrosine-based inhibition motif). FcγR was classified into activating FcγR (FcγRI, FcγRIIaR, FcγRIIaH, FcγRIIIa, and FcγRIIIb) and inhibitory FcγR (FcγRIIb).

[0219] 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 disclosed in BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIb are disclosed in BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIa are disclosed in BC033678.1 and AAH33678.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIb are disclosed in BC128562.1 and AAI28563.1, respectively (RefSeq accession numbers). FcγRIIa exhibits two types of genotypes, with the 131st amino acid of FcγRIIa being replaced by either histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990). FcγRIIb exhibits two types of genotypes, with the 232nd amino acid of FcγRIIb being replaced by either isoleucine (I type) or threonine (T type) (Arthritis. Reheum. 46: 1242-1254 (2002)). FcγRIIIa exhibits two types of genotypes, with either valine (type V) or phenylalanine (type F) replacing the 158th amino acid of FcγRIIIa (J. Clin. Invest. 100(5):1059-1071 (1997). FcγRIIIb exhibits two types of genotypes (NA1 type and NA2 type) (J. Clin. Invest. 85:1287-1295 (1990)).

[0220] The reduced binding activity of Fcγ receptors can be confirmed by conventional methods such as FACS, ELISA format, ALPHAScreen (amplified luminescent proximity homogeneous assay screen), or BIACORE method based on surface plasma resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0221] The ALPHA technique, using two types of beads (donor and recipient), implements ALPHA Screen based on the following principle: luminescence is detected only when the two beads are brought close together through interactions between molecules bound to the donor bead and molecules bound to the recipient bead. The laser-excited photosensitizer in the donor bead converts ambient oxygen into excited singlet oxygen. This singlet oxygen diffuses around the donor bead and contacts the nearby recipient bead, thereby inducing chemiluminescence within the bead, ultimately emitting light. When interactions between molecules bound to the donor and recipient beads are absent, the singlet oxygen generated by the donor bead does not contact the recipient bead. Therefore, no chemiluminescence reaction occurs.

[0222] For example, biotin-tagged antigen-binding molecules are allowed to bind to donor beads, while glutathione S-transferase (GST)-tagged Fcγ receptor systems are allowed to bind to recipient beads. In the absence of competitive antigen-binding molecules with mutant Fc regions, antigen-binding molecules with wild-type Fc regions interact with Fcγ receptors to generate a signal at 520 to 620 nm. Tagged antigen-binding molecules with mutant Fc regions and antigen-binding molecules with wild-type Fc regions compete for interaction with Fcγ receptors. The fluorescence reduction resulting from the competition can be quantified to determine relative binding activity. Biotinylation of antigen-binding molecules such as thio-NHS-biotin is known in the art. Fcγ receptors can be tagged with GST by appropriate methods, for example, involving: fusing a polynucleotide encoding the Fcγ receptor in the framework with a polynucleotide encoding GST; and expressing the resulting fusion gene in cells carrying a vector capable of expression, followed by purification using a glutathione column. The obtained signal is preferably analyzed using, for example, software such as GraphPad PRISM (GraphPad Software, Inc., San Diego), which is suitable for one-site competition models based on nonlinear regression analysis.

[0223] One of the substances whose interaction is to be observed (the ligand) is immobilized on a thin gold film of the sensing chip. The sensing chip is backlit, causing total internal reflection at the interface between the gold film and the glass. Therefore, a point of decrease in the reflected intensity (SPR signal) is formed on a portion of the reflected light. The other substance whose interaction is to be observed (the analyte) is injected onto the surface of the sensing chip. Once the analyte binds to the ligand, the mass of the immobilized ligand molecules increases, changing the refractive index of the solvent on the sensing chip surface. This change in refractive index shifts the position of the SPR (relative to which the dissociation of the bound molecules returns the signal to its original position). The Biacore system plots this shift on an axis representing the mass change on the sensing chip surface, showing the time-dependent mass change as measurement data (sensing map). The amount of analyte bound to the ligand and captured on the sensing chip surface (the change in the sensing map response between before and after the analyte interaction) can be determined by the sensing map. However, since the amount bound also depends on the amount of ligand, comparisons must be performed under conditions where the amount of ligand used is substantially the same. The kinetics, namely the association rate constant (ka) and dissociation rate constant (kd), can be determined from the sensing curve, and the affinity (KD) can be determined from the ratio between these constants. Inhibition assays are also preferred for the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.

[0224] In this specification, reduced binding activity against the Fcγ receptor means that, based on the above analytical methods, the antigen-binding molecule to be tested exhibits, 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 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 binding activity compared to the binding activity of a control antigen-binding molecule containing the Fc region.

[0225] Antigen-binding molecules containing the Fc region of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be appropriately used as control antigen-binding molecules. The Fc region structures are described in sequence number 94 (RefSeq accession No. AAC82527.1 with A-to-N terminal), sequence number 95 (RefSeq accession No. AAB59393.1 with A-to-N terminal), sequence number 96 (RefSeq accession No. CAA27268.1 with A-to-N terminal), or sequence number 97 (RefSeq accession No. AAB59394.1 with A-to-N terminal). In cases where an antigen-binding molecule containing a variant of a certain isotype antibody's Fc region is used as the test substance, an antigen-binding molecule containing the Fc region of this isotype antibody is used as a control to test the effect of mutations in the variant on binding activity against the Fcγ receptor. Therefore, antigen-binding molecules containing Fc region variants that demonstrate reduced binding activity against the Fcγ receptor are appropriately prepared.

[0226] For example, variants of 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 numbers) are known in the art as such variants.

[0227] Preferred examples include antigen-binding molecules having an Fc region derived from an isoantibody substituted with any of the following constituent amino acids: amino acids defined by EU numbers at 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. The isoantibody from which the Fc region is derived is not particularly limited, and Fc regions derived from monoclonal antibodies of IgG1, IgG2, IgG3, or IgG4 may be used appropriately. Fc regions derived from naturally occurring human IgG1 antibodies are preferred.

[0228] For example, antigen-binding molecules with Fc regions may also be appropriately used, wherein the aforementioned Fc region is derived from the Fc region of an IgG1 antibody comprising any of the following substitution groups of amino acids (the numbers indicate the positions of the amino acid residues as defined by EU numbers; the one-letter amino acid code preceding the number indicates the amino acid residue before substitution; and the one-letter amino acid code following the number indicates the amino acid residue after substitution): (a) L234F, L235E, and P331S, (b) C226S, C229S, and P238S, (c) C226S and C229S, and (d) C226S, C229S, E233P, L234V, and L235A.

[0229] Or derived by deletion of amino acid sequences from positions 231 to 238 as defined by EU numbering.

[0230] The Fc region of the antigen-binding molecule may also be used appropriately, wherein the aforementioned Fc region is derived from the Fc region of an IgG2 antibody comprising any of the following substitution groups of amino acids (the numbers indicate the position of the amino acid residue as defined by the EU number; the one-letter amino acid code preceding the number indicates the amino acid residue before substitution; and the one-letter amino acid code following the number indicates the amino acid residue after 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

[0231] Defined according to EU numbering.

[0232] Antigen-binding molecules having an Fc region may also be used, wherein the aforementioned Fc region is derived from the Fc region of an IgG3 antibody comprising any of the following substitution groups of amino acids (the numbers indicate the position of the amino acid residue as defined by the EU number; the one-letter amino acid code preceding the number indicates the amino acid residue before substitution; and the one-letter amino acid code following the number indicates the amino acid residue after substitution): (k) F241A, (l) D265A, and (m) V264A

[0233] Defined according to EU numbering.

[0234] Antigen-binding molecules having an Fc region may also be used appropriately, wherein the aforementioned Fc region is derived from the Fc region of an IgG4 antibody comprising any of the following substitution groups of amino acids (the numbers indicate the position of the amino acid residue as defined by the EU number; the one-letter amino acid code preceding the number indicates the amino acid residue before substitution; and the one-letter amino acid code following the number indicates the amino acid residue after substitution): (n) L235A, G237A and E318A, (o) L235E, and (p) F234A and L235A

[0235] Defined according to EU numbering.

[0236] Other preferred examples include antigen-binding molecules having an Fc region, wherein the aforementioned Fc region is derived from the Fc region of an IgG1 antibody comprising any of the following groups of substitutions of amino acids: amino acids defined by EU numbers at positions 233, 234, 235, 236, 237, 327, 330, and 331, derived from the amino acids at the corresponding EU number positions in the Fc region of the corresponding IgG2 or IgG4.

[0237] Other preferred examples include antigen-binding molecules having an Fc region, wherein the aforementioned Fc region is derived from the Fc region of an IgG1 antibody comprising any of the following groups of substitutions for amino acids: amino acids defined at positions 234, 235, and 297 according to EU designations, by different amino acids. The type of amino acid present after substitution is not particularly limited. Particularly preferred are antigen-binding molecules having an Fc region in which one or more amino acids at positions 234, 235, and 297 are substituted with alanine.

[0238] Other preferred examples include antigen-binding molecules having an Fc region, wherein the aforementioned Fc region is derived from the Fc region of an IgG1 antibody by substitution of the constituent amino acid at position 265 as defined by EU numbering, using different amino acids. The type of amino acid present after substitution is not particularly limited. Particularly preferred are antigen-binding molecules having an Fc region where the amino acid at position 265 is substituted with alanine.

[0239] A preferred form of the "antigen-binding molecule" of the present invention may be, for example, a multispecific antibody comprising the antibody variable region of the present invention.

[0240] The technique of suppressing unwanted associations between H chains by introducing charge repulsion into the interface between the second constant domain (CH2) or the third constant domain (CH3) of the antibody H chain (WO2006 / 106905) can be applied to the association of multispecific antibodies.

[0241] In techniques that suppress undesirable associations between H chains by introducing charge repulsion to the CH2 or CH3 interface, examples of amino acid residues that are in contact with each other at the interface between constant domains of H chains may include residues at EU position 356, EU position 439, EU position 357, EU position 370, EU position 399, and EU position 409 in one CH3 domain, and partner residues in another CH3 domain.

[0242] More specifically, for example, an antibody containing two H-chain CH3 domains can be prepared as an antibody, wherein one to three pairs of amino acid residues selected from the first H-chain CH3 domain, namely (1) to (3), carry the same charge: (1) amino acid residues contained at EU number positions 356 and 439 of the H-chain CH3 domain; (2) amino acid residues contained at EU number positions 357 and 370 of the H-chain CH3 domain; and (3) amino acid residues contained at EU number positions 399 and 409 of the H-chain CH3 domain.

[0243] The antibody can be further prepared into an antibody, wherein one to three pairs of amino acid residues are selected from amino acid residue pairs (1) to (3) in a second H chain CH3 domain that is different from the first H chain CH3 domain, so as to correspond to the amino acid pairs (1) to (3) in the first H chain CH3 domain that have the same charge and have the opposite charge to the amino acid residues that correspond to the first H chain CH3 domain.

[0244] The amino acid residues described in (1) to (3) are located near partners in the associated H chain. Those skilled in the art can use commercially available software to find the positions of the amino acid residues described in (1) to (3) such as the desired H chain CH3 domain or H chain constant domain by means of homology models, and can appropriately change the amino acid residues at these positions.

[0245] In the antibodies described above, the "charged amino acid residues" are preferably selected from, for example, the amino acid residues included in any of the following groups (a) and (b): (a) glutamic acid (E) and aspartic acid (D); and (b) lysine (K), arginine (R) and histidine (H).

[0246] In the aforementioned antibodies, the term "with the same charge" means, for example, that all two or more amino acid residues are amino acid residues included in either group (a) or (b). The term "with opposite charge" means, for example, that at least one of the two or more amino acid residues may be an amino acid residue included in either group (a) or (b), while the remaining amino acid residues are amino acid residues included in other groups.

[0247] In a preferred embodiment, the antibody may have a first H-chain CH3 domain and a second H-chain CH3 domain cross-linked via disulfide bonds.

[0248] The amino acid residues to be modified according to the present invention are not limited to the amino acid residues in the antibody variable region or antibody constant region described above. Those skilled in the art can use commercially available software to find the amino acid residues at the compositional interface of peptide variants or heteropolyploids using homology models, and can modify the amino acid residues at these positions to regulate association.

[0249] The association of the multispecific antibodies of the present invention can also be performed using alternative techniques known in the art. Amino acid side chains present in the variable domains of an antibody H chain are replaced by larger side chains (buttons), and partner amino acid side chains present in the variable domains of other H chains are replaced by smaller side chains (buttons). Buttons can be placed within buttons to efficiently associate peptides with Fc domains of different amino acid sequences (WO1996 / 027011; Ridway JB et al., Protein Engineering (1996) 9, 617-621; and Merchant AM et al., Nature Biotechnology (1998) 16, 677-681).

[0250] In addition to this technique, another alternative technique known in the art can be used to form the multispecific antibody of the present invention. A portion of the CH3 region of an antibody H chain is converted to its corresponding IgA-derived sequence, and the complementary portion of the CH3 region of other H chains is converted to its corresponding IgA-derived sequence. The resulting strand-exchange engineered CH3 region can be used to induce efficient association between polypeptides with different sequences via complementary CH3 association (Protein Engineering Design & Selection, 23; 195-202, 2010). By using this technique known in the art, multispecific antibodies of interest can also be formed efficiently.

[0251] Alternatively, multispecific antibodies can be formed, for example, by using antibody preparation techniques such as CH1-CL association and VH-VL association as described in WO2011 / 028952, by using separately prepared monoclonal antibodies (Fab arm exchange) as described in WO2008 / 119353 and WO2011 / 131746, by using techniques to control association between CH3 domains of antibody heavy chains as described in WO2012 / 058768 and WO2013 / 063702, by using techniques to prepare bispecific antibodies composed of two types of light chains and one type of heavy chain as described in WO2012 / 023053, or by using two bacterial cell lines that each express an antibody half-molecule composed of an H chain and an L chain as described by Christoph et al. (Nature Biotechnology Vol.31, p.753-758 (2013)) to prepare bispecific antibodies. In addition to these association techniques, the CrossMab technique, a conventional hybrid light chain association technique (Scaefer et al., Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192) involving the association of a light chain forming a variable region bound to a first antigenic determinant and a light chain forming a variable region bound to a second antigenic determinant, respectively, to a heavy chain forming a variable region bound to a first antigenic determinant and a heavy chain forming a variable region bound to a second antigenic determinant, can also be used to prepare the multispecific or multiparatopic antigen-binding molecules provided by this invention. Examples of techniques for preparing bispecific antibodies using separately prepared monoclonal antibodies may include methods involving promoting antibody heterodiploidization by placing a monoclonal antibody with a specific amino acid substituted in the CH3 domain of the heavy chain under reducing conditions to obtain the desired bispecific antibody. Preferred amino acid substitution sites for this method may include residues at EU position 392 and EU position 397 in the CH3 domain. Furthermore, bispecific antibodies can also be prepared by using antibodies containing one to three pairs of amino acid residues selected from the following pairs of amino acid residues (1) to (3) in the CH3 domain of the first H chain, which carry the same charge: (1) amino acid residues at EU positions 356 and 439 in the CH3 domain of the H chain; (2) amino acid residues at EU positions 357 and 370 in the CH3 domain of the H chain; and (3) amino acid residues at EU positions 399 and 409 in the CH3 domain of the H chain.Bispecific antibodies can also be prepared by using one to three pairs of amino acid residues selected from amino acid residue pairs (1) to (3) in the CH3 domain of the second H chain, which are different from the CH3 domain of the first H chain, to correspond to amino acid residue pairs (1) to (3) with the same charge in the CH3 domain of the first H chain, and with an opposite charge to the amino acid residues corresponding to the CH3 domain of the first H chain.

[0252] Even if the multispecific antibody of interest cannot be efficiently formed, the multispecific antibody of the present invention can be obtained from the manufactured antibody by isolating and purifying the multispecific antibody of interest. For example, previously reported methods involve introducing amino acid substitutions into the variable domains of two types of H chains to impart different isoelectric points, so that the two types of homodiploid and heterodiploid antibodies of interest can be separated and purified by ion exchange chromatography (WO2007114325). Methods for purifying heterodiploid antibodies composed of mouse IgG2a H chains that can bind to protein A and rat IgG2b H chains that cannot bind to protein A have been reported as methods for purifying heterodiploids (WO98050431 and WO95033844). Alternatively, amino acid residues at EU positions 435 and 436 of the protein A binding site constituting IgG can be substituted with amino acids, such as Tyr and His, which provide different protein A binding strengths, and the resulting H chains are used to modify the interaction between each H chain and protein A. Therefore, only heterodiploidized antibodies can be efficiently purified using protein A columns.

[0253] Multiple, such as two or more, techniques can be used in combination. Furthermore, these techniques can be suitably applied separately to the two H chains to be associated. Based on, but separately from the thus modified type, the antigen-binding molecule of the present invention can be prepared as an antigen-binding molecule having the same amino acid sequence.

[0254] The amino acid sequence can be altered using various methods known in the relevant technical field. Examples of such methods that can be performed include, but are not limited to, site-directed mutations (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-dirested mutagenesis of DNA fragments cloned into M13 vectors. Methods Enxymo. 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 HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Method. Enzymol. 154, 350-367; and Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 82, 488-492), PCR mutation, and cassette mutagenesis methods.

[0255] The "antigen-binding molecule" of the present invention can be either the heavy chain or the light chain contained in a single polypeptide chain constituting the "antibody variable region" of the present invention, but lacking a constant region. Such antibody fragments can be, for example, double-chain antibodies (diabody, Db), single-chain antibodies, or sc (Fab')2.

[0256] The Db system is a diploid composed of two polypeptide chains (e.g., Holliger P et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); EP404,097 and WO93 / 11161). These polypeptide chains can be linked by linkers as short as, for example, about 5 residues, such that the L-chain variable domain (VL) and H-chain variable domain (VH) of the same polypeptide chain cannot pair with each other.

[0257] Because of this short linker, VL and VH, which encode the same polypeptide chain, cannot form a single-chain Fv. Instead, they are diploidized with another polypeptide chain, VH and VL, respectively, to form two antigen-binding sites.

[0258] Examples of single-chain antibodies include sc(Fv)2. sc(Fv)2 is a single-chain antibody having a chain consisting of four variable domains, i.e., two VLs and two VHs linked by a linker, for example, a peptide linker (J Immunol. Methods (1999) 231(1-2), 177-189). These two VHs and VLs can be derived from different monoclonal antibodies. A preferred example includes bispecific sc(Fv)2, which recognizes two types of antigenic determinants present in the same antigen, as described in the Journal of Immunology (1994) 152(11), 5368-5374. sc(Fv)2 can be prepared by methods commonly known to those skilled in the art. For example, sc(Fv)2 can be prepared by linking two scFvs via a linker, for example, a peptide linker.

[0259] Examples of the configurations constituting the antigen-binding domain of sc(Fv)2 described herein include antibodies in which the two VH and two VL lines are aligned as VH, VL, VH, and VL lines aligned (i.e., [VH]-linker-[VL]-linker-[VH]-linker-[VL]), starting from the N-terminus of the single-chain polypeptide in this order. The order of the two VH and two VL lines is not particularly limited to the above configuration and can be any arrangement. Examples may also include the following arrangements: [VL]-connector-[VH]-connector-[VH]-connector-[VL], [VH]-connector-[VL]-connector-[VL]-connector-[VH], [VH]-connector-[VH]-connector-[VL]-connector-[VL], [VL]-connector-[VL]-connector-[VH]-connector-[VH], and [VL]-connector-[VH]-connector-[VL]-connector-[VH].

[0260] The molecular form of this sc(Fv) is also described in detail in WO2006 / 132352. Based on the description in that document, those skilled in the art can appropriately prepare the desired sc(Fv)2 to prepare the antigen-binding molecule disclosed in this invention.

[0261] The antigen-binding molecule of the present invention can be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Furthermore, it is preferable that the glycan chain can be added to the antigen-binding molecule of the present invention by inserting a glycosylation sequence to produce the desired effect.

[0262] For example, any peptide linker introduced by genetic engineering, or a synthetic compound linker (e.g., a linker disclosed in Protein Engineering, 9(3), 299-305, 1996) can be used as a linker for linking the variable domain of an antibody. In this invention, peptide linkers are preferred. The length of the peptide linker is not particularly limited and can be suitably selected by those skilled in the art as appropriate. The length is preferably 5 or more amino acids (the upper limit is not particularly limited, and it is generally 30 or fewer amino acids, preferably 20 or fewer amino acids), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, all such peptide linkers used may have the same length or may have different lengths.

[0263] Examples of peptide linkers may include Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (sequence number: 162). Ser-Gly-Gly-Gly(sequence number: 163), Gly-Gly-Gly-Gly-Ser(sequence number: 164), Ser-Gly-Gly-Gly-Gly(sequence number: 165), Gly-Gly-Gly-Gly-Gly-Ser(sequence number: 166), Ser-Gly-Gly-Gly-Gly-Gly(sequence number: 167), Gly-Gly-Gly-Gly-Gly-Gly-Ser(sequence number: 168), Ser-Gly-Gly-Gly-Gly-Gly-Gly(sequence number: 169), (Gly-Gly-Gly-Gly-Ser(sequence number: 164))n, and (Ser-Gly-Gly-Gly-Gly(sequence number: 165))n, where n is an integer of 1 or greater.

[0264] However, the length or sequence of the peptide linker can be appropriately selected by someone with ordinary knowledge in the relevant technical field, depending on the purpose.

[0265] Synthetic linkers (chemical cross-linking agents) are cross-linking agents commonly used for peptide cross-linking, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), and ethylene glycol bis(sulfosuccinimidyl) Crosslinking agents include succinate, sulfo-EGS, disuccinimidyl tartrate (DST), dithiosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfonate (BSOCOES), or bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfonate (sulfo-BSOCOES). These crosslinking agents are commercially available.

[0266] Typically, three linkers are needed to link the four antibody variable domains. All such linkers used can be the same or different linkers.

[0267] F(ab')2 comprises two light chains and two heavy chains containing constant regions (part of the CH1 and CH2 domains), such that interchain disulfide bonds are formed between the two heavy chains. Preferably, F(ab')2 constituting the polypeptide complex disclosed herein can be obtained by partially cleaving, for example, a whole monoclonal antibody having the desired antigen-binding domain, with a proteolytic enzyme such as pepsin, followed by removal of the Fc fragment adsorbed onto a protein A column. Such proteolytic enzymes are not particularly limited, as long as the enzyme, under appropriately set reaction conditions (e.g., pH), can cleave the whole antibody to restrictively form F(ab')2. Examples include pepsin and ficin.

[0268] In addition to the aforementioned amino acid alterations, the antigen-binding molecule of the present invention may contain further modifications. These additional modifications may be selected from, for example, amino acid substitutions, deletions, modifications, and combinations thereof.

[0269] For example, the antigen-binding molecule of the present invention can be further modified arbitrarily without substantially altering the intended function of the molecule. Such mutations can be achieved, for example, by conserved substitution of amino acid residues. Alternatively, changes can even be made to alter the intended function of the antigen-binding molecule of the present invention, as long as the altered function is within the objectives of the present invention.

[0270] The alteration of the amino acid sequence according to the present invention also includes post-translational modifications. Specifically, post-translational modifications may refer to the addition or deletion of glycans. For example, the antigen-binding molecule of the present invention having an IgG1-type constant region may have a glycan-modified amino acid residue at EU number position 297. The glycan structure used for modification is not particularly limited. Generally, antibodies expressed by eukaryotic cells involve glycan modifications in their constant regions. Therefore, antibodies expressed by cells such as mammalian antibody-producing cells and eukaryotic cells transfected with an expression vector containing antibody-encoding DNA are typically modified with a glycan.

[0271] In this article, eukaryotic cells include yeast and animal cells. For example, CHO cells or HEK293H cells are typical animal cells used for transfection with expression vectors containing antibody-encoding DNA. On the other hand, the antibodies of the present invention also include antibodies lacking glycan modification at this location. Antibodies with constant regions without glycan modification can be obtained by expressing genes encoding such antibodies in prokaryotic cells such as Escherichia coli (E. coli).

[0272] More specifically, an additional modification according to the invention may be, for example, the addition of sialic acid to the sugar chain at the Fc region (mAbs. 2010 Sep-Oct; 2(5): 519-27).

[0273] When the antigen-binding molecule of the present invention has an Fc region, for example, it can be modified by adding amino acid substitutions that target the binding activity against FcRn (J Immunol.2006 Jan1;176(1):346-56;J Biol Chem.2006 Aug 18;281(33):23514-24;Int Immunol.2006 Dec;18(12):1759-69;Nat Biotechnol.2000 Feb;28(2):157-9;WO2006 / 053301;andWO2009 / 086320)) or by adding amino acid substitutions that improve antibody heterogeneity or stability (WO2009 / 041613)).

[0274] In this invention, the term "antibody" is used in the broadest sense and includes any antibody, such as monoclonal antibodies (including holoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, and humanized antibodies, as long as the antibody exhibits the desired biological activity.

[0275] The antibodies of this invention are not limited by the type or source of their antigens, and can be any antibody. Examples of antibody sources may include, but are not particularly limited to, human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

[0276] Antibodies can be prepared using methods known to those skilled in the art. For example, monoclonal antibodies can be prepared using the fusion tumor method (Kohler and Milstein, Nature 256:495 (1975)) or the recombinant method (US Patent No. 4,816,567). Alternatively, monoclonal antibodies can be isolated from a phage-displayed antibody library (Clackson et al., Nature 352:624-628 (1991); and Marks et al., J.Mol.Biol.222:581-597 (1991)). Furthermore, monoclonal antibodies can be isolated from a single pure B cell line (N.Biotechnol.28(5):253-4572011).

[0277] Humanized antibodies are also known as reshaped human antibodies. Specifically, for example, humanized antibodies are composed of antibodies grafted with a non-human animal (e.g., mouse) antibody CDR, a method known in the art. General gene recombination protocols for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is a method known in the art for grafting a mouse antibody CDR onto a human FR.

[0278] DNA encoding antibody variable domains, each containing three CDRs and four FRs, and DNA encoding human antibody constant domains, can be inserted into an expression vector, such that the variable domain DNA and the constant domain DNA are fused within a framework to prepare a vector for humanized antibody expression. These vectors with the inserts are transferred to a host to establish recombinant cells. The recombinant cells are then cultured to express DNA encoding humanized antibodies, producing a culture of humanized antibodies in the cultured cells (see European Patent Publication No. EP239400 and International Patent Publication No. WO1996 / 002576).

[0279] If necessary, the amino acid residues of FR can be substituted so that the CDR of the reconstituted human antibody forms an appropriate antigen-binding site. For example, the amino acid residues of FR can be mutated by applying a PCR method used for grafting mouse CDRs onto human FR.

[0280] Genetically modified animals with all strains of human antibody genes can be used as immunizing animals to obtain the desired human antibodies through DNA immunization (see International Patent Publication Nos. WO1993 / 012227, WO1992 / 0039185, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096 and WO1996 / 033735).

[0281] Furthermore, techniques for obtaining human antibodies through panning using human antibody libraries are also well-known. For example, using phage display methods, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage. Phages that express antigen-binding scFv can be selected. The genes of the selected phages 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 within a framework with the desired human antibody C region sequence and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is transferred to the preferred expression cells listed above to express the gene encoding the human antibody to obtain the human antibody. These methods are known in the art (see International Patent Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438 and WO1995 / 015388).

[0282] Besides phage display technology, techniques such as those using cell-free translation systems, displaying antigen-binding molecules on cell or viral surfaces, and emulsification techniques are known for obtaining human antibodies through panning of human antibody libraries. For example, ribosome display methods involving the formation of mRNA and translated protein complexes via ribosomes by removing stop codons, cDNA or mRNA display methods involving the covalent binding of translated proteins to gene sequences using compounds such as puromycin, and CIS display methods involving the formation of gene and translated protein complexes using nucleic acid-binding proteins are all techniques that can be used with cell-free translation systems. Phage display methods, as well as E. coli display methods, Gram-positive bacteria display methods, yeast display methods, mammalian cell display methods, and virus display methods, can be used as techniques for displaying antigen-binding molecules on cell or viral surfaces. For example, in vitro virus display methods using genes and translation-related molecules contained in emulsions can be used as emulsification techniques. These methods are known in the relevant technical field (Nat Biotechnol.2000 Dec;18(12):1287-92;Nucleic Acids Res.2006;34(19):e27;Proc Natl Acad Sci USA.2004 Mar 2;101(9):2806-10;Proc Natl Acad Sci USA.2004 Jun 22;101(25):9193-8;Protein Eng Des Sel.2008 Apr;21(4):247-55;Pron Natl Acad Sci USA.2000 Sep 26;97(20):10701-5;MAbs.2010 Sep-Oct;2(5):508-18;andMethods Mol Biol.2012;911:183-98).

[0283] The variable region binding to the third antigen in this invention can be a variable region that recognizes any antigen. The variable region binding to the third antigen in this invention can be a variable region that recognizes molecules specifically expressed in cancer tissue.

[0284] In this instruction manual, "third antigen" is not specifically limited and can be any antigen. Examples of antigens include 17-IA, 4Dc, 6-keto-PGFIa, 8-iso-PGF2a, 8-side-oxy-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, adiponectin, ADP-ribosylcyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergens, α1-antichymotrypsin (alpha 1-antichemotrypsin), α1-antitrypsin, α-synuclein, α-V / β-1 antagonists, aminin, amylin, amyloid β, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, androgens, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, apo A1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, artesunate, ASPARTIC, atrial natriuretic peptide factor Factor), atrial diuretic peptide, atrial diuretic peptide A and atrial diuretic peptide B, atrial diuretic peptide C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCAM, BDNF, β-ECGF, β-2-microglobulin, β-endoramicin, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, B-lymphocyte stimulatorStimulator (BIyS), 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), BMPRII (BRK-3), BMPs, BOK, Bombesin, bone-derived neutrophils, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte 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 (CEA), myocardial nutrient-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 / EotaxinCCL12 / 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 / C TACK, CCL28 / MEC, CCL3 / M1P-1-α, CCL31 / LD-78-β, CCL4 / MIP-1-β, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CC L9 / 10 / MTP-1-γ, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c , CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD37, CD38, CD3E, CD4, CD40, CD4 0L, 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, clotting protein 18, CLC, botulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV, CMVUL, CNTF, CNTN-1, Complement Factor 3 (C3), Complement Factor D, Corticosteroid-binding globulin, Community-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-1-α / β, CXCL13 / BCA -1, CXCL14 / BRAK, CXCL15 / Lungkine, CXCL16, CXCL2 / Gro-β, CXCL3 / Gro-γ, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCL10 / IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cystatin C, Cytokeratin Tumor-Associated Antigen (CTA) antigen), DAN, DCC, DcR3, DC-SIGN, decay accelerator, delta-like protein ligand 4, de(1-3)-IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, dipeptidyl peptidase IV, DK1, DNAM-1, deoxyribonuclease, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), protein containing EGF-like domain 7, elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, endothelial sialic acid protein, endothelin receptor, endotoxin, enkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2, eotaxini, EpCAM, EphrinB2 / 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, RSV F protein, F10, F11, F12, F13, F5, F9, factor Ia, factor IX, factor Xa, factor VII, factor VIII, factor VIIIc, Fas, FcαR, FcεRI, FcγIIb, FcγRI, FcγRIIa, FcγRIIIa, FcγRIIIb, F cRn, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic, Fibrin, Fibroblast activating protein (FAP), Fibroblast growth factor, Fibroblast growth factor-10, Reticulin, FL, FLIP, Flt-3, FLT3 ligand, Folic acid receptor, Follicle-stimulating hormone (FSH), Actin (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, gellingin, GFAP, GF-CSF, GFR-α1, GFR-α2, GFR-α3, GF-β1, gH outer membrane glycoprotein, GITR, glucagon, glucagon receptor, glucagon-like peptide-1 receptor, Glut4, fumonisin carboxypeptidase II, glycoprotein hormone receptor, glycoprotein IIb / IIIa (GP IIb / IIIa), phosphatidylinositol glycan-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulosa-CSF (G-CSF), GRO / MGSA, growth hormone-releasing factor, GRO-β, GRO-γ, Helicobacter pylori, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMV gB outer membrane glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep Bgp120, heparinase, heparin cofactor II, liver 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 outer membrane proteins such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human myocardial coagulant, human cytomegalovirus (human cytomegalovirus) cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasmin activator (t-PA), Huntington's protein, 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-1R, 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-16, 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 complexes, immunoglobulin, INF-α, INF-α receptor, INF-β, INF-β receptor, INF-γ, INF-γ receptor, IFN-I, IFN-1 receptor, influenza virus, 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-TAC, JE, kalliklein, kalliklein 11, kalliklein 12, kalliklein 14, kalliklein 15, kalliklein 2, kalliklein 5, kalliklein 6, kalliklein L1, kalliklein L2, kalliklein L3, kalliklein L4, kallistatin, KC, KDR, keratinocyte growth factor (KGF), keratinocyte growth factor-2 (KGF-2), KGF, killer-like immunoglobulin receptor, Kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (amyloid, pancreatic islet amyloid polypeptide), LAP (TGF-1), potential related peptide, potential TGF-1, potential TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, leptin, luteinizing hormone (LBP)LH hormone, 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, lymphocyte chemokine, lymphotoxin β receptor, hemolysin receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, mammary serine protease inhibitor (maspin), MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-1 (MCAF), M-CSF, MDC, MDC (67 amino acids), MDC (69 amino acids), serine protease inhibitor antibody (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, mononuclear spheroid aspirant protein, mononuclear spheroid colony inhibitor factor, mouse gonadotropin-related protein, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Müller's inhibitory substance, Mug, Musk, myelin-related glycoprotein, myeloid progenitor inhibitor factor-1 (MPIF-1), NAIP, nanosomes, NAP, NAP-2, NCA 90, NCAD, N-cadherin, NCAM, enkephalin, neural cell adhesion molecule, neuroserine, neuronal growth factorNeurotrophic factor (NGF), neurotrophic molecule-3, neurotrophic molecule-4, neurotrophic molecule-6, neurotrophic molecule-1, neurotrophin, NGF-β, NGFR, NKG20, N-methionine-based human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural protein type 3 (NS3) from hepatitis C virus, NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, tumor suppressor M, OP-2, OPG, OPN, OSM, OSM receptor, bone-inducing factor, osteopontin, OX 40L, 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 hormone, Placental alkaline phosphatase Alkaline phosphatase (PLAP), placental prolactin, plasminogen activator inhibitor-1 (PAI-1), platelet-growth factor, plcR, PLP, polyol chains of different sizes (e.g., PEG-20, PEG-30, PEG-40), PP14, prokinin-releasing peptide, prion protein, procalcitonin, planned cell death protein 1 (PC-1), proinsulin, prolactin, proprotein convertase PC9 (PC-9), prochaskinin, prostate-specific membrane antigen (PSMA), protein A, protein C, protein D, protein S, protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PIN, P-selectin glycoprotein ligand-1 (P-SELECTEN), R51, RAGE, RANK, RANKL, RANTES, relaxin, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV). Virus (RSV)F, Ret, endoplasmic reticulum protein 4, rheumatoid factor, RLI P76, RPA2, RPK-1, RSK, RSVFgp, S100, RON-8, SCF / KL, SCGF, sclerosingin, SDF-1, SDF1α, SDF1β, SERINE, serum amyloid protein P, serum albumin, sFRP-3, Shh, Shiga toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, ceramine 1-phosphate receptor, Staphylococcus aureus lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptococcal kinase, superoxide dismutase, ligand proteoglycan 1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor α / β, TdT TECK, TEM1, TEM5, TEM7, TEM8, tendinin, TERT, testicular-like PLAP alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β panspecific, TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-βRI (ALK-5), TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thymic stromal lymphoprotein receptor, thymic Ck-1, thyroid stimulating hormone (TSH). TSH, thyroxine, thyroxine-binding protein, Tie, TIMP, TIQ, tissue factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-α, TNF-β, 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 ODFR / TRANCE R), TNFRSF11B (OPG OCIF / TR1), TNFRSF12 (TWEAK R) FN14), TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF14(HVEM ATAR / HveA / LIGHT R / TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ / TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF R1 CD20a / p55-60), TNFRSF1B (TNF RII CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3 / LARD / TR-3 / TRAMP / WSL-1), THFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII / TNFC R), TNFRSF4(OX40 ACT35 / TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6 (Fas Apo-1 / APT1 / CD95), TNFRSF6B (DcR3 M68 / TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137 / ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 ligand / TL2), TNFSF11 (TRANCERANK 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 / VEG1), TNFSF18 (GITR ligand AITR ligand / TL6), TNFSF1A (TNF-α) Conectin / DIF / TNFSF2), TNFSF1B (TNF-b LTa / TNFSF1), TNFSF3 (LTb TNFC / p33), TNFSF4 (OX40 ligand p34TXGP1), 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 (4-1BB ligand CD137 ligand), TNF-α, TNF-β, TNIL-1, toxic metabolites, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factor (TGF) such as TGF-α and TGF-β, transmembrane glycoprotein NMB, transthyretin, TRF, Trk, TROP-2, trophoblast glycoprotein, TSG, TSLP, tumor necrosis factor (TNF), tumor-associated antigen CA125, Lewis Y-related carbohydrates expressing tumor-associated antigens, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VAP-1, vascular endothelial growth factor (VEGF). VEGF factor, visceral fat-specific serine protease inhibitor (vaspin), VCAM, VCAM-1, VECAD, VE-calponin, VE-calponin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, viral antigen, vitamin B12 receptor, vitronectin receptor, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor Factors (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-1-β, XCL1 / lymphocyte chemokine, XCR1, XEDAR, XIAP, and XPD.

[0285] Specific examples of T cell specificity include CD3 and T cell receptors. CD3 is particularly preferred. For example, in the case of human CD3, the binding site of the antigen-binding molecule of the present invention in CD3 can be any antigenic determinant present in the γ, δ, or ε chain constituting human CD3. In particular, antigenic determinants present in the extracellular region of the ε chain in the human CD3 complex are preferred. The polynucleotide sequences constituting the γ, δ, or ε chain structure of CD3 are shown in sequence numbers: 170 (NM_000073.2), 172 (NM_000732.4), and 174 (NM_000733.3), and their polypeptide sequences are shown in sequence numbers: 171 (NP_000064.1), 173 (NP_000723.1), and 175 (NP_000724.1) (RefSeq accession numbers are shown in parentheses).

[0286] The antigen-binding molecule of the present invention includes one of the two variable regions of an antigen that binds to a "third antigen" different from "CD3" and "CD137" described above. In some embodiments, the third antigen is derived from humans, mice, rats, monkeys, rabbits, or dogs. In some embodiments, the third antigen is a molecule specifically expressed in cells or organs derived from humans, mice, rats, monkeys, rabbits, or dogs. Preferably, the third antigen is a molecule that is not systemically expressed in cells or organs. Preferably, the third antigen is, for example, a tumor cell-specific antigen and also includes antigens expressed in conjunction with malignant changes in cells and abnormal glycan chains appearing on the cell surface or protein molecules during malignant transformation of cells. Specific examples include ALK receptor (pleiotropic growth factor receptor), pleiotropic growth factor, KS1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate-specific antigen (PSA), melanoma-associated antigen p97, melanoma antigen gp75, high-molecular-weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, rectal tumor-associated antigens (e.g., CEA, TAG-72, CO17-1A, GICA, CTA-1, and LEA), and Birch's lymphoma antigen 38.13. CD19, human B-cell lymphoma antigen CD20, CD33, melanoma-specific antigens (e.g., ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3), tumor-specific transplantation antigen (TSTA), T antigen, virus-induced tumor antigens (e.g., outer membrane antigens of DNA tumor viruses and RNA tumor viruses), colorectal CEA, carcinoembryonic antigen alpha-fetoprotein (e.g., carcinoembryonic trophoblast glycoprotein 5T4 and carcinoembryonic bladder tumor antigen), differentiation antigens (e.g., human lung cancer antigens L6 and L20), fibrosarcoma antigen, human T-cell leukemia-associated antigen Gp37, neonatal glycoprotein, sphingomyelin, breast cancer antigens (e.g., EGFR (epithelial growth factor receptor)), NY-BR-16, NY-BR-16 and HER2 antigen (p185HER2), polymorphic epithelial mucin. mucin (PEM), malignant human lymphocyte antigen APO-1, differentiation antigens such as I antigen found in fetal erythrocytes, protoendoderm I antigen found in adult erythrocytes, I (Ma) found in pre-implantation embryos or in gastric cancer, M18 and M39 found in mammary epithelial cells, SSEA-1, VEP8, VEP9, Myl, and VIM-D5 found in bone marrow cells, D156-22 and TRA-1-85 (blood group H) found in colorectal cancer, SCP-1 found in testicular and uterine cancer, C14 found in colorectal cancer, F3 found in lung cancer, AH6 found in gastric cancer, Y hapten, and other antigens found in embryonic cancer cells. The following are observed: Ley, TL5 (blood group A), EGF receptor found in A4312 cells, E1 series found in pancreatic cancer (blood group B), FC10.2 found in embryonic cancer cells, gastric cancer antigen, CO-514 found in adenocarcinoma (blood group Lea), NS-10 and CO-43 found in adenocarcinoma (blood group Leb), G49 found in EGF receptor in A431 cells, MH2 (blood group ALeb / Ley) found in colorectal cancer, 19.9 found in colorectal cancer, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2, GD3, and 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 embryos, cutaneous T-cell lymphoma-associated antigen, MART-1 antigen, sialic acid Tn (STn) antigen, colorectal cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, paraneoplastic brain-testicular cancer antigens (oncogenic neuron antigen MA2 and paraneoplastic neuron antigen), neuroncological ventral antigen 2. 2, NOVA2), hematologic malignancy antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigen MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b, MAGE-X2, cancer-testis antigen (NY-EOS-1), YKL-40, and any fragments of these polypeptides, as well as their modified structures (the aforementioned modified phosphate groups, glycans, etc.), EpCAM, EREG, CA19-9, CA15-3, sialic acid SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A.

[0287] The term "CD137" used in this article, also known as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. Examples of factors belonging to the TNF superfamily or TNF receptor superfamily include CD137, CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL.

[0288] In one embodiment, the antigen-binding molecule of the present invention has at least one feature selected from the group consisting of (1) to (4) below: (1) the variable region binds to the extracellular domain of CD3ε (epsilon) containing the amino acid sequence of sequence number: 91; (2) the antigen-binding molecule has activating activity against CD137; (3) the antigen-binding molecule induces CD3 activation of T cells against cells expressing the third antigen, but does not induce activation of T cells against cells expressing CD137; and (4) the antigen-binding molecule does not induce the release of cytokines from PBMCs when it is absent from cells expressing the third antigen.

[0289] In one embodiment, the antigen-binding molecule of the present invention has at least one feature selected from the group consisting of (1) to (4) below: (1) the variable region binds to the extracellular domain of CD3ε (epsilon) containing the amino acid sequence of sequence number: 91; (2) the antigen-binding molecule has activating activity against CD137; (3) the antigen-binding molecule induces cytotoxicity of T cells against cells expressing the third antigen, but does not induce activation of T cells against cells expressing CD137; and (4) the antigen-binding molecule does not induce the release of cytokines from PBMCs when it is absent from cells expressing the third antigen.

[0290] In some embodiments, the antigen-binding molecule of the present invention has at least one feature selected from the group consisting of (1) to (2) below: (1) the antigen-binding molecule does not compete with the CD137 ligand for binding to CD137, and (2) the antigen-binding molecule induces cytotoxicity of T cells against cells expressing a third antigen, but does not induce cytotoxicity of T cells against cells expressing CD137.

[0291] In this invention, the "CD137 agonist antibody" or "antigen-binding molecule having agonist activity against CD137" means, when added to cells, tissues, or living organisms expressing CD137, an antibody or antigen-binding molecule that activates at least about 5%, specifically at least about 10%, or more specifically at least about 15% of the cells expressing CD137, wherein 0% activation represents the background level of inactive cells expressing CD137 (e.g., IL-6 secretion). In various specific examples, the CD137 agonist antibody used as a pharmaceutical component of this invention can activate cell activity 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%.

[0292] In this embodiment, the "CD137 agonist antibody" or "antigen-binding molecule having agonist activity against CD137" of the present invention also means, when added to cells, tissues, or living organisms expressing CD137, an antibody or antigen-binding molecule that activates at least about 5%, specifically at least about 10%, or more specifically at least about 15% of the cells expressing CD137, wherein 100% activation is the level of activation achieved under physiological conditions by an equal molar amount of binding partners. In various specific examples, the CD137 agonist antibody used as a pharmaceutical component of the present invention can activate cell activity 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%. In some embodiments, the term "binding partner" used herein refers to molecules known to bind to CD137 and induce activation of CD137-expressing cells. In other embodiments, examples of binding partners include Urelumab (CAS Registration No. 934823-49-1) disclosed in WO2005 / 035584A1 and its variants, Utomilumab (CAS Registration No. 1417318-27-4) disclosed in WO012 / 032433A1 and its variants, and various known CD137 agonist antibodies. In some embodiments, examples of binding partners include CD137 ligands. In other embodiments, activation of CD137-expressing cells by anti-CD137 agonist antibodies can be determined using ELISA to characterize IL6 secretion (see, for example, Examples 10-2 herein). The anti-CD137 antibody used as a binding partner and the antibody concentration used for assay can be referred to Example 10-2, wherein 100% activation is the degree of activation achieved by the antibody. In other embodiments, an antibody containing the heavy chain amino acid sequence of sequence number 69 and the light chain amino acid sequence of sequence number 71 can be used at 30 μg / mL as a binding partner (see, for example, Examples 10-2 herein).

[0293] As a non-limiting embodiment, the present invention provides a "CD137 agonist antibody" comprising an Fc region, wherein the Fc region has enhanced binding activity to the inhibitory Fcγ receptor.

[0294] As a non-limiting example, CD137 agonist activity can be demonstrated using B cells known to express CD137 on their surface. As a non-limiting example, the HDLM-2 B cell line can be used as the B cell line. Since IL-6 expression is induced by CD137 activation, CD137 agonist activity can be assessed by the amount of human interleukin-6 (IL-6) produced. In this assessment, it is possible to determine the percentage of CD137 agonist activity of the molecule being evaluated by assessing the increase in IL-6 expression using the amount of IL-6 from inactive B cells as a 0% background level.

[0295] In some embodiments, the antigen-binding molecule of the present invention induces CD3 activation of T cells against cells expressing a third antigen, but does not induce CD3 activation of T cells against cells expressing CD137. Whether the antigen-binding molecule induces CD3 activation of T cells against cells expressing a third antigen can be determined, for example, by co-culturing T cells and cells expressing a third antigen in the presence of the antigen-binding molecule and measuring the CD3 activation of the T cells. T cell activation can be measured, for example, by using recombinant T cells that respond to CD3 signaling and express a reporter gene (e.g., luciferase), 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 that respond to CD3 signaling and express a reporter gene are co-cultured with cells expressing a third antigen in the presence of the antigen-binding molecule, the detection of the reporter gene expression or the activity of the reporter gene product in a dose-dependent manner indicates that the antigen-binding molecule induces T cell activation against cells expressing a third antigen. Similarly, whether an antigen-binding molecule does not induce CD3 activation of T cells against CD137-expressing cells can be determined, for example, by co-culturing T cells and CD137-expressing cells in the presence of the antigen-binding molecule and measuring T cell CD3 activation as described above. When recombinant T cells that respond to CD3 signaling and express a reporter gene are co-cultured with CD137-expressing cells in the presence of the antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is absent or below the detection limit or below the negative control, it is determined that the antigen-binding molecule does not induce T cell activation against CD137-expressing cells. In a single-state sample, when recombinant T cells expressing a reporter gene that responds 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%, and the activation achieved by the antigen-binding molecule that simultaneously binds to CD3 and CD137 is 100%, then the antigen-binding molecule does not induce T cell activation against cells expressing CD137. In a single-state sample, when recombinant T cells that express 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%, wherein the activation achieved by the same antigen-binding molecule of cells expressing a third antigen is 100%, then it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137.

[0296] In some embodiments, the antigen-binding molecule of the present invention does not induce the release of interleukins from PBMCs in the absence of cells expressing the third antigen. Whether the antigen-binding molecule does not induce the release of interleukins in the absence of cells expressing the third antigen can be determined, for example, by culturing PBMCs with the antigen-binding molecule in the absence of cells expressing the third antigen and measuring the interleukins such as IL-2, IFNγ, and TFNα released from the PBMCs into the culture supernatant using methods known in the art. If no significant level of interleukins is detected in the absence of cells expressing the third antigen, or if no significant cytokine expression occurs in the culture supernatant of PBMCs already cultured with the antigen-binding molecule, it is determined that the antigen-binding molecule does not induce the release of interleukins from PBMCs in the absence of cells expressing the third antigen. In a single-state sample, "no significant level of interleukin" also means that the level of interleukin concentration is approximately 50%, 30%, 20%, 10%, 5%, or 1%, where the interleukin concentration achieved by antigen-binding molecules that simultaneously bind to CD3 and CD137 is 100% activated. In a single-state sample, "no significant induced interleukin" also means that the level of interleukin concentration is approximately 50%, 30%, 20%, 10%, 5%, or 1%, where 100% activation is the interleukin concentration achieved in the presence of cells expressing the third antigen. In a single-state sample, "no significant induced interleukin expression" also means that the level of interleukin concentration increases to at most 5 times, 2 times, or 1 times the concentration of each interleukin before the addition of the antigen-binding molecule.

[0297] In some embodiments, the antigen-binding molecule of the present invention competes with antibodies selected from the group consisting of: (a) an antibody comprising a VH sequence having an amino acid sequence of sequence number 30 and a VL sequence having an amino acid sequence of sequence number 51; (b) an antibody comprising a VH sequence having an amino acid sequence of sequence number 46 and a VL sequence having an amino acid sequence of sequence number 53; (c) an antibody comprising a VH sequence having an amino acid sequence of sequence number 40 and a VL sequence having an amino acid sequence of sequence number 56; (d) an antibody comprising a VH sequence having an amino acid sequence of sequence number 30 and a VL sequence having an amino acid sequence of sequence number 58; and (e) an antibody comprising a VH sequence having an amino acid sequence of sequence number 40 and a VL sequence having an amino acid sequence of sequence number 61.

[0298] In some embodiments, the antigen-binding molecule of the present invention binds to the same antigenic determinant with an antibody selected from the group consisting of: [1] an antibody comprising an amino acid sequence of sequence number 98 as a heavy chain variable region and an amino acid sequence of sequence number 99 as a light chain variable region; [2] an antibody comprising an amino acid sequence of sequence number 100 as a heavy chain variable region and an amino acid sequence of sequence number 101 as a light chain variable region; [3] an antibody comprising an amino acid sequence of sequence number 102 as a heavy chain variable region and an amino acid sequence of sequence number 103 as a light chain variable region; [4] an antibody comprising an amino acid sequence of sequence number 104 as a heavy chain variable region and an amino acid sequence of sequence number 105 as a light chain variable region; [5] [1] An antibody containing an amino acid sequence of sequence number 106 as the heavy chain variable region and an amino acid sequence of sequence number 107 as the light chain variable region; [6] An antibody containing an amino acid sequence of sequence number 108 as the heavy chain variable region and an amino acid sequence of sequence number 109 as the light chain variable region; [7] Of any of the antibodies in [1] to [6], an antibody containing an amino acid sequence of sequence number 110 as the heavy chain constant region and an amino acid sequence of sequence number 111 or an amino acid sequence of sequence number 112 as the light chain constant region; and [8] An antibody having an activity equivalent to any of the antibodies in [1] to [7]; and [9] An antibody that binds to the same antigenic determinant as any of the antibodies in [1] to [7].

[0299] In the antibodies of [8], "equivalent activity" means that the CD137 agonist activity is 70% or more, preferably 80% or more, and even more preferably 90% or more of the binding activity of any antibody of [1] to [7].

[0300] Whether a test antibody shares a common antigenic determinant with another antibody can be assessed based on the competition between the two antibodies for the same antigenic determinant. Competition between antibodies can be detected using methods such as cross-blocking tests. For example, a competitive ELISA test is a preferred cross-blocking test. Specifically, in a cross-blocking test, the CD137 protein used to coat the wells of a microtiter pan is pre-cultured in the presence or absence of a candidate competing antibody, and then the anti-CD137 antibody of the present invention is added. The amount of the anti-CD137 antibody of the present invention bound to the CD137 protein in the wells is not directly related to the binding activity of the candidate competing antibody (test antibody) that binds to the same antigenic determinant. That is, the greater the affinity of the test antibody for the same antigenic determinant, the lower the amount of the anti-CD137 antibody of the present invention bound to the CD137 protein-coated wells, and the higher the amount of the test antibody bound to the CD137 protein-coated wells.

[0301] The amount of antibody bound to the well can be easily determined using pre-labeled antibodies. For example, avidin / peroxidase conjugates and suitable receptors can be used to measure biotin-labeled antibodies. In particular, cross-blocking assays using enzyme-labeled substances such as peroxidase are called "competitive ELISA assays." Other labeling substances that can be detected or measured can be used to label antibodies. Specifically, radiolabeling, fluorescent labeling, etc., are known.

[0302] Furthermore, when the test antibody has a constant region derived from a species different from the species of the anti-CD137 antibody of the present invention, the amount of antibody bound to the well can be measured by using a labeled antibody that identifies the constant region of the antibody. Alternatively, if the antibody is derived from the same species but belongs to a different class, antibodies that distinguish between classes can be used to determine the amount of antibody bound to the well.

[0303] Compared to the binding activity obtained in a control experiment conducted in the absence of a candidate competing antibody, if the candidate antibody can block the binding of the CD137 antibody by at least 20%, preferably at least 20% to 50%, and even more preferably at least 50%, the candidate competing antibody is an antibody that binds substantially to the same antigenic determinant as the anti-C137 antibody of the present invention or an antibody that competitively binds to the same antigenic determinant.

[0304] In another embodiment, a standard binding assay, such as BIAcore analysis or fluid cytometry known in the art, can be used to appropriately determine the ability of a test antibody to bind competitively or cross-competitively with another antibody by a person of ordinary skill in the art.

[0305] Methods for determining the spatial configuration of antigenic determinants include, for example, X-ray crystallization and two-dimensional nuclear magnetic resonance (see, Epitope Mapping Protocols in Methods in Molecular Biology, GEMorris (ed.), Vol. 66 (1996)).

[0306] The ability of a test antibody to competitively or cross-competitively bind to a CD137 ligand can also be assessed based on the competition between the test antibody and the CD137 ligand for the same antigenic determinant. Competition between the antibody and the CD137 ligand can be detected using methods such as the cross-blocking test described above. In another embodiment, standard binding assays such as BIAcore analysis or fluid cytometry, known in the art, can be used to appropriately determine the competitive or cross-competitive binding ability of the test antibody to the CD137 ligand by someone skilled in the art.

[0307] In some embodiments, preferred examples of the antigen-binding molecules of the present invention include antigen-binding molecules that bind to the same antigenic determinant as that bound by antibodies selected from the group consisting of: antibodies that recognize regions in the human CD137 protein containing the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (Sequence No.: 81), antibodies that recognize regions containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (Sequence No.: 76), antibodies that recognize regions containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (Sequence No.: 79), and antibodies that recognize regions containing the sequence LQDPCSNCPAGTFCDNNRNQIC (Sequence No.: 74).

[0308] Depending on the target cancer antigen, those skilled in the art can appropriately select the heavy chain variable region sequence and the light chain variable region sequence that bind to the cancer antigen for inclusion in the heavy chain variable region and the light chain variable region of the cancer-specific antigen-binding domain. When the antigenic determinant bound by the antigen-binding domain is contained in a plurality of different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to multiple antigens containing that antigenic determinant.

[0309] "Antigenic determinant" refers to an antigenic determinant in an antigen, and specifically refers to the antigenic site to which it binds to the various binding domains of the antigen-binding molecules disclosed herein. Therefore, for example, an antigenic determinant can be defined based on its structure. Alternatively, an antigenic determinant can be defined based on the antigen-binding activity of the antigen-binding molecule that identifies the antigenic determinant. When the antigen is a peptide or polypeptide, the antigenic determinant can be concretized by the amino acid residues that form the antigenic determinant. Alternatively, when the antigenic determinant is a glycan, the antigenic determinant can be concretized by its specific glycan structure.

[0310] A linear antigenic determinant is an antigenic determinant containing an antigenic determinant whose primary amino acid sequence is identified. Such a linear antigenic determinant typically contains at least 3, and most commonly at least 5, amino acids in its specific sequence, for example, about 8 to 10 or 6 to 20.

[0311] In contrast to linear antigenic determinants, "configurational antigenic determinants" are antigenic determinants in which the primary amino acid sequence containing the antigenic determinant is not the only determinant of the recognized antigenic determinant (e.g., the primary amino acid sequence of a configurational antigenic determinant is not necessarily recognized by antigenic determinant-defining antibodies). Compared to linear antigenic determinants, configurational antigenic determinants can contain a larger number of amino acids. Configurational antigenic determinant-defining antibodies recognize the three-dimensional structure of peptides or proteins. For example, when a protein molecule folds and forms a three-dimensional structure, the amino acids and / or polypeptide backbone forming the configurational antigenic determinant become aligned, making the antigenic determinant recognizable by the antibody. Methods for determining conformational antigen determinants include, for example, X-ray crystallization, two-dimensional nuclear magnetic resonance spectroscopy, site-specific spin labeling, and electron paramagnetic resonance spectroscopy, but are not limited to these. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed).

[0312] An example of an assessment method for evaluating the binding of antigenic determinants in cancer-specific antigens to antigen-binding molecules is shown below. Based on the example below, an appropriate assessment method for evaluating the binding of antigenic determinants in target antigens to another binding domain can also be performed.

[0313] For example, whether a test antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen recognizes a linear antigenic determinant in an antigen molecule can be verified, for example, as described below. For example, a linear peptide containing an amino acid sequence forming 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 domain in cDNA encoding the cancer-specific antigen corresponding to the amino acid sequence of the extracellular domain. Then, the test antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen is evaluated for its binding activity to a linear antigenic determinant containing the amino acid sequence constituting the extracellular domain. For example, an immobilized linear peptide can be used as an antigen to evaluate the binding activity of the antigen-binding molecule to the peptide by ELISA. Alternatively, the binding activity of the linear peptide can be assessed based on the degree to which the linear peptide inhibits the binding of the antigen-binding molecule to the cancer-specific antigen-expressing cell. The binding activity of the antigen-binding molecule to the linear peptide can be demonstrated by such tests.

[0314] Whether the aforementioned test antigen-binding molecules containing an antigen-binding domain for an antigen recognize conformational antigenic determinants can be verified as described below. For example, an antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen binds strongly to cancer-specific antigen-expressing cells upon contact, but does not substantially bind to immobilized linear peptides containing amino acid sequences that form the extracellular domain of the cancer-specific antigen. In this context, "does not substantially bind" means that the binding activity using antigen-expressing cells for antigen ELISA or fluorescence-activated cell sorting (FACS) is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less, compared to the binding activity to antigen-expressing cells.

[0315] In ELISA formats, the binding activity of test antigen-binding molecules to the antigen-binding domain of antigen-expressing cells can be quantitatively assessed by comparing the signal intensity produced by the enzyme reaction. Specifically, the test antigen-binding molecule is added to an ELISA disc on which antigen-expressing cells are immobilized. Then, an enzyme-labeled antibody that recognizes the test antigen-binding molecule is used to detect the test antigen-binding molecule bound to the cell. Alternatively, when using FACS, serially diluted test antigen-binding molecules are prepared, and the antibody binding titer to antigen-expressing cells can be determined to compare the binding activity of the test antigen-binding molecule to antigen-expressing cells.

[0316] Flow cytometry can be used to detect the binding of antigen-binding molecules to antigens on the surface of cells suspended in a buffer solution or similar medium. Conventional flow cytometry includes, for example, the following device: FACSCanto™ II.

[0317] FACSAria™

[0318] FACSArray™

[0319] FACSVantage™ SE

[0320] FACSClibur™ (all are trade names of BD Biosciences)

[0321] EPICS ALTRA HyPreSort

[0322] Cytomics FC 500

[0323] EPICS XL-MCL ADC EPICS XL ADC

[0324] Cell Lab Quanta / Cell Lab Quanta SC (all are Beckman Coulter product names)

[0325] Suitable methods for assessing the binding activity of the aforementioned test antigen-binding molecules, which contain an antigen-binding domain, for antigens include, for example, the following method. First, antigen-expressing cells react with the test antigen-binding molecule, followed by secondary staining with FITC-labeled FACSCalibur (BD). The resulting fluorescence intensity, i.e., the geometric mean, is analyzed using CELL QUEST software (BD) to quantify the antibody bound to the cells. That is, the binding activity of the test antigen-binding molecule can be determined by measuring this geometric mean, expressed as a quantification of the bound test antigen-binding molecule.

[0326] Whether an antigen-binding molecule containing the antigen-binding domain of this invention shares a common antigenic determinant with another antigen-binding molecule can be assessed based on competition between the two molecules for the same determinant. Competition between antigen-binding molecules can be detected by methods such as cross-blocking assays. For example, competitive ELISA is a preferred cross-blocking assay.

[0327] Specifically, in the cross-blocking test, the antigen in the wells of the microtiter plate is pre-cultured with or without candidate competing antigen-binding molecules, and then the test antigen-binding molecule is added. The quantification of the test antigen-binding molecule binding to the antigen in the well is not directly related to the binding activity of the candidate competing antigen-binding molecules competing for the same antigenic determinant. That is, the greater the affinity of the competing antigen-binding molecule for the same antigenic determinant, the lower the binding activity of the test antigen-binding molecule to the antigen-coated well.

[0328] The quantity of test antigen-binding molecules that bind to the well can be easily measured by pre-labeling antigen-binding molecules. For example, avidin / peroxidase conjugates and appropriate receptors can be used to measure biotin-labeled antigen-binding molecules. In particular, cross-blocking assays using enzyme-labeled substances such as peroxidase are called "competitive ELISA assays". Other detectable or measurable labeling substances can also be used to label antigen-binding molecules. Specifically, radiolabeling, fluorescent labeling, etc., are known.

[0329] Compared to the binding activity in a control test conducted in the absence of a competing antigen-binding molecule, when a candidate competing antigen-binding molecule can block the binding activity of a test antigen-binding molecule containing an antigen-binding domain by at least 20%, preferably at least 20% to 50%, and more preferably at least 50%, it is determined that the test antigen-binding molecule substantially binds to the same antigenic determinant bound by the competing antigen-binding molecule, or competes for binding to the same antigenic determinant.

[0330] When the structure of the antigenic determinant bound by the antigen-binding molecule containing the antigen-binding domain of the present invention has been identified, the antigen-binding molecules can be tested and compared to see if they share a common antigenic determinant by comparing the binding activity of the two antigen-binding molecules to peptides prepared by introducing amino acid mutations to form antigenic determinants.

[0331] As a method for measuring this binding activity, for example, the binding activity of test and control antigen-binding molecules to the introduced mutant linear peptide can be measured by comparison in the ELISA format described above. Besides the ELISA method, the binding activity of the mutant peptide bound to the column can be achieved by passing test and control antigen-binding molecules through the column and then quantitatively extracting the antigen-binding molecules from the extract. Methods for absorbing mutant peptides into the column, for example, in the form of GST fusion peptides, are commonly known.

[0332] Alternatively, when the identified antigenic determinant is a conformational antigenic determinant, whether the test and control antigen-binding molecules share a common antigenic determinant can be assessed by the following method. First, cells expressing the antigen targeted by the antigen-binding molecule and cells expressing the antigenic determinant with the introduced mutation are prepared. The test and control antigen-binding molecules are added to a cell suspension prepared by suspending these cells in an appropriate buffer, such as PBS. The cell suspension is then appropriately washed with buffer and FITC-labeled antibodies that can identify the test and control antigen-binding molecules are added. The fluorescence intensity and number of cells stained with the labeled antibody are determined using FACSCalibur (BD). The test and control antigen-binding molecules are diluted with an appropriate buffer and used at the desired concentration. For example, they can be used at concentrations in the range of 10 μ / ml to 10 ng / ml. The determined fluorescence intensity, i.e., the geometric mean, is analyzed using CELL QUEST software (BD), reflecting the quantification of the labeled antibody bound to the cells. That is, the binding activity of the test and control antigen-binding molecules can be determined by measuring the geometric mean, which is expressed by the quantitative amount of the bound labeled antibody.

[0333] In some specific embodiments, the antigen-binding molecule of the present invention comprises an amino acid sequence resulting from the introduction of one or more amino acids into the template sequence consisting of the heavy chain variable domain sequence of sequence number 92 and / or the light chain variable domain sequence of sequence number 93, wherein the one or more amino acids comprise at least one amino acid selected from the following positions: H chain: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 1 00, 100a, 100b, 100c, 100d, 100e, 100f, and 100g (Kabat numbers); 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 numbers), among which the modified heavy chains are... The variable-domain sequence HVR-H3 contains at least one amino acid selected from the following: Ala, Pro, Ser, Arg, His, or Thr at amino acid position 98; Ala, Ser, Thr, Gln, His, or Leu at amino acid position 99; Tyr, Ala, Ser, Pro, or Phe at amino acid position 100; Tyr, Val, Ser, Leu, or Gly at amino acid position 100a; Asp, S er, Thr, Leu, Gly, or Tyr at amino acid position 100b; Val, Leu, Phe, Gly, His, or Ala at amino acid position 100c; Leu, Phe, Ile, or Tyr at amino acid position 100d; Gly, Pro, Tyr, Gln, Ser, or Phe at amino acid position 100e; Tyr, Ala, Gly, Ser, or Lys at amino acid position 100f; Gly, Tyr, Phe, or Val at amino acid position 100g (Kabat number).

[0334] In some embodiments, the antigen-binding molecule of the present invention comprises (a) a VH sequence having at least 95% sequence identity with an amino acid sequence of sequence number 41, 30, 46 or 40; (b) a VL sequence having at least 95% sequence identity with an amino acid sequence of sequence number 51, 52, 53, 54, 55, 56 or 57; or (c) the VH sequence of (a) and the VL sequence of (b).

[0335] The antigen-binding molecule of the present invention can be manufactured by methods known to those skilled in the art. For example, antibodies can be prepared by the methods described below, but the methods for preparing the antibodies of the present invention are not limited thereto. Many combinations of host cells and expression vectors are known in the art for the preparation of antibodies by transferring a single gene encoding a polypeptide to a suitable host. All such expression systems can be applied to isolate the antigen-binding molecule of the present invention. In cases where eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells may be suitably used. Specifically, examples of animal cells may include the following: (1) mammalian cells such as CHO (Chinese hamster ovary cell line), COS (monkey kidney cell line), myeloma cells (Sp2 / O, NSO, etc.), BHK (baby hamster kidney cell line), HEK293 (human embryonic kidney cell line carrying cleaved adenovirus (Ad)5 DNA), PER.C6 cells (human embryonic kidney 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 toad egg cells; and (3) insect cells such as sf9, sf21, and Tn5.

[0336] Antibodies can also be prepared using *E. coli* (mAbs 2012 Mar-Apr;4(2):217-225) or yeast (WO2000023579). Antibodies prepared using *E. coli* are non-glycosylated. On the other hand, antibodies prepared using yeast are glycosylated.

[0337] DNA encoding a heavy chain of an antibody, wherein the variable domain of the antibody is represented by a heavy chain having one or more amino acid residues replaced by different amino acids of interest, and DNA encoding a light chain. For example, DNA encoding a heavy chain or light chain having one or more amino acid residues replaced by different amino acids of interest in the variable domain can be obtained by obtaining DNA encoding an antibody variable domain against an antigen prepared by methods known in the art, and by appropriately introducing substitutions such that codons encoding specific amino acids in the domain encode different amino acids of interest.

[0338] Alternatively, DNA can be pre-designed to encode proteins in which one or more amino acid residues in the variable domain of an antibody prepared against an antigen by methods known in the art are replaced by different amino acids of interest, and can be chemically synthesized to obtain DNA encoding a heavy chain in the variable domain having one or more amino acid residues replaced by different amino acids of interest. The amino acid substitution sites and the type of substitution are not particularly limited. Examples of preferred regions for amino acid alteration include solvent-exposed regions and loops in the variable domain. Among these, CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, Kabat positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the H-chain variable domain and Kabat positions 24 to 34, 50 to 56, and 89 to 97 in the L-chain variable domain are preferred. More preferably, the Kabat numbering positions are 31, 52a to 61, 71 to 74 and 97 to 101 in the variable field of the H chain, and the Kabat numbering positions are 24 to 34, 51 to 56 and 89 to 96 in the variable field of the L chain.

[0339] Amino acid changes are not limited to substitution and can include deletion, addition, insertion, modification, or a combination thereof.

[0340] DNA encoding a heavy chain having one or more amino acid residues in the variable domain substituted with different amino acids of interest can also be prepared as separate partial DNA sequences. Examples of combinations of partial DNA sequences 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 DNA sequences.

[0341] These DNAs can be expressed by, for example, integrating DNA encoding the heavy chain variable domain together with DNA encoding the heavy chain constant domain into an expression vector to construct a heavy chain expression vector. Similarly, integrating DNA encoding the light chain variable domain together with DNA encoding the light chain constant domain into an expression vector to construct a light chain expression vector. These heavy chain and light chain genes can be integrated into a single vector.

[0342] The DNA encoding the antibody of interest is integrated into an expression vector for expression under the control of expression control regions such as enhancers and promoters. Next, the resulting expression vector is used to transform host cells, allowing antibody expression. In this case, appropriate host cells and expression vectors can be used in combination.

[0343] Examples of vectors include the M13 series, pUC series, pBR322, pBluescript, and pCR-Script. In addition to these vectors, pGEM-T, pDIRECT, or pT7 can also be used for cDNA secondary selection and cleavage purposes.

[0344] Specifically, the expression vector serves the purpose of using the vector to manufacture the antibodies of the present invention. For example, when the host is Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue, the expression vector indispensablely has a promoter that allows 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, the full text of which is incorporated herein by reference), the araB promoter (Better et al., Science (1988) 240, 1041-1043, the full text of which is incorporated herein by reference), or the T7 promoter. Examples of such vectors include the vectors mentioned above, as well as pGEX-5X-1 (manufactured by Pharmacia), the "QIAexpress system" (manufactured by Qiagen NV), pEGFP, and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase).

[0345] The vector may contain a signal sequence for peptide secretion. In the case of vectors manufactured in the periplasm of *E. coli*, the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4397, the full text of which is incorporated herein by reference) can be used as the signal sequence for peptide secretion. The vector can be transferred to the host cell using, for example, lipid staining, calcium phosphate method, or DEAE-glucan method.

[0346] In addition to expression vectors used in *Escherichia coli*, examples of vectors used to manufacture the peptides of the present invention include mammalian-derived expression vectors (e.g., pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p. 5322, the entire text of which is incorporated herein by reference), pEF and pCDM8), insect cell-derived expression vectors (e.g., the "Bac-to-BAC Baculovirus Expression System" (manufactured by GIBCOL BRL) and pBacPAK8), plant-derived expression vectors (e.g., pMH1 and pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLew), retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derived expression vectors (e.g., the "Pichia Expression Kit" (manufactured by Invitrogen), pNV11, and SP-Q01), and *Bacillus subtilis*. Subtilis-derived expression vectors (e.g., pPL608 and pKTH50).

[0347] For the purpose of expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, or HEK293 cells, the vector indispensablely contains the promoter required for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, the full text of which is incorporated herein by reference), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, the full text of which is incorporated herein by reference), the CAG promoter (Gene. (1991) 108, 193, the full text of which is incorporated herein by reference), or the CMV promoter, and more preferably, contains genes for screening transfected cells (e.g., drug resistance genes that can be marked by drugs (neomycin, G418, etc.). Examples of vectors with this property include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13. Furthermore, the EBNA1 protein can be co-expressed to increase the number of gene clones. In this case, a vector with the origin of replication, OriP, is used (Biotechnol Bioeng. 2001 Oct 20;75(20:197-203; and Biotechnol Bioeng. 2005 Sep 20;1(6):670-7).

[0348] Exemplary methods for stably expressing genes and increasing the number of gene sets within cells involve transforming CHO cells with defective nucleic acid synthesis pathways using a vector (e.g., pCHOI) containing a DHFR gene as its complement, and using methotrexate (MTX) in gene amplification. Exemplary methods for transiently expressing genes involve transforming COS cells with the SV40 T antigen gene on their chromosomes using a vector (pcD, etc.) containing the SV40 replication origin. Replication origins derived from polyomaviruses, adenoviruses, bovine papillomaviruses (BPV), etc., can also be used. To increase the number of gene sets in the host cell system, the expression vector may contain selection markers such as aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene.

[0349] Antibodies can be recovered, for example, by culturing transfected cells and then isolating them from the molecularly transfected cells or from their culture solution. Methods such as centrifugation, ammonium sulfate separation, salting out, ultrafiltration, C1q, FcRn, protein A and protein G columns, affinity columns, ion exchange chromatography, and gel filtration chromatography can be appropriately combined to separate and purify antibodies.

[0350] The aforementioned techniques, such as the knots-into-holes technique (WO1996 / 027011; Ridway JB et al., Protein Engineering (1996) 9, 617-621; and Merchant AM et al., Nature Biotechnology (1998) 16, 677-681) or the technique of suppressing unintentional association between H chains by introducing charge repulsion (WO2006 / 106905), can be applied to methods for the efficient preparation of multispecific antibodies.

[0351] The present invention further provides a method for manufacturing the antigen-binding molecule of the present invention, and more specifically, a method for manufacturing the antigen-binding molecule, the aforementioned antigen-binding molecule comprising: an antibody variable region capable of binding to two different antigens (a first antigen and a second antigen), but not simultaneously binding to CD3 and CD137 (this variable region is also referred to as the first variable region); and a variable region binding to a third antigen different from CD3 and CD137 (this variable region is also referred to as the second variable region), the method comprising the step of preparing an antigen-binding molecule library containing a variety of amino acid sequences of the first variable region.

[0352] Examples may include a manufacturing method comprising the steps of: (i) preparing an antigen-binding molecule library having at least one amino acid alteration in the variable regions of antibodies, each of which binds to CD3 or CD137, wherein the altered variable regions each contain at least one amino acid; (ii) selecting from the prepared library an antigen-binding molecule comprising a variable region having binding activity against CD3 and CD137 but not simultaneously binding to CD3 and CD137; (iii) culturing a host cell comprising a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable region of an antigen-binding molecule binding to a third antigen, to express an antigen-binding molecule comprising an antibody variable region capable of binding to CD3 and CD137 but not simultaneously binding to CD3 and CD137 and a variable region binding to a third antigen; and (iv) recovering the antigen-binding molecule from the host cell culture.

[0353] In this manufacturing method, step (ii) may be the following selection step: (v) selecting from the prepared library an antigen-binding molecule that contains variable regions of CD3 and CD137 having binding activity against CD3 and CD137 but not simultaneously binding to CD3 and CD137 individually expressed in different cells.

[0354] The antigen-binding molecules used in step (i) are not particularly limited, as long as each of these molecules contains an antibody variable region. The antigen-binding molecules may be antibody fragments such as Fv, Fab, or Fab', or may be antibodies containing an Fc region.

[0355] The amino acid to be modified is selected from, for example, the amino acid whose binding to the antigen is not canceled in the variable region of the antibody that binds to CD3 or CD137.

[0356] In this invention, a single amino acid can be modified, or multiple amino acid modifications can be used in combination.

[0357] In cases where multiple amino acid changes are used in combination, the number of changes to be combined is not particularly limited, and is, for example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, or 2 or more and 3 or less.

[0358] The desired combination of multiple amino acid changes can be added only to the antibody heavy chain variable domain or the light chain variable domain, or can be appropriately distributed in both the heavy chain variable domain and the light chain variable domain.

[0359] Examples of preferred regions for amino acid alteration include solvent-exposed regions and rings within the variable region. Among these, CDR1, CDR2, CDR3, FR3, and rings are preferred. Specifically, Kabat numbering positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the H-chain variable domain, and Kabat numbering positions 24 to 34, 50 to 56, and 89 to 97 in the L-chain variable domain. More preferably, Kabat numbering positions 31, 52a to 61, 71 to 74, and 97 to 101 in the H-chain variable domain, and Kabat numbering positions 24 to 34, 51 to 56, and 89 to 96 in the L-chain variable domain.

[0360] Modifications to amino acid residues also include: random alterations to the amino acids in the aforementioned regions of the antibody variable domain that binds to CD3 or CD137; and insertions of peptides previously known to have binding activity against CD3 or CD137 into the aforementioned regions. The antigen-binding molecules of the present invention can be obtained by selecting from the thus modified antigen-binding molecules those that can bind to both CD3 and CD137, but not simultaneously to the variable regions of these antigens.

[0361] The above methods can be used to verify whether the variable region can bind to CD3 and CD137, but not to these antigens at the same time, and further, whether the variable region can bind to both CD3 and CD137 at the same time, but not to these antigens that are expressed in different cells, when either CD3 or CD137 is present in a cell and the other antigen is present alone, when both antigens are present alone, or when both antigens are present in the same cell.

[0362] The inventors have successfully developed a more efficient method for binding to the antigen-binding domain of two or more different antigens.

[0363] In some embodiments, the screening method of the present invention for antigen-binding domains bound to at least two or more different antigens of interest includes: (a) providing a library containing a plurality of antigen-binding domains, (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains bound to the first antigen, (c) contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting antigen-binding domains bound to the second antigen, and (d) amplifying the gene encoding the antigen-binding domain collected in step (c) and identifying candidate antigen-binding domains, wherein the method does not include amplifying the nucleic acid encoding the antigen-binding domain collected in step (b) between steps (b) and (c).

[0364] In the above methods, the number of steps involving contacting the antigen-binding domain with the antigen is not particularly limited. In some embodiments, when the number of antigens of interest is two or more, the screening method of the present invention may include three or more contact steps. In other embodiments, the screening method of the present invention may include two or more steps involving contacting the antigen-binding domain with one or more antigens of interest. In this case, the antigen-binding domain may be contacted with each antigen in any order. For example, the antigen-binding domain may be contacted with each antigen two or more times in sequence, or it may be contacted with one antigen one or more times first, and then contacted with other antigens before being contacted with the same antigen again. Even when the screening method of the present invention includes three or more steps involving contacting the antigen-binding domain with the antigen, the method does not include amplifying the nucleic acid encoding the collected antigen-binding domain between any two consecutive contact steps.

[0365] In some embodiments, the antigen-binding domain of the present invention is Fab, scFv, Fab'2, VHH, VH, or VL.

[0366] In some embodiments, the antigen-binding domain of the present invention is formed by fusing the antigen-binding domain with a scaffold to crosslink the antigen-binding domain with the nucleic acid encoding the antigen-binding domain.

[0367] In some embodiments, the scaffold of the present invention is a bacteriophage. In some embodiments, the scaffold of the present invention is a ribosome, RepA protein, or DNA puromycin linker.

[0368] In some embodiments, an extraction solution, such as an acidic solution, an alkaline solution, DTT, or IdeS, is used for extraction in steps (b) and (c) above.

[0369] In some embodiments, the extraction solution used in steps (b) and (c) of the present invention is EDTA or IdeS.

[0370] In some embodiments, the screening method of the present invention for antigen-binding domains bound to at least two or more different antigens of interest includes: (a) providing a library containing a plurality of antigen-binding domains; (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains bound to the first antigen; (c) translating nucleic acids encoding the antigen-binding domains collected in step (b); (d) contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting antigen-binding domains bound to the second antigen; and (e) amplifying the gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains, wherein the method does not include amplifying the nucleic acid encoding the antigen-binding domains collected in step (b) between steps (b) and (c).

[0371] In some embodiments, the method of manufacturing antigen-binding domains of the present invention, which bind to at least two or more different antigens of interest, comprises: (a) providing a library comprising a plurality of antigen-binding domains; (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains bound to the first antigen; (c) contacting the antigen-binding domains collected in step (b) with a second antigen of interest and collecting antigen-binding domains bound to the second antigen; (d) amplifying a gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; (e) linking a polynucleotide encoding a candidate antigen-binding domain selected in step (d) with a polynucleotide encoding a polypeptide comprising an Fc region; (f) culturing cells introduced into a vector in which the polynucleotides obtained in step (d) are operatively linked; and (g) collecting antigen-binding molecules from the culture solution of the cells cultured in step (f), wherein the method does not include, between steps (b) and (c), amplifying a nucleic acid encoding the antigen-binding domains collected in step (b).

[0372] In some embodiments, the antigen-binding molecule of the present invention is an antibody prepared by the above method.

[0373] In a single-state sample, the screening method of the present invention makes it possible to obtain antigen-binding domains that bind more efficiently to at least two or more different antigens of interest.

[0374] In this specification, "library" refers to a plurality of antigen-binding molecules or a plurality of fusion polypeptides comprising antigen-binding molecules or encoding such sequences of nucleic acids or polynucleotides. The plurality of antigen-binding molecules or fusion polypeptides comprising antigen-binding molecules included in the library are antigen-binding molecules that do not have a single sequence and whose sequences are mutually exclusive, or fusion polypeptides comprising antigen-binding molecules. In some embodiments, the library of the present invention is a design library. In other embodiments, the design library is the design library disclosed in WO2016 / 076345.

[0375] In one embodiment of the present invention, fusion polypeptides and heterologous polypeptides of the antigen-binding molecule of the present invention can be prepared. In one embodiment, the fusion polypeptide may comprise the antigen-binding molecule of the present invention fused to at least a portion of a viral capsid protein selected from the group consisting of, for example, viral capsid proteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, and variants thereof.

[0376] In one embodiment, the antigen-binding molecule of the present invention may be ScFv, Fab fragment, F(ab)2, or F(ab')2. In another embodiment, the present invention provides a library consisting primarily of a plurality of fusion peptides with sequences distinct from each other, each fusion peptide comprising any of the antigen-binding molecules and a heterologous peptide. Specifically, the present invention provides a library consisting primarily of a plurality of fusion peptides with sequences distinct from each other, the fusion peptides comprising any of the antigen-binding molecules fused to at least a portion of a viral capsid protein selected from the group consisting of, for example, viral outer membrane proteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, and variants thereof. The antigen-binding molecule of the present invention may further comprise a diploidization domain. In one embodiment, the diploidization domain may be located between the antibody heavy chain or light chain variable domain and at least a portion of the viral capsid protein. This diploidization domain may comprise at least one diploidization sequence and / or a sequence comprising one or more cysteine ​​residues. This diploidization domain may preferably be linked to the C-terminus of the heavy chain variable domain or constant domain. The diploidization domain can assume various structures, depending on whether the antibody variable domain is prepared as a fusion polypeptide component containing viral outer membrane proteins (where the amber stop codon following the diploidization domain is absent) or whether the antibody variable domain is predominantly prepared without containing viral capsid proteins (e.g., where the amber stop codon following the diploidization domain is present). When the antibody variable domain is predominantly prepared as a fusion polypeptide containing viral capsid proteins, bivalent display is generated by one or more disulfide bonds and / or a single diploidization sequence.

[0377] The term "different sequences" in the context of multiple antigen-binding molecules with distinct sequences refers to individual antigen-binding molecules in the library having different sequences. Specifically, the number of different sequences in the library reflects the number of distinct sequences among individual pure strains in the library, and can also be referred to as the "library size." Typically, the size of a phage display library is 10⁶ to 10¹², and can be expanded to 10¹⁴ using techniques known in the art, such as ribosome display methods. However, the actual number of phage particles selected using panning for phage libraries is typically 10 to 10,000 times larger than the library size. This excessive multiple, also known as the "number of equalities in the library," indicates that 10 to 10,000 individual pure strains may have the same amino acid sequence. Therefore, the term "different sequences" as used in this invention means that individual antigen-binding molecules in a library, excluding equal numbers of molecules in the library, have different sequences, and more specifically means that the library has 10⁶ to 10¹⁴, preferably 10⁷ to 10¹², more preferably 10⁸ to 10¹¹, and particularly preferably 10⁸ to 10¹⁰ antigen-binding molecules with different sequences.

[0378] As described herein, "phage display" refers to a scheme in which variant peptides are displayed as fusion proteins having at least a portion of the coat protein on the surface of phage particles, such as filamentous phages. Phage display is useful because a large library of randomized protein variants can be rapidly and efficiently screened for sequences that bind to target antigens with high affinity. Display of phage peptide and protein libraries has been used to screen millions of peptides for those with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins via fusion with filamentous phage gene III or gene VIII (Wells and Lowman, Curr. Opin. Struct. Biol. (1992) 3, 335-362; and the references cited herein). Monovalent phage display involves fusing a protein or peptide library to gene III or a portion thereof, and expressing the fusion protein to a low degree in the presence of wild-type gene III protein, such that each phage particle displays a set or no fusion protein. Monovalent phages have lower avidity than multivalent phages, so phagemid vectors are used for screening based on endogenous ligand affinity, which simplifies DNA manipulation (Lowman and Wells, Methods: A Companion to Methods in Enzymology (1991) 3, 205-216).

[0379] "Phleumsome" refers to a plastid vector, such as ColE1, containing the origin of bacterial replication, and a set of intergenic regions of a bacteriophage. Phleumsomes derived from any bacteriophage known in the art may be used appropriately, such as filamentous or lambdoid phages. Typically, plastids also contain selectivity markers for antibiotic resistance. DNA fragments selected into such vectors can grow into plastids. When cells carrying such plastids possess all the genes necessary for the production of phage particles, the replication mode of the plastids shifts to rolling circle replication to form multiple sets of plastid DNA strands and packaging phage particles. Phleumsomes can form infectious or non-infectious phage particles. This terminology includes phleumsomes containing a phage coat protein gene or fragment thereof bound to a heterologous polypeptide gene via gene fusion, such that the heterologous polypeptide is displayed on the surface of the phage particle.

[0380] The term "phage vector" refers to a double-stranded replicating phage containing heterologous genes. A phage vector has a phage replication origin that allows for phage replication and phage particle formation. The phage is preferably a filamentous phage, such as M13, f1, fd, or Pf3 phage or its derivatives, or a λ-like phage, such as λ, 21, phi80, phi81, 82, 424, 434, or any other phage or its derivatives.

[0381] The term "coat protein" refers to a protein, at least a portion of which is present on the surface of a viral particle. From a functional standpoint, a coat protein is any protein that binds to a viral particle during viral construction within a host cell and remains bound until the virus infects another host cell. A coat protein can be a major coat protein or a minor coat protein. Minor coat proteins are typically present in the viral capsid at a number of at least 5, more preferably at least 7, and even more preferably at least 10 or more protein sets per virion. Major coat proteins can be present in tens, hundreds, or thousands of protein sets per virion. An example of a major coat protein is the filamentous phage p8 protein.

[0382] The term "ribosome display" as used herein refers to a scheme by which a variant peptide is displayed on a ribosome (Nat. Methods 2007 Mar;4(3):269-79, Nat. Biotechnol.2000 Dec;18(12): 1287-92, Methods Mol. Biol.2004;248:177-89). Preferably, the ribosome display method requires that the nucleic acid encoding the variant peptide has a suitable ribosome stalling sequence like that of E. coli secM (J. Mol. Biol.2007 Sep 14;372(2):513-24) or does not have a stop codon. Preferably, the nucleic acid encoding the variant peptide also has a spacer sequence. As used herein, the term "spacer sequence" means a sequence of nucleic acids encoding a peptide fused to the variant peptide so that the variant peptide passes through a ribosomal tunnel after translation and allows the variant peptide to perform its function. Any in vivo translation system can be used for ribosome display, such as the E. coli S30 system, the PURE system, the rabbit reticulocyte lysate system, or the cereal-free germ cell translation system.

[0383] The term "oligonucleotide" refers to a short, single- or double-stranded polydeoxynucleotide synthesized chemically by methods known in the art (e.g., using phosphotriester, phosphorous acid, or phosphoamine chemistry via solid-phase methods as described in EP266032; or the method via a deoxynucleotide H-phosphate intermediate as described in Froeshler et al., Nucl. Acids. Res. (1986) 14, 5399-5407). Other methods for oligonucleotide synthesis include polymerase chain reaction and other automated primer methods described below, as well as oligonucleotide synthesis on solid supports. All such methods are described in Engels et al., Agnew. Chem. Int. Ed. Engl. (1989) 28, 716-734. These methods are used if the complete nucleic acid sequence of the gene is known or if a nucleic acid sequence complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, the known and preferred residues encoding each amino acid residue can be used to appropriately predict the possible nucleic acid sequence. Oligonucleotides can be purified using polyacrylamide gel or molecular sizing column or by precipitation.

[0384] The term "nucleic acid amplification" refers to the experimental process of increasing the molar number of nucleic acids. As a non-limiting example, nucleic acids include single-stranded RNA (ssRNA), double-stranded DNA (dsDNA), or single-stranded DNA (ssDNA). As a non-limiting example, PCR (polymerase chain reaction) is generally used as a method for amplifying nucleic acids, but any method capable of amplifying nucleic acids can be used. Alternatively, when a nucleic acid vector is introduced into these host cells, the nucleic acid can be amplified in the host cells. As a non-limiting example, electroporation, heat shock, phage or viral infection with a vector, or chemical agents can be used to introduce nucleic acids into cells. Alternatively, transcription of DNA or mRNA followed by reverse transcription can also amplify nucleic acids. As a non-limiting example, the introduction of phage vectors into *E. coli* is generally used to amplify nucleic acids encoding binding domains, but PCR can also be used with phage display technology. In ribosome display, cDNA display, mRNA display, and CIS display, PCR or transcription methods are commonly used to amplify nucleic acids.

[0385] The terms "fusion protein" and "fusion polypeptide" refer to polypeptides having two segments linked together. These segments in the polypeptide have different properties. For example, these properties can be biological properties such as in vitro or in vivo activity. Alternatively, the properties can be a single chemical or physical property, such as binding to a target antigen or catalysis of a reaction. The two segments can be linked directly via a single peptide bond or via a peptide linker containing one or more amino acid residues. Typically, the two segments and the linker are located in the same reading frame. Preferably, the two segments of the polypeptide are obtained from heterologous or different polypeptides.

[0386] In the phrase "fusion protein formed by fusing an antigen-binding domain and a scaffold," the term "scaffold" refers to a molecule that crosslinks an antigen-binding domain with a nucleic acid encoding that domain. As a non-limiting example, phage capsid proteins in phage display, ribosomes in ribosome display, puromycin in mRNA or cDNA display, RepA proteins in CIS display, viral capsid proteins in virus display, mammalian cell membrane anchoring proteins in mammalian cell display, yeast cell membrane anchoring proteins in yeast display, and bacterial cell membrane anchoring proteins in bacterial or E. coli display can be used as scaffolds in various display methods.

[0387] In this invention, the term "one or more amino acids" is not limited to a specific number of amino acids and can be 2 or more types of amino acids, 5 or more types of amino acids, 10 or more types of amino acids, 15 or more types of amino acids, or 20 or more types of amino acids.

[0388] Regarding the display of fusion peptides, fusion peptides of variable regions of antigen-binding molecules can be displayed in various forms on the surface of cells, viruses, ribosomes, DNA, RNA, or bacteriophage particles. These forms include single-chain Fv fragments (scFv), F(ab) fragments, and multivalent forms of these fragments. Preferred multivalent forms are scFv, Fab, and F(ab') diploids, referred to herein as (ScFv)2, F(ab)2, and F(ab')2, respectively. Display in multivalent forms is preferred, possibly in part because displayed multivalent forms typically allow for the identification of low-affinity purebreds and / or provide multiple antigen-binding sites during selection, allowing for efficient selection of rare purebreds.

[0389] Methods for displaying fusion peptides containing antibody fragments on the surface of bacteriophages are well known in the art and described in, for example, WO1992001047 and this specification. Other related methods are disclosed in WO1992020791, WO1993006213, WO1993011236 and WO1993019172. These methods can be appropriately used by those skilled in the art. Other publications (HR Hoorenboom & G. Winter (1992) J. Mol. Biol. 227, 381-388, WO1993006213 and WO1993011236) disclose the use of artificially rearranged variable regions of the genome against various antigens displayed on the surface of bacteriophages to identify antibodies.

[0390] In constructing vectors for display in scFv format, the vector contains nucleic acid sequences encoding both the light chain variable domain and the heavy chain variable domain of an antigen-binding molecule. Generally, the nucleic acid sequence encoding the heavy chain variable domain of the antigen-binding molecule is fused with a nucleic acid sequence encoding a component of the viral capsid protein. The nucleic acid sequence encoding the light chain variable domain of the antigen-binding molecule is linked to the heavy chain variable domain nucleic acid of the antigen-binding molecule via a nucleic acid sequence encoding a peptide linker. The peptide linker typically contains approximately 5 to 15 amino acids. If necessary, additional sequences encoding, for example, tags for purification or detection, may be fused to the 3' end of the nucleic acid sequence encoding the light chain variable domain of the antigen-binding molecule or the 3' end of the nucleic acid sequence encoding the heavy chain variable domain of the antigen-binding molecule, or both.

[0391] In constructing a vector for display in F(ab) form, the vector contains nucleic acid sequences encoding variable and constant domains of an antigen-binding molecule. The nucleic acid sequence encoding the light chain variable domain is fused with a nucleic acid sequence encoding the light chain constant domain. The nucleic acid sequence encoding the heavy chain variable domain of the antigen-binding molecule is fused with a nucleic acid sequence encoding the heavy chain constant CH1 domain. Generally, the nucleic acid sequences encoding the heavy chain variable and constant domains are fused with a nucleic acid sequence encoding a whole or a portion of a viral capsid protein. Preferably, the heavy chain variable and constant domains are fused products containing at least a portion of the viral capsid protein, while the light chain variable and constant domains are separately represented by the heavy chain viral capsid fusion protein. The heavy and light chains can associate with each other via covalent or non-covalent bonds. If necessary, additional sequences encoding, for example, tags for purification or detection, can be fused to the 3' end of the nucleic acid sequence encoding the light chain constant domain of the antigen-binding molecule or the 3' end of the nucleic acid sequence encoding the heavy chain constant domain of the antigen-binding molecule, or both.

[0392] Regarding the transfer of the vector to host cells, the vector system constructed as described above is transferred to host cells for amplification and / or expression. Transformation methods known in the art, including electroporation and calcium phosphate precipitation, can be used to transfer the vector to host cells. When the vector is an infectious particle such as a virus, the vector itself invades the host cell. Host cells are transfected with a reproducible expression vector containing a polypeptide insert encoding the fusion protein, and phage particles are manufactured using methods known in the art to display the fusion protein on the surface of the phage particles.

[0393] Reproducible expression vectors can be transferred to host cells using various methods. In a non-limiting embodiment, the vector can be transferred to host cells via electroporation as described in WO2000106717. Cells are cultured in standard medium at 37°C for approximately 6 to 48 hours as needed (or until the OD at 600 nm reaches 0.6 to 0.8). The medium is then centrifuged, and the culture supernatant is removed (e.g., by decantation). At the initial stage of purification, the cell pellet is preferably resuspended in a buffer solution (e.g., 1.0 mM HEPES (pH 7.4)). The suspension is then centrifuged again to remove the supernatant. The resulting cell pellet is resuspended in glycerol diluted, for example, to 5 to 20% v / v. The suspension is centrifuged again to remove the supernatant to obtain the cell pellet. The cell pellet is resuspended in water or diluted glycerol. Based on the measured cell density of the resulting suspension, the final cell density is adjusted to the desired density using water or diluted glycerol.

[0394] Preferred examples of accepting cells include the *E. coli* strain SS320 (Siduh et al., Methods Enzymol. (2000) 328, 333-363), which is responsive to electroporation. *E. coli* strain SS320 was prepared by coupling MC1061 cells with XL1-BLUE under conditions sufficient for the transfer of reproductive episomes (F' plastids) or XL1-BLUE into MC1061 cells. *E. coli* strain SS320 is registered at ATCC (10801 University Boulevard, Manassas, Virginia) under accession number No. 98795. Any F' episome in this strain that allows phage replication can be used in this invention. Appropriate appendages can be obtained from strains stored in ATCC or from commercially available products (TG1, CJ236, CSH18, DHF', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18, etc.).

[0395] Electroporation uses higher DNA concentrations (nearly 10-fold) to improve transmutation frequency and increase the amount of DNA in the transfected host cells. Using high cell density also improves efficiency (nearly 10-fold). Increasing the amount of transferred DNA can generate a library of independent pure strains with greater diversity and more sequence differences. Transfected cells are typically selected based on the presence or absence of growth in a medium containing antibiotics.

[0396] The present invention further provides a nucleic acid encoding the antigen-binding molecule of the present invention. The nucleic acid of the present invention may be in any form, such as DNA or RNA.

[0397] The present invention further provides a vector comprising the nucleic acid of the present invention. Depending on the host cell receiving the vector, the type of vector may be appropriately selected by those skilled in the art. For example, any of the vectors described above may be used.

[0398] This invention relates more to host cells transformed with the vector of this invention. The host cell can be suitably selected by those skilled in the art to which this invention pertains. For example, any of the host cells described above can be used.

[0399] This invention also provides pharmaceutical compositions comprising the antigen-binding molecule of this invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions of this invention can be formulated by supplementing the antigen-binding molecule of this invention with a pharmaceutically acceptable carrier, according to methods known in the art. For example, the pharmaceutical compositions can be used in the form of a sterile solution or suspension of water or other pharmaceutically acceptable solutions for parenteral injection. For example, the pharmaceutical compositions can be formulated into unit dosage forms required for generally acceptable pharmaceutical administration, mixed with the antigen-binding molecule, and appropriately combined with a medically acceptable carrier or media, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, medium, preservative, binder, etc. Specific examples of carriers may include anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, polysaccharides, carboxymethyl cellulose, corn starch, and inorganic salts. The amount of the active ingredient in this formulation is determined to achieve an appropriate dosage within the dosage range.

[0400] A medium such as injectable distilled water can be used to prepare sterile compositions for injection according to conventional medical practices. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These solutions can be used in combination with suitable solubilizers, such as alcohols (specifically, ethanol) or polyols (e.g., propylene glycol and ethylene glycol) or nonionic surfactants, such as polysorbate 80(TM) or HCO-50.

[0401] Examples of oily solutions include sesame oil and soybean oil. These solutions can be used in combination with benzyl benzoate or benzyl alcohol as a cosolvent. The solutions can be further mixed with buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), tranquilizers (e.g., benzyl alcohol and phenol), and antioxidants. The resulting injectable solutions are typically filled into appropriate ampoules. The pharmaceutical composition of the present invention is preferably administered parenterally. Specific examples of dosage forms include injection, intranasal administration, pulmonary administration, and transdermal administration. Examples of injection include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, through which the pharmaceutical composition can be administered systemically or locally.

[0402] The method of administration can be appropriately selected based on the patient's age and symptoms. The dosage of pharmaceutical compositions containing polypeptides or polynucleotides encoding polypeptides can be selected, for example, in the range of 0.0001 to 1000 mg / kg body weight per dose. Alternatively, the dosage can be selected, for example, in the range of 0.001 to 100,000 mg / kg body weight of the patient; however, the dosage is not necessarily limited to these values. Although the dosage and method of administration vary depending on the patient's weight, age, symptoms, etc., those skilled in the art can appropriately select the dosage and method.

[0403] The present invention also provides a method for treating cancer including the step of administering the antigen-binding molecule of the present invention, the antigen-binding molecule of the present invention for treating cancer, the use of the antigen-binding molecule of the present invention in the manufacture of a therapeutic agent for cancer, and a process for manufacturing a therapeutic agent for cancer including the step of using the antigen-binding molecule of the present invention.

[0404] The three-letter codes and corresponding one-letter codes for amino acids used in this article are defined as follows: alanine: Ala and A; arginine: Arg and R; asparagine: Asn and N; aspartic acid: Asp and D; cysteine: Cys and C; glutamine: Gln and Q; glutamic acid: ... acid): Glu and E, glycine: Gly and G, histidine: His and H, isoleucine: Ile and I, leucine: Leu and L, lysine: Lys and K, methionine: Met and M, phenylalanine: Phe and F, proline: Pro and P, serine: Ser and S, threonine: Thr and T, tryptophan: Trp and W, tyrosine: Tyr and Y, and valine: Val and V.

[0405] Those skilled in the art will understand that one or more combinations of states described herein are also included in this invention, unless a technical contradiction arises based on the common technical knowledge of those skilled in the art to which this invention pertains.

[0406] All references cited in this paper are incorporated herein by reference in their full text.

[0407] The present invention will be further described with reference to the following embodiments. However, the present invention is not limited to the following embodiments.

[0408] [Example]

[0409] [Example 1] Concept of altered immunoglobulin variable (Fab) regions that bind to CD3 and CD137, but not simultaneously bind to CD3 and CD137.

[0410] T cells play a crucial role in tumor immunity and are known to be activated by the following two signals: 1) T cell receptor (TCR) binding to antigenic peptides presented by major histocompatibility complex (MHC) type I molecules and activating the TCR; and 2) costimulators located on the T cell surface binding to ligands located on antigen-presenting cells and activating the stimulators. Furthermore, the activation of molecules belonging to the tumor necrosis factor (TNF) superfamily and the TNF receptor superfamily, such as CD137 (4-1BB) located on the T cell surface, has been described as important for T cell activation (Vinay, 2011, Cellular & Molecular Immunology, 8, 281-284).

[0411] CD137 agonist antibodies have demonstrated anti-tumor efficacy, experimentally shown to be primarily mediated by activation of CD8-positive and NK cells (Houot, 2009, Blood, 114, 3431-8). It is also understood that engineered T cells (CAR-T cells) with chimeric antigen receptor molecules (CAR-T cells) comprising an extracellular domain of tumor antigen-binding domain and an intracellular domain of CD3 and CD137 signaling domains can enhance the duration of drug efficacy (Porter, N ENGL J MED, 2011, 365; 725-733). However, the side effects of these CD137 agonist antibodies due to their non-specific hepatotoxicity have become a clinical and non-clinical problem, and there has been no progress in the development of pharmaceutical formulations (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22). The primary cause of this side effect has been suggested to involve the antibody binding to Fcγ via its constant region (Schabowsky, Vaccine, 2009, 28, 512-22). Furthermore, it has been reported that in order for agonist antibodies targeting receptors belonging to the TNF receptor superfamily to exert their in vivo agonist activity, antibody cross-linking between Fcγ receptor-expressing cells (FcγRII-expressing cells) is necessary (Li, Proc Natl Acad Sci USA. 2013, 110(48), 19501-6). WO2015 / 156268 describes that bispecific antibodies possessing both a CD137 agonist binding domain and a tumor-specific antigen binding domain can exert CD137 agonist activity to activate immune cells only in the presence of cells expressing tumor-specific antigens, thereby avoiding the hepatotoxic adverse events associated with CD137 agonist antibodies while retaining the antibody's antitumor activity. WO2015 / 156268 further describes how combining this bispecific antibody with another bispecific antibody possessing a binding domain with CD3 activating activity and a binding domain targeting tumor-specific antigens can further enhance the antitumor activity and avoid these adverse events. A trispecific antibody with three binding domains targeting CD137, CD3, and tumor-specific antigen (EGFR) has been reported (WO2014 / 116846). However, it is expected that this molecule will crosslink CD3ε-expressing T cells and CD137-expressing cells (T cells, B cells, NK cells, DCs, etc.) even in the absence of tumor-specific antigen-expressing cells, as it will bind to both CD3ε and CD137 simultaneously (Figure 2).In fact, it has been reported that bispecific antibodies targeting CD3 and CD8 cross-link CD8-positive T cells and induce cytotoxicity in these cells (Wong, Clin. Immunol. Immunopathol. 1991, 58(2), 236-250). Therefore, it is also expected that cross-linking of CD3 and antigens expressed on T cells will induce cross-linking of T cells and cause T cells to kill each other.

[0412] Catumaxomab is known to be a bispecific antibody that recognizes proteins expressed on T cells and proteins expressed on cancer cells (cancer antigens). It binds to the cancer antigen (EpCAM) and the CD3ε chain expressed on T cells at two Fabs, respectively. It is known to bind to both CD3ε and FcγR even in the absence of cancer antigens. Therefore, even in a cancer-free environment, CD3ε-expressing T cells are cross-linked to Fcγ-expressing cells to produce large amounts of various intercytokines. The induction of cancer antigen-independent intercytokine production has limited the administration of this trifunctional antibody to the intraperitoneal route (Cancer Treat Rev. 2010 36(6), 458-67 (Non-Patent Literature 16)). Therefore, due to the severe adverse reactions of intercytokine storm, this trifunctional antibody is very difficult to administer systemically (Cancer Immunol Immunother. 2007 Sep; 56(9): 1397-406 (Non-Patent Literature 18)).

[0413] Meanwhile, traditional multispecific antibodies bind to multiple antigens simultaneously. Depending on the antigen combination, simultaneous binding to multiple antigens may be undesirable. Antibodies exhibiting both T-cell-mediated cytotoxic activity and cancer antigen-specific activation activity via CD137-mediated T-cell and other immune cell activation remain unknown.

[0414] Therefore, a possible method for regulating poorly controlled cross-linking reactions is dual-binding Fab, which is a variable (Fab) region that binds to CD3 via one portion and to CD137 via a different portion that does not participate in this binding to the first antigen (Figure 1). If two adjacent portions of a variable (Fab) region are necessary for binding to their respective antigens, as shown in Figure 1, binding to CD3 inhibits binding to CD137, and binding to CD137 also inhibits binding to CD3. Therefore, modified antibodies with this dual-binding Fab property cannot simultaneously bind to CD3 and CD137, and thus it is presumed that no cross-linking reaction will occur between CD3 and CD137 (Figure 2). Furthermore, when CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or when both are present in the same cell, it is thought that dual-binding Fab can simultaneously bind to CD3 and CD137, but not simultaneously bind to these antigens expressed in different cells, and does not cross-link these two cells (Figure 3). On the other hand, the antigen bound to another variable (Fab) region (the third antigen) can cross-link with CD3 and CD137 on T cells (Figure 4) or with CD137 on CD137-positive immune cells (Figure 5). For this antibody, the Fc region bound to FcγR can be used as the constant region, or the Fc region with reduced binding activity against FcγR can be used as the constant region.

[0415] By utilizing the properties of this dual CD3 / CD137 binding Fab, for example, it is possible to further enhance the activation of T cells, NK cells, and / or immune cells in techniques that use antibody-mediated retargeting of T cells to damage cancer cells expressing cancer antigens, thereby achieving higher anti-cancer potential.

[0416] In short, if the variable (Fab) region can be modified into a dual-binding Fab to impart the following properties, then an antibody with the following efficacy, as shown in Figure 1, can be developed: 1. It has binding activity against CD3; 2. It has binding activity against C137; and 3. It does not bind to both CD3 and CD137 simultaneously.

[0417] The phrase "not simultaneously binding to CD3 and CD137" also includes not cross-linking CD3-expressing cells to CD137-expressing cells, or not simultaneously binding to CD3 and CD137, which are expressed on different cells. This phrase further includes situations where CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or where both are located on the same cell, and the variable region can bind to both CD3 and CD137 simultaneously, but not simultaneously to CD3 and CD137 expressed on different cells.

[0418] Similarly, if the variable (Fab) region can be modified to be a dual-binding Fab to impart the following properties, antibodies with, for example, the effects shown in Figures 3, 4 and 5 can be developed: 1. having binding activity against CD3 located on T cells; 2. having binding activity against CD137 located on CD137-expressing cells; and 3. not binding to both CD3 and CD137 simultaneously.

[0419] [Example 2] Construction of a dual scFv library for ribosome display

[0420] The antibody library fragment synthesized in Reference Example 3 was used to construct a dual scFv library for ribosome display. This dual library was prepared such that the H chain was diversified as shown in Table 38 (in Reference Example 4), while the L chain was immobilized with the original sequence GLS3000 (sequence number: 1).

[0421] The design of the ribosome-displayed dual antibody library is shown in Figure 6. A portion of the phage λgpD gene and the *E. coli* secM gene were used as spacer genes to efficiently display the scFv library on ribosomes (Sequence No. 2). The VL fragment of GLS3000 was assembled with the spacer gene and Gly / Ser enriched linker gene via PCR (Sequence No. 3). The synthesized antibody VH library fragment was then amplified by PCR, fused at the 3' end to the VL-spacer gene, and fused at the 5' end to the T7 promoter containing the 5' untranslated region (UTR) (Sequence No. 4).

[0422] [Example 3] Obtaining scFv domains bound to CD3ε and human CD137 from dual scFv libraries

[0423] (3-1) Obtain the scFv domain bound to human CD137

[0424] The scFv domain bound to human CD137 was identified from the dual scFv library designed and constructed in Example 2. A biotinylated human CD137 fusion fused to the human IgG1 Fc fragment (referred to as human CD137-Fc, sequence number: 16) was used as the antigen.

[0425] The scFv ribosome display library constructed in Example 2 was used for in vitro transcription (T7 RiboMAX™ Express Large scale RNA production system, P1320, Promega) to prepare an mRNA scFv library. The synthesized mRNA was purified using an RNAeasy mini kit (Cat. No. 74104, QIAGEN). The obtained mRNA library was translated using a PUREfrex 1.0 (PF001-025, Genefroniter) in a cell-free in vitro translation system with DnaK GroE Mix and DS supplements (PF003-0.5, PF004-0.5, PF005-0.5, Genefroniter). After that, WBTH buffer (50 mM Tris-Acetate, 150 mM NaCl, 70 mM Mg-Acetate, 0.1% Tween 20, 2.5 mg / mL Heparin) was added to stop translation, and blocking buffer (one package of SuperBlock Dry Blend Blocking Buffer in TBS (Cat No. 37545, Pierce) in 200 mM milliQ) was also added. The panning method was carried out with magnetic beads according to the general panning method (Nat Methods. 2007 Mar; 4(3); 269-79; Methods Mol Biol. 2012; 805: 261-86). The magnetic beads used are NeutrAvidin-coated beads (Sera-Mag Speed ​​Beads NeutrAvidin-corted or FG NeutrAvidin beads) or Streptavidin-coated beads (Dynabeads M-280 Streptavidin or Dynabeads MyOne Streptavidin T1 beads).

[0426] Specifically, 250 pmol of biotin-labeled antigen and 2 nmol of free human IgG Fc domains (“free” means “biotin-free”) were added to the prepared ribosome display library solution, and the solution was incubated at 4°C for 60 minutes. After adding SuperBlock-Blocked magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed three times with WBT buffer (50 mM Tris-Acetate, 150 mM NaCl, 50 mM Mg-Acetate, 0.1% Tween). The beads were then suspended at 50°C for 15 minutes with extraction buffer (50 mM Tris-Acetate, 150 mM NaCl, 50 mM EDTA and 50 μg / mL S. cerevisiae RNA (SIGMA)), and immediately separated using a magnetic base to recover the mRNA solution. Using primer 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), the purified mRNA library was converted to cDNA via reverse transcription, followed by PCR amplification using primer 148 and KOD-FX polymerase (TOYOBO). Using primers 149 and 150, the T7 promoter gene was added to the amplified DNA library via PCR. This cycle, called panning, was repeated several times. In the second and subsequent rounds of panning, human CD137-Fc was labeled with 150 to 50 pmol of biotin, and mRNA was recovered using either EDTA-labeled buffer (EDTA-labeled transport phase) or FabRICATOR (IdeS, a protease for the IgG hinge region, GENOVIS) (IdeS-labeled transport phase). In this process, 5 μL of Fabricator (10 units / μL) and 95 μL of WBT buffer were added, and the beads were suspended at 37°C for 10 minutes. Immediately afterward, the beads were separated using a magnetic base to recover the mRNA solution. Then, 100 μL of extraction buffer was added to the recovered mRNA, and the mixture was incubated at 50°C for 10 minutes.

[0427] (3-2) Binding of the scFv domain to CD3ε or CD137 (scFv ELISA)

[0428] To assess scFv domain binding by ELISA, FLAG-tags were added to the recovered DNA library via PCR in rounds 5 and 6 using primers 148 and 151. The resulting scFv-FLAG DNA fragments were conjugated into a TOPO TA selection manifold dual-promoter (Invitrogen) vector, and DH5α *E. coli* were transduced. The VH sequences from individual *E. coli* colonies were analyzed. Five to seven pure clones with VH sequences distinct from those obtained during the EDTA and IdeA extraction cycles in rounds 5 and 6 were then selected. Each scFv-FLAG gene line was amplified from the colonies using primers 148 and 151. PUREfrex 1.0ss was added to each amplified scFv gene and the cells were incubated at 37°C for 2 hours. After adding 2% skim milk buffer, the solution containing scFv was subjected to ELISA using the following procedure: A StreptaWell 96 microtiter plate (F. Hoffmann-La Roche Ltd.) was coated with biotinylated CD3ε peptide (Sequence No.: 6) or biotinylated human CD137-Fc at room temperature for 30 minutes. Each well of the plate was washed with TBST (TBS containing 0.1% Tween 20; TBS is available from Takara Bio Inc.) to remove unbound antigen. The well was then blocked with 250 μL of 2% skim milk-TBS for 1 hour or longer. After removing the 2% skim milk-TBS, the prepared scFv solution was added to each well, and the plate was incubated at room temperature for 1 hour to allow scFv to bind to the antigen in each well. Each well was washed with TBST, and then MONOCLONAL ANTI-FLAG® M2, ANTIBODY (SIGMA, diluted 1000-fold with TBS) was added to each well. Incubation trays for 1 hour. Each well was washed with TBST, and then goat anti-mouse IgG, IgA, IgM (H+L), and HRP conjugate (Invitrogen, diluted 2000-fold with TBS) were added. Incubation trays for 1 hour. After washing with TBST, a single TMB solution (ZYMED Laboratories, Inc.) was added to the wells. The colorimetric reaction in each well was terminated by adding sulfuric acid. Colorimetric analysis was then performed based on absorbance at 450 nm. The results are shown in Figure 7. Most scFvs bound to CD3ε or CD137, but two scFvs from the IdeS extraction run, highlighted in black in Figure 7, showed binding to both human CD137 and CD3ε. In other words, a pure strain exhibiting binding activity against the second antigen (human CD137) was successfully selected using a dual scFv library.

[0429] (3-3) Analysis of IgG binding to CD3ε or CD137

[0430] Eleven homologous strains (dBBDu_001 to 011) were selected for further evaluation. These homologous strains were converted to IgG (with the VH and VL sequences of each homologous strain ligated to the human H and L chain constant domains, respectively), and their binding activity against CD3ε and CD137 was evaluated. The VH fragments of each homologous strain were amplified by PCR using primers specifically bound to the H chain in the library. The amplified VH fragments were assembled into the CH1 gene of human IgG1 and integrated into animal phenoplastics. The prepared phenoplastics were expressed in animal cells using the method described in Reference Example 1. The GLS3000 strain was used as the light chain, and its phenoplastics were prepared as shown in Reference Examples 4-2.

[0431] The antigen binding of each molecule was tested using an electrochemiluminescence (ECL) method. Specifically, a solution containing 0.1% Tween 20 TBS (TBST), diluted to 18 pmol / mL with biotinylated CD3ε peptide or biotinylated human CD137 and adjusted to 2 μg / mL, along with 25 μL / well of SULFO-TAG-labeled (MESO SCALE DIAGNOSTICS, ruthenium(II)-tri-bipyridine, N-hydroxysuccinimide) anti-human IgG antibody (Invitrogen #628400) adjusted to 18 pmol / mL, was added to a Nunc-Immuno™ MicroWell™ 96-well round-bottom dish (Nunc), and the mixture was incubated at room temperature for 1 hour to form antibody-antigen complexes. 150 μL / well of TBST solution containing 0.5% BSA was added to streptomycin trays (MSD KK, L15SA-1), and the trays were incubated overnight at 4°C. After removing the blocking solution, each well was washed three times with 250 μL of TBST solution. 75 μL / well of antibody-antigen complex solution was added, and the trays were incubated at room temperature for 1 hour to allow biotin-anti-human IgG Ab to bind to the streptomycin trays. After removing the antibody-antigen complex solution, each well was washed three times with TBST solution, and 150 μL / well of readout buffer (MSD KK) was added. The luminescence signal of the sulfo-tag was then detected using a Sector Imager 2400 (MSD KK).

[0432] Of the 11 pure strains, pure strain 011 (sequence number: 5) showed clear binding to both CD3ε and human CD137, and some other pure strains also showed binding to both CD3ε and human CD137 (Figure 8). Therefore, this result proves that these dual antibodies binding to two different antigens can be obtained from this designed dual scFv library.

[0433] [Example 4] Obtaining an scFv domain bound to CD3ε and human CD137 using a dual-scFv library with a dual-round selection.

[0434] (4-1) A selection scheme to improve the efficiency of obtaining the scFv domain bound to human CD137.

[0435] The scFv domains binding to CD3ε and CD137 were successfully obtained in Example 3, but the acquisition efficiency was not very high. One possible solution to improve efficiency is alternative panning, in which different antigens are used in different panning rounds. By this method, the selection pressure for both CD3ε and CD137 can be placed on the dual scFv library in different rounds, but not simultaneously. To address this deficiency, the inventors used a dual-round selection, which has been reported to perform a second panning of antigens in one round (one round means, in phage display, the recovery of phages from E. coli to phage infection of E. coli, and in ribosome display, the recovery of phages from in vitro transcription to PCR amplification) (J Mol Biol. 1992 Aug 5; 226(3): 889-96). Only one type of antigen was used in each panning round, but the inventors believe that by using a dual-round selection in which two different antigens are used in each panning process, antibodies specific to the two different antigens can be recovered more efficiently.

[0436] (4-2) Obtain the scFv domain bound to human CD137 through a double-turn selection.

[0437] The selection criteria are shown in Table 1. Runtimes 1 and 2 are alternative selection criteria, and runtime 3 is a dual-round selection criterion in which dual-round selection is performed in runs 3, 5, and 7. Biotin-labeled CD3ε peptide antigen (amino acid sequence: sequence number: 6) and biotin-labeled human CD137 fused to a human IgG Fc fragment (named human CD137-Fc) were used as antigens. In Table 1, CD3 refers to selection using biotin-labeled CD3 peptide, CD137 refers to selection using biotin-labeled human CD137-Fc, and Duble refers to dual-round selection.

[0438]

[0439] The scFv ribosome display library constructed in Example 2 was used for in vitro transcription (T7 RiboMAX™ Express Large scale RNA Production system, P1320, Progema) to prepare an mRNA scFv library. The synthesized mRNA was purified using the RNeasy mini kit (Cat. No. 74104, QIAGEN). The resulting mRNA library was translated using the PUREfrex 1.0 (PF001-0.25, Genefroniter) cell-free in vitro translation system with DnaK GroE Mix and DS supplements (PF003-0.5, PF004-0.5, PF005-0.5, Genefroniter). After that, WBTH buffer (50 mM Tris-Acetate, 150 mM NaCl, 70 mM Mg-Acetate, 0.1% Tween, 2.5 mg / mL Heparin) was added to stop translation, and blocking buffer (one package of SuperBlock Dry Blend Blocking Buffer in TBS (Cat No. 37545, Pierce) in 200 mM milliQ) was also added. The panning method was carried out with magnetic beads according to the general panning method (Nat Methods. 2007 Mar; 4(3); 269-79; Methods Mol Biol. 2012; 805: 261-86). The magnetic beads used are Neutr biotin-cored beads (Sera-Mag Speed ​​Beads Neutr Avidin-cored or FG Neutr Avidin beads) or streptomycin-cored beads (Dynabeads M-280 Streptavidin or Dynabeads MyOne Strptavidin T1 beads).

[0440] Specifically, 250 pmol of biotin-labeled antigen and 2 nmol of free human IgG Fc domain (when the biotin-labeled antigen is human CD137-Fc) were added to the prepared ribosome display library solution, and the solution was then in contact with the library solution at 4°C for 60 minutes. After adding SuperBlock-blocking magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed two or three times with WBT buffer (50 mM Tris-Acetate, 150 mM NaCl, 50 mM Mg-Acetate, 0.1% Tween). After adding extraction buffer (50 mM Tris-Acetate, 150 mM NaCl, 50 mM EDTA, and 50 μg / mL S. cerevisiae RNA (SIGMA)), the beads were suspended at 50°C for 15 minutes. Immediately afterward, the beads were separated using a magnetic base to recover the mRNA solution. The recovered mRNA was purified using a High Pure RNA Isolation kit (Roche). Primers with sequence number 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) were used. Scientifically, the purified mRNA library was converted into cDNA via reverse transcription, and then amplified by PCR using primer 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR using primers 149 and 150. This cycle was repeated several times. In the second and subsequent rounds of panning, 150 to 50 pmol of biotin-labeled human CD137-Fc or 250 pmol of biotin-labeled CD3ε peptide were used.

[0441] In rounds 3, 5, and 7 of cycle 3, a dual-round selection was performed. Specifically, 250 pmol of biotin-labeled CD3ε peptide was added to the prepared ribosome display library solution, and the solution was incubated at 4°C for 60 minutes. After adding SuperBlock-blocking magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed six to ten times with WBT buffer (50 mM Tris-Acetate, 150 mM NaCl, 50 mM Mg-Acetate, 0.1% Tween) (depending on the panning round). After adding extraction buffer, the beads were suspended at 50°C for 15 minutes, and immediately thereafter separated using a magnetic base to recover the mRNA solution. The recovered mRNA was purified using a High Pure RNA Isolation kit (Roche). The purified mRNA was subsequently translated again using PUREfrex 1.0 (Genefroniter). 250 pmol or 100 pmol of biotin-labeled CD137-Fc and 2 nmol of free human IgG Fc domains were added to the prepared ribosome display library solution, and the solution was incubated at 4°C for 60 minutes. After adding SuperBlock-blocking magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed three to ten times with WBT buffer (depending on the panning rounds). After adding extraction buffer, the beads were suspended at 50°C for 15 minutes, and immediately thereafter separated using a magnetic base to recover the mRNA solution. The recovered mRNA was purified using a High Pure RNA Isolation kit (Roche). The purified mRNA library was converted to cDNA by reverse transcription using primer 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR using primer 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene line was added to the amplified DNA library by PCR using primers of SEQ ID NO:149 and 150.

[0442] (XX-2) binding of the scFv domain to CD3ε or CD137 (scFv ELISA)

[0443] To assess scFv domain binding by ELISA, FLAG-tags were added to the recovered DNA libraries by PCR using primers 149 and 151 in rounds 6 and 7. The resulting scFv-FLAG DNA fragments were conjugated to the TOPO TA selection mantle dual promoter (Invitrogen) and transduced into DH5α *E. coli*. VH sequences from individual *E. coli* colonies were analyzed. Then, certain pure strains with different VH sequences from each panning stage in rounds 6 and 7 were selected. The scFv-FLAG genes were amplified from each colony using primers 149 and 151. PUREfrex 1.0ss was added to each amplified scFv gene and incubated at 37°C for 2 hours. After adding 2% skim milk buffer, the solution containing scFv was subjected to ELISA using the following procedure: A StreptaWell 96 microtiter plate (F. Hoffmann-La Roche Ltd) was coated with biotinylated CD3ε peptide or biotinylated human CD137-Fc at room temperature for 30 minutes. Each well of the plate was washed with TBST (TBS containing 0.1% Tween 20; TBS is available from Takara Bio Inc.) to remove unbound antigen. The well was then blocked with 250 μL of 2% skim milk-TBS for 1 hour or longer. After removing the 2% skim milk-TBS, the prepared scFv solution was added to each well, and the plate was incubated at room temperature for 1 hour to allow scFv to bind to the antigen in each well. Each well was washed with TBST, and then MONOCLONAL ANTI-FLAG® M2, ANTIBODY (SIGMA, diluted 1000-fold with TBS) was added to each well. The plate was incubated for 1 hour. Each well was washed with TBST, and then goat anti-mouse IgG, IgA, IgM (H+L), and HRP conjugate (Invitrogen, diluted 2000 times with TBS) were added. The wells were incubated for 1 hour. After washing with TBST, a single TMB solution (ZYMED Laboratories, Inc.) was added to the wells. The colorimetric reaction of the solutions in each well was terminated by adding sulfuric acid. Colorimetric analysis was then performed based on absorbance at 450 nm. The results are shown in Figure 9. All scFVs in runners 1 and 2 bound to either CD3ε or CD137. On the other hand, many scFVs in runner 3 showed binding to both human CD137 and CD3ε. In other words, by using a dual-round selection with two different antigens in each panning round, the efficiency of obtaining pure strains exhibiting binding activity against CD3ε and the second antigen (human CD137) was successfully improved.

[0444] (4-3) Gain additional selections by using a double-round selection to combine more scFv domains into human CD137.

[0445] To create more scFv domains that combine with human CD137 and CD3ε, additional rounds of selection are performed in runs 2 and 3. In run 8, both conventional selection and double-round selection are used in the run 3 run 7 output library, and only double-round selection is performed in the run 3 run 7 output library. Each selection process is the same as in Example 4-2.

[0446] (4-4) Obtain the scFv domain bound to human CD137 by double-round selection and IdeS boost.

[0447] The selection criteria are shown in Table 2. Runtimes 4 and 5 are alternative selection criteria, and runtime 6 is a double-round selection criterion, where double-round selection is performed in runs 3, 5, and 7. Biotin-labeled CD3ε peptide antigen (amino acid sequence: sequence number: 6) and biotin-labeled human CD137 fused to the human IgG Fc fragment (named human CD137-Fc) were used as antigens. In Table 2, CD3 refers to selection using biotin-labeled CD3 peptide, CD137 refers to selection using biotin-labeled human CD137-Fc, and Double refers to double-round selection.

[0448]

[0449] The scFv ribosome display library constructed in Example 2 was used for in vitro transcription (T7 RiboMAX™ Express Large scale RNA Production system, P1320, Progema) to prepare an mRNA scFv library. The synthesized mRNA was purified using the RNeasy mini kit (Cat. No. 74104, QIAGEN). The resulting mRNA library was translated using the PUREfrex 1.0 (PF001-0.25, Genefroniter) cell-free in vitro translation system with DnaK GroE Mix and DS supplements (PF003-0.5, PF004-0.5, PF005-0.5, Genefroniter). After that, WBTH buffer (50 mM Tris-Acetate, 150 mM NaCl, 70 mM Mg-Acetate, 0.1% Tween, 2.5 mg / mL Heparin) was added to stop translation, and blocking buffer (one package of SuperBlock Dry Blend Blocking Buffer in TBS (Cat No. 37545, Pierce) in 200 mM milliQ) was also added. The panning method was carried out with magnetic beads according to the general panning method (Nat Methods. 2007 Mar; 4(3); 269-79; Methods Mol Biol. 2012; 805: 261-86). The magnetic beads used are Neutr biotin-cored beads (Sera-Mag Speed ​​Beads Neutr Avidin-cored or FG Neutr Avidin beads) or streptomycin-cored beads (Dynabeads M-280 Streptavidin or Dynabeads MyOne Strptavidin T1 beads).

[0450] Specifically, 250 pmol of biotin-labeled antigen and 2 nmol of free human IgG Fc domain (when the biotin-labeled antigen was human CD137-Fc) were added to the prepared ribosome display library solution, and the solution was incubated at 4°C for 60 minutes. After adding SuperBlock-blocking magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed two or three times with WBT buffer. After adding extraction buffer, the beads were suspended at 50°C for 15 minutes, and then immediately separated using a magnetic base to recover the mRNA solution. The recovered mRNA was purified using a High Pure RNA Isolation kit (Roche). Using primer 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), the purified mRNA library was converted to cDNA by reverse transcription, and then amplified by PCR using primer 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR using primers of SEQ ID NO: 149 and 150. This cycle was repeated several times. In the second and subsequent rounds of panning, 150 to 50 pmol of biotin-labeled human CD137-Fc or 250 pmol of biotin-labeled CD3ε peptide was used. When the antigen was biotin-labeled human CD137-Fc in the second and subsequent rounds, mRNA was recovered using a Fabricator (IdeS, a protease for the IgG hinge region, GENOVIS). During this process, 5 μL of Fabricator (10 units / μL) and 95 μL of WBT buffer were added, and the beads were suspended at 37°C for 10 minutes. Immediately afterward, the beads were separated using a magnetic base to recover the mRNA solution. Then, 100 μL of extraction buffer was added to the recovered mRNA and incubated at 50°C for 10 minutes. When the antigen was biotin-labeled CD3ε peptide, only the extraction buffer was used in the same manner as in round 1.

[0451] Double-round selection was performed in rounds 3, 5, and 7 of cycle 6. Specifically, 250 pmol of biotin-labeled CD3ε peptide was added to the prepared ribosome display library solution and incubated at 4°C for 60 min. After adding SuperBlock-blocking magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 min. The beads were washed six to ten times with WBT buffer (depending on the panning round). After adding extraction buffer, the beads were suspended at 50°C for 15 min, and immediately thereafter separated using a magnetic base to recover the mRNA solution. Ribosomes (GeneFroniter) were added to the recovered mRNA, and the mRNA was purified using the High Pure RNA Isolation kit (Roche). The purified mRNA was subsequently translated again using PUREfrex 1.0 (GeneFroniter). 250 pmol or 100 pmol of biotin-labeled CD137-Fc and 2 nmol of free human IgG Fc domains were added to the prepared ribosome display library solution, and the solution was incubated at 4°C for 60 minutes. After adding SuperBlock-blocking magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed three to ten times with WBT buffer (depending on the panning rounds). 5 μL of 10 units / μL Fabricator and 95 μL of WBT buffer were added, and the beads were suspended at 37°C for 10 minutes. Immediately afterward, the beads were separated using a magnetic base to recover the mRNA solution. Then, 100 μL of extraction buffer was added to the recovered mRNA, and the solution was incubated at 50°C for 10 minutes. The recovered mRNA was purified using a High Pure RNA Isolation Kit (Roche). The purified mRNA library was converted into cDNA via reverse transcription using primer SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR using primer SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR using primers SEQ ID NO: 149 and 150.

[0452] (4-5) Use double-round selection and IdeS boost to gain more additional panning for the scFv domain bound to human CD137.

[0453] To create more scFv domains that bind to human CD137 and CD3ε, additional rounds of selection are performed in runs 4, 5, and 6, as in Examples 4-3. Both conventional and double-round selections are used in the output library in runs 5 and 6, and a double-round selection is performed in the output library in run 4. All selection processes are identical to those in Examples 4-4.

[0454] (4-6) Binding of the scFv domain to CD3ε or CD137 (scFv ELISA)

[0455] To assess scFv domain binding by ELISA, FLAG-tags were added to the recovered DNA libraries from rounds 6 to 8 of Examples 4-3, 4-4, and 4-5 by PCR using primers with sequence numbers 149 and 151. The resulting scFv-FLAG DNA fragments were conjugated to the TOPO TA selection mantle dual promoter (Invitrogen) and transduced into DH5α *E. coli*. VH sequences from individual *E. coli* colonies were analyzed. The inventors then selected certain pure strains with different VH sequences from each panning stage. Each scFv-FLAG gene was amplified from each colony using primers with sequence numbers 149 and 151. PUREfrex 1.0ss was added to each amplified scFv gene and incubated at 37°C for 2 hours. After adding 2% skim milk buffer, the solution containing scFv was subjected to ELISA using the following procedure: A StreptaWell 96 microtiter plate (F. Hoffmann-La Roche Ltd) was coated with biotinylated CD3ε peptide or biotinylated human CD137-human IgG1 Fc at room temperature for 30 minutes. Each well of the plate was washed with TBST (containing 0.1% Tween 20 TBS; TBS is available from Takara Bio Inc.) to remove unbound antigen. The well was then blocked with 250 μL of 2% skim milk-TBS for 1 hour or longer. After removing the 2% skim milk-TBS, the prepared scFv solution was added to each well, and the plate was incubated at room temperature for 1 hour to allow scFv to bind to the antigen in each well. Each well was washed with TBST, and then MONOCLONAL ANTI-FLAG® M2, ANTIBODY (SIGMA, diluted 1000-fold with TBS) was added to each well. Incubation trays for 1 hour. Each well was washed with TBST, then goat anti-mouse IgG, IgA, IgM (H+L), and HRP conjugate (Invitrogen, diluted 2000-fold with TBS) were added. Incubation trays for 1 hour. After washing with TBST, a single TMB solution (ZYMED Laboratories, Inc.) was added to each well. The colorimetric reaction in each well was terminated by adding sulfuric acid. Color development was then measured based on absorbance at 450 nm.

[0456] The results are shown in Figure 10. Many scFvs showed binding to human CD137 and CD3ε during various panning runs.

[0457] (4-7) Converting antibody format to IgG1 and preparing various IgG1 molecules

[0458] Eleven pools (shown in Table 3) were selected for further evaluation. The scFv lines included in the pools were converted into IgG (VH and VL sequences of each pure strain were ligated to the human H and L chain constant domains, respectively), and their binding activity against CD3ε and CD138 was evaluated. The VH fragments from each pool were amplified by PCR using primers (sequence numbers: 152 and 153) that specifically bind to the H chain in the library. The amplified VH fragments were integrated into animal phenoplasts already possessing the human IgG1 CH1-Fc region. The prepared phenoplasts were used for animal cell expression according to the method described in Example 1. GLS3000 (sequence number: 1) was used as the light chain, and its phenoplasts were prepared as shown in Examples 4-2.

[0459]

[0460] (4-8) Evaluation of the CD3ε and human CD137 binding activities of the obtained antibodies

[0461] The prepared antibodies were subjected to ELISA to evaluate their binding ability to CD3ε and human CD137.

[0462] First, streptomycin-coated microplates (384 wells, Greiner) were coated with 20 μL of TBS containing biotinylated CD3ε peptide or biotinylated human CD137-Fc at room temperature for 1 hour or more. After washing each well with TBST to remove unbound biotinylated antigen, the wells were blocked with 20 μL of blocking buffer (2% skim milk / TBS) for 1 hour or more. The blocking buffer was then removed from each well. 10 μL of mammalian cell supernatant containing IgG, diluted twice with 1% skim milk / TBS, was added to each well, and the plates were incubated at room temperature for 1 hour to allow the IgG to bind to the biotinylated antigen in each well. The wells were then washed with TBST. Goat anti-human kappa light chain alkaline phosphatase conjugate (BETHYL, A80-115AP), diluted with TBS, was added to each well. The plates were incubated for 1 hour. After cleaning with TBST, the color development reaction in each well with the solution containing Blue Phos Microwell Phosphatase Substrate System (KPL) was terminated by adding Blue Phos Stop Solution (KPL). The color development was then measured using the absorbance at 615 nm. The measurement results are shown in Figure 11.

[0463] Even in the 1gG1 format, many pure strains showed binding to CD3ε and human CD137. VH sequences from individual E. coli colonies were then analyzed. Among these antibodies binding to CD3ε and CD137, a total of 19 different HCDR3 sequences were found, with numerous HCDR1 or HCDR2 sequence variants present.

[0464] This suggestion combines the antibody systems for two different antigens, CD3ε and human CD137, obtained from a rationally designed library constructed using CD3-binding antibodies as templates as described in Reference Example 4, with the dual-round selection significantly improving the efficiency of obtaining these antibodies. In traditional alternative panning, the selection pressure changes in each panning round, so the concentration of the ideal pure strain becomes increasingly slow or may result in the loss of the ideal pure strain. By using two different antigens and employing dual-round selection, the inventors can uniformize the selection pressure in each panning round, making it easier and more direct to collect the ideal pure strain.

[0465] [Example 5] Affinity maturation of a dual scFv library with a designed light chain library binding to the antibody domains of CD3ε and human CD137.

[0466] (5-1) Construct a library of light chains containing the obtained heavy chains.

[0467] In Example 4, many antibodies that bind to CD3ε and human CD137 were obtained, but their affinity for human CD137 was still weak, so affinity maturation was performed to improve their affinity.

[0468] To achieve this, the light chain library designed as described in Reference Example 4 was combined with the heavy chain of a candidate antibody that binds to both CD3ε and human CD137. The inventors selected an antibody, named dBBDu_115 (sequence number: 7), as a candidate antibody for affinity maturation because it preferably binds to both CD3ε and human CD137 among the 19 antibodies described in Example 4.

[0469] The inventors constructed two different antibody library formats: VL-GS-linker-VH scFv format and Fab format. The design of the ribosome-displayed dual antibody library is shown in Figure 12. As in Example 2, a portion of the phage λgpD gene and the E. coli secM gene were used as spacer genes to efficiently display the scFv or Fab library on ribosomes.

[0470] The synthetic antibody VL library fragment described in Example 4 was amplified by PCR and fused at the 3' end with the Gly / Ser enriched linker-dBBU_VH spacer gene (sequence number: 8) and at the 5' end with the T7 promoter (sequence number: 4) with a 5' untranslated region to create a VL-VH scFv format library.

[0471] Referring to Example 4, the synthetic antibody VL library fragment was amplified by PCR, fused at the 3' end with the CL-spacer gene (sequence number: 9) and at the 5' end with the T7 promoter (sequence number: 4) containing a 5' untranslated region, to create a Fab format library. The VH gene fragment of dBBDu_115 was also amplified by PCR, fused at the 3' end with the CH1 gene (sequence number: 0) and at the 5' end with the T7 promoter (sequence number: 4) containing a 5' untranslated region, to create a Fab format Hch fragment.

[0472] (5-2) Obtain antibody domains that bind to both CD3ε and human CD137.

[0473] The selection criteria are shown in Table 4. In Table 4, "double" indicates double-round selection and "CD137Fc" indicates conventional selection against biotinylated human CD137-Fc. Biotinylated CD3ε peptide antigen (amino acid sequence: sequence number: 6) and biotinylated human CD137 fused to the human IgG1 Fc fragment (named human CD137-Fc) were used as antigens.

[0474]

[0475] The scFv ribosome display library, Fab light chain ribosome display library, and Fab heavy chain line constructed in Example 5-1 were used for in vitro transcription (T7 RiboMAX™ Express Large scale RNA production system, P1320, Promega) to prepare mRNA libraries and heavy chain RNA. The synthesized mRNA lines were purified using the RNeasy mini kit (Cat. No. 74104, QIAGEN). The obtained mRNA libraries and Fab heavy chain lines were translated using a PUREfrex 1.0 (PF001-025, Genefroniter) in a cell-free in vitro translation system with DnaK GroE Mix and DS supplements (PF003-0.5, PF004-0.5, PF005-0.5, Genefroniter). Afterward, WBTH buffer was added to stop translation, and blocking buffer (2x, c-block-e, Beacle) was also added. The magnetic beads used are Neutr biotin-cored beads (Sera-Mag Speed ​​Beads NeutrAvidin-cored or FG NeutrAvidin beads) or streptomycin-cored beads (Dynabeads M-280 Streptavidin or Dynabeads MyOne Strptavidin T1 beads).

[0476] Specifically, 60 pmol of biotin-labeled human CD137-Fc was added to magnetic beads at 4°C for 60 minutes, followed by c-block-e (Beacle) addition at 4°C for 60 minutes to block the beads. These antigen-coated magnetic beads and 2 nmol of free human IgG Fc domains were added to the prepared ribosome display library solution, thereby contacting the library solution at 4°C for 75 minutes. The beads were incubated with WBT buffer for 2 minutes (runtime 10) or 10 minutes (runtimes 2, 6, and 9), after which the WBT buffer was discarded. This cleanup process was repeated 10 times with WBT buffer. After adding extraction buffer, the beads were suspended at 50°C for 15 minutes, after which the beads were immediately separated using a magnetic base to recover the mRNA solution. The recovered RNA was purified using a High Pure RNA Isolation Kit (Roche). The purified mRNA library was converted into cDNA via reverse transcription using primer 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR using primer 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene line was then added to the amplified DNA library by PCR using primers 149 and 150. This cycle, called panning, was repeated several times.

[0477] The changes in the amount of biotin-labeled human CD137-Fc in the second and subsequent rounds of selection are shown in Table 5, and the changes in the number of washes are also shown in Table 6. When the antigen in the second and subsequent rounds of runs 6, 9, and 10 was biotin-labeled human CD137-Fc, a fabricator (IdeS, a protease for the IgG hinge region, GENOVIS) was used to recover mRNA. In this process, 10 units / μL of fabricator 5 μL and 95 μL of WBT buffer were added, and the beads were suspended at 37°C for 10 minutes. Immediately afterward, the beads were separated using a magnetic base to recover the mRNA solution. Then, 100 μL of extraction buffer was added to the recovered mRNA and incubated at 50°C for 10 minutes.

[0478]

[0479]

[0480] A dual-round selection was performed during certain runs. Specifically, 150 pmol of biotin-labeled CD3ε peptide was added to magnetic beads at 4°C for 60 minutes, followed by c-block-e (Beacle) addition at 4°C for 60 minutes to block the beads. These antigen-coated magnetic beads were then added to the prepared ribosome display library solution, thereby contacting the library solution at 4°C for 60 minutes. The beads were washed ten times with WBT buffer. After adding extraction buffer, the beads were suspended at 50°C for 15 minutes, and immediately thereafter separated using a magnetic base to recover the mRNA solution. The recovered mRNA was purified using a High Pure RNA Isolation kit (Roche). The purified mRNA was subsequently translated again using PUREfrex 1.0 (Genefroniter). Biotin-labeled CD137-Fc was added to the magnetic beads at 4°C for 60 minutes, followed by c-block-e (Beacle) addition at 4°C for 60 minutes to block the beads. These antigen-coated magnetic beads and 2 nmol of free human IgG Fc domains were added to the prepared ribosome display library solution, and the solution was in contact with the library solution at 4°C for 60 minutes. The beads were incubated with WBT buffer for 10 minutes, and then the WBT buffer was discarded. This purification process was repeated 10 times with WBT buffer. During runs 6, 9, and 10, 5 μL of 10 units / μL Fabricator and 95 μL of WBT buffer were added, and the beads were suspended at 37°C for 10 minutes. Immediately afterward, the beads were separated using a magnetic base to recover the mRNA solution. Then, 100 μL of extraction buffer was added to the recovered mRNA, and the solution was incubated at 50°C for 10 minutes. The recovered mRNA was purified using a High Pure RNA Isolation Kit (Roche). The purified mRNA library was converted into cDNA via reverse transcription using primer 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR using primer 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR using primers 149 and 150.

[0481] The antibody was converted to IgG1 and individual IgG1 molecules were prepared.

[0482] Light chain genes from the affinity-matured panned scFv or Fab domain libraries described in Examples 5-2 were converted into IgG, and their binding activity against CD3ε and CD137 was evaluated. VL-CL fragments from pools 5, 6, and 9 of the training phase were amplified by PCR using primers (sequence numbers: 154 and 155) from the library that specifically bind to the L chain. The amplified VL-CL fragments were integrated into animal phenoplastids and transformed into DH5α *E. coli* strains. The resulting phenoplastids were also used to construct light chain expression vectors from training phase 10. The VL region genes were removed from the obtained expression vectors using the restriction enzymes SfiI and kpnI. VL fragments from the training phase 10 pool were amplified by PCR using primers (sequence numbers: 154 and 156) from the library that specifically bind to the VL region. The prepared VL fragments were introduced into degraded phenoplastids. Colony counts are shown in Table 7. The prepared plasma system was used for expression in animal cells by referring to the method of Example 1. The dBBDu_115 heavy chain constructed in Example 4 was also used to express full-length IgG.

[0483]

[0484] (5-4) Evaluation of the CD3ε and human CD137 binding activities of the obtained antibodies

[0485] The prepared antibodies were subjected to ELISA to evaluate their binding ability to CD3ε and human CD137.

[0486] First, streptomycin-coated microplates (384 wells, Greiner) were coated with 20 μL of TBS containing biotinylated CD3ε peptide, biotinylated human CD137-Fc, and biotinylated human IgG1 Fc region at room temperature for 1 hour or more. After washing each well with TBST to remove unbound biotinylated antigen, the wells were blocked with 20 μL of blocking buffer (2% skim milk / TBS) for 1 hour or more. The blocking buffer was then removed from each well. 10 μL of mammalian cell supernatant containing IgG, diluted twice with 2% skim milk / TBS, was added to each well, and the plates were incubated at room temperature for 1 hour to allow the IgG to bind to the biotinylated antigen in each well. The wells were then washed with TBST. Goat anti-human kappa light chain alkaline phosphatase conjugate (BETHYL, A80-115AP), diluted with TBS, was added to each well. The plates were incubated for 1 hour. After cleaning with TBST, the color development reaction in each well with the solution containing Blue Phos Microwell Phosphatase Substrate System (KPL) was terminated by adding Blue Phos Stop Solution (KPL). The color development was then measured using the absorbance at 615 nm. The measurement results are shown in Figure 13.

[0487] (5-5) Evaluation of the binding of the obtained Fab domain to both CD3ε and human CD137

[0488] Five antibodies (shown in Table 8) were selected for further evaluation. These antibody systems were purified according to Examples 5-3 and Reference Example 1. The purified antibodies were subjected to ELISA to assess their simultaneous binding ability to CD3ε and human CD137.

[0489]

[0490] First, MyOne-T1 streptomycin beads were mixed with 0.625 pmol of biotin-labeled human CD137-Fc or biotin-labeled human Fc and incubated at room temperature for 10 minutes. Then, 2% skim milk / TBS was added to block the magnetic beads. The mixed solution was dispensed into each well of a 96-well dish (Corning, 3792 black round-bottom PS dish) and incubated at room temperature for 60 minutes or longer. After that, the magnetic beads were washed once with TBS. 100 ng of purified IgG was mixed with 62.5, 6.25, or 0.625 pmol of free human CD3ε or 62.5 pmol of free human Fc (in this example, "free" means "biotin-free") or TBS, and then added to the magnetic beads in each well. The dish was incubated at room temperature for one hour to allow the IgG to bind to the biotin-labeled antigen in each well. After that, the wells were washed with TBST. Goat anti-human kappa light chain alkaline phosphatase conjugate (BETHYL, A80-115AP) diluted with TBS was added to each well. The plates were incubated for 1 hour. After washing with TBST, APS-5 (Lumigen) was added to each well. Fluorescence was measured in each well after 2 minutes. The results are shown in Figure 14 and Table 9.

[0491]

[0492] Inhibition of human CD134-Fc binding was observed in all tested antibodies via the free CD3ε peptide, but not via the free Fc domain. This result indicates that the obtained antibodies cannot bind to human CD137-Fc in the presence of the CD3ε peptide; in other words, these antibodies cannot bind to both human CD137 and the CD3ε peptide simultaneously. Therefore, this demonstrates that by using a designed library and ribosomes to exhibit dual-round selection, a Fab domain capable of binding to two different antigens, CD137 and CD3ε peptide, but not simultaneously, was successfully obtained.

[0493] [Example 6] Obtaining the Fab domain that binds to CD3ε peptide and human CD137 from a dual-Fab phage display library.

[0494] (6-1) Construct a heavy-chain phage display library with GLS3000 light chains

[0495] The antibody library fragment synthesized in Reference Example 4 was used to construct a dual-Fab library for phage display. The dual-library system was prepared as follows: the H chain was diversified as shown in Reference Example 4, while the L chain was fixed to the original sequence GLS3000 (sequence number: 1). The H chain library sequence derived from CE115HA000 was obtained by adding a V11L / L78I mutation to the FR (framework) and further diversifying the CDRs as shown in Table 38 (in Reference Example 4). This was entrusted to DNA2.0, Inc., a DNA synthesis company, to obtain the antibody library fragment (DNA fragment). The obtained antibody library fragment was inserted into a phage plasmid for phage display amplified by PCR. GLS3000 was selected as the L chain. The constructed phage plasmid for phage display was transferred to *E. coli* via electroporation to prepare *E. coli* containing the antibody library fragment.

[0496] A phage library displaying the Fab domain was created by infecting the helper phage M13KO7TC / FkpA, which encodes the FkpA chaperone protein gene, with the phage then incubated overnight at 25°C in the presence of 0.002% arabinose (this phage library is named DA library) or at 20°C in the presence of 0.02% arabinose (this phage library is named DX library). The phage library was constructed from *E. coli* carrying the constructed phage plasmid. M13KO7TC is a helper phage containing an insert of a trypsin-cleaving sequence between the N2 and CT domains of the pIII protein (see National Publication of International Patent Application No. 2002-514413). The insertion of the insert gene into the M13KO7TC gene has been disclosed elsewhere (see National Publication of International Patent Application No. WO2015016554).

[0497] (6-2) Obtain the Fab domain by combining CD3ε and human CD137 through a double-round selection.

[0498] The Fab domains binding to CD3ε and human CD137 were identified from the dual-Fab library constructed in Example 6-1. Biotinylated CD3ε peptide antigen (amino acid sequence: sequence number: 6), biotinylated CD3ε peptide antigen via disulfide linkers (Figure 15, referred to as C3NP1-27; amino acid sequence: sequence number: 145, synthesized by Genscript), biotinylated human CD137 fused to the human IgG1 Fc fragment (named human CD137-Fc), and SS-biotinylated human CD137 fused to the human IgG1 Fc fragment (named ss-human CD137-Fc) were used as antigens. ss-human CD137-Fc was prepared by using the EZ-Link Sulfo-NHS-SS-Biotinylation Kit (PIERCE, Cat. No. 21445) on human CD137 fused to the human IgG1 Fc fragment. Biotinylation was performed according to the instruction manual.

[0499] The phage system was created from *E. coli* carrying phage plasmids constructed for phage display. 2.5 M NaCl / 10% PEG was added to the culture solution of the *E. coli* containing the phages, and the resulting precipitated phage pool was diluted with TBS to obtain a phage library solution. Subsequently, BSA (final concentration: 4%) was added to the phage library solution. Panning was performed using antigens immobilized on magnetic beads, following general panning methods (J. Immunol. Methods. (2008) 332(1-2), 2-9; J. Immunol. Methods (2001) 247(1-2), 191-203; Biotechnol. Prog (2002) 18(2) 212-20; and Mol. Cell Proteomics (2003) 2(2), 61-9). The magnetic beads used were NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptomycin-coated beads (Dynabeads M-280 Streptavidin). To remove antibody-displaying phages bound to the magnetic beads themselves or the human IgG1 Fc region, the magnetic beads and biotin-labeled human Fc were subtracted.

[0500] Specifically, the phage solution was mixed with 250 pmol of human CD137-Fc and 4 nmol of free human IgG1 Fc domains and incubated at room temperature for 60 minutes. Magnetic beads were blocked with 2% skim milk / TBS containing free streptomycin (Roche) at room temperature for 60 minutes or longer and washed three times with TBS, then mixed with the incubated phage solution. After incubating at room temperature for 15 minutes, the beads were washed three times with TBST (TBS containing 0.1% Tween 20; TBS is available from Takara Bio Inc.) and then further washed twice with 1 mL of TBS. 5 μL of 100 mg / mL trypsin and 495 μL of TBS were added and incubated at room temperature for 15 minutes, after which the beads were immediately separated using a magnetic base to recover the phage solution. The strain was incubated with the phage by gentle rotation at 37°C for 1 hour to infect *E. coli*. The infected *E. coli* were inoculated into 225 mm × 225 mm plates. Next, the bacteriophages were recovered from the culture solution of the inoculated E. coli to prepare a bacteriophage library solution.

[0501] In this first round of panning, the antibody-bound phage display system was concentrated. In the second round of panning, 250 pmol of ss-human CD137-Fc was used as the biotinylated antigen and washed three times with TBSTR, followed by two washes with TBS. Extraction was performed at room temperature with 25 mM DTT for 15 minutes, followed by trypsin degradation.

[0502] In the third and sixth rounds of panning, 62.5 pmol of C3NP1-27 was used as the biotinylated antigen, and the samples were washed three times with TBST and then twice with TBS. Extraction was carried out at room temperature with 25 mM DTT for 15 minutes, followed by trypsin digestion.

[0503] In the fourth, fifth, and seventh rounds of selection, 62.5 pmol of ss-human CD137-Fc was used as the biotinylated antigen, and the samples were washed three times with TBST and then twice with TBS. Extraction was carried out at room temperature with 25 mM DTT for 15 minutes, followed by trypsin digestion.

[0504] (6-3) Binding of CD3ε or human CD137 via the Fab domain displayed by bacteriophages

[0505] The culture supernatant containing phages was recovered from 96 single colonies of *E. coli* obtained by the above method according to standard procedures (Methods Mol. Biol. (2002) 178, 133-145). The culture supernatant containing phages was used for ELISA as follows: Streptomycin-coated microplates (384 wells, Greiner, Cat#781990) were coated overnight at 4°C or for 1 hour at room temperature with 10 μL of TBS containing biotinylated antigen (biotinylated CD3ε peptide or biotinylated human CD137-Fc). Each well was washed with TBST to remove unbound antigen. The wells were then blocked with 80 μL of TBS / 2% skim milk for 1 hour or longer. After removing the TBS / 2% skim milk, the prepared culture supernatant was added to each well, and the plate was incubated at room temperature for 1 hour to allow the antibodies displayed by the phages to bind to the antigens in each well. Each well was washed with TBST, and then HRP / anti-M13 (GE Healthcare 27-9421-010) was added. The wells were incubated for 1 hour. After washing with TBST, a single TMB solution (ZYMED Laboratories, Inc.) was added to the wells. The chromogenic reaction in each well was terminated by adding sulfuric acid. The colorimetric system was then measured based on the absorbance at 450 nm. The results are shown in Figure 16.

[0506] As shown in Figure 16, all pure strains showed binding to human CD3ε but not to human CD137, even after five panning processes for human CD137. This may be due to the low sensitivity of the phage ELISA with streptomycin-coated microplates, so phage ELISA with streptomycin-coated beads was also performed.

[0507] (6-4) Binding of human CD137 via the Fab domain displayed by bacteriophages (phage bead ELISA)

[0508] First, streptomycin-coated MyOne-T1 magnetic beads were washed three times with a blocking buffer containing 0.5x block Ace, 0.02% Tween, and 0.05% ProClin 300, and then blocked at room temperature for 60 minutes or longer with the same blocking buffer. After washing once with TBST, 0.625 pmol of ss-human CD137-Fc was added to the magnetic beads, and they were incubated at room temperature for 10 minutes or longer before being applied to each well of a 96-well plate (Corning, 3792 black round-bottom PS plate). 12.5 μL of Fab display phage solution and 12.5 μL of TBS were added to each well, and the plate was allowed to stand at room temperature for 30 minutes to allow the Fab to bind to the biotinylated antigen in each well. The wells were then washed with TBST. Anti-M13(p8)Fab-HRP diluted with blocking buffer containing 0.5x block Ace, 0.02% Tween, and 0.05% ProClin 300 was added to each well. The wells were incubated for 10 minutes. After washing three times with TBST, LumiPhos-HRP (Lumigen) was added to each well. Fluorescence was detected after 2 minutes. The results are shown in Figure 17.

[0509] Some pure strains showed significant binding to human CD137. This result indicates that the Fab domains binding to both human CD3ε and CD137 were also obtained from this library with a phage display panning scheme. However, the binding to human CD137 remained weak compared to the CD3ε peptide. The VH fragments of each human CD137-binding pure strain were amplified by PCR using primers specifically bound to phage vectors (sequence numbers: 157 and 158), and the DNA sequence was analyzed. The results showed that all binding pure strains had the same VH sequence, meaning that only one Fab pure strain bound to both human CD137 and CD3ε. To improve this, a two-round selection to phage display scheme was also applied in the next experiment.

[0510] [Example 7] Fab domains binding to CD3ε and human CD137 were obtained from a dual-Fab phage display library using a two-round selection method.

[0511] (7-1) Construct a heavy-chain phage display library with GLS3000 light chains

[0512] The phage display library exhibiting the Fab domain was created by infecting *E. coli* with the constructed phage particles via infection with the helper phage M13KO7TC / FkpA, which encodes the FkpA chaperone protein (sequence number: 17), and then incubating overnight at 25°C in the presence of 0.002% arabinose (this phage library is named DA library) or at 20°C in the presence of 0.02% arabinose (this phage library is named DX library). M13KO7TC is a helper phage with an insertion of a trypsin cleavage sequence between the N2 and CT domains of the pIII protein (see Japanese Patent Application Publication No. 2002-514413). The insertion of the gene into the M13KO7TC gene has been disclosed elsewhere (see WO2015 / 046554).

[0513] (7-2) Obtain the Fab domain by double-round selection, which is combined with CD3ε and human CD137.

[0514] The Fab domains binding to CD3ε and human CD137 were identified from the Fab library constructed in Example 7-1. Biotinylated CD3ε peptide antigen (amino acid sequence: sequence number: 6), biotinylated CD3ε peptide antigen via a disulfide linker (C3NP1-27: sequence number: 145), and biotinylated human CD137 fused to a human IgG1 Fc fragment (named human CD137-Fc) were used as antigens.

[0515] To generate more Fab domains that bind to human CD137 and CD3ε, dual-round selection was also applied in phage display panning in panning round 2 and subsequent rounds.

[0516] The phage system was created from *E. coli* with phage particles constructed for phage display. 2.5 M NaCl / 10% PEG was added to the culture solution of *E. coli* containing the phage, and the precipitated phage pool was diluted with TBS to obtain a phage library solution. Next, BSA (final concentration: 4%) was added to the phage display solution. Panning was performed according to a general panning method using antigens immobilized on magnetic beads (J. Immunol. Methods. (2008) 332(1-2), 2-9; J. Immunol. Methods (2001) 247(1-2), 191-203; Biotechnol. Prog. (2002) 18(2) 212-20; and Mol. Cell Proteomics (2003) 2(2), 61-9). The magnetic beads used were NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptomycin-coated beads (Dynabeads M-280 Streptavidin). To remove antibody-displaying phages bound to the magnetic beads themselves or the human IgG1 Fx region, the magnetic beads and biotin-labeled human Fc were depleted.

[0517] Specifically, in Round 1, magnetic beads were blocked at room temperature with 2% skim milk / TBS for 60 minutes or longer and washed three times with TBS. Phage solutions from the DA or DX library were added to the blocked magnetic beads and incubated at room temperature for 60 minutes or longer, after which the supernatant was recovered. 500 pmol of biotin-labeled human IgG1 Fc was added to new magnetic beads and incubated at room temperature for 15 minutes, followed by the addition of 2% skim milk / TBS. After blocking at room temperature for 60 minutes or longer, the magnetic beads were washed three times with TBS. The recovered phage solution was added to the blocked magnetic beads and incubated at room temperature for 60 minutes or longer, after which the supernatant was recovered. 500 pmol of biotin-labeled human CD137-Fc was added to new magnetic beads and incubated at room temperature for 15 minutes, followed by the addition of 2% skim milk / TBS. After blocking at room temperature for 60 minutes or longer, the magnetic beads were washed three times with TBS. The recovered phage solution was added to blocked magnetic beads, along with 8 nmol of free human IgG1 Fc domain, and incubated at room temperature for 60 minutes. The beads were washed twice with TBST (TBS containing 0.1% Tween 20; TBS was available from Takara Bio Inc.), followed by a further wash with 1 mL of TBS. After adding 0.5 mL of 1 mg / mL trypsin, the beads were suspended at room temperature for 15 minutes. Immediately afterward, the beads were separated using a magnetic base to recover the phage solution. The recovered phage solution was added to *E. coli* strain ER2738 in the logarithmic growth phase (OD 600: 0.4–0.5). The strain was incubated at 37°C with gentle rotation for 1 hour to infect *E. coli* with phage. The infected *E. coli* were inoculated into 225 mm × 225 mm plates. The phage was then recovered from the culture solution of the inoculated *E. coli* to prepare a phage library solution.

[0518] During this first round of selection, the antibody-display phage system bound to human CD137 is concentrated, so a double-round selection is performed from the second round of the selection process to recover antibody-display phages bound to both CD3ε and human CD137.

[0519] Specifically, in Round 2, magnetic beads were blocked with 2% skim milk / TBS at room temperature for 60 minutes or longer, and washed three times with TBS. Phage solution was added to the blocked magnetic beads and incubated at room temperature for 60 minutes or longer, after which the supernatant was recovered. 500 pmol of biotin-labeled human IgG1 Fc was added to new magnetic beads and incubated at room temperature for 15 minutes, followed by the addition of 2% skim milk / TBS. After blocking at room temperature for 60 minutes or longer, the magnetic beads were washed three times with TBS. The recovered phage solution was added to the blocked magnetic beads and incubated at room temperature for 60 minutes or longer, after which the supernatant was recovered. 500 pmol of biotin-labeled human CD137-Fc was added to new magnetic beads and incubated at room temperature for 15 minutes, followed by the addition of 2% skim milk / TBS. After blocking at room temperature for 60 minutes or longer, the magnetic beads were washed three times with TBS. The recovered phage solution was added to blocked magnetic beads and incubated at room temperature for 60 minutes. The beads were washed three times with TBST (TBS containing 0.1% Tween; TBS was available from Takara Bio Inc.) and then twice with 1 mL of TBS. Antibody-displaying phages were recovered using a fabricator (IdeS, the protease of the IgG hinge region, GENOVIS) (named IdeS flushing transport phase). In this process, 20 μL of Fabricator (10 units / μL) and 80 μL of TBS buffer were added, and the beads were suspended at 37°C for 30 minutes. Immediately afterward, the beads were separated using a magnetic base to recover the phage solution.

[0520] In the first cycle of this panning process, the antibody-display phage system bound to human CD1378 is concentrated and then proceeds to the second panning cycle to recover antibody-display phages also bound to CD3ε before phage infection and amplification. 500 pmol of biotin-labeled CD3ε is added to new magnetic beads and incubated at room temperature for 15 minutes, followed by the addition of 2% skim milk / TBS. After blocking at room temperature for 60 minutes or longer, the magnetic beads are washed three times with TBS. The recovered phage solution, 50 μL of TBS, and 250 μL of 8% BSA blocking buffer are added to the blocked magnetic beads, and the mixture is incubated at 37°C for 30 minutes, at room temperature for 60 minutes, at 4°C overnight, and then at room temperature for 60 minutes to transfer the antibody-display phage from human CD137 to CD3ε. The beads were washed three times with TBST (TBS containing 0.1% Tween 20; TBS was available from Takara Bio Inc.), followed by two more washes with 1 mL of TBS. The beads were then suspended at room temperature for 15 minutes with 0.5 mL of 1 mg / mL trypsin, and immediately separated using a magnetic base to recover the phage solution. The phage recovered from the trypsin-treated phage solution was added to *E. coli* strain ER2738 in the logarithmic growth phase (OD600: 0.4–0.7). The strain was incubated with the phage at 37°C with gentle rotation for 1 hour to infect *E. coli*. The infected *E. coli* were inoculated into 225 mm × 225 mm plates. The phage was then recovered from the culture solution of the inoculated *E. coli* to recover the phage library solution.

[0521] In the third and fourth rounds of selection, the number of washes was increased to five with TBST fol...

Claims

1. An antigen-binding molecule comprising: an antibody variable region capable of binding to CD3 and CD137, but not simultaneously binding to CD3 and CD137; and a variable region capable of binding to a third antigen different from CD3 and CD137, wherein the antibody variable region is selected from the group consisting of: (a) an antibody variable region comprising a VH sequence having an amino acid sequence of sequence number 30 and a VL sequence having an amino acid sequence of sequence number 51, (b) an antibody variable region comprising a VH sequence having an amino acid sequence of sequence number 46 and a VL sequence having an amino acid sequence of sequence number 53, (c) an antibody variable region comprising a VH sequence having an amino acid sequence of sequence number 40 and a VL sequence having an amino acid sequence of sequence number 53. The region includes: (d) an antibody variable region containing a VL sequence with an amino acid sequence of sequence number 56; (e) an antibody variable region containing a VH sequence with an amino acid sequence of sequence number 30 and a VL sequence with an amino acid sequence of sequence number 50; (f) an antibody variable region containing a VH sequence with an amino acid sequence of sequence number 46 and a VL sequence with an amino acid sequence of sequence number 54; and (g) an antibody variable region containing a VH sequence with an amino acid sequence of sequence number 40 and a VL sequence with an amino acid sequence of sequence number 57.

2. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule further has at least one feature selected from the group consisting of (3) and (4) below: (3) the antibody variable region binds to the extracellular domain of CD3ε containing the amino acid sequence of sequence number 91, and (4) the antigen-binding molecule does not induce the release of cytokines from PBMCs in the absence of a cell expressing a third antigen.

3. The antigen-binding molecule as claimed in claim 1 or 2, wherein the third antigen is a molecule specifically expressed in cancerous tissue.

4. The antigen-binding molecule of claim 1, wherein the antibody variable region that does not bind to CD3 and CD137 simultaneously is an antibody variable region that does not bind to CD3 and CD137 respectively expressed in different cells.

5. The antigen-binding molecule in claim 1 further includes an antibody Fc region.

6. The antigen-binding molecule of claim 5, wherein the antibody Fc region is an antibody Fc region that has reduced binding activity against FcγR compared to the antibody Fc region of naturally occurring human IgG1 antibody.

7. A pharmaceutical composition comprising an antigen-binding molecule as described in any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. A method for manufacturing an antigen-binding molecule, the antigen-binding molecule comprising an antibody variable region capable of binding to CD3 and CD137, but not simultaneously binding to CD3 and CD137; and a variable region binding to a third antigen different from CD3 and CD137, the method comprising: (i) preparing a vector wherein a polynucleotide encoding the antibody variable region is linked to a polynucleotide encoding a polypeptide comprising one of the Fc regions, wherein the antibody variable region is selected from the group consisting of: (a) an antibody variable region comprising a VH sequence having an amino acid sequence having sequence number 30 and a VL sequence having an amino acid sequence having sequence number 51. (b) An antibody variable region comprising a VH sequence having an amino acid sequence of sequence number 46 and a VL sequence having an amino acid sequence of sequence number 53; (c) An antibody variable region comprising a VH sequence having an amino acid sequence of sequence number 40 and a VL sequence having an amino acid sequence of sequence number 56; (d) An antibody variable region comprising a VH sequence having an amino acid sequence of sequence number 30 and a VL sequence having an amino acid sequence of sequence number 50; (e) An antibody variable region comprising a VH sequence having an amino acid sequence of sequence number 46 and a VL sequence having an amino acid sequence of sequence number 53. (i) A VL sequence of amino acid sequence number 54, (f) an antibody variable region comprising a VH sequence having amino acid sequence number 46 and a VL sequence having amino acid sequence number 55, and (g) an antibody variable region comprising a VH sequence having amino acid sequence number 40 and a VL sequence having amino acid sequence number 57; (ii) culturing cells containing the vector prepared in step (i) above; and (iii) collecting antigen-binding molecules from a culture medium containing one of the cells cultured in step (ii) above.

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