An antigen-binding molecule comprising a modified antibody variable region that binds to CD3 and CD137

Abstract

To provide antigen-binding domains binding to CD3 and CD137 and methods of using the same, and also provide methods to obtain antigen binding domains which bind to two or more different antigens more efficiently.SOLUTION: The present invention provides antigen-binding molecules, comprising: an antibody variable region that is capable of binding to CD3 and CD137, but does not bind to CD3 and CD137 at the same time; and a variable region binding to a third antigen different from CD3 and CD137.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to antigen-binding molecules that bind to CD3 and CD137 (4-1BB), and methods of using the same.

Background Art

[0002] Antibodies have attracted attention as pharmaceuticals because of their high stability in plasma and few adverse reactions (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-Patent Document 1) and Eur J Pharm Biopharm. (2005) 59 (3), 389-396 (Non-Patent Document 2)). Antibodies not only have the function of binding to antigens and agonist or antagonist functions, but also induce effector cell-mediated cytotoxic activities (also referred to as effector functions) such as ADCC (antibody-dependent cell-mediated cytotoxicity), ADCP (antibody-dependent cell phagocytosis), or CDC (complement-dependent cytotoxicity). In particular, antibodies of the IgG1 subclass exhibit effector functions against cancer cells, and thus many antibody pharmaceuticals have been developed in the field of oncology.

[0003] For an antibody to exert ADCC, ADCP, or CDC, its Fc region must bind to antibody receptors (FcγRs) present on effector cells (such as NK cells or macrophages) and various complement components. In humans, the protein family of FcγRs includes isoforms of FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and their respective allotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (Non-Patent Document 3)). Among these isoforms, FcγRIa, FcγRIIa, and FcγRIIIa have a domain called ITAM (immunoreceptor tyrosine-based activation motif) in their intracellular domain, which transmits activation signals. In contrast, only FcγRIIb has a domain called ITIM (immunoreceptor tyrosine-based inhibitory motif) in its intracellular domain, which transmits inhibitory signals. All of these isoforms of FcγR are known to transmit signals through cross-linking by immune complexes and the like (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-Patent Document 4)). In fact, when an antibody exerts an effector function against cancer cells, FcγR molecules on the effector cell membrane cluster due to the Fc regions of multiple antibodies bound to the cancer cell membrane, and activation signals are thereby transmitted through the effector cells. As a result, a cytotoxic effect is exerted. In this regard, the cross-linking of FcγR is limited to effector cells located near cancer cells, indicating that the activation of immunity is localized to cancer cells (Ann. Rev. Immunol. (1988). 6. 251-81 (Non-Patent Document 5)).

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

[0005] Natural IgG-type antibodies typically recognize and bind only one antigen because they recognize and bind one epitope by their variable regions (Fab). On the other hand, in cancer or inflammation, it is known that multiple types of proteins are involved, and these proteins may cross-talk with each other. For example, in immune diseases, it is known that several inflammatory cytokines (TNF, IL1, and IL6) are involved (Nat. Biotech., (2011) 28, 502-10 (Non-Patent Document 11)). Also, as one mechanism underlying the acquisition of drug resistance by cancer, it is known that other receptors are activated (Endocr Relat Cancer (2006) 13, 45-51 (Non-Patent Document 12)). In such cases, normal antibodies that recognize one epitope cannot inhibit multiple proteins.

[0006] As molecules that inhibit multiple targets, antibodies that bind to two or more antigens with a single molecule (these antibodies are called bispecific antibodies) have been studied. By modifying natural IgG antibodies, it is possible to confer binding activity against two different antigens (the first antigen and the second antigen) (mAbs. (2012) Mar 1, 4(2)). Therefore, such antibodies not only neutralize these two or more antigens with a single molecule, but also have the effect of enhancing antitumor activity by bridging cells with cytotoxic activity to cancer cells. As molecular forms of bispecific antibodies, molecules with antigen-binding sites added to the N-terminus or C-terminus of the antibody (DVD-Ig, TCB, and scFv-IgG), molecules in which the two Fab regions of the antibody have different sequences (common light chain bispecific antibodies and hybrid hybridomas), molecules in which one Fab region recognizes two antigens (Two-in-one IgG and DutaMab), and molecules having a CH3 domain loop as another antigen-binding site (Fcab) have been reported so far (Nat. Rev. (2010), 10, 301-316 (Non-Patent Document 13) and Peds(2010), 23(4), 289-297 (Non-Patent Document 14)). Since all of these bispecific antibodies interact with FcγR in their Fc regions, the effector functions of the antibodies are conserved therein.

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

[0008] For example, catumaxomab is known as a bispecific antibody that recognizes a protein expressed on T cells and a protein expressed on cancer cells (cancer antigen). Catumaxomab has two Fabs, each of which binds to the cancer antigen (EpCAM) and the CD3ε chain expressed on T cells. By simultaneously binding to the cancer antigen and CD3ε, catumaxomab induces T cell-mediated cytotoxic activity, and by simultaneously binding to the cancer antigen and FcγR, it induces NK cell- or antigen-presenting cell (e.g., macrophage)-mediated cytotoxic activity. By using these two cytotoxic activities, catumaxomab has shown a high therapeutic effect against malignant ascites by intraperitoneal administration and has thus been approved in Europe (Cancer Treat Rev. (2010) Oct 36(6), 458-67 (Non-Patent Document 16)). Furthermore, cases have been reported in which antibodies reactive against cancer cells appeared following administration of catumaxomab, demonstrating that acquired immunity is induced (Future Oncol. (2012) Jan 8(1), 73-85 (Non-Patent Document 17)). From these results, such antibodies having both T cell-mediated cytotoxic activity and the action brought about by cells such as NK cells or macrophages via FcγR (these antibodies are particularly referred to as trifunctional antibodies) are attracting attention because a strong antitumor effect and induction of acquired immunity can be expected.

[0009] However, since trifunctional antibodies simultaneously bind to CD3ε and FcγR even in the absence of a cancer antigen, they crosslink T cells expressing CD3ε to cells expressing FcγR in an environment where cancer cells are absent, causing the massive production of various cytokines. Due to the induction of the production of various cytokines independent of cancer antigens in this way, the administration of trifunctional antibodies is currently limited to the intraperitoneal route (Cancer Treat Rev. 2010 Oct 36(6), 458 - 67 (Non-Patent Document 16)). Trifunctional antibodies are very difficult to administer systemically due to severe cytokine storm-like adverse reactions (Cancer Immunol Immunother. 2007 Sep; 56(9): 1397 - 406 (Non-Patent Document 18)). Conventional bispecific antibodies can simultaneously bind to both antigens, namely, the cancer antigen (EpCAM) as the first antigen and CD3ε as the second antigen, simultaneously with binding to FcγR. Therefore, such adverse reactions caused by simultaneously binding to FcγR and the second antigen, CD3ε, cannot be avoided due to their molecular structure. In recent years, by using an Fc region with reduced binding activity to FcγR, improved antibodies have been provided that cause cytotoxic activity mediated by T cells while avoiding adverse reactions (WO2012 / 073985). However, even such antibodies cannot, due to their molecular structure, act on two immune receptors, namely, CD3ε and FcγR while binding to a cancer antigen. Antibodies that exhibit both cytotoxic activity mediated by T cells and cytotoxic activity mediated by cells other than T cells in a cancer antigen-specific manner while avoiding adverse reactions are not yet known.

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

[0011] CD137 agonist antibodies have already been demonstrated to exhibit antitumor effects, which have been experimentally shown to be mainly due to the activation of CD8-positive T cells and NK cells (Houot, 2009, Blood, 114, 3431-8 (Non-Patent Document 20)). T cells engineered to have a chimeric antigen receptor molecule consisting of a tumor antigen-binding domain as an extracellular domain and CD3 and CD137 signaling domains as intracellular domains (CAR-T cells) can enhance the durability of efficacy (Porter, N ENGL J MED, 2011, 365;725-733 (Non-Patent Document 21)). However, the side effects of such CD137 agonist antibodies due to their non-specific hepatotoxicity are clinical and preclinical problems, and drug development has not advanced (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22 (Non-Patent Document 22)). It has been suggested that the main cause of the side effects involves the binding of the antibody to Fcγ receptors via the antibody constant region (Schabowsky, Vaccine, 2009, 28, 512-22 (Non-Patent Document 23)). Furthermore, it has been reported that antibody cross-linking by Fcγ receptor-expressing cells (FcγRII-expressing cells) is required for agonist antibodies targeting receptors belonging to the TNF receptor superfamily to exert agonist activity in vivo (Li, Proc Natl Acad Sci USA. 2013, 110(48), 19501-6 (Non-Patent Document 24)). WO2015 / 156268 (Patent Document 3) describes that a bispecific antibody having a binding domain with CD137 agonist activity and a binding domain for a tumor-specific antigen 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 while retaining the antitumor activity of the antibody.WO2015 / 156268 further describes that the anti-tumor activity can be further enhanced and these adverse events can be avoided by using this bispecific antibody in combination with another bispecific antibody having a binding domain with CD3 agonist activity and a binding domain for a tumor-specific antigen. A trispecific antibody having three binding domains for CD137, CD3, and a tumor-specific antigen (EGFR) has also been reported (WO2014 / 116846 (Patent Document 4)). However, an antibody that exhibits both cytotoxic activity mediated by T cells and activation activity of T cells and other immune cells via CD137 in a cancer antigen-specific manner while avoiding adverse reactions has not yet been known.

[0012] Techniques for obtaining binding domains for any antigen using libraries are well known (Clackson et al., Nature 352:624-628 (1991)(Non-Patent Document 25); Marks et al., J. Mol. Biol. 222:581-597(1991)(Non-Patent Document 26)). For example, phage display, ribosome display, mRNA display, CIS display, Escherichia coli (E. coli) display, cell display, and yeast display are known as techniques for obtaining binding domains using libraries (Nat Biotechnol. 1996 Mar;14(3):309-14(Non-Patent Document 27); Nat Biotechnol. 2000 Dec; 18 (12): 1287-92(Non-Patent Document 28); Nucleic Acids Res. 2006; 34 (19): e127(Non-Patent Document 29); Proc Natl Acad Sci U S A. 2004 Mar 2; 101 (9): 2806-10(Non-Patent Document 30); Proc Natl Acad Sci U S A. 2004 Jun 22; 101 (25): 9193-8(Non-Patent Document 31); Protein Eng Des Sel. 2008 Apr; 21 (4): 247-55(Non-Patent Document 32); Proc Natl Acad Sci U S A. 2000 Sep 26; 97 (20): 10701-5(Non-Patent Document 33); MAbs. 2010 Sep-Oct; 2 (5): 508-18(Non-Patent Document 34); and Methods Mol Biol. 2012; 911: 183-98(Non-Patent Document 35)).

[0013] Binding domains that bind to two different antigens have also been obtained by library methods (Bostrom et al., Science 323:1610-4 (2009) (Non-Patent Document 36)). To obtain such domains that bind to two different antigens, several reported techniques exist, for example, a method of alternately using different antigens in different panning rounds, and a method of first obtaining a binding domain for a first antigen and then obtaining a binding domain for a second antigen from a library created by randomization of the binding domain for the first antigen. However, those strategies require a gene amplification step after recovery of the first antigen-binding domain in order to amplify the recovered polynucleotide.

[0014] A phage display method called double round selection has been reported, in which the selection pressure for one antigen is applied continuously twice without an intervening step of amplifying nucleic acids (Hawkins et al., J. Mol. Biol. 226:889-96 (1992) (Non-Patent Document 37)). However, a method for more efficiently collecting binding domains for two or more different antigens by applying the selection pressure two or more times continuously to two or more different antigens is not known.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0016]

Non-Patent Document 1

Non-Patent Document 28

Non-Patent Document 29

Non-Patent Document 30

Non-Patent Document 31

Non-Patent Document 32

Non-Patent Document 33

Non-Patent Document 34

Non-Patent Document 35

Non-Patent Document 36

Non-Patent Document 37

Summary of the Invention

Problems to be Solved by the Invention

[0017] A trispecific antibody comprising a tumor-specific antigen (EGFR) binding domain, a CD137 binding domain, and a CD3 binding domain has already been reported (WO2014116846). However, since antibodies having such a molecular format can bind to three different antigens simultaneously, the inventors speculated that those trispecific antibodies could bring about cross-linking between CD3ε-expressing T cells and CD137-expressing cells (e.g., T cells, B cells, NK cells, DCs, etc.) by binding to CD3 and CD137 simultaneously. Furthermore, bispecific antibodies against CD8 and CD3ε have already been reported to induce mutual cytotoxic activity among CD8-positive T cells in order to cross-link them (Wong, Clin. Immunol. Immunopathol. 1991, 58(2), 236-250). Therefore, the inventors speculated that bispecific antibodies against molecules expressed on T cells and CD3ε would also induce mutual cytotoxic activity among T cells in order to cross-link cells expressing that molecule and cells expressing CD3ε.

[0018] To obtain antigen domains that bind to two different antigens, several previously reported techniques exist, such as methods of alternatively using different antigens in different panning rounds, and methods of first obtaining a binding domain for a first antigen and then obtaining a binding domain for a second antigen from a library generated by randomization of the binding domain for the first antigen. However, those strategies require the steps of recovering the binding domain for the first antigen and then amplifying the recovered nucleotides encoding the binding domain for the first antigen, and further recovering a binding domain that can also bind to the second antigen and amplifying its nucleic acid. As a result of this process, the inventors considered that each panning round process would ultimately specifically enrich a binding domain that shows stronger binding to one of the various antigens used therein than to other antigens, over binding domains that show binding to each of the various antigens, thus preventing the desired molecule from being efficiently recovered.

[0019] In some methodologies such as cell display, yeast display, or bacterial display where FACS (fluorescence-activated cell sorting) can be used for selection, it is understood that it is possible to apply two or more selection pressures simultaneously to two or more different antigens. However, the inventors believe that in methodologies such as phage display, ribosome display, mRNA display, or CIS display where FACS cannot be used, it has been difficult to apply two or more selection pressures simultaneously to two or more different antigens.

Means for Solving the Problems

[0020] The present invention provides an antigen-binding domain that binds to CD3 and CD137, and methods of using the same. The present invention also provides a method for more efficiently obtaining an antigen-binding domain that binds to two or more different antigens.

[0021] In some embodiments, 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 does not bind to CD3 and CD137 simultaneously, and a variable region that binds to a third antigen different from CD3 and CD137. In some embodiments, the antigen-binding molecule of the present invention is an antigen-binding molecule comprising an antibody variable region that can bind to a T cell receptor and CD137 (4-1BB) but does not bind to the T cell receptor and CD137 simultaneously; and a variable region that binds to a third antigen different from the T cell receptor and CD137. In some embodiments, 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 but does not bind to CD3 and CD137 simultaneously, and a variable region that binds to a molecule specifically expressed in cancer tissue.

[0022] In some embodiments, the antigen-binding domain of the present invention is a variable region that can bind to CD3 and CD137, but does not bind to CD3 and CD137 simultaneously. In some embodiments, the antibody variable region of the present invention is a variable region that can bind to CD3 and CD137, but does not bind to CD3 and CD137 simultaneously.

[0023] In some embodiments, the present invention also provides an antigen-binding domain that is a variable region that does not bind to CD3 and CD137 simultaneously, and is expressed on different cells respectively.

[0024] In some embodiments, the antigen-binding molecule of the present invention comprises an antibody Fc region. In a further embodiment, the antigen-binding molecule of the present invention comprises an antibody Fc region with reduced binding activity to FcγR as compared to the Fc region of a native human IgG1 antibody.

[0025] In some embodiments, the antigen-binding molecule of the present invention has at least one characteristic selected from the group consisting of the following (1) to (4): (1) The variable region binds to the extracellular domain of CD3ε comprising the amino acid sequence of SEQ ID NO: 91. (2) The antigen-binding molecule has agonist activity against CD137. (3) The antigen-binding molecule induces CD3 activation of T cells against cells expressing a third antigen molecule, but does not induce activation of T cells against cells expressing CD137, and (4) The antigen-binding molecule does not induce cytokine release from PBMCs in the absence of cells expressing a third antigen molecule.

[0026] In some embodiments, the antigen-binding molecule of the present invention has at least one characteristic selected from the group consisting of the following (1) to (2): (1) The antigen-binding molecule does not compete with the CD137 ligand for binding to CD137, and (2) The antigen-binding molecule induces the cytotoxic activity of T cells against cells expressing the molecule of the third antigen, but does not induce the cytotoxic activity of T cells against cells expressing CD137.

[0027] In some embodiments, the antigen-binding molecule of the present invention competes for binding to CD137 with an antibody selected from the group consisting of: (a) an antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 30 and a VL sequence having the amino acid sequence of SEQ ID NO: 51, (b) an antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 46 and a VL sequence having the amino acid sequence of SEQ ID NO: 53, (c) an antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 40 and a VL sequence having the amino acid sequence of SEQ ID NO: 56, (d) an antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 30 and a VL sequence having the amino acid sequence of SEQ ID NO: 58, and (e) an antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 40 and a VL sequence having the amino acid sequence of SEQ ID NO: 61.

[0028] In some embodiments, the antigen-binding molecule of the present invention comprises an amino acid sequence generated by introducing one or more amino acid modifications into a template sequence consisting of the heavy chain variable domain sequence set forth in SEQ ID NO: 92 and / or the light chain variable domain sequence set forth in SEQ ID NO: 93, wherein the one or more amino acids are at the following positions: H chain: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, and 100g (Kabat numbering); and L chain: 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52, 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, and 96 (Kabat numbering) and comprise at least one amino acid selected from: The HVR-H3 of the modified heavy chain variable domain sequence is 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 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 (Kabat numbering) at amino acid position 100g and contains at least one amino acid selected from

[0029] In some embodiments, the antigen-binding molecule of the invention comprises a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 41, 30, 46, or 40; a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, or 57; or (c) the VH sequence of (a) and the VL sequence of (b).

[0030] In some embodiments, the antigen-binding molecule of the invention is a monoclonal antibody. In some embodiments, the antigen-binding molecule of the invention is a human antibody, a humanized antibody, or a chimeric antibody. In further embodiments, the antigen-binding molecule of the invention is a full-length IgG1, IgG2, IgG3, or IgG4 antibody.

[0031] The present invention also provides an isolated nucleic acid encoding the antigen-binding molecule of the present invention. The present invention also provides a host cell comprising the nucleic acid of the present invention. The present invention also provides a method for producing an antibody, which comprises the step of culturing the host cell of the present invention so that an antibody is produced.

[0032] The present invention also provides a pharmaceutical preparation comprising the antigen-binding molecule of the present invention and a pharmaceutically acceptable carrier.

[0033] The antigen-binding molecule of the present invention may be for use as a drug. The antigen-binding molecule of the present invention may be for use in the treatment of various types of cancer. The antigen-binding molecule of the present invention may be used in the manufacture of a drug. In some embodiments, the drug is for the treatment of various types of cancer. The present invention also provides a method for treating an individual having various types of cancer. In some embodiments, the method comprises the step of administering to the individual an effective amount of the antigen-binding molecule of the present invention.

[0034] The inventors have succeeded in preparing an antigen-binding molecule comprising an antibody variable region having binding activity to two different antigens (CD3 and CD137) but not binding to these antigens simultaneously, and a variable region binding to an antigen different from these antigens (a third antigen), and have found that it enhances the activity induced by this antigen-binding molecule through the use of its binding activity to three different antigens. In addition, the inventors have succeeded in preparing an antigen-binding molecule that can avoid cross-linking between different cells resulting from the binding of a conventional multispecific antigen-binding molecule to antigens expressed on different cells, which is considered to cause adverse reactions when the multispecific antigen-binding molecule is used as a medicine.

[0035] The inventors have also succeeded in developing a method for more efficiently obtaining an antigen-binding domain that binds to two or more different antigens. In some embodiments, the method for screening for an antigen-binding domain that binds to at least two or more different antigens of interest of the present invention is (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 gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains. The method does not include, between step (b) and step (c), amplifying the nucleic acid encoding the antigen-binding domains collected in step (b).

[0036] In some embodiments, the antigen-binding domain of the present invention is a Fab, scFv, Fab'2, VHH, VH, or VL. In some embodiments, the antigen-binding domain of the present invention is a fusion polypeptide formed by fusing an antigen-binding domain with a scaffold for cross-linking the antigen-binding domain and the nucleic acid encoding the antigen-binding domain.

[0037] In some embodiments, the scaffold of the present invention is a bacteriophage. In some embodiments, the scaffold of the present invention is a ribosome, a RepA protein, or a DNA puromycin linker.

[0038] In some embodiments, in steps (b) and (c) above, elution is performed using an elution solution that is an acidic solution, a basic solution, DTT, or IdeS. In some embodiments, the elution solution used in steps (b) and (c) of the present invention is EDTA or IdeS.

[0039] In some embodiments, a method for screening an antigen-binding domain of the present invention that binds to at least two or more different antigens of interest is (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; (b)' translating the nucleic acid encoding the antigen-binding domain collected in step (b); (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 gene encoding the antigen-binding domain collected in step (c) and identifying candidate antigen-binding domains, and the method does not include a step of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b) between step (b) and step (c).

[0040] In some embodiments, a method for generating an antigen-binding domain of the present invention that binds to at least two or more different antigens of interest is (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 gene encoding the antigen-binding domain collected in step (c) and identifying candidate antigen-binding domains; (e) ligating the polynucleotide encoding the candidate antigen-binding domain selected in step (d) with a polynucleotide encoding a polypeptide comprising an Fc region; (f) culturing a cell into which a vector in which the polynucleotide obtained in the above step (d) is functionally linked is introduced, and (g) collecting the antigen-binding molecule from the culture solution of the cells cultured in the above step (f) comprising, and the method does not include a step of amplifying a nucleic acid encoding the antigen-binding domain collected in step (b) between step (b) and step (c).

[0041] In some embodiments, the library provided in step (a) of the present invention is a design library.

[0042] In some embodiments, the antigen-binding molecule of the present invention is an antibody prepared by the above method.

[0043] More specifically, the present invention relates to the following. [1] An antibody variable region capable of binding to CD3 and CD137 but not binding to CD3 and CD137 simultaneously; and A variable region that binds to a third antigen different from CD3 and CD137 An antigen-binding molecule comprising. [2] The antigen-binding molecule of [1], wherein the third antigen is a molecule specifically expressed in cancer tissue. [3] The antigen-binding molecule of [1] or [2], wherein the variable region that does not bind to CD3 and CD137 simultaneously is a variable region that does not bind to CD3 and CD137 simultaneously expressed on different cells. [4] The antigen-binding molecule of any one of [1] to [3], further comprising an antibody Fc region. [5] The antigen-binding molecule of [4], wherein the Fc region has a reduced binding activity to FcγR compared to the Fc region of a native human IgG1 antibody. [6] The antigen-binding molecule of any one of [1] to [5], having at least one characteristic selected from the group consisting of the following (1) to (4): (1) The variable region binds to the extracellular domain of CD3ε containing the amino acid sequence of SEQ ID NO: 91 (2) The antigen-binding molecule has agonist activity against CD137. (3) The antigen-binding molecule induces CD3 activation of T cells against cells expressing the molecule of 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 cytokine release from PBMCs in the absence of cells expressing the molecule of the third antigen. [7] An antigen-binding molecule according to any one of [1] to [6] that competes with an antibody selected from the group consisting of the following for binding to CD137: (a) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 30 and a VL sequence having the amino acid sequence of SEQ ID NO: 51. (b) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 46 and a VL sequence having the amino acid sequence of SEQ ID NO: 53. (c) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 40 and a VL sequence having the amino acid sequence of SEQ ID NO: 56. (d) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 30 and a VL sequence having the amino acid sequence of SEQ ID NO: 58, and (e) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 40 and a VL sequence having the amino acid sequence of SEQ ID NO: 61. [8] Comprising an amino acid sequence generated by introducing one or more amino acid modifications into a template sequence consisting of the heavy chain variable domain sequence described in SEQ ID NO: 92 and / or the light chain variable domain sequence described in SEQ ID NO: 93, wherein the one or more amino acids are at the following positions: H chain: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, and 100g (Kabat numbering); and L chain: 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52, 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, and 96 (Kabat numbering) comprising at least one amino acid selected from the HVR-H3 of the modified heavy chain variable domain sequence is Ala, Pro, Ser, Arg, His, or Thr at position 98 of the amino acid; Ala, Ser, Thr, Gln, His, or Leu at position 99 of the amino acid; Tyr, Ala, Ser, Pro, or Phe at position 100 of the amino acid; Tyr, Val, Ser, Leu, or Gly at position 100a of the amino acid; Asp, Ser, Thr, Leu, Gly, or Tyr at position 100b of the amino acid; Val, Leu, Phe, Gly, His, or Ala at position 100c of the amino acid; Leu, Phe, Ile, or Tyr at position 100d of the amino acid; Gly, Pro, Tyr, Gln, Ser, or Phe at position 100e of the amino acid; Tyr, Ala, Gly, Ser, or Lys at position 100f of the amino acid; Gly, Tyr, Phe, or Val at position 100g of the amino acid (Kabat numbering) comprising at least one amino acid selected from the antigen-binding molecule of any one of [1] to [7]. [9] (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 41, 30, 46, or 40; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, or 57; or (c) the VH sequence of (a) and the VL sequence of (b) comprising the antigen-binding molecule of any one of [1] to [8]. A pharmaceutical composition comprising any one of the antigen-binding molecules [1] to [9] and a pharmaceutically acceptable carrier.

[11] A method for screening antigen-binding domains that bind to at least two or more different antigens of interest, (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 gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains comprising, A method that does not include a step of amplifying the nucleic acid encoding the antigen-binding domains collected in step (b) between step (b) and step (c).

[12] The antigen-binding domain is A fusion polypeptide formed by fusing the antigen-binding domain with a scaffold in order to crosslink the antigen-binding domain and the nucleic acid encoding the antigen-binding domain is the method of

[11] .

[13] The method of

[12] , wherein the scaffold is a bacteriophage.

[14] The method of

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

[15] The method of

[12] or

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

Brief Description of the Drawings

[0044]

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Mode for Carrying Out the Invention

[0045] Description of Embodiments In one aspect, 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 does not bind to CD3 and CD137 simultaneously, and a variable region that binds to a third antigen different from CD3 and CD137. In one aspect, the antigen-binding molecule of the present invention can bind to the T cell receptor and CD137 (4-1BB), but does not bind to the T cell receptor and CD137 simultaneously, and comprises an antibody variable region that binds to a third antigen different from the T cell receptor and CD137, and a variable region that binds to the third antigen. In one aspect, the antigen-binding molecule of the present invention can bind to CD3 and CD137, but does not bind to CD3 and CD137 simultaneously, and comprises an antibody variable region that binds to a molecule specifically expressed in cancer tissue.

[0046] In one aspect, the antigen-binding domain of the present invention is a variable region that can bind to CD3 and CD137, but does not bind to CD3 and CD137 simultaneously. In one aspect, the antibody variable region of the present invention is a variable region that can bind to CD3 and CD137, but does not bind to CD3 and CD137 simultaneously.

[0047] In some embodiments, the antigen-binding molecule of the present invention can activate T cells by its agonistic activity against CD3, and can induce the cytotoxic activity of T cells against target cells, and can enhance the activation, survival, and differentiation of T cells into memory T cells by its co-stimulatory agonistic activity against CD137 and CD3. On the other hand, since the antigen-binding molecule of the present invention does not bind to CD3 and CD137 simultaneously, it can avoid the harmful effects caused by the cross-linking of CD137 and CD3.

[0048] In some embodiments, the antigen-binding molecule of the present invention can also activate immune cells expressing CD137 and enhance the immune response against target cells by its agonistic activity against CD137.

[0049] In the present invention, the "antibody variable region" generally means a region containing a domain composed of four framework regions (FRs) and three adjacent complementarity-determining regions (CDRs), and also includes a partial sequence thereof as long as the partial sequence has an activity of binding to a part or all of an antigen. In particular, a region containing an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH) is preferred. The antibody variable region of the present invention may have any sequence, and may be a variable region derived from any antibody such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, a humanized antibody obtained by humanizing any of these non-human antibodies, and a human antibody. A "humanized antibody" is also called a reshaped human antibody, and is obtained by transplanting a complementarity-determining region (CDR) of an antibody derived from a non-human mammal, for example, a mouse antibody, into a CDR of a human antibody. Methods for identifying CDRs are known in the art (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; and Chothia et al., Nature (1989) 342: 877). General genetic recombination techniques therefor are also known in the art (see European Patent Application Publication No. EP 125023 and WO 96 / 02576).

[0050] "Does not bind to CD3 and CD137 (4-1BB) simultaneously" The "antibody variable region" of the present invention means that when the antibody variable region of the present invention is bound to CD3, it cannot bind to CD137, and conversely, when the variable region is bound to CD137, it cannot bind to CD3. Here, the phrase "does not bind to CD3 and CD137 simultaneously" includes not cross-linking cells expressing CD3 and cells expressing CD137, or not binding to CD3 and CD137 expressed on different cells simultaneously. This phrase further includes that when the variable region is such that CD3 and CD137 are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell, the variable region can bind to both CD3 and CD137 simultaneously, but does not include the case where it cannot bind to CD3 and CD137 expressed on different cells simultaneously. Such antibody variable regions are not particularly limited as long as they have these functions. Examples thereof may include variable regions derived from IgG-type antibody variable regions in which a part of the amino acids has been modified to bind to a desired antigen. The amino acids to be modified are selected, for example, from amino acids in the antibody variable region that binds to CD3 or CD137 and whose modification does not cause loss of binding to the antigen. Here, the phrase "expressed on different cells" simply means that the antigens are expressed on separate cells. Such combinations of cells may be, for example, the same type of cells such as T cells and another T cell, or different types of cells such as T cells and NK cells.

[0051] In the present invention, one amino acid modification may be used alone, or a plurality of amino acid modifications may be used in combination. When using a plurality of amino acid modifications in combination, the number of modifications to be combined is not particularly limited and can be appropriately set within the range that can achieve the object of the invention. The number of modifications 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. The multiple amino acid modifications to be combined may be added only to the heavy chain variable domain or the light chain variable domain of the antibody, or may be appropriately distributed to both the heavy chain variable domain and the light chain variable domain.

[0052] One or more amino acid residues in the variable region are acceptable as the amino acid residues to be modified as long as the antigen-binding activity is maintained. When 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 a binding activity that is, for example, 50% or more, more preferably 80% or more, even more preferably 100% or more higher than that before modification, but the variable regions according to the present invention are not limited thereto. The binding activity may be increased by amino acid modification, for example, it may be 2-fold, 5-fold, or 10-fold the binding activity before modification.

[0053] Examples of preferred regions for amino acid modification include regions and loops exposed to the solvent in the variable region. Among them, CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, positions 31-35, 50-65, 71-74, and 95-102 according to Kabat numbering in the heavy chain variable domain, and positions 24-34, 50-56, and 89-97 according to Kabat numbering in the light chain variable domain are preferred. Positions 31, 52a-61, 71-74, and 97-101 according to Kabat numbering in the heavy chain variable domain, and positions 24-34, 51-56, and 89-96 according to Kabat numbering in the light chain variable domain are more preferred. Also, when modifying amino acids, amino acids that increase the antigen-binding activity may be further introduced.

[0054] As used herein, the terms "hypervariable region" or "HVR" refer to each region of an antibody variable domain in which the sequences ("complementary determining regions" or "CDRs") are hypervariable and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact an antigen ("antigen contact sites"). Generally, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contact sites present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and / or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domains (e.g., FR residues) are numbered herein according to Kabat et al. as described above.

[0055] In the present invention, "loop" means a region containing residues not involved in maintaining the β-barrel structure of an immunoglobulin. In the present invention, amino acid modification means substitution, deletion, addition, insertion, or modification, or a combination thereof. In the present invention, amino acid modification can be used interchangeably with amino acid mutation and can be used in the same meaning.

[0056] Substitution of an amino acid residue is carried out, for example, by replacement with another amino acid residue for the purpose of modifying any of the following (a) to (c): (a) the polypeptide backbone structure of a region having a sheet structure or a helical structure; (b) the charge or hydrophobicity of the target site; and (c) the size of the side chain. Amino acid residues are classified into the following groups based on the nature of their general side chains: (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 affecting the orientation of the chain: Gly and Pro; and (6) aromatic residues: Trp, Tyr, and Phe.

[0057] Substitution of an amino acid residue within each of these groups is called a conservative substitution, while substitution of an amino acid residue in one of these groups with an amino acid residue in another group is called a non-conservative substitution. The substitution according to the present invention may be a conservative substitution or a non-conservative substitution. Alternatively, a combination of conservative substitution and non-conservative substitution may be used.

[0058] Modification of the amino acid residues also includes selection of variable regions that can bind to CD3 and CD137 but cannot bind to these antigens simultaneously, from those obtained by random modification of amino acids in the antibody variable regions that bind to CD3 or CD137 and do not cause loss of binding to the antigen; and modification by inserting a peptide previously known to have binding activity to a desired antigen into the above-mentioned region.

[0059] In the antibody variable regions of the present invention, the above-mentioned modifications may be combined with modifications known in the art. For example, modification to pyroglutamic acid by pyroglutamylation of the N-terminal glutamine of the variable region is a modification well known to those skilled in the art. Therefore, an antibody of the present invention having glutamine at the N-terminus of its heavy chain may contain a variable region in which this N-terminal glutamine is modified to pyroglutamic acid.

[0060] Such antibody variable regions may further have amino acid modifications, for example, to improve antigen binding, pharmacokinetics, stability, or antigenicity. The antibody variable regions of the present invention may be modified to have pH-dependent binding to the antigen, thereby enabling repeated binding to the antigen (WO2009 / 125825).

[0061] Also, for example, amino acid modifications may be added to such antibody variable regions that bind to a third antigen to change the antigen binding activity according to the concentration of a target tissue-specific compound (WO2013 / 180200).

[0062] The variable regions may be further modified, for example, for the purpose of enhancing binding activity, improving specificity, lowering pI, imparting pH-dependent antigen binding characteristics, improving thermal stability of binding, improving solubility, improving stability against chemical modification, improving heterogeneity derived from sugar chains, avoiding T cell epitopes identified by in silico prediction or use of in vitro T cell-based assays for reducing immunogenicity, or introducing T cell epitopes for activating regulatory T cells (mAbs 3:243-247, 2011).

[0063] Whether the antibody variable region of the present invention "can bind to CD3 and CD137" can be determined by methods known in the art. This can be determined, for example, by the electrochemiluminescence method (ECL method) (BMC Research Notes 2011, 4:281). Specifically, for example, a region of a biotin-labeled test antigen-binding molecule that can bind to CD3 and CD137, such as a small antibody composed of a Fab region, or a monovalent antibody thereof (an antibody lacking one of the two Fab regions of a normal antibody), is mixed with CD3 or CD137 labeled with a sulfo-tag (Ru complex), and the mixture is added onto a streptavidin-immobilized plate. In this operation, the biotin-labeled test antigen-binding molecule binds to streptavidin on the plate. Light is generated from the sulfo-tag, and the luminescence signal is detected using, for example, a Sector Imager 600 or 2400 (MSD K.K.), whereby the binding of the above-described region of the test antigen-binding molecule to CD3 or CD137 can be confirmed. Alternatively, this assay may be carried out by ELISA, FACS (fluorescence-activated cell sorting), ALPHAScreen (amplified luminescence proximity homogeneous assay screen), the BIACORE method based on the surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0064] Specifically, the assay can be carried out using, for example, a Biacore (GE Healthcare Japan Corp.), which is an interaction analysis instrument based on the surface plasmon resonance (SPR) phenomenon. The Biacore analysis instrument includes any model such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any Biacore sensor chip such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, or Au chip can be used as the sensor chip. A protein for capturing the antigen-binding molecule of the present invention, such as protein A, protein G, protein L, anti-human IgG antibody, anti-human IgG-Fab, anti-human light chain antibody, anti-human Fc antibody, antigen protein, or antigen peptide, is immobilized on the sensor chip by a coupling method such as amine coupling, disulfide coupling, or aldehyde coupling. Then, CD3 or CD137 is injected as an analyte, and the interaction is measured to obtain a sensorgram. In this operation, the concentration of CD3 or CD137 can be selected within the range of several μM to several pM according to the strength (e.g., KD) of the interaction of the assay sample.

[0065] Alternatively, CD3 or CD137 may be immobilized on the sensor chip instead of the antigen-binding molecule, and then the antibody sample to be evaluated is allowed to interact. Whether the antibody variable region of the antigen-binding molecule of the present invention has binding activity to CD3 or CD137 can be confirmed based on the dissociation constant (KD) value calculated from the interaction sensorgram or based on the degree of increase in the sensorgram after the action, which exceeds the level before the action of the antigen-binding molecule sample.

[0066] ALPHAScreen is carried out based on the following principle by ALPHA technology using two types of beads (donor and acceptor): A luminescence signal is detected only when the two beads are in close proximity due to a biological interaction between the molecule bound to the donor bead and the molecule bound to the acceptor bead. The photosensitizer in the donor bead excited by a laser converts the surrounding oxygen into singlet oxygen in an excited state. When the singlet oxygen diffuses around the donor bead and reaches the acceptor bead located in its vicinity, it thereby causes a chemiluminescence reaction in the bead, and finally light is emitted. If there is no interaction between the molecule bound to the donor bead and the molecule bound to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead. Therefore, the chemiluminescence reaction does not occur.

[0067] One of the substances (ligand) whose interaction is to be observed is immobilized on the gold thin film of the sensor chip. Light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass. As a result, a portion of the reflected light forms a site where the reflection intensity decreases (SPR signal). The other substance (analyte) whose interaction is to be observed is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the sensor chip surface changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the bound molecules dissociate, the signal returns to its original position). The Biacore system plots the amount of the shift, i.e., the mass change on the sensor chip surface, on the vertical axis and displays the time-dependent change in mass as assay data (sensorgram). The amount of analyte bound to the ligand captured on the sensor chip surface (the amount of change in the response on the sensorgram before and after the analyte is allowed to interact) can be determined from the sensorgram. However, since the amount of binding also depends on the amount of ligand, comparisons must be made under conditions where substantially the same amount of ligand is used. Kinetics, i.e., the association rate constant (ka) and the dissociation rate constant (kd), can be determined from the curve of the sensorgram, while the affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also suitably used in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0068] Whether the antigen-binding molecule of the present invention "does not bind to CD3 and CD137 simultaneously" can be confirmed by: confirming that the antigen-binding molecule has binding activity to both CD3 and CD137; then, pre-binding either CD3 or CD137 to the antigen-binding molecule containing the variable region having this binding activity; and then determining the presence or absence of its binding activity to the other one by the method described above. Alternatively, this can also be confirmed by determining whether the binding of the antigen-binding molecule to either CD3 or CD137 immobilized on an ELISA plate or a sensor chip is inhibited by the addition of the other one into the solution. In some embodiments, the binding of the antigen-binding molecule of the present invention to either CD3 or CD137 is inhibited by the binding of the antigen-binding molecule to the other one by at least 50%, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, or even more preferably 95% or more.

[0069] In one aspect, while immobilizing one antigen (e.g., CD3), the inhibition of the binding of the antigen-binding molecule to CD3 can be determined by a method known in the prior art (i.e., ELISA, BIACORE, etc.) in the presence of another antigen (e.g., CD137). In another aspect, while immobilizing CD137, the inhibition of the binding of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is implemented, if the binding is inhibited by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, or even more preferably 95% or more, the antigen-binding molecule of the present invention is determined not to bind to CD3 and CD137 simultaneously. In some embodiments, the concentration of the antigen injected as an analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen to be immobilized. In a preferred mode, the concentration of the antigen injected 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%. In one aspect, the ratio of the KD value for the CD3 (analyte) binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized) binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the KD value ratio (KD(CD3) / KD(CD137)) of the CD137 (immobilized) concentration can be used for the above-described competition measurement. (For example, when the KD value ratio is 0.1, concentrations that are 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Further, when the KD value ratio is 10, concentrations that are 100-fold, 500-fold, 1000-fold, or 2000-fold higher can be selected.)

[0070] In one aspect, while one antigen (e.g., CD3) is being 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 prior art (i.e., ELISA, ECL, etc.). In another aspect, while CD137 is being immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is implemented, if the binding signal is attenuated by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, or even more preferably 95% or more, the antigen-binding molecule of the present invention is determined not to bind to CD3 and CD137 simultaneously (see Examples 5-5, 7-5, 8-9, 9-4). In some aspects, the concentration of the antigen injected as an analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen to be immobilized. In a preferred mode, the concentration of the antigen injected 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%. In one aspect, the ratio of the KD value for the CD3 (analyte) binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized) binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the KD value ratio (KD(CD3) / KD(CD137)) of the CD137 (immobilized) concentration can be used for the above measurements. (For example, when the KD value ratio is 0.1, a concentration 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Further, when the KD value ratio is 10, a concentration 100-fold, 500-fold, 1000-fold, or 2000-fold higher can be selected.)

[0071] Specifically, for example, when using the ECL method, a biotin-labeled test antigen-binding molecule, CD3 labeled with sulfo-tag (Ru complex), and unlabeled CD137 are prepared. If the test antigen-binding molecule can bind to CD3 and CD137 but not simultaneously to CD3 and CD137, a mixture of the test antigen-binding molecule and labeled CD3 is added onto a streptavidin-immobilized plate, and the luminescence signal of the sulfo-tag is detected in the absence of unlabeled CD137 by subsequent luminescence. In contrast, the luminescence signal decreases in the presence of unlabeled CD137. The decrease in this luminescence signal can be quantified to determine the relative binding activity. This analysis can be similarly performed using labeled CD137 and unlabeled CD3.

[0072] In the case of ALPHAScreen, the test antigen-binding molecule interacts with CD3 in the absence of competing CD137 to generate a signal at 520 - 620 nm. Untagged CD137 competes with CD3 for interaction with the test antigen-binding molecule. The decrease in fluorescence caused as a result of the competition can be quantified, thereby determining the relative binding activity. Biotinylation of polypeptides using, for example, sulfo-NHS-biotin is known in the art. For example, CD3 can be tagged with GST by an appropriately employed method including fusing a polynucleotide encoding CD3 and a polynucleotide encoding GST in-frame; and expressing the resulting fusion gene in cells or the like carrying a vector capable of its expression, and then purifying it using a glutathione column. The obtained signal is preferably analyzed using software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) that fits a one-site competition model based on, for example, non-linear regression analysis. This analysis can be similarly performed using tagged CD137 and untagged CD3. Alternatively, a method using fluorescence resonance energy transfer (FRET) may be used. FRET is a phenomenon in which excitation energy directly moves between two fluorescent molecules located in proximity to each other by electron resonance. When FRET occurs, the excitation energy of the donor (a fluorescent molecule in an excited state) moves to the acceptor (another fluorescent molecule located near the donor), so the fluorescence emitted from the donor disappears (more precisely, the fluorescence lifetime is shortened), and instead fluorescence is emitted from the acceptor. By using this phenomenon, it is possible to analyze whether or not it binds to CD3 and CD137 simultaneously. For example, when CD3 having a fluorescent donor and CD137 having a fluorescent acceptor bind to a test antigen-binding molecule simultaneously, the fluorescence of the donor disappears, while fluorescence is emitted from the acceptor. Therefore, a change in the fluorescence wavelength is observed. Such an antibody is confirmed to bind to CD3 and CD137 simultaneously. On the other hand, if the mixture of CD3, CD137, and the test antigen-binding molecule does not change the fluorescence wavelength of the fluorescent donor bound to CD3, this test antigen-binding molecule can be regarded as an antigen-binding domain that can bind to CD3 and CD137 but does not bind to CD3 and CD137 simultaneously.

[0073] For example, a biotin-labeled test antigen-binding molecule is bound to streptavidin on donor beads, while CD3 tagged with glutathione S-transferase (GST) is bound to acceptor beads. The test antigen-binding molecule interacts with CD3 in the absence of a competing second antigen to generate a signal at 520 - 620 nm. The untagged second antigen competes with CD3 for interaction with the test antigen-binding molecule. The decrease in fluorescence caused as a result of the competition can be quantified, whereby the relative binding activity can be determined. Biotinylation of polypeptides using, for example, sulfo-NHS-biotin is known in the art. For example, CD3 can be tagged with GST by appropriately employed methods including fusing a polynucleotide encoding CD3 and a polynucleotide encoding GST in-frame; and expressing the resulting fusion gene in cells or the like carrying a vector capable of its expression and then purifying using a glutathione column. The resulting signal is preferably analyzed using software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) that fits a one-site competition model based on, for example, non-linear regression analysis.

[0074] Tagging is not limited to GST tagging and may be carried out with any tag such as, but not limited to, a histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc. Binding of the test antigen-binding molecule to the donor beads is not limited to binding using the biotin-streptavidin reaction. In particular, when the test antigen-binding molecule contains Fc, possible methods include binding the test antigen-binding molecule via an Fc-recognizing protein such as protein A or protein G on the donor beads.

[0075] Also, when CD3 and CD137 are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell, the variable region can bind to CD3 and CD137 simultaneously, but when CD3 and CD137 are expressed on different cells, it cannot bind to them simultaneously. This can also be assayed by methods known in the art. Specifically, a test antigen-binding molecule that has been confirmed to be positive in an ECL-ELISA for detecting simultaneous binding to CD3 and CD137 is also mixed with cells expressing CD3 and cells expressing CD137. It can be shown that the test antigen-binding molecule cannot bind simultaneously to CD3 and CD137 expressed on different cells unless the antigen-binding molecule and these cells bind to each other simultaneously. This assay can be performed, for example, by cell-based ECL-ELISA. Cells expressing CD3 are immobilized on a plate in advance. After binding the test antigen-binding molecule thereto, cells expressing CD137 are added to the plate. Different antigens expressed only on cells expressing CD137 are detected using an antibody labeled with a sulfo-tag against this antigen. A signal is observed when the antigen-binding molecule binds simultaneously to the two antigens expressed on the two cells respectively. No signal is observed when the antigen-binding molecule does not bind to these antigens simultaneously. Alternatively, this assay may be performed by the ALPHAScreen method. The test antigen-binding molecule is mixed with cells expressing CD3 bound to donor beads and cells expressing CD137 bound to acceptor beads. A signal is observed when the antigen-binding molecule binds simultaneously to the two antigens expressed on the two cells respectively. No signal is observed when the antigen-binding molecule does not bind to these antigens simultaneously. Alternatively, this assay may be performed by the Octet interaction analysis method. First, cells expressing CD3 with a peptide tag are bound to a biosensor that recognizes the peptide tag. Cells expressing CD137 and the test antigen-binding molecule are placed in a well and analyzed for interaction. When the antigen-binding molecule simultaneously binds to two antigens expressed on two cells respectively, a large wavelength shift caused by the binding of the test antigen-binding molecule and the cells expressing CD137 to the biosensor is observed. When the antigen-binding molecule does not simultaneously bind to these antigens, a small wavelength shift caused by the binding of only the test antigen-binding molecule to the biosensor is observed.

[0076] Instead of these methods based on binding activity, an assay based on biological activity may be performed. For example, cells expressing CD3 and cells expressing CD137 are mixed with the test antigen-binding molecule and cultured. The two antigens expressed on two cells respectively are mutually activated via the test antigen-binding molecule when the antigen-binding molecule simultaneously binds to these two antigens. Therefore, changes in activation signals such as an increase in the phosphorylation level downstream of each antigen can be detected. Alternatively, cytokine production is induced as a result of activation. Therefore, the amount of cytokine produced can be measured, and thereby it can be confirmed whether the antigen-binding molecule binds to the two cells simultaneously. Alternatively, cytotoxic activity against cells expressing CD137 is induced as a result of activation. Alternatively, the expression of a reporter gene is induced by a promoter activated downstream of the signal transduction pathway of CD137 or CD3 as a result of activation. Therefore, the cytotoxic activity or the amount of reporter protein produced can be measured, and thereby it can be confirmed whether the antigen-binding molecule binds to the two cells simultaneously.

[0077] In the present invention, the "Fc region" refers to a region in an antibody molecule that includes a fragment consisting of a hinge or a part thereof, and CH2 and CH3 domains. The Fc region of the IgG class means, for example, the region from cysteine 226 (EU numbering (also referred to as EU index in this specification)) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus, but is not limited thereto. The Fc region can preferably be obtained, for example, by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a proteolytic enzyme such as pepsin and then redissolving the fraction adsorbed to a protein A column or a protein G column. Such a proteolytic enzyme is not particularly limited as long as it can digest the full-length antibody to form Fab or F(ab')2 restrictively under appropriately set enzyme reaction conditions (for example, pH). Examples thereof may include pepsin and papain.

[0078] In some embodiments, the "antigen-binding molecule" is not particularly limited as long as it includes the "antibody variable region" of the present invention. The antigen-binding molecule may further include a peptide or protein having a length of approximately 5 amino acids or more. The peptide or protein is not limited to a peptide or protein derived from an organism, and may be, for example, a polypeptide consisting of an artificially designed sequence. Also, natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. may be used.

[0079] In some embodiments, the "antigen-binding molecule" of the present invention is not particularly limited to a molecule containing an "antibody variable region". In certain embodiments, an antigen-binding molecule other than an antibody containing a variable region can bind to two different antigens. For example, an affibody, etc., may be obtained by methods generally 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 U S A. 2011 Aug 23;108(34):14067-72).

[0080] Preferred examples of the antigen-binding molecule of the present invention may include an antigen-binding molecule containing an antibody Fc region.

[0081] For example, an Fc region derived from native IgG can be used as the "Fc region" of the present invention. Here, native IgG means a polypeptide that contains the same amino acid sequence as that found in nature and belongs to the class of antibodies substantially encoded by the immunoglobulin γ gene. Native human IgG means, for example, native human IgG1, native human IgG2, native human IgG3, or native human IgG4. Native IgG also includes naturally occurring variants derived therefrom. Multiple allotype sequences based on gene polymorphisms are described as the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies in Sequences of proteins of immunological interest, NIH Publication No.91-3242, and any of them can be used in the present invention. In particular, the sequence of human IgG1 may have DEL or EEM as the amino acid sequence at positions 356 to 358 of EU numbering.

[0082] The Fc region of the antibody is found, for example, as the Fc region of the IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM type. For example, the Fc region derived from a native human IgG antibody can be used as the antibody Fc region of the present invention. For example, the constant region of native IgG, specifically, the constant region originating from native human IgG1 (SEQ ID NO: YY004), the constant region originating from native human IgG2 (SEQ ID NO: YY005), the constant region originating from native human IgG3 (SEQ ID NO: YY006), or the Fc region derived from the constant region originating from native human IgG4 (SEQ ID NO: YY007) can be used as the Fc region of the present invention. The constant region of native IgG also includes naturally occurring variants derived therefrom and the like.

[0083] The Fc region of the present invention is particularly preferably an Fc region with reduced binding activity to Fcγ receptors. Here, the Fcγ receptor (also referred to as FcγR in this specification) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4, and means any member of the protein family substantially encoded by the Fcγ receptor gene. In humans, this family includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); and also includes any undiscovered human FcγR or FcγR isoform or allotype, but is not limited thereto. FcγRs include those derived from humans, mice, rats, rabbits, and monkeys. FcγR is not limited to these molecules and may be derived from any organism. Mouse FcγRs include FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), and also includes any undiscovered mouse FcγR or FcγR isoform or allotype, but is not limited thereto. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).

[0084] FcγRs are found in the form of activating receptors having an ITAM (immunoreceptor tyrosine-based activation motif) and inhibitory receptors having an ITIM (immunoreceptor tyrosine-based inhibitory motif). FcγRs are classified into activating FcγRs (FcγRI, FcγRIIa R, FcγRIIa H, FcγRIIIa, and FcγRIIIb) and inhibitory FcγR (FcγRIIb). The polynucleotide and amino acid sequences of FcγRI are described in NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIa are described in BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIb are described in BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIa are described in BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIb are described in BC128562.1 and AAI28563.1, respectively (RefSeq accession numbers). There are two genetic polymorphisms in FcγRIIa in which the 131st amino acid of FcγRIIa is substituted by histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19 - 25, 1990). There are two genetic polymorphisms in FcγRIIb in which the 232nd amino acid of FcγRIIb is substituted by isoleucine (I type) or threonine (T type) (Arthritis. Rheum. 46: 1242 - 1254 (2002)). There are two genetic polymorphisms in FcγRIIIa in which the 158th amino acid of FcγRIIIa is substituted by valine (V type) or phenylalanine (F type) (J. Clin. Invest. 100(5): 1059 - 1070 (1997)). There are two genetic polymorphisms (NA1 type and NA2 type) in FcγRIIIb (J. Clin. Invest. 85: 1287 - 1295 (1990)).

[0085] The reduced binding activity to the Fcγ receptor can be confirmed by well-known methods such as FACS, ELISA format, ALPHAScreen (amplified luminescence proximity homogeneous assay screen), or the BIACORE method based on the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHAScreen method is carried out based on the following principle by the ALPHA technology using two types of beads (donor and acceptor): A luminescence signal is detected only when the two beads are positioned in proximity due to a biological interaction between a molecule bound to the donor bead and a molecule bound to the acceptor bead. The photosensitizer in the donor bead excited by a laser converts the surrounding oxygen to singlet oxygen in an excited state. The singlet oxygen diffuses around the donor bead and, when it reaches the acceptor bead positioned in proximity thereto, causes a chemiluminescence reaction in the bead, and finally light is emitted. When there is no interaction between the molecule bound to the donor bead and the molecule bound to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead. Therefore, the chemiluminescence reaction does not occur.

[0086] For example, a biotin-labeled test antigen-binding molecule is bound to a donor bead, while an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to an acceptor bead. In the absence of an antigen-binding molecule having a competing mutant Fc region, an antigen-binding molecule having a wild-type Fc region interacts with the Fcγ receptor to generate a signal at 520 to 620 nm. An antigen-binding molecule having an untagged mutant Fc region competes with the antigen-binding molecule having a wild-type Fc region for interaction with the Fcγ receptor. The decrease in fluorescence caused as a result of the competition can be quantified, whereby the relative binding affinity can be determined. Biotinylation of an antigen-binding molecule (e.g., an antibody) using, for example, sulfo-NHS-biotin is known in the art. For example, tagging the Fcγ receptor with GST can be appropriately employed by methods including fusing a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST in-frame; and expressing the resulting fusion gene by, for example, a cell carrying a vector capable of expressing it, and then purifying it using a glutathione column. The obtained signal is preferably analyzed using software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) that fits a one-site competition model based on, for example, non-linear regression analysis.

[0087] One of the substances (ligand) for observing the interaction therebetween is immobilized on the gold thin film of the sensor chip. Light is applied from the back side of the sensor chip so as to totally reflect at the interface between the gold thin film and the glass. As a result, a portion with a reduced reflection intensity (SPR signal) is formed in a part of the reflected light. The other substance (analyte) for observing the interaction therebetween is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecules increases, and the refractive index of the solvent on the sensor chip surface changes. Due to this change in the refractive index, the position of the SPR signal shifts (conversely, when the bound molecules dissociate, the signal returns to its original position). The Biacore system plots the amount of the shift, i.e., the mass change on the sensor chip surface, on the vertical axis and displays the time-dependent change in mass as assay data (sensorgram). Kinetics, i.e., the association rate constant (ka) and the dissociation rate constant (kd), can be determined from the curve of the sensorgram, while the affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also preferably used in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0088] As used herein, a decrease in the binding activity to the Fcγ receptor means that the test antigen-binding molecule exhibits a binding activity, based on the above-described analysis method, of, for example, 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, as compared to the binding activity of a control antigen-binding molecule containing an Fc region. An antigen-binding molecule having an Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be appropriately used as a control antigen-binding molecule. The structure of the Fc region is described in SEQ ID NO: 94 (A added to the N-terminus of RefSeq accession number AAC82527.1), SEQ ID NO: 95 (A added to the N-terminus of RefSeq accession number AAB59393.1), SEQ ID NO: 96 (A added to the N-terminus of RefSeq accession number CAA27268.1), or SEQ ID NO: 97 (A added to the N-terminus of RefSeq accession number AAB59394.1). When an antigen-binding molecule having a variant of the Fc region of an antibody of a specific isotype is used as a test substance, an antigen-binding molecule having the Fc region of this specific isotype antibody is used as a control to test the effect of the mutation in the variant on the binding activity to the Fcγ receptor. An antigen-binding molecule having an Fc region variant thus confirmed to have a reduced binding activity to the Fcγ receptor is appropriately prepared.

[0089] For example, 231A-238S deletion (WO 2009 / 011941), C226S, C229S, P238S, (C220S) (J.Rheumatol (2007) 34, 11), C226S, C229S (Hum.Antibod.Hybridomas (1990) 1(1), 47-54), C226S, C229S, E233P, L234V, or L235A (Blood (2007) 109, 1185-1192) (these amino acids are defined according to EU numbering) variants are known in the art as such variants. Preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of a particular isotype of antibody by substitution of any of the following constituent amino acids: amino acids 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 as defined according to EU numbering. The isotype of the antibody from which the Fc region is derived is not particularly limited, and an Fc region derived from an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be used as appropriate. An Fc region derived from a native human IgG1 antibody is preferably used. For example, the following groups of substitutions of constituent amino acids, where the numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid notation preceding the number represents the amino acid residue before substitution; and the single-letter amino acid notation following the number represents the amino acid residue before substitution: (a) L234F, L235E, and P331S, (b) C226S, C229S, and P238S, (c) C226S and C229S, and (d) C226S, C229S, E233P, L234V, and L235A Antigen-binding molecules having an Fc region derived from the Fc region of an IgG1 antibody, either by any of the above or by deletion of the amino acid sequence at positions 231-238, can also be used as appropriate.

[0090] The following groups of substitutions of constituent amino acids, where the numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid notation preceding the number represents the amino acid residue before substitution; and the single-letter amino acid notation following the number represents the amino acid residue before substitution: (e) H268Q, V309L, A330S, and P331S, (f) V234A, (g) G237A, (h) V234A and G237A, (i) A235E and G237A, and (j) V234A, A235E, and G237A Antigen-binding molecules having an Fc region derived from the Fc region of an IgG2 antibody, by any of the above, may also be used as appropriate.

[0091] The following groups of substitutions of constituent amino acids, defined according to EU numbering (the numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid notation preceding the number represents the amino acid residue before substitution; and the single-letter amino acid notation following the number represents the amino acid residue before substitution): (k) F241A, (l) D265A, and (m) V264A Antigen-binding molecules having an Fc region derived from the Fc region of an IgG3 antibody, by any of the above, may also be used as appropriate.

[0092] The following groups of substitutions of constituent amino acids, defined according to EU numbering (the numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid notation preceding the number represents the amino acid residue before substitution; and the single-letter amino acid notation following the number represents the amino acid residue before substitution): (n) L235A, G237A, and E318A, (o) L235E, and (p) F234A and L235A Antigen-binding molecules having an Fc region derived from the Fc region of an IgG4 antibody, by any of the above, may also be used as appropriate.

[0093] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of a native human IgG1 antibody by substitution of the amino acid at the corresponding EU numbering position in the Fc region of the counterpart IgG2 or IgG4 with any of the following constituent amino acids: the amino acids at positions 233, 234, 235, 236, 237, 327, 330, and 331 as defined according to EU numbering.

[0094] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of a native human IgG1 antibody by substitution of any one or more of the following constituent amino acids: the amino acids at positions 234, 235, and 297 as defined according to EU numbering with different amino acids. The type of amino acid present after substitution is not particularly limited. Antigen-binding molecules having an Fc region in which any one or more of the amino acids at positions 234, 235, and 297 are substituted with alanine are particularly preferred.

[0095] Other preferred examples include antigen-binding molecules having an Fc region derived from the Fc region of an IgG1 antibody by substitution of the constituent amino acid at position 265 as defined according to EU numbering with a different amino acid. The type of amino acid present after substitution is not particularly limited. Antigen-binding molecules having an Fc region in which the amino acid at position 265 is substituted with alanine are particularly preferred.

[0096] One preferred form of the "antigen-binding molecule" of the present invention can be, for example, a multispecific antibody containing the antibody variable region of the present invention.

[0097] For the association of multispecific antibodies, techniques for suppressing unintended H-chain association by introducing charge repulsion at the interface between the second constant domain (CH2) or the third constant domain (CH3) of the antibody H-chain can be applied (WO2006 / 106905). In a technique for suppressing unintended H-chain association by introducing charge repulsion at the interface of CH2 or CH3, examples of amino acid residues that contact each other at the interface between H-chain constant domains include the residue at position 356 of EU numbering, the residue at position 439 of EU numbering, the residue at position 357 of EU numbering, the residue at position 370 of EU numbering, the residue at position 399 of EU numbering, and the residue at position 409 of EU numbering in one CH3 domain, and their partner residues in another CH3 domain.

[0098] More specifically, for example, as an antibody in which one to three pairs of amino acid residues selected from the following pairs (1) to (3) of amino acid residues in the first H-chain CH3 domain have the same charge, an antibody containing two H-chain CH3 domains can be prepared: (1) the amino acid residues at positions 356 and 439 of EU numbering contained in the H-chain CH3 domain; (2) the amino acid residues at positions 357 and 370 of EU numbering contained in the H-chain CH3 domain; and (3) the amino acid residues at positions 399 and 409 of EU numbering contained in the H-chain CH3 domain.

[0099] The antibody can further be prepared as an antibody in which one to three pairs of amino acid residues are selected from the pairs (1) to (3) of amino acid residues in a second H-chain CH3 domain different from the first H-chain CH3 domain such that the one to three pairs of amino acid residues have the same charge as the pairs (1) to (3) of amino acid residues with the same charge in the first H-chain CH3 domain and have a charge opposite to that of the corresponding amino acid residues in the first H-chain CH3 domain.

[0100] Each amino acid residue described in pairs (1) to (3) is located near its partner in the associated H-chain. A person skilled in the art can find the positions corresponding to the amino acid residues described in each of pairs (1) to (3) according to homology modeling using commercially available software, etc., for the desired H-chain CH3 domain or H-chain constant domain, and can appropriately modify the amino acid residues at those positions.

[0101] In the above antibody, each of the "amino acid residues having a charge" is preferably selected from amino acid residues contained in any one of the following groups (a) and (b): (a) Glutamic acid (E) and aspartic acid (D); and (b) Lysine (K), arginine (R), and histidine (H).

[0102] In the above antibody, the phrase "having the same charge" means, for example, that all of two or more amino acid residues are amino acid residues contained in any one of groups (a) and (b). The phrase "having opposite charges" means, for example, that at least one of the two or more amino acid residues may be an amino acid residue contained in any one of groups (a) and (b), while the remaining amino acid residues are amino acid residues contained in the other group.

[0103] In a preferred embodiment, the antibody may have the first H-chain CH3 domain and the second H-chain CH3 domain crosslinked by a disulfide bond. The amino acid residues modified according to the present invention are not limited to the amino acid residues in the above-mentioned antibody variable region or antibody constant region. Those skilled in the art can find the amino acid residues constituting the interface according to homology modeling using commercially available software for polypeptide variants or heteromultimers, and can modify the amino acid residues at that position so as to control the association.

[0104] The association of the multispecific antibodies of the present invention can also be carried out by alternative techniques known in the art. The amino acid side chains present in the variable domain of one antibody H chain are replaced with larger side chains (knobs), and the amino acid side chains of its partner present in the variable domain of another H chain are replaced with smaller side chains (holes). The knob can be placed into the hole so that polypeptides of different Fc domains associate efficiently (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; and Merchant AM et al. Nature Biotechnology (1998) 16, 677-681).

[0105] In addition to this technique, further alternative techniques known in the art may be used to form the multispecific antibodies of the present invention. A part of the CH3 of one antibody H chain is converted to the corresponding IgA-derived sequence, and the complementary part in the CH3 of another H chain is converted to the corresponding IgA-derived sequence. By using the resulting chain exchange operation domain CH3, efficient association between polypeptides with different sequences can be caused by complementary CH3 association (Protein Engineering Design & Selection, 23; 195-202, 2010). By using this technique known in the art, the multispecific antibody of interest can also be formed efficiently.

[0106] Alternatively, the multispecific antibody can be formed by, for example, an antibody preparation technique using the CH1-CL association and VH-VL association of antibodies as described in WO2011 / 028952, a technique for preparing a bispecific antibody using separately prepared monoclonal antibodies as described in WO2008 / 119353 and WO2011 / 131746 (Fab arm exchange), a technique for controlling the association between the CH3 domains of antibody heavy chains as described in WO2012 / 058768 and WO2013 / 063702, a technique for preparing a bispecific antibody composed of two types of light chains and one type of heavy chain as described in WO2012 / 023053, or a technique for preparing a bispecific antibody using two bacterial cell lines each expressing a half molecule of an antibody consisting of one H chain and one L chain as described in Christoph et al. (Nature Biotechnology Vol. 31, p 753-758 (2013)). In addition to these association techniques, the CrossMab technology (Scaefer et al., Proc. Natl. Acad. Sci. U.S.A. (2011) 108, 11187-11192), a known heterologous light chain association technique in which a light chain forming a variable region that binds to a first epitope and a light chain forming a variable region that binds to a second epitope are each associated with a heavy chain forming a variable region that binds to the first epitope and a heavy chain forming a variable region that binds to the second epitope, can also be used to prepare the multispecific or multiparatopic antigen-binding molecules provided by the present invention. Examples of techniques for preparing a bispecific antibody using separately prepared monoclonal antibodies can include methods that include promoting the heterodimerization of the antibodies by placing a monoclonal antibody in which specific amino acids in the heavy chain CH3 domain have been substituted under reducing conditions to obtain the desired bispecific antibody. Examples of preferred amino acid substitution sites for this method can include the residue at position 392 and the residue at position 397 of the EU numbering in the CH3 domain.Furthermore, the bispecific antibody can also be prepared by using an antibody in which one to three pairs of amino acid residues selected from the following pairs (1) to (3) of amino acid residues in the first H-chain CH3 domain have the same charge: (1) the amino acid residues at positions 356 and 439 contained in the H-chain CH3 domain; (2) the amino acid residues at positions 357 and 370 contained in the H-chain CH3 domain; and (3) the amino acid residues at positions 399 and 409 contained in the H-chain CH3 domain. The bispecific antibody can also be prepared by using an antibody in which one to three pairs of amino acid residues are selected from the pairs (1) to (3) of amino acid residues in the second H-chain CH3 domain different from the first H-chain CH3 domain such that the one to three pairs of amino acid residues have the same charge as the pairs (1) to (3) of amino acid residues having the same charge in the first H-chain CH3 domain and have the opposite charge to the corresponding amino acid residues in the first H-chain CH3 domain.

[0107] Even when it is not possible to efficiently form a multispecific antibody of interest, the multispecific antibody of the present invention can be obtained by separating and purifying the multispecific antibody of interest from the produced antibodies. For example, in previously reported methods, amino acid substitutions are introduced into the variable domains of two types of heavy chains to impart a difference in isoelectric point so that two types of homodimers and the heterodimerizing antibody of interest can be separately purified by ion exchange chromatography (WO2007114325). A method using protein A to purify a heterodimerizing antibody consisting of a heavy chain of mouse IgG2a that can bind to protein A and a heavy chain of rat IgG2b that cannot bind to protein A has been previously reported as a method for purifying heterodimers (WO98050431 and WO95033844). Alternatively, the amino acid residues at positions 435 and 436 of the EU numbering that constitute the protein A binding site of IgG may be substituted with amino acids such as Tyr and His that provide different strengths of protein A binding, and the resulting heavy chains are used to change the interaction between each heavy chain and protein A. As a result, only the heterodimerizing antibody can be efficiently purified by using a protein A column.

[0108] Multiple of these techniques, for example two or more, may be used in combination. Also, these techniques can be appropriately applied separately to the two heavy chains to be associated. The antigen-binding molecule of the present invention may be prepared as an antigen-binding molecule having the same amino acid sequence, apart from being based on such a modified form.

[0109] Modification of the amino acid sequence can be carried out by various methods known in the art. Examples of these methods that may be performed include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M.(1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500; Kramer,W, Drutsa,V, Jansen,HW, Kramer,B, Pflugfelder,M, and Fritz,HJ(1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz HJ(1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; and Kunkel,TA(1985) Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc Natl Acad Sci U S A. 82, 488-492), PCR mutagenesis, and cassette mutagenesis, etc.

[0110] The "antigen-binding molecule" of the present invention may be an antibody fragment that includes both a heavy chain and a light chain that constitute the "antibody variable region" of the present invention in a single polypeptide chain but lacks a constant region. Such antibody fragments may be, for example, a diabody (Db), a single-chain antibody, or sc(Fab')2.

[0111] Db is a dimer composed of two polypeptide chains (e.g., Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; and W093 / 11161). These polypeptide chains are linked through a linker, for example, approximately 5-residue linker, that is short enough so that the L-chain variable domain (VL) and the H-chain variable domain (VH) on the same polypeptide chain cannot pair with each other. Due to this short linker, VL and VH encoded on the same polypeptide chain cannot form a single-chain Fv. Instead, they dimerize with VH and VL, respectively, on another polypeptide chain to form two antigen-binding sites.

[0112] Examples of single-chain antibodies include sc(Fv)2. sc(Fv)2 is a single-chain antibody having one chain composed of four variable domains, i.e., two VLs and two VHs, linked via a linker such as a peptide linker (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VHs and VLs may be derived from different monoclonal antibodies. Preferred examples include bispecific sc(Fv)2 that recognizes two epitopes present in the same antigen as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be prepared by methods generally known to those skilled in the art. For example, sc(Fv)2 can be prepared by connecting two scFvs via a linker such as a peptide linker.

[0113] Examples of the composition of the antigen-binding domains that make up the sc(Fv)2 described in this specification include antibodies in which two VHs and two VLs are aligned in the order of VH, VL, VH, and VL (i.e., [VH]-linker-[VL]-linker-[VH]-linker-[VL]) starting from the N-terminus of the single-chain polypeptide. The order of the two VHs and two VLs is not particularly limited to the above composition and may be in any arrangement order. Examples thereof may also include the following arrangements: [VL]-linker-[VH]-linker-[VH]-linker-[VL], [VH]-linker-[VL]-linker-[VL]-linker-[VH], [VH]-linker-[VH]-linker-[VL]-linker-[VL], [VL]-linker-[VL]-linker-[VH]-linker-[VH], and [VL]-linker-[VH]-linker-[VL]-linker-[VH].

[0114] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352. Based on the description therein, those skilled in the art can appropriately prepare the desired sc(Fv)2 for preparing the antigen-binding molecules disclosed in this specification.

[0115] The antigen-binding molecules of the present invention may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Also, sugar chains can be suitably added to the antigen-binding molecules of the present invention by inserting a sugar chain addition sequence for the purpose of producing a desired effect.

[0116] For example, any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (e.g., the linker disclosed in Protein Engineering, 9 (3), 299-305, 1996), can be used as a linker for linking antibody variable domains. In the present invention, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. The length is preferably 5 amino acids or more (the upper limit is not particularly limited and is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, all of these peptide linkers used may have the same length or may have different lengths.

[0117] Examples of peptide linkers include Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 162), Ser-Gly-Gly-Gly (SEQ ID NO: 163), Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 164), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 165), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 166), Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 167), Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 168), Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 169), (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 164))n, and (Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 165))n, (Here, n is an integer of 1 or more) may be included. However, the length or sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose.

[0118] Synthetic compound linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, 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), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidyloxycarbonyloxy)ethyl] sulfone (BSOCOES), or bis[2-(sulfosuccinimidyloxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES). These crosslinking agents are commercially available. To link four antibody variable domains, three linkers are usually required. All of these linkers used may be the same linker or different linkers.

[0119] F(ab')2 contains two light chains and two heavy chains that contain the constant regions (a portion of the CH1 domain and the CH2 domain) such that inter-chain disulfide bonds are formed between the two heavy chains. The F(ab')2 that constitutes the polypeptide aggregate disclosed herein can preferably be obtained, for example, by partially digesting a full-length monoclonal antibody having a desired antigen-binding domain with a proteolytic enzyme such as pepsin and then removing the adsorbed Fc fragment on a protein A column. Such proteolytic enzymes are not particularly limited as long as they can digest the full-length antibody to form F(ab')2 restrictively under appropriately set enzyme reaction conditions (e.g., pH). Examples thereof may include pepsin and ficin.

[0120] In addition to the above-described amino acid modifications, the antigen-binding molecule of the present invention can further contain additional modifications. The additional modifications can be selected, for example, from amino acid substitutions, deletions, and modifications, and combinations thereof. For example, the antigen-binding molecule of the present invention can be further optionally modified without substantially changing the intended function of the molecule. Such mutations can be made, for example, by conservative substitution of amino acid residues. Alternatively, even a modification that changes the intended function of the antigen-binding molecule of the present invention may be carried out as long as the function changed by such modification is within the scope of the object of the present invention.

[0121] The modification of the amino acid sequence according to the present invention also includes post-translational modification. Specifically, the post-translational modification can refer to the addition or deletion of sugar chains. For example, the antigen-binding molecule of the present invention having a constant region of the IgG1 type can have an amino acid residue modified with a sugar chain at position 297 of the EU numbering. The sugar chain structure for use in the modification is not limited. Generally, antibodies expressed by eukaryotic cells contain sugar chain modifications in the constant region. Therefore, antibodies expressed by the following cells are usually modified with several sugar chains: antibody-producing cells of mammals; and A eukaryotic cell transformed with an expression vector containing DNA encoding an antibody. Here, eukaryotic cells include yeast and animal cells. For example, CHO cells or HEK293H cells are typical animal cells for transformation with an expression vector containing DNA encoding an antibody. On the other hand, the antibodies of the present invention also include antibodies without sugar chain modification at that position. Antibodies having a constant region not modified with sugar chains can be obtained by expression of the genes encoding these antibodies in prokaryotic cells such as Escherichia coli.

[0122] An additional modification according to the present invention may more specifically be, for example, the addition of sialic acid to the sugar chain in the Fc region (mAbs. 2010 Sep - Oct;2(5):519 - 27).

[0123] When the antigen - binding molecule of the present invention has an Fc region, for example, amino acid substitutions that improve the binding activity to FcRn (J Immunol. 2006 Jan 1;176(1):346 - 56; J Biol Chem. 2006 Aug 18;281(33):23514 - 24; Int Immunol. 2006 Dec;18(12):1759 - 69; Nat Biotechnol. 2010 Feb;28(2):157 - 9; WO2006 / 019447; WO2006 / 053301; and WO2009 / 086320), or amino acid substitutions for improving the heterogeneity or stability of the antibody ((WO2009 / 041613)) may be added.

[0124] In the present invention, the term "antibody" is used in the broadest sense and includes any antibody such as monoclonal antibodies (including full - length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, and humanized antibodies as long as they exhibit the desired biological activity.

[0125] The antibody of the present invention is not limited by the type of its antigen, its origin, etc., and may be any antibody. Examples of the origin of the antibody may include, but are not particularly limited to, human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

[0126] Antibodies can be prepared by methods well known to those skilled in the art. For example, monoclonal antibodies may be produced by the hybridoma method (Kohler and Milstein, Nature 256:495 (1975)) or the recombinant method (U.S. Patent No. 4,816,567). Alternatively, monoclonal antibodies may be isolated from a phage display antibody library (Clackson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991)). Monoclonal antibodies may also be isolated from a single B cell clone (N. Biotechnol. 28(5): 253-457 (2011)).

[0127] Humanized antibodies are also referred to as recombinant human antibodies. Specifically, for example, humanized antibodies composed of human antibodies with CDRs of non-human animal (e.g., mouse) antibodies transplanted are known in the art. General genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known in the art as a method for transplanting the CDRs of mouse antibodies into human FRs.

[0128] DNA encoding an antibody variable domain containing three CDRs and four FRs linked thereto, respectively, and DNA encoding a human antibody constant domain are inserted into an expression vector such that the variable domain DNA is fused in-frame with the constant domain DNA to prepare a vector for expressing a humanized antibody. These vectors having the insert are introduced into a host to establish recombinant cells. Then, the recombinant cells are cultured for the expression of the DNA encoding the humanized antibody to produce the humanized antibody in the culture of the cultured cells (see European Patent Publication No. EP 239400 and International Publication No. WO1996 / 002576).

[0129] If necessary, the amino acid residues of the FRs may be substituted so that the CDRs of the reconstructed human antibody form an appropriate antigen-binding site. For example, the amino acid sequence of the FRs can be mutated by applying the PCR method used in the transplantation of mouse CDRs to human FRs.

[0130] The desired human antibody can be obtained by DNA immunization using a transgenic animal having all repertoires of human antibody genes (see International Publication Nos. WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) as an immunized animal.

[0131] In addition, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, the human antibody V region is expressed as a single-chain antibody (scFv) on the surface of phage by the phage display method. Phage expressing the antigen-binding scFv can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the V region of the antigen-binding human antibody can be determined. After determining the DNA sequence of the antigen-binding scFv, the V region sequence is fused in-frame with the sequence of the desired human antibody C region, and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is transferred into the preferred expression cells listed above for the expression of the gene encoding the human antibody to obtain a human antibody. These methods are known in the art (see International Publication Nos. WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0132] In addition to phage display technology, for example, technologies using cell-free translation systems, technologies for presenting antigen-binding molecules on the surface of cells or viruses, and technologies using emulsions are known as techniques for obtaining human antibodies by panning using a human antibody library. For example, the ribosome display method, which includes forming a complex of mRNA and a protein translated via ribosomes by removing a stop codon, etc., the cDNA or mRNA display method, which includes covalently binding a translated protein to a gene sequence using a compound such as puromycin, or the CIS display method, which includes forming a complex of a gene and a translated protein using a nucleic acid-binding protein, can be used as a technology using a cell-free translation system. The phage display method, the Escherichia coli display method, the Gram-positive bacteria display method, the yeast display method, the mammalian cell display method, the virus display method, etc. can be used as a technology for presenting antigen-binding molecules on the surface of cells or viruses. For example, the in vitro virus display method using genes and translation-related molecules encapsulated in an emulsion can be used as a technology using an emulsion. These methods are known in the art (Nat Biotechnol. 2000 Dec; 18 (12): 1287-92; Nucleic Acids Res. 2006; 34 (19): e127; Proc Natl Acad Sci U S A. 2004 Mar 2; 101 (9): 2806-10; Proc Natl Acad Sci U S A. 2004 Jun 22; 101 (25): 9193-8; Protein Eng Des Sel. 2008 Apr; 21 (4): 247-55; Proc Natl Acad Sci U S A. 2000 Sep 26; 97 (20): 10701-5; MAbs. 2010 Sep-Oct; 2 (5): 508-18; and Methods Mol Biol. 2012; 911: 183-98).

[0133] The variable region that binds to the third antigen of the present invention can be a variable region that recognizes any antigen. The variable region that binds to the third antigen of the present invention can be a variable region that recognizes a molecule specifically expressed in cancer tissue.

[0134] As used herein, the "third antigen" is not particularly limited and may be any antigen. Examples of antigens include 17-IA, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, adiponectin, ADP ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, α1-antichemotrypsin, α1-antitrypsin, α-synuclein, α-V / β-1 antagonist, aminin, amylin, amyloid β, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, apo A1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, β-2-microglobulin, β-lactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, B lymphocyte stimulator (BlyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR,BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, bombesin, bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte cell adhesion molecule, C10, C1 inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, cadherin-3, calcitonin, cAMP, carbonic anhydrase-IX, carcinoembryonic antigen (CEA), cancer-associated antigen, cardiotrophin-1, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1 / I-309, CCL11 / eotaxin, CCL12 / MCP-5, CCL13 / MCP-4, CCL14 / HCC-1, CCL15 / HCC-2, CCL16 / HCC-4, CCL17 / TARC, CCL18 / PARC, CCL19 / ELC, CCL2 / MCP-1, CCL20 / MIP-3-α, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF-1, CCL24 / eotaxin-2, CCL25 / TECK, CCL26 / eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CCL3 / MIP-1-α, CCL3Ll / LD-78-β, CCL4 / MIP-1-β, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / 10 / MTP-1-γ, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, 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, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52,CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, Claudin 18, CLC, Clostridium botulinum toxin, Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, Complement factor 3 (C3), Complement factor D, Corticosteroid-binding globulin, Colony-stimulating factor-1 receptor, COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1 / Fractalkine, CX3CR1, CXCL, CXCL1 / Gro-α, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF-l-α / β, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15 / Lungkine, CXCL16, CXCL16, CXCL2 / Gro-β, CXCL3 / Gro-γ, CXCL3, CXCL4 / PF4, CXCL5 / ENA-78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCLlO / IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cystatin C, Cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay-accelerating factor, Delta-like protein ligand 4, des(1-3)-IGF-1 (Brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, Digoxin, Dipeptidyl peptidase IV, DKl, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EGF-like domain-containing protein 7, Elastase, Elastin, EMA, EMMPRIN, ENA, ENA-78, Endocan, Endothelin receptor, Endotoxin, Enkephalinase, eNOS, Eot,Eotaxin, Eotaxin-2, Eotaxini, EpCAM, Ephrin B2 / EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO), erythropoietin receptor, E-selectin, ET-1, Exodus-2, F protein of RSV, F10, F11, F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, Factor VIII, Factor VIIIc, Fas, FcαR, FcεRI, FcγIIb, FcγRI, FcγRIIa, FcγRIIIa, FcγRIIIb, FcRn, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic, Fibrin, fibroblast activation protein (FAP), fibroblast growth factor, fibroblast growth factor-10, Fibronectin, FL, FLIP, Flt-3, FLT3 ligand, folate receptor, follicle-stimulating hormone (FSH), fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-1), GDF-3 (Vgr-2), GDF-5 (BMP-14 / CDMP-1), GDF-6 (BMP-13 / CDMP-2), GDF-7 (BMP-12 / CDMP-3), GDF-8 (Myostatin), GDF-9, GDNF, Gelsolin, GFAP, GF-CSF, GFR-α1, GFR-α2, GFR-α3, GF-β1, gH envelope glycoprotein, GITR, Glucagon, Glucagon receptor, Glucagon-like peptide 1 receptor, Glut 4, Glutamic acid carboxypeptidase II, glycoprotein hormone receptor, glycoprotein IIb / IIIa (GP IIb / IIIa), Glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO / MGSA, growth hormone releasing factor, GRO-β, GRO-γ, H. pyloriHapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, heparin cofactor II, hepatic growth factor, anthrax protective antigen, hepatitis C virus E2 glycoprotein, hepatitis E, hepcidin, Her1, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, high molecular weight melanoma associated antigen (HMW-MAA), HIV envelope protein, e.g., GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), hantigen, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-α, IFN-β, IFN-γ, IgA, IgA receptor, IgE, IGF, IGF binding protein, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptor, IL-11, IL-11 receptor, IL-12, IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-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 complex, immunoglobulin, INF-α, INF-α receptor, INF-β, INF-β receptor, INF-γ, INF-γ receptor, type I IFN,Type I IFN receptor, influenza, inhibin, inhibin α, inhibin β, iNOS, insulin, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein, integrin, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α-V / β-3, integrin α-V / β-6, integrin α4 / β7, integrin α5 / β1, integrin α5 / β3, integrin α5 / β6, integrin ασ(αV), integrin αθ, integrin β1, integrin β2, integrin β3 (GPIIb-IIIa), IP-10, I-TAC, JE, kalliklein, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, calistatin, KC, KDR, keratinocyte growth factor (KGF), keratinocyte growth factor 2 (KGF-2), KGF, killer immunoglobulin-like receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (amylin, pancreatic islet amyloid polypeptide), LAP (TGF-1), latent associated peptide, latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, leptin, luteinizing hormone (LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptor, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-α, LT-β, LTB4, LTBP-1, pulmonary surfactant, Luteinizing hormone, lymphotactin, lymphotoxin β receptor, lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 a.a.), MDC (69 a.a.), megsin, Mer,MET tyrosine kinase receptor family, metalloproteases, membrane glycoprotein OX2, mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial proteins, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte chemoattractant protein, monocyte colony inhibitory factor, mouse gonadotropin-related peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Müllerian duct inhibitory substance, Mug, MuSK, myelin-associated glycoprotein, myeloid progenitor cell inhibitory factor-1 (MPIF-I), NAIP, nanobody, NAP, NAP-2, NCA 90, NCAD, N-cadherin, NCAM, neprilysin, neural cell adhesion molecule, neuroserpin, nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin-1, neuritin, NGF-β, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, hepatitis C virus-derived non-structural protein type 3 (NS3), NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM, OSM receptor, osteogenic factor, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet-derived growth factor, plgR, PLP, polyglycol chains of various sizes (e.g., PEG-20, PEG-30, PEG40), PP14, prekallikrein,Prion protein, procalcitonin, , Programmed cell death protein 1, proinsulin, prolactin, proprotein convertase PC9, prolaxin, prostate-specific membrane antigen (PSMA), Protein A, Protein C, Protein D, Protein S, Protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, rheumatoid factor, RLIP76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF / KL, SCGF, sclerostin, SDF-1, SDF1α, SDF1β, SERINE, serum amyloid P, serum albumin, sFRP-3, Shh, Shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor α / β, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, Pan-specific TGF-β, TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-βRl (ALK-5), TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thymic stromal lymphopoietin (Thymic stromallymphoprotein) receptor, thymic Ck-1, thyroid-stimulating hormone (TSH), thyroxine, thyroxine-binding globulin, 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 ODF R / TRANCE R), TNFRSF11B (OPG OCIF / TR1), TNFRSF12 (TWEAK R FN14), TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR / HveA / LIGHT R / TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ / TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF Rl CD120a / p55-60), TNFRSF1B (TNF RII CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3 / LARD / TR-3 / TRAMP / WSL-1), TNFRSF26 (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 (TRAILApo-2 ligand / TL2), TNFSF11 (TRANCE / RANK ligand / ODF / OPG ligand), TNFSF12 (TWEAK / Apo-3 ligand / DR3 ligand), TNFSF13 (APRIL / TALL2), TNFSF13B (BAFF / BLYS / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT / HVEM ligand / LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand / AITR ligand / TL6), TNFSF1A (TNF-α connectin (Conectin) / DIF / TNFSF2), TNFSF1B (TNF-β / LTa / TNFSF1), TNFSF3 (LTb / TNFC / p33), TNFSF4 (OX40 ligand / gp34 / TXGP1), TNFSF5 (CD40 ligand / CD154 / gp39 / HIGM1 / IMD3 / TRAP), TNFSF6 (Fas ligand / Apo-1 ligand / APT1 ligand), TNFSF7 (CD27 ligand / CD70), TNFSF8 (CD30 ligand / CD153), TNFSF9 (4-1BB ligand / CD137 ligand), TNF-α, TNF-β, TNIL-I, toxic metabolites, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factor (TGF), e.g., TGF-α and TGF-β, transmembrane glycoprotein NMB, transthyretin, TRF, Trk, TROP-2, trophoblast glycoprotein, TSG, TSLP, tumor necrosis factor (TNF), tumor-associated antigen CA125. Tumor-associated antigens expressing Lewis Y-related carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VitB12 receptor, vitronectin receptor, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2 / SCM-l-β, XCLl / lymphotactin, XCR1, XEDAR, XIAP, and XPD are included.

[0135] Specific examples of molecules specifically expressed on T cells include CD3 and the T cell receptor. In particular, CD3 is preferred. For example, in the case of human CD3, the site in CD3 to which the antigen molecule of the present invention binds may be any epitope present in the sequences of the γ chain, δ chain, or ε chain constituting human CD3. In particular, an epitope present in the extracellular region of the ε chain in the human CD3 complex is preferred. The polynucleotide sequences of the structures of the γ chain, δ chain, and ε chain constituting CD3 are shown in SEQ ID NOs: 170 (NM_000073.2), 172 (NM_000732.4), and 174 (NM_000733.3), and the polypeptide sequences thereof are shown in SEQ ID NOs: 171 (NP_000064.1), 173 (NP_000723.1), and 175 (NP_000724.1) (RefSeq accession numbers are shown in parentheses).

[0136] One of the two variable regions of the antibody contained in the antigen-binding molecule of the present invention binds to a "third antigen" different from the above-mentioned "CD3" and "CD137". In some embodiments, the third antigen is derived from human, mouse, rat, monkey, rabbit, or dog. In some embodiments, the third antigen is a molecule specifically expressed on cells or organs derived from human, mouse, rat, monkey, rabbit, or dog. The third antigen is preferably a molecule that is not expressed systemically on cells or organs. The third antigen is preferably, for example, a tumor cell-specific antigen, and also includes antigens that are expressed along with the malignancy of cells, and abnormal sugar chains that appear on the cell surface or protein molecules during the malignant transformation of cells. Specific examples thereof include ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate-specific antigen (PSA), melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigens (e.g., CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1, and LEA), Burkitt lymphoma antigen 38.13, CD19, human B lymphoma antigen CD20, CD33, melanoma-specific antigens (e.g., gangliosides GD2, GD3, GM2, and GM3), tumor-specific transplantation antigen (TSTA), T antigen, virus-induced tumor antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), colon CEA, tumor fetal antigen α-fetoprotein (e.g., tumor fetal trophoblast glycoprotein 5T4 and tumor fetal bladder tumor antigen), differentiation antigens (e.g., human lung cancer antigens L6 and L20), fibrosarcoma antigen, human T cell leukemia-associated antigen Gp37, neonatal glycoproteins, sphingolipids, breast cancer antigens (e.g., EGFR (epidermal growth factor receptor)), NY-BR-16, NY-BR-16 and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen APO-1, differentiation antigens such as I antigen found in fetal erythrocytes, early endoderm I antigen found in adult erythrocytes, I(Ma) found in pre-transplant embryos or gastric cancer, M18 found in mammary epithelium, M39, SSEA-1 found in bone marrow cells, VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), SCP-1 found in testicular and ovarian cancer, C14 found in colon cancer, F3 found in lung cancer, AH6 found in gastric cancer, Y hapten, Ley found in embryonic cancer cells, TL5 (blood group A), EGF receptor found in A431 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 found in adenocarcinoma, CO-43 (blood group Leb), G49 found in the EGF receptor of A431 cells, MH2 found in colon cancer (blood group ALeb / Ley), 19.9 found in colon cancer, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2 found in embryonic cancer cells, GD3, D1.1. OFA-1, GM2, OFA-2, GD2, and M1:22:25:8, SSEA-3 and SSEA-4 found in embryos at the 4-cell to 8-cell stage, skin T cell lymphoma-related antigen, MART-1 antigen, sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, tumor-associated brain-testicular cancer antigen (tumor neural antigen MA2 and tumor-associated neural antigen), neuro-oncological abdominal antigen 2 (NOVA2), blood cell cancer 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 fragment of these polypeptides, as well as their modified structures (such as the aforementioned modified phosphate groups, sugar chains, etc.), EpCAM, EREG, CA19-9, CA15-3, sialyl SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A are included.

[0137] The term "CD137" as used herein, 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 the TNF receptor superfamily include CD137, CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL.

[0138] In one aspect, the antigen-binding molecule of the present invention has at least one characteristic selected from the group consisting of the following (1) to (4): (1) The variable region binds to the extracellular domain of CD3ε containing the amino acid sequence of SEQ ID NO: 91. (2) The antigen-binding molecule has agonist activity against CD137. (3) The antigen-binding molecule induces the CD3 activation of T cells against cells expressing the molecule of the third antigen, but does not induce the 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 molecule of the third antigen.

[0139] In one aspect, the antigen-binding molecule of the present invention has at least one characteristic selected from the group consisting of the following (1) to (4): (1) The variable region binds to the extracellular domain of CD3ε containing the amino acid sequence of SEQ ID NO: 91, (2) The antigen-binding molecule has agonist activity against CD137, (3) The antigen-binding molecule induces the cytotoxic activity of T cells against cells expressing the molecule of the third antigen, but does not induce the 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 molecule of the third antigen. In some embodiments, the antigen-binding molecule of the present invention has at least one characteristic selected from the group consisting of the following (1) to (2): (1) The antigen-binding molecule does not compete with the CD137 ligand for binding to CD137, and (2) The antigen-binding molecule induces the cytotoxic activity of T cells against cells expressing the molecule of the third antigen, but does not induce the cytotoxic activity of T cells against cells expressing CD137.

[0140] In one aspect, the "CD137 agonist antibody" or "antigen-binding molecule having agonist activity against CD137" of the present invention refers to 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 when added to cells, tissues, or organisms expressing CD137, where 0% activation is the background level of non-activated cells expressing CD137 (e.g., IL6 secretion, etc.). In various specific examples, the CD137 agonist antibody for use as a pharmaceutical composition of the present 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%. In one aspect, the "CD137 agonist antibody" or "antigen-binding molecule having agonist activity against CD137" of the present invention also refers to 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 when added to cells, tissues, or organisms expressing CD137, where 100% activation is the level of activation achieved by an equimolar amount of binding partner under physiological conditions. In various specific examples, the CD137 agonist antibody for use as a pharmaceutical composition 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 "binding partner" as used herein is a molecule known to bind to CD137 and induce activation of cells expressing CD137. In a further aspect, examples of binding partners include Urelumab (CAS registration number 934823-49-1) and its variants described in WO2005 / 035584A1, Utomilumab (CAS registration number 1417318-27-4) and its variants described in WO2012 / 032433A1, and various known CD137 agonist antibodies. In a particular aspect, examples of binding partners include the CD137 ligand. In a further aspect, the activation of cells expressing CD137 by an anti-CD137 agonist antibody can be determined using an ELISA that characterizes IL6 secretion (see, for example, Example 10-2 herein). The anti-CD137 antibody used as a binding partner and the antibody concentration for measurement can refer to Example 10-2, where 100% activation is the level of activation achieved by the antibody.In a further aspect, an antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 69 and the light chain amino acid sequence of SEQ ID NO: 71 can be used at 30 μg / mL as a binding partner for measurement (see, for example, Example 10-2 herein).

[0141] In a non-limiting aspect, the present invention provides a "CD137 agonist antibody" comprising an Fc region, wherein the Fc region has enhanced binding activity to inhibitory Fcγ receptors.

[0142] In a non-limiting aspect, CD137 agonist activity can be confirmed using B cells, which are known to express CD137 on their surface. In a non-limiting aspect, the HDLM-2 B cell line can be used as the B cells. As a result of the activation of CD137, the expression of IL-6 is induced, so CD137 agonist activity can be evaluated by the amount of human interleukin-6 (IL-6) produced. In this evaluation, by using the amount of IL-6 to evaluate the increase in IL-6 expression from non-activated B cells as the 0% background level, it is possible to determine what percentage of CD137 agonist activity the molecule being evaluated has.

[0143] 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 with cells expressing the third antigen in the presence of the antigen-binding molecule and assaying for CD3 activation of the T cells. T cell activation can be assayed, for example, by using recombinant T cells that express a reporter gene (e.g., luciferase) in response to CD3 signaling and detecting the expression of the reporter gene or the activity of the reporter gene product as an indicator of T cell activation. Detection of the expression of the reporter gene or the activity of the reporter gene product in a dose-dependent manner of the antigen-binding molecule when recombinant T cells that express a reporter gene in response to CD3 signaling are co-cultured with cells expressing a third antigen in the presence of the antigen-binding molecule indicates that the antigen-binding molecule induces activation of T cells against cells expressing the third antigen. Similarly, whether the antigen-binding molecule does not induce CD3 activation of T cells against cells expressing CD137 can be determined, for example, by co-culturing T cells with cells expressing CD137 in the presence of the antigen-binding molecule and assaying for CD3 activation of the T cells as described above. 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 the antigen-binding molecule, if there is no expression of the reporter gene or the activity of the reporter gene product, or if it is below the detection limit, or if it is below that of the negative control, then the antigen-binding molecule is determined not to induce activation of T cells against cells expressing CD137.In one aspect, when a recombinant T cell expressing a reporter gene in response to CD3 signaling is co-cultured with a cell expressing CD137 in the presence of an antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is at most about 50%, 30%, 20%, 10%, 5%, or 1%, then the antigen-binding molecule is determined not to induce activation of the T cell against the cell expressing CD137, where 100% activation is the level of activation achieved by an antigen-binding molecule that binds simultaneously to CD3 and CD137. In one aspect, when a recombinant T cell expressing a reporter gene in response to CD3 signaling is co-cultured with a cell expressing CD137 in the presence of an antigen-binding molecule, if the expression of the reporter gene or the activity of the reporter gene product is at most about 50%, 30%, 20%, 10%, 5%, or 1%, then the antigen-binding molecule is determined not to induce activation of the T cell against the cell expressing CD137, where 100% activation is the level of activation achieved by the same antigen-binding molecule against a cell expressing a third antigen molecule.

[0144] In some embodiments, the antigen-binding molecule of the invention does not induce cytokine release from PBMCs in the absence of cells expressing a third antigen. Whether an antigen-binding molecule induces cytokine release in the absence of cells expressing a third antigen can be determined, for example, by incubating PBMCs with the antigen-binding molecule in the absence of cells expressing the third antigen, and measuring cytokines such as IL-2, IFNγ, and TNFα released into the culture supernatant from the PBMCs using methods known in the art. If no significant level of cytokine is detected, or no induction of significant cytokine expression occurs, in the culture supernatant of PBMCs incubated with the antigen-binding molecule in the absence of cells expressing the third antigen, the antigen-binding molecule is determined not to induce cytokine release from PBMCs in the absence of cells expressing the third antigen. In one aspect, "no significant level of cytokine is detected" also refers to the level of cytokine concentration being at most about 50%, 30%, 20%, 10%, 5%, or 1%, where 100% is the cytokine concentration achieved by an antigen-binding molecule that binds simultaneously to CD3 and CD137. In one aspect, "no significant level of cytokine is detected" also refers to the level of cytokine concentration being at most about 50%, 30%, 20%, 10%, 5%, or 1%, where 100% is the cytokine concentration achieved in the presence of cells expressing the molecule of the third antigen. In one aspect, "no induction of significant cytokine expression occurs" also refers to the level of increase in cytokine concentration being at most 5-fold, 2-fold, or 1-fold the concentration of each cytokine before addition of the antigen-binding molecule.

[0145] In some embodiments, the antigen-binding molecule of the invention competes for binding to CD137 with an antibody selected from the group consisting of: (a) an antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 30 and a VL sequence having the amino acid sequence of SEQ ID NO: 51, (b) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 46 and a VL sequence having the amino acid sequence of SEQ ID NO: 53, (c) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 40 and a VL sequence having the amino acid sequence of SEQ ID NO: 56, (d) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 30 and a VL sequence having the amino acid sequence of SEQ ID NO: 58, and (e) An antibody comprising a VH sequence having the amino acid sequence of SEQ ID NO: 40 and a VL sequence having the amino acid sequence of SEQ ID NO: 61.

[0146] In some embodiments, the antigen-binding molecule of the present invention binds to the same epitope as an antibody selected from the group consisting of: [1] An antibody comprising the amino acid sequence of SEQ ID NO: 98 as the heavy chain variable region and the amino acid sequence of SEQ ID NO: 99 as the light chain variable region; [2] An antibody comprising the amino acid sequence of SEQ ID NO: 100 as the heavy chain variable region and the amino acid sequence of SEQ ID NO: 101 as the light chain variable region; [3] An antibody comprising the amino acid sequence of SEQ ID NO: 102 as the heavy chain variable region and the amino acid sequence of SEQ ID NO: 103 as the light chain variable region; [4] An antibody comprising the amino acid sequence of SEQ ID NO: 104 as the heavy chain variable region and the amino acid sequence of SEQ ID NO: 105 as the light chain variable region; [5] An antibody comprising the amino acid sequence of SEQ ID NO: 106 as the heavy chain variable region and the amino acid sequence of SEQ ID NO: 107 as the light chain variable region; [6] An antibody comprising the amino acid sequence of SEQ ID NO: 108 as the heavy chain variable region and the amino acid sequence of SEQ ID NO: 109 as the light chain variable region; [7] An antibody comprising the amino acid sequence of SEQ ID NO: 110 as the heavy chain constant region and the amino acid sequence of SEQ ID NO: 111 or the amino acid sequence of SEQ ID NO: 112 as the light chain constant region, of any one of [1] to [6]; and [8] An antibody having an activity equivalent to that of any one of the antibodies of [1] to [7]; and An antibody that binds to the same epitope to which any one of the antibodies of [1] to [7] binds.

[0147] In the antibody of [8], "equivalent activity" refers to a CD137 agonist activity that is 70% or more, preferably 80% or more, and more preferably 90% or more of the binding activity of any one of the antibodies of [1] to [7].

[0148] Whether a test antibody shares a common epitope with a specific antibody can be evaluated based on the competition between two antibodies for the same epitope. The competition between antibodies can be detected by a cross-blocking assay or the like. For example, a competitive ELISA assay is a preferred cross-blocking assay. Specifically, in a cross-blocking assay, the CD137 protein used to coat the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antibody, and then the anti-CD137 antibody of the present invention is added thereto. The amount of the anti-CD137 antibody of the present invention bound to the CD137 protein in the well is indirectly correlated with the binding ability of the candidate competing antibody (test antibody) that competes for binding to the same epitope. That is, the higher the affinity of the test antibody for the same epitope, the less the amount of the anti-CD137 antibody of the present invention bound to the well coated with the CD137 protein, and the more the amount of the test antibody bound to the well coated with the CD137 protein.

[0149] The amount of antibody bound to the well can be easily determined by pre-labeling the antibody. For example, a biotin-labeled antibody can be measured using an avidin / peroxidase conjugate and an appropriate substrate. In particular, a cross-blocking assay using an enzyme label such as peroxidase is called a "competitive ELISA assay". The antibody can be labeled with other labeling substances that enable detection or measurement. Specifically, radiolabels, fluorescent labels, etc. are known.

[0150] Furthermore, when the test antibody has a constant region derived from a species different from that 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 recognizes the constant region of the antibody. Alternatively, if the antibody is from the same species but belongs to a different class, the amount of antibody bound to the well can be measured using an antibody that distinguishes the individual classes.

[0151] If the candidate competing antibody can block the binding of the anti-CD137 antibody by at least 20%, preferably at least 20% to 50%, and even more preferably at least 50%, as compared to the binding activity obtained in a control experiment conducted in the absence of the candidate competing antibody, the candidate competing antibody is either an antibody that binds to substantially the same epitope as the anti-CD137 antibody of the present invention or an antibody that competes for binding to the same epitope.

[0152] In another aspect, the ability of the test antibody to bind competitively or cross-competitively with another antibody can be determined as appropriate by those skilled in the art using standard binding assays such as BIAcore analysis or flow cytometry known in the art.

[0153] Methods for determining the spatial three-dimensional structure of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance (see Epitope Mapping Protocols in Methods in Molecular Biology, G. E. Morris (ed.), Vol. 66 (1996)).

[0154] Whether the test antibody shares an epitope common with CD137 ligand can also be evaluated based on competition between the test antibody and CD137 ligand for the same epitope. Competition between the antibody and CD137 ligand can be detected by, for example, the cross-blocking assay described above. In another aspect, the ability of the test antibody to bind competitively or cross-competitively with CD137 ligand can be determined as appropriate by those skilled in the art using standard binding assays such as BIAcore analysis or flow cytometry known in the art.

[0155] In some aspects, advantageous examples of the antigen-binding molecules of the present invention include antigen-binding molecules that bind to the same epitope as the epitope on human CD137 to which an antibody selected from the group consisting of the following binds: In the human CD137 protein An antibody that recognizes a region containing the SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC sequence (SEQ ID NO: 81), An antibody that recognizes a region containing the DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC sequence (SEQ ID NO: 76), An antibody that recognizes a region containing the LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC sequence (SEQ ID NO: 79), and An antibody that recognizes a region containing the LQDPCSNCPAGTFCDNNRNQIC sequence (SEQ ID NO: 74).

[0156] Depending on the target cancer antigen, those skilled in the art can appropriately select the heavy chain variable region and the light chain variable region contained in the cancer-specific antigen-binding domain for the heavy chain variable region sequence and the light chain variable region sequence that bind to the cancer antigen. When the epitope to which the antigen-binding domain binds is contained in a plurality of different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to various antigens having the epitope.

[0157] An "epitope" means an antigenic determinant in an antigen and refers to the antigenic site to which various binding domains in the antigen-binding molecules disclosed herein bind. Thus, for example, an epitope can be defined according to its structure. Alternatively, an epitope may be defined according to the antigen-binding activity of the antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that form the epitope. Alternatively, when the epitope is a sugar chain, the epitope can be specified by its specific sugar chain structure.

[0158] A linear epitope is an epitope that contains an epitope whose primary amino acid sequence is recognized. Such linear epitopes typically contain at least 3, and most commonly at least 5, for example, about 8 - 10 or 6 - 20 amino acids in their specific sequence.

[0159] In contrast to a linear epitope, a "conformational epitope" is an epitope in which the primary amino acid sequence containing the epitope is not the only determinant of the recognized epitope (for example, the primary amino acid sequence of a conformational epitope is not necessarily recognized by the antibody that defines the epitope). A conformational epitope can contain a greater number of amino acids compared to a linear epitope. Antibodies that recognize conformational epitopes recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, the amino acids and / or polypeptide backbone that form the conformational epitope align, making the epitope recognizable by the antibody. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-directed spin labeling, and electron paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0160] An example of a method for evaluating the binding of an epitope in a cancer-specific antigen by a test antigen-binding molecule is shown below. According to the following examples, a method for evaluating the binding of an epitope in a target antigen by another binding domain can also be appropriately implemented.

[0161] For example, whether a test antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen recognizes a linear epitope in the antigen molecule can be confirmed as described below. For example, a linear peptide containing the 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 region in the cDNA of the cancer-specific antigen that encodes the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the test antigen-binding molecule containing the antigen-binding domain for the cancer-specific antigen to the linear peptide containing the amino acid sequence constituting the extracellular domain is evaluated. For example, using the immobilized linear peptide as an antigen, the binding activity of the antigen-binding molecule to the peptide can be evaluated by ELISA. Alternatively, the binding activity to the linear peptide can be evaluated based on the level at which the linear peptide inhibits the binding of the antigen-binding molecule to cancer-specific antigen-expressing cells. The binding activity of the antigen-binding molecule to the linear peptide can be demonstrated by these tests.

[0162] Whether a test antigen molecule containing an antigen-binding domain for the above antigen recognizes a conformational epitope can be confirmed as follows. For example, an antigen-binding molecule containing an antigen-binding domain for a cancer-specific antigen strongly binds to cancer-specific antigen-expressing cells upon contact, but does not substantially bind to an immobilized linear peptide containing the amino acid sequence forming the extracellular domain of the cancer-specific antigen. As used herein, "does not substantially bind" means that the binding activity is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less compared to the binding activity of the antigen-expressing cells in ELISA or fluorescence-activated cell sorting (FACS) using the antigen-expressing cells as the antigen.

[0163] In the ELISA format, the binding activity of a test antigen-binding molecule containing an antigen-binding domain for antigen-expressing cells can be quantitatively evaluated by comparing the levels of signals generated by the enzyme reaction. Specifically, the test antigen-binding molecule is added to an ELISA plate on which antigen-expressing cells are immobilized. Then, the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, when using FACS, a dilution series of the test antigen-binding molecule can be prepared, and the antigen-binding titer for antigen-expressing cells can be determined to compare the binding activity of the test antigen-binding molecule for antigen-expressing cells.

[0164] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices: FACSCanto (trademark) II FACSAria (trademark) FACSArray (trademark) FACSVantage (trademark) SE FACSCalibur (trademark) (all are product names of BD Biosciences) EPICS ALTRA HyPerSort Cytomics FC 500 EPICS XL-MCL ADC, EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (all are product names of Beckman Coulter).

[0165] Suitable methods for assaying the binding activity of a test antigen-binding molecule containing an antigen-binding domain against the above antigen include, for example, the following methods. First, antigen-expressing cells are reacted with the test antigen-binding molecule, and then this is stained with an FITC-labeled secondary antibody and FACSCalibur (BD) is used. The fluorescence intensity, i.e., the geometric mean value, obtained by analysis using CELL QUEST Software (BD) reflects the amount of antibody bound to the cells. That is, the binding activity of the test antigen-binding molecule represented by the amount of the bound test antigen-binding molecule can be measured by determining the geometric mean value.

[0166] Whether a test antigen-binding molecule containing the antigen-binding domain of the present invention shares a common epitope with another antigen-binding molecule can be evaluated based on the competition between two molecules for the same epitope. The competition between antigen-binding molecules can be detected by a cross-blocking assay or the like. For example, a competitive ELISA assay is a preferred cross-blocking assay.

[0167] Specifically, in a cross-blocking assay, the antigen coating the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antigen-binding molecule, and then the test antigen-binding molecule is added thereto. The amount of the test antigen-binding molecule bound to the antigen in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule competing for binding to the same epitope. That is, the higher the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule for the well coated with the antigen.

[0168] The amount of test antigen-binding molecules bound to the well via the antigen can be easily determined by pre-labeling the antigen-binding molecules. For example, biotin-labeled antigen-binding molecules can be measured using avidin / peroxidase conjugate and an appropriate substrate. In particular, a cross-blocking assay using an enzyme label such as peroxidase is called a "competitive ELISA assay". Antigen-binding molecules can also be labeled with other labeling substances that enable detection or measurement. Specifically, radiolabels, fluorescent labels, etc. are known. If a candidate competitive antigen-binding molecule can block the binding of a test antigen-binding molecule containing an antigen-binding domain by at least 20%, preferably at least 20 - 50%, and more preferably at least 50%, compared to the binding activity in a control experiment conducted in the absence of the competitive antigen-binding molecule, the test antigen-binding molecule is determined to bind substantially to the same epitope to which the competitive antigen-binding molecule binds or to compete for binding to the same epitope.

[0169] If the structure of the epitope to which the test antigen-binding molecule containing the antigen-binding domain of the present invention binds has already been identified, whether the test antigen-binding molecule and the control antigen-binding molecule share a common epitope can be evaluated by comparing the binding activities of the two antigen-binding molecules to a peptide prepared by introducing amino acid mutations into the peptide forming the epitope.

[0170] As a method for measuring such binding activity, for example, the binding activities of the test antigen-binding molecule and the control antigen-binding molecule to a linear peptide into which mutations have been introduced are measured by comparison in the above ELISA format. In addition to the ELISA method, the binding activity to the mutant peptide bound to the column can be determined by passing the test antigen-binding molecule and the control antigen-binding molecule through a column and then quantifying the eluted antigen-binding molecules in the eluate. For example, methods for adsorbing mutant peptides to a column in the form of GST fusion peptides are known.

[0171] Alternatively, when the identified epitope is a conformational epitope, whether the test antigen-binding molecule and the control antigen-binding molecule share a common epitope can be evaluated by the following method. First, cells expressing the antigen targeted by the antigen-binding domain and cells expressing the antigen having the mutated epitope are prepared. The test antigen-binding molecule and the control antigen-binding molecule are added to a cell suspension prepared by suspending these cells in an appropriate buffer such as PBS. Next, the cell suspension is washed appropriately with the buffer, and an FITC-labeled antibody capable of recognizing the test antigen-binding molecule and the control antigen-binding molecule is added thereto. The fluorescence intensity and number of the cells stained with the labeled antibody are determined using FACSCalibur (BD). The test antigen-binding molecule and the control antigen-binding molecule are appropriately diluted using a suitable buffer and used at a desired concentration. For example, they may be used at a concentration within the range of 10 μg / ml to 10 ng / ml. The fluorescence intensity, i.e., the geometric mean value, determined by analysis using CELL QUEST Software (BD) reflects the amount of the labeled antibody bound to the cells. That is, the binding activities of the test antigen-binding molecule and the control antigen-binding molecule represented by the amount of the labeled antibody bound thereto can be measured by determining the geometric mean value.

[0172] In some embodiments, the antigen-binding molecule of the present invention contains an amino acid sequence generated by introducing one or more amino acid modifications into a template sequence consisting of the heavy chain variable domain sequence set forth in SEQ ID NO: 92 and / or the light chain variable domain sequence set forth in SEQ ID NO: 93, and the one or more amino acids to be modified are at the following positions: H chain: 31, 52b, 52c, 53, 54, 56, 57, 61, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, and 100g (Kabat numbering); and L chains: 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52, 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, and 96 (Kabat numbering) selected from the HVR-H3 of the modified heavy chain variable domain array is 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 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 numbering) and contains at least one amino acid selected from

[0173] In some embodiments, the antigen-binding molecule of the invention comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 41, 30, 46, or 40; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, or 57; or (c) the VH sequence of (a) and the VL sequence of (b).

[0174] The antigen-binding molecules of the present invention can be produced by methods generally known to those skilled in the art. For example, antibodies can be prepared by the methods shown below, but the methods for preparing the antibodies of the present invention are not limited thereto. For the preparation of antibodies by transferring an isolated gene encoding a polypeptide into a suitable host, many combinations of host cells and expression vectors are known in the art. All of these expression systems can be applied to the isolation of the antigen-binding molecules of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specifically, examples of animal cells may include the following cells: (1) Mammalian cells, such as CHO (Chinese hamster ovary cell line), COS (monkey kidney cell line), myeloma cells (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), HEK293 (human fetal kidney cell line with sheared adenovirus (Ad) 5 DNA), PER.C6 cells (human fetal retinal cell line transformed with adenovirus type 5 (Ad5) E1A and E1B genes), Hela, and Vero (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1)); (2) Amphibian cells, such as Xenopus oocytes; and (3) Insect cells, such as sf9, sf21, and Tn5. Antibodies can also be prepared using Escherichia coli (mAbs 2012 Mar-Apr; 4(2): 217-225) or yeast (WO2000023579). Antibodies prepared using Escherichia coli do not have sugar chains added. On the other hand, antibodies prepared using yeast have sugar chains added.

[0175] DNA encoding an antibody heavy chain encoding a heavy chain in which one or more amino acid residues in the variable domain are replaced by different amino acids of interest, and DNA encoding the light chain of the antibody are expressed. DNA encoding a heavy chain or a light chain in which one or more amino acid residues in the variable domain are replaced by different amino acids of interest can be obtained, for example, by obtaining DNA encoding an antibody variable domain prepared by a method known in the art for a specific antigen, and appropriately introducing substitutions so that the codons encoding specific amino acids in the domain encode different amino acids of interest.

[0176] Alternatively, DNA encoding a protein in which one or more amino acid residues in an antibody variable domain prepared by a method known in the art for a specific antigen are replaced by different amino acids of interest may be pre-designed and chemically synthesized to obtain DNA encoding a heavy chain in which one or more amino acid residues in the variable domain are replaced by different amino acids of interest. The amino acid substitution sites and the types of substitutions are not particularly limited. Examples of preferred regions for amino acid modification include regions and loops exposed to the solvent in the variable region. Among them, CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, positions 31 to 35, 50 to 65, 71 to 74, and 95 to 102 in the Kabat numbering of the H chain variable domain, and positions 24 to 34, 50 to 56, and 89 to 97 in the Kabat numbering of the L chain variable domain are preferred. Positions 31, 52a to 61, 71 to 74, and 97 to 101 in the Kabat numbering of the H chain variable domain, and positions 24 to 34, 51 to 56, and 89 to 96 in the Kabat numbering of the L chain variable domain are more preferred. The amino acid modification is not limited to substitution, and may be deletion, addition, insertion, or modification, or a combination thereof.

[0177] DNA encoding a heavy chain in which one or more amino acid residues in the variable domain are substituted by different amino acids of interest can also be prepared as separate partial DNAs. Examples of combinations of partial DNAs include, but are not limited to, DNA encoding a variable domain and DNA encoding a constant domain; and DNA encoding a Fab domain and DNA encoding an Fc domain. Similarly, DNA encoding a light chain can also be prepared as separate partial DNAs.

[0178] These DNAs can be expressed by the following method: for example, DNA encoding a heavy chain variable domain is incorporated into an expression vector together with DNA encoding a heavy chain constant domain to construct a heavy chain expression vector. Similarly, DNA encoding a light chain variable domain is incorporated into an expression vector together with DNA encoding a light chain constant domain to construct a light chain expression vector. The genes for these heavy and light chains may be incorporated into a single vector.

[0179] DNA encoding the antibody of interest is incorporated into an expression vector so as to be expressed under the control of an expression control region, such as an enhancer and a promoter. Next, the resulting expression vector is used to transform a host cell to express the antibody. In this case, an appropriate host and expression vector can be used in combination.

[0180] Examples of vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, and pCR-Script. In addition to these vectors, for example, pGEM-T, pDIRECT, or pT7 can also be used for the purpose of subcloning and excision of cDNA.

[0181] In particular, for the purpose of producing the antibody of the present invention using a vector, an expression vector is useful. For example, when the host is Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue, the expression vector has a promoter that enables efficient expression in Escherichia coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546, which is incorporated herein by reference in its entirety; and FASEB J. (1992) 6, 2422-2427), the araB promoter (Better et al., Science (1988) 240, 1041-1043, which is incorporated herein by reference in its entirety), or the T7 promoter. Examples of such vectors include the vectors described above, as well as pGEX-5X-1 (manufactured by Pharmacia), the "QIAexpress system" (manufactured by Qiagen N.V.), pEGFP, and pET (in this case, the host is preferably BL21 that expresses T7 RNA polymerase).

[0182] The vector may contain a signal sequence for polypeptide secretion. In the case of production in the periplasm of Escherichia coli, the pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4397, which is incorporated herein by reference in its entirety) can be used as the signal sequence for polypeptide secretion. The vector can be introduced into host cells, for example, by using the lipofectin method, the calcium phosphate method, or the DEAE-dextran method.

[0183] In addition to the expression vector for Escherichia coli, examples of vectors for producing the polypeptide of the present invention include expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen Corp.), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, which is incorporated herein by reference in its entirety), pEF, and pCDM8), expression vectors derived from insect cells (for example, "Bac-to-BAC baculovirus expression system" (manufactured by GIBCO BRL), and pBacPAK8), expression vectors derived from plants (for example, pMH1 and pMH2), expression vectors derived from animal viruses (for example, pHSV, pMV, and pAdexLcw), expression vectors derived from retroviruses (for example, pZIPneo), expression vectors derived from yeast (for example, "Pichia Expression Kit" (manufactured by Invitrogen Corp.), pNV11, and SP-Q01), and expression vectors derived from Bacillus subtilis (for example, pPL608 and pKTH50).

[0184] For the purpose of expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, or HEK293 cells, the vector indispensably has a promoter necessary for intracellular expression, for example, the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, which is incorporated herein by reference in its entirety), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, which is incorporated herein by reference in its entirety), CAG promoter (Gene. (1991) 108, 193, which is incorporated herein by reference in its entirety), or CMV promoter, and more preferably has a gene for screening transformed cells (for example, a drug resistance gene that can act as a marker by a drug (such as neomycin, G418)). Examples of vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13. In addition, for the purpose of increasing the gene copy number, EBNA1 protein may be co-expressed. In this case, a vector having the origin of replication OriP is used (Biotechnol Bioeng. 2001 Oct 20;75(2):197-203; and Biotechnol Bioeng. 2005 Sep 20;91(6):670-7).

[0185] Exemplary methods intended to stably express a gene and increase the gene copy number in a cell include transforming CHO cells deficient in a nucleic acid synthesis pathway with a vector having a DHFR gene (e.g., pCHOI) that serves as a complement thereto, and using methotrexate (MTX) in gene amplification. Exemplary methods intended to transiently express a gene include transforming cells with a vector having an SV40 origin of replication (such as pcD) using COS cells having the SV40 T antigen gene on their chromosomes. Origins of replication derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can also be used. To increase the gene copy number in a host cell line, an expression vector can contain a selection marker such as the aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene.

[0186] Antibodies can be recovered, for example, by culturing the transformed cells and then separating the antibody from within the molecularly transformed cells or from their culture medium. Antibodies can be separated and purified by appropriately combining methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, C1q, FcRn, protein A and protein G columns, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0187] Techniques such as knob-into-hole technology (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; and Merchant AM et al., Nature Biotechnology (1998) 16, 677-681) or techniques for suppressing unintended association between heavy chains by introducing charge repulsion (WO2006 / 106905) can be applied to methods for efficiently preparing multispecific antibodies.

[0188] The present invention further provides a method for producing an antigen-binding molecule of the present invention. Specifically, an antibody variable region that can bind to two different antigens (a first antigen and a second antigen) but does not bind to CD3 and CD137 simultaneously (this variable region is referred to as the first variable region); and a variable region that binds to a third antigen different from CD3 and CD137 (this variable region is referred to as the second variable region). Provided is a method for producing an antigen-binding molecule comprising a step of preparing an antigen-binding molecule library containing diverse amino acid sequences of the first variable region.

[0189] Examples thereof may include a production method comprising the following steps: (i) A step of preparing a library of antigen-binding molecules in which at least one amino acid is modified in its antibody variable region that binds to CD3 or CD137, wherein at least one amino acid of the modified variable region is different from each other; (ii) A step of selecting from the prepared library an antigen-binding molecule comprising a variable region that has binding activity to CD3 and CD137 but does not bind to CD3 and CD137 simultaneously; (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 that binds to a third antigen, to express an antigen-binding molecule comprising 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 the third antigen; and (iv) recovering the antigen-binding molecule from the host cell culture.

[0190] In this production method, step (ii) may be the following selection step: (v) selecting, from the prepared library, an antigen-binding molecule comprising a variable region that has binding activity to CD3 and CD137 but does not bind simultaneously to CD3 and CD137 expressed on different cells.

[0191] The antigen-binding molecules used in step (i) are not particularly limited as long as these molecules each contain the variable region of an antibody. The antigen-binding molecule may be an antibody fragment such as Fv, Fab, or Fab', or may be an antibody containing an Fc region.

[0192] The amino acids to be modified are selected from amino acids in the antibody variable region that binds to CD3 or CD137, and the modification does not cause loss of binding to the antigen.

[0193] In the present invention, one amino acid modification may be used alone, or a plurality of amino acid modifications may be used in combination. When a plurality of amino acid modifications are used in combination, the number of modifications to be combined is not particularly limited. For example, it is 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. The plurality of amino acid modifications to be combined may be added only to the heavy chain variable domain or the light chain variable domain of the antibody, or may be appropriately distributed to both the heavy chain variable domain and the light chain variable domain.

[0194] Examples of preferred regions for amino acid modifications include regions and loops exposed to the solvent in the variable region. Among them, CDR1, CDR2, CDR3, FR3, and loops are preferred. Specifically, positions 31-35, 50-65, 71-74, and 95-102 in the Kabat numbering in the H chain variable domain, and positions 24-34, 50-56, and 89-97 in the Kabat numbering in the L chain variable domain are preferred. Positions 31, 52a-61, 71-74, and 97-101 in the Kabat numbering in the H chain variable domain, and positions 24-34, 51-56, and 89-96 in the Kabat numbering in the L chain variable domain are more preferred.

[0195] Modification of amino acid residues also includes random modification of amino acids in the above-mentioned regions in the antibody variable region that binds to CD3 or CD137; and insertion of peptides previously known to have binding activity to CD3 or CD137 into the above-mentioned regions. The antigen-binding molecule of the present invention can be obtained by selecting from among such modified antigen-binding molecules a variable region that can bind to CD3 and CD137 but cannot bind to these antigens simultaneously.

[0196] Whether the variable region can bind to CD3 and CD137 but cannot bind to these antigens simultaneously, and further, whether the variable region can bind to CD3 and CD137 simultaneously when either one of CD3 and CD137 is present on the cell surface and the other antigen is present alone, or both antigens are present alone, or both antigens are present on the same cell, but cannot bind to these antigens expressed on different cells simultaneously can also be confirmed according to the above-mentioned method.

[0197] The present inventors have also succeeded in developing a method for more efficiently obtaining antigen-binding domains that bind to two or more different antigens. In some embodiments, a method for screening for antigen-binding domains that bind to at least two or more different antigens of the present invention 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 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 gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains, wherein the method does not include a step of amplifying the nucleic acid encoding the antigen-binding domains collected in step (b) between step (b) and step (c). In the above method, the number of steps of contacting the antigen-binding domain with an antigen is not particularly limited. In some embodiments, the screening method of the present invention may include three or more contacting steps when the number of antigens of interest is two or more. In a further embodiment, the screening method of the present invention may include two or more steps of contacting the antigen-binding domain with each one or more of the antigens of interest. In this case, the antigen-binding domain can be contacted with each antigen in any order. For example, the antigen-binding domain may be contacted with each antigen two or more times continuously, or first contacted with one antigen one or more times, and then contacted with other antigens before contacting with the same antigen again. Even when the screening method of the present invention includes three or more steps of contacting the antigen-binding domain with an antigen, the method does not include a step of amplifying the nucleic acid encoding the antigen-binding domains collected between any two consecutive contacting steps.

[0198] In some embodiments, the antigen-binding domain of the present invention is Fab, scFv, Fab'2, VHH, VH, or VL. In some embodiments, the antigen-binding domain of the present invention is a fusion polypeptide formed by fusing the antigen-binding domain to a scaffold and cross-linking the antigen-binding domain and the nucleic acid encoding the antigen-binding domain.

[0199] In some embodiments, the scaffold of the present invention is a bacteriophage. In some embodiments, the scaffold of the present invention is a ribosome, a RepA protein, or a DNA puromycin linker.

[0200] In some embodiments, elution is performed using an elution solution that is an acid solution, a base solution, DTT, or IdeS in steps (b) and (c) above. In some embodiments, the elution solution used in steps (b) and (c) of the present invention is EDTA or IdeS.

[0201] In some embodiments, a method for screening an antigen-binding domain that binds to at least two or more different antigens of interest of the present invention is (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; (b)' translating the nucleic acid encoding the antigen-binding domain collected in step (b); (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 gene encoding the antigen-binding domain collected in step (c) and identifying candidate antigen-binding domains. comprising, and between steps (b) and (c), the method does not include a step of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b).

[0202] In some embodiments, a method for generating antigen-binding domains that bind to at least two different antigens of interest of the present invention 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 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 gene encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; (e) ligating the polynucleotide encoding the candidate antigen-binding domain selected in step (d) to a polynucleotide encoding a polypeptide comprising an Fc region; (f) culturing a cell into which a vector into which the polynucleotide obtained in step (d) is operably linked has been introduced; and (g) collecting an antigen-binding molecule from the culture supernatant of the cells cultured in step (f). comprising, and between steps (b) and (c), the method does not include a step of amplifying the nucleic acid encoding the antigen-binding domains collected in step (b).

[0203] In some embodiments, the antigen-binding molecule of the present invention is an antibody prepared by the above method.

[0204] In one aspect, the screening method of the present invention enables more efficient acquisition of antigen-binding domains that bind to at least two different antigens of interest.

[0205] As used herein, "library" refers to a plurality of antigen-binding molecules, or a plurality of fusion polypeptides comprising antigen-binding molecules, or nucleic acids or polynucleotides encoding these sequences. The plurality of antigen-binding molecules, or the plurality of fusion polypeptides comprising antigen-binding molecules, contained in the library are antigen-binding molecules or fusion polypeptides comprising antigen-binding molecules that have different sequences from each other and do not have a single sequence. In some embodiments, the library of the present invention is a designed library. In a further embodiment, the designed library is the designed library disclosed in WO2016 / 076345.

[0206] In one embodiment of the present invention, a fusion polypeptide of the antigen-binding molecule of the present invention and a heterologous polypeptide can be prepared. In one embodiment, the fusion polypeptide can comprise, for example, at least a part of a viral coat protein selected from the group consisting of viral coat proteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, and variants thereof, fused to the antigen-binding molecule of the present invention.

[0207] In one aspect, the antigen-binding molecule of the present invention can be a ScFv, Fab fragment, F(ab)2, or F(ab')2. In another aspect, the present invention provides a library consisting essentially of a plurality of fusion polypeptides having different sequences, each containing any one of these antigen-binding molecules and a heterologous polypeptide. Specifically, the present invention provides, for example, a library consisting essentially of a plurality of fusion polypeptides having different sequences, each containing any one of these antigen-binding molecules fused to at least a part of a viral coat protein selected from the group consisting of viral coat proteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, and variants thereof. The antigen-binding molecule of the present invention may further contain a dimerization domain. In one aspect, the dimerization domain can be located between the variable region domain of the heavy or light chain of the antibody and at least a part of the viral coat protein. This dimerization domain may contain at least one dimerization sequence and / or a sequence containing one or more cysteine residues. This dimerization domain can preferably be linked to the C-terminus of the heavy chain variable domain or constant domain. The dimerization domain can adopt various structures depending on whether the antibody variable domain is prepared as a fusion polypeptide component with the viral coat protein component (in the absence of an amber stop codon after the dimerization domain), or whether the antibody variable domain is prepared mainly without containing the viral coat protein component (for example, in the presence of an amber stop codon after the dimerization domain). When the antibody variable domain is prepared mainly as a fusion polypeptide with the viral coat protein component, bivalent presentation is brought about by one or more disulfide bonds and / or a single dimerization sequence.

[0208] In a plurality of antigen-binding molecules in which the sequences are different from each other as described in this specification, the term "the sequences are different from each other" means that individual antigen-binding molecules in the library have distinct sequences. Specifically, the number of distinct sequences in the library reflects the number of independent clones with different sequences in the library, and may be referred to as the "library size". The library size of a normal phage display library is 10 6 ~10 12 and can be expanded up to 10 14 by applying known techniques in the art such as the ribosome display method. However, the actual number of phage particles used in the panning selection of a phage library is usually 10 to 10,000 times larger than the library size. This excess multiple is also called the "library equivalent number" and represents that 10 to 10,000 individual clones can have the same amino acid sequence. Therefore, the term "the sequences are different from each other" described in the present invention means that individual antigen-binding molecules in the library excluding the library equivalent number have distinct sequences, and more specifically, the library has antigen-binding molecules with sequences different from each other in the range of 10 6 ~10 14 , preferably 10 7 ~10 12 , more preferably 10 8 ~10 11 , particularly preferably 10 8 ~10 10 .

[0209] As used herein, "phage display" refers to a technique in which variant polypeptides are presented as fusion proteins with at least a portion of a coat protein on the surface of phage particles, such as filamentous phage particles. Phage display is useful because large libraries of randomized protein variants can be rapidly and efficiently screened for sequences that bind with high affinity to a target antigen. The display of peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties. Multivalent phage display methods have been used to display small random peptides and small proteins through fusion to gene III or gene VIII of filamentous phage (Wells and Lowman, Curr. Opin. Struct. Biol. (1992) 3, 355-362; and references cited therein). Monovalent phage display involves fusing a library of proteins or peptides to gene III or a portion thereof such that each phage particle presents one or zero copies of the fusion protein, and expressing the fusion protein at low levels in the presence of wild-type gene III protein. Monovalent phage have a lower avidity effect than multivalent phage and are therefore screened using phagemid vectors based on the intrinsic ligand affinity, which simplifies DNA manipulation (Lowman and Wells, Methods: A Companion to Methods in Enzymology (1991) 3, 205-216).

[0210] "Phagemid" refers to a plasmid vector having a copy of a bacterial origin of replication, such as ColE1, and an intergenic region of a bacteriophage. Phagemids derived from any bacteriophage known in the art, such as filamentous bacteriophage or lambdoid bacteriophage, can be used as appropriate. Usually, the plasmid also contains a selectable marker for antibiotic resistance. DNA fragments cloned into these vectors can be propagated as plasmids. When the cells carrying these vectors possess all the genes necessary for the production of phage particles, the replication pattern of the plasmid shifts to rolling circle replication to form copies of a single plasmid DNA strand and packaged phage particles. Phagemids can form infectious or non-infectious phage particles. This term includes phagemids containing a phage coat protein gene or a fragment thereof linked to a heterologous polypeptide gene by gene fusion such that the heterologous polypeptide is presented on the surface of the phage particle.

[0211] The term "phage vector" means a double-stranded replicative bacteriophage containing a heterologous gene and capable of replication. The phage vector has a phage origin of replication that permits phage replication and phage particle formation. The phage is preferably a filamentous bacteriophage, such as M13, f1, fd, or Pf3 phage or derivatives thereof, or a lambdoid phage, such as λ, 21, phi80, phi81, 82, 424, 434, or any other phage or derivatives thereof.

[0212] The term "coat protein" refers to a protein that is present, at least in part, on the surface of a viral particle. From a functional perspective, a coat protein is any protein that binds to a viral particle during the process of virus assembly in a host cell and remains bound to it until infection of another host cell by the virus. A coat protein may be a larger coat protein or a smaller coat protein. Smaller coat proteins are typically, preferably, present in the viral capsid at at least approximately 5, more preferably at least approximately 7, even more preferably at least approximately 10 or more protein copies per billion. Larger coat proteins may be present in tens, hundreds, or thousands of copies per billion. Examples of larger coat proteins include the filamentous phage p8 protein.

[0213] "Ribosome display" as described herein refers to a technique by which variant polypeptides are presented on ribosomes (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 polypeptide has an appropriate ribosome stalling sequence such as E. coli secM (J. Mol. Biol. 2007 Sep14;372(2):513-24) or has no stop codon. Preferably, the nucleic acid encoding the variant polypeptide also has a spacer sequence. As used herein, the term "spacer sequence" refers to a series of nucleic acids that fuse to the variant polypeptide to allow the variant polypeptide to pass through the ribosome tunnel after translation and to enable the variant polypeptide to express its function. Any in vitro translation system, for example, the E. coli S30 system, the PUREsystem, the rabbit reticulocyte lysate system, or the wheat germ cell-free translation system can be used for ribosome display.

[0214] The term "oligonucleotide" refers to short single-stranded or double-stranded polydeoxynucleotides that are chemically synthesized by methods known in the art (e.g., phosphotriester, phosphite, or phosphoramidite chemistry using solid-phase methods such as those described in EP266032; or methods via deoxynucleotide H-phosphonate intermediates as described in Froeshler et al., Nucl. Acids. Res. (1986) 14, 5399-5407). Other methods for oligonucleotide synthesis include the following polymerase chain reaction and other autoligation methods, as well as oligonucleotide synthesis on solid supports. All of these methods are described in Engels et al., Agnew. Chem. Int. Ed. Engl. (1989) 28, 716-734. These methods are used when the entire nucleic acid sequence of a gene is known or when a nucleic acid sequence complementary to the coding strand is available. Alternatively, when the target amino acid sequence is known, possible nucleic acid sequences can be appropriately deduced using known and preferred residues encoding each amino acid residue. Oligonucleotides can be purified using polyacrylamide gels or molecular sizing columns, or by precipitation methods.

[0215] The term "amplification of nucleic acid" refers to an experimental procedure for increasing the number of moles of nucleic acid. In a non-limiting aspect, nucleic acids include single-stranded RNA (ssRNA), double-stranded DNA (dsDNA), or single-stranded DNA (ssDNA). In a non-limiting aspect, the PCR (polymerase chain reaction) method is generally used as a method for amplifying nucleic acid, but any method capable of amplifying nucleic acid can be used. Alternatively, when a nucleic acid vector is introduced into a host cell, the nucleic acid can be amplified in the host cell. In a non-limiting aspect, electroporation, heat shock, infection with a phage or virus having a vector, or chemical reagents can be used to introduce nucleic acid into cells. Alternatively, DNA transcription, or reverse transcription of mRNA and subsequent transcription thereof can also amplify nucleic acid. In a non-limiting aspect, introduction of a phagemid vector into Escherichia coli is generally used for amplifying nucleic acid encoding a binding domain, but PCR can also be used in phage display technology. In ribosome display, cDNA display, mRNA display, and CIS display, the PCR method or transcription is generally used for amplifying nucleic acid.

[0216] The terms "fusion protein" and "fusion polypeptide" refer to a polypeptide having two segments linked to each other. These segments in the polypeptide have different properties. This property can be, for example, a biological property such as in vitro or in vivo activity. Alternatively, this property can be a single chemical or physical property, for example, binding to a target antigen or catalysis of a reaction. These two segments may be linked either directly through a single peptide bond or via a peptide linker containing one or more amino acid residues. Usually, these two segments and the linker are located in the same reading frame. Preferably, the two segments of the polypeptide are obtained from different or distinct polypeptides.

[0217] The term "scaffold" in "fusion polypeptide formed by fusing an antigen-binding domain to a scaffold" refers to a molecule that crosslinks an antigen-binding domain and a nucleic acid encoding the antigen-binding domain. As a non-limiting embodiment, phage coat proteins in phage display, ribosomes in ribosome display, puromycin in mRNA or cDNA display, RepA protein in CIS display, virus coat proteins in virus display, mammalian cell membrane anchor proteins in mammalian cell display, yeast cell membrane anchor proteins in yeast display, and bacterial cell membrane anchor proteins in bacterial display or E. coli display can be used as scaffolds in each display methodology.

[0218] In the present invention, the term "one or more amino acids" is not limited to a specific number of amino acids and may be two or more types of amino acids, five or more types of amino acids, ten or more types of amino acids, fifteen or more types of amino acids, or twenty types of amino acids.

[0219] Regarding the presentation of fusion polypeptides, the fusion polypeptides of the variable regions of antigen-binding molecules can be presented in various forms on the surface of cells, viruses, ribosomes, DNA, RNA, or phagemid particles. These forms include single-chain Fv fragments (scFv), F(ab) fragments, and multivalent forms of these fragments. The multivalent forms are preferably dimers of ScFv, Fab, and F(ab'), which are referred to herein as (ScFv)2, F(ab)2, and F(ab')2, respectively. The presentation of multivalent forms is preferred, probably for one reason that the presented multivalent forms usually have multiple antigen-binding sites that enable the identification of low-affinity clones and / or enable more efficient selection of rare clones in the selection process.

[0220] Methods for presenting fusion polypeptides containing antibody fragments on the surface of bacteriophages are known in the art and are described, for example, in WO1992001047 and herein. Other related methods are described in WO1992020791, WO1993006213, WO1993011236, and 1993019172. Those skilled in the art can use these methods as appropriate. Other published documents (H.R. Hoogenboom & G. Winter (1992) J. Mol. Biol. 227, 381-388, WO1993006213, and WO1993011236) disclose the identification of antibodies using artificially rearranged variable region gene repertoires against various antigens presented on the surface of phages.

[0221] When constructing a vector for presentation in the form of scFv, the vector contains nucleic acid sequences encoding the light chain variable domain and the heavy chain variable domain of the antigen-binding molecule. Generally, the nucleic acid sequence encoding the heavy chain variable domain of the antigen-binding molecule is fused to the nucleic acid sequence encoding the viral coat protein component. The nucleic acid sequence encoding the light chain variable domain of the antigen-binding molecule is linked to the nucleic acid of the heavy chain variable domain of the antigen-binding molecule through a nucleic acid sequence encoding a peptide linker. The peptide linker generally contains approximately 5 to 15 amino acids. Optionally, for example, additional sequences encoding tags useful 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 nucleic acid sequence encoding the heavy chain variable domain of the antigen-binding molecule, or both.

[0222] When constructing a vector for presentation in the form of F(ab), the vector contains nucleic acid sequences encoding the variable domain of the antigen-binding molecule and the constant domain of the antigen-binding molecule. The nucleic acid sequence encoding the light chain variable domain is fused with the 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 the nucleic acid sequence encoding the heavy chain constant CH1 domain. Generally, the nucleic acid sequences encoding the heavy chain variable domain and the constant domain are fused with the nucleic acid sequence encoding all or part of the viral coat protein. The heavy chain variable domain and the constant domain are preferably expressed as a fusion product with at least part of the viral coat protein, while the light chain variable domain and the constant domain are expressed separately from the heavy chain-viral coat fusion protein. The heavy chain and the light chain may associate with each other through covalent or non-covalent bonds. Optionally, for example, an additional sequence encoding a polypeptide tag useful for purification or detection is fused to the 3' end of the nucleic acid sequence encoding the light chain constant domain of the antigen-binding molecule or the nucleic acid sequence encoding the heavy chain constant domain of the antigen-binding molecule, or both.

[0223] Regarding the transfer of the vector into the host cell, the vector constructed as described above is transferred into the host cell for amplification and / or expression. The vector can be transferred into the host cell by transformation methods known in the art, including electroporation, calcium phosphate precipitation, etc. When the vector is an infectious particle such as a virus, the vector itself penetrates into the host cell. The fusion protein is presented on the surface of the phage particle by transfection of the host cell with a replicable expression vector having an insert of the polynucleotide encoding the fusion protein and production of phage particles by methods known in the art.

[0224] Replicable expression vectors can be introduced into host cells by use of a variety of methods. In a non-limiting embodiment, the vector can be introduced into cells by electroporation as described in WO2000106717. The cells are cultured in a standard culture medium at 37° C. for optionally approximately 6 to 48 hours (or until the OD at 600 nm reaches 0.6 to 0.8). Next, the culture medium is centrifuged and the culture supernatant is removed (e.g., by decantation). In the initial stage of purification, the cell pellet is preferably resuspended in a buffer (e.g., 1.0 mM HEPES (pH 7.4)). Next, the suspension is centrifuged again to remove the supernatant. The resulting cell pellet is resuspended in, for example, glycerol diluted to 5 to 20% V / V. For removal of the supernatant, the suspension is centrifuged again to obtain a 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.

[0225] Examples of preferred recipient cells include the E. coli strain SS320 (Sidhu et al., Methods Enzymol. (2000) 328, 333-363) which can respond to electroporation. The E. coli strain SS320 was prepared by coupling of MC1061 cells and XL1-BLUE cells under conditions sufficient to transfer a fertility episome (F' plasmid) or XL1-BLUE into MC1061 cells. The E. coli strain SS320 is deposited with the ATCC (10801 University Boulevard, Manassas, Virginia) under the accession number 98795. Any F' episome that allows phage replication in this strain can be used in the present invention. Suitable episomes may be obtained from strains deposited with the ATCC or may be obtained as commercially available products (TG1, CJ236, CSH18, DHF', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18, etc.).

[0226] By using a higher DNA concentration (about 10-fold) in electroporation, the transformation frequency is improved and the amount of DNA for transforming host cells is increased. The use of a high cell density also improves the efficiency (about 10-fold). An increase in the amount of DNA introduced can result in a library with greater diversity and a larger number of independent clones with different sequences. Transformed cells are usually selected based on the presence or absence of growth on a medium containing an antibiotic.

[0227] 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.

[0228] The present invention further provides a vector containing the nucleic acid of the present invention. The type of vector can be appropriately selected by those skilled in the art according to the host cell receiving the vector. For example, any of the above-described vectors can be used.

[0229] The present invention further relates to a host cell transformed with the vector of the present invention. The host cell can be appropriately selected by those skilled in the art. For example, any of the above-described host cells can be used.

[0230] The present invention also provides a pharmaceutical composition comprising the antigen-binding molecule of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be formulated according to methods known in the art by adding a pharmaceutically acceptable carrier to the antigen-binding molecule of the present invention. For example, the pharmaceutical composition can be used in the form of a parenteral injection of a sterile solution or suspension with water or any other pharmaceutically acceptable solution. For example, the pharmaceutical composition can be formulated by mixing the antigen-binding molecule with a pharmacologically acceptable carrier or vehicle, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc. in an appropriate combination in a unit dosage form required by generally recognized pharmaceutical practice. Specific examples of carriers can include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carmellose, sodium carmellose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, saccharide, carboxymethyl cellulose, corn starch, and inorganic salts. The amount of the active ingredient in such a preparation is determined so that an appropriate dosage within the indicated range can be achieved.

[0231] The sterile composition for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride). These solutions can be used in combination with appropriate solubilizing agents, such as alcohol (specifically, ethanol) or polyalcohol (for example, propylene glycol and polyethylene glycol), or nonionic surfactants, such as polysorbate 80 (trademark) or HCO-50.

[0232] Examples of the oily solutions include sesame oil and soybean oil. These solutions can be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. The solutions can further be mixed with a buffer (e.g., phosphate buffer and sodium acetate buffer), an anesthetic (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol), and an antioxidant. The injection solution thus prepared is usually filled into a suitable ampoule. The pharmaceutical composition of the present invention is preferably administered parenterally. Specific examples of its dosage form include injections, intranasal administration agents, transpulmonary administration agents, and transdermal administration agents. Examples of injections include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, through which the pharmaceutical composition can be administered systemically or locally.

[0233] The administration method can be appropriately selected according to the age and symptoms of the patient. The dosage of the pharmaceutical composition containing a polypeptide or a polynucleotide encoding the polypeptide can be selected, for example, within the range of 0.0001 to 1000 mg per kg of body weight per dose. Alternatively, the dosage can be selected, for example, within the range of 0.001 to 100000 mg per patient, but is not necessarily limited to these numerical values. The dosage and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can appropriately select the dosage and method.

[0234] The present invention also provides a method for treating cancer, which includes the step of administering the antigen-binding molecule of the present invention, the antigen-binding molecule of the present invention for use in the treatment of cancer, the use of the antigen-binding molecule of the present invention in the preparation of a therapeutic agent for cancer, and a process for preparing a therapeutic agent for cancer, which includes the step of using the antigen-binding molecule of the present invention.

[0235] The three-letter notations and corresponding one-letter notations of amino acids used in this specification 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: 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.

[0236] One of the aspects described in this specification, or any combination of two or more thereof, should also be understood by those skilled in the art to be included in the present invention, provided that no technical contradiction occurs based on the common general knowledge of those skilled in the art.

[0237] All reference documents cited in this specification are hereby incorporated by reference in their entirety.

[0238] The present invention is further illustrated in connection with the following examples. However, the present invention is not intended to be limited by the following examples.

Examples

[0239] [Example 1] Concept of a modified immunoglobulin variable (Fab) region that binds to CD3 and CD137 but not simultaneously to CD3 and CD137 T cells play an important role in tumor immunity and are known to be activated by two signals: 1) the binding of the T cell receptor (TCR) to antigen peptides presented by major histocompatibility complex (MHC) class I molecules and the activation of the TCR; and 2) the binding of co-stimulatory molecules on the surface of T cells to ligands on antigen-presenting cells and the activation of 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 surface of T cells, has been described as important for T cell activation (Vinay, 2011, Cellular & Molecular Immunology, 8, 281-284).

[0240] CD137 agonist antibodies have already been demonstrated to exhibit anti-tumor effects, which have been experimentally shown to be mainly due to the activation of CD8-positive T cells and NK cells (Houot, 2009, Blood, 114, 3431-8). It is also understood that T cells engineered to have a chimeric antigen receptor molecule consisting of a tumor antigen-binding domain as an extracellular domain and CD3 and CD137 signaling domains as intracellular domains (CAR-T cells) can enhance the durability of efficacy (Porter, N ENGL J MED, 2011, 365;725-733). However, the side effects of such CD137 agonist antibodies due to their non-specific hepatotoxicity are clinical and preclinical problems, and drug development has not advanced (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22). It has been suggested that the main cause of the side effects is involved in the binding of the antibody to the Fcγ receptor via the antibody constant region (Schabowsky, Vaccine, 2009, 28, 512-22). Furthermore, it has been reported that antibody cross-linking by Fcγ receptor-expressing cells (FcγRII-expressing cells) is required for agonist antibodies targeting receptors belonging to the TNF receptor superfamily to exert agonist activity in vivo (Li, Proc Natl Acad Sci USA. 2013, 110(48), 19501-6). WO2015 / 156268 describes that a bispecific antibody having a binding domain with CD137 agonist activity and a binding domain for a tumor-specific antigen can exert CD137 agonist activity only in the presence of cells expressing the tumor-specific antigen, activate immune cells, and thereby avoid the hepatotoxic adverse events of CD137 agonist antibodies while retaining the anti-tumor activity of the antibody. WO2015 / 156268 further describes that by using this bispecific antibody in combination with another bispecific antibody having a binding domain with CD3 agonist activity and a binding domain for a tumor-specific antigen, the anti-tumor activity can be further enhanced and these adverse events can be avoided.Tri-specific antibodies having three binding domains against CD137, CD3, and tumor-specific antigen (EGFR) have also been reported (WO2014 / 116846). However, since this molecule binds to CD3ε and CD137 simultaneously, it is expected to crosslink CD3ε-expressing T cells and CD137-expressing cells (T cells, B cells, NK cells, DC, etc.) even in the absence of tumor-specific antigen-expressing cells (Figure 2). In fact, it has been reported that a bispecific antibody against CD3 and CD8 crosslinked CD8-positive T cells and induced cytotoxic activity between those cells (Wong, Clin. Immunol. Immunopathol. 1991, 58(2), 236-250). Therefore, crosslinking of CD3 and an antigen expressed on T cells is also expected to induce crosslinking of T cells and cause T cells to kill each other.

[0241] Catumaxomab is known as a bispecific antibody that recognizes a protein expressed on T cells and a protein expressed on cancer cells (cancer antigen), with two Fabs, each binding to the cancer antigen (EpCAM) and the CD3ε chain expressed on T cells, and simultaneously binding to CD3ε and FcγR even in the absence of the cancer antigen. Therefore, it is known to crosslink CD3ε-expressing T cells to FcγR-expressing cells and produce a large amount of various cytokines even in an environment without cancer cells. Due to the induction of the production of various cytokines independent of the cancer antigen, the administration of trifunctional antibodies is currently limited to the intraperitoneal route (Cancer Treat Rev. 2010 Oct 36(6), 458-67 (Non-Patent Document 16)). Therefore, trifunctional antibodies are very difficult to administer systemically due to severe cytokine storm-like adverse reactions (Cancer Immunol Immunother. 2007 Sep; 56(9): 1397-406 (Non-Patent Document 18)).

[0242] On the one hand, conventional multispecific antibodies bind to multiple antigens simultaneously. Depending on the combination of antigens, there are cases where it is not preferable to bind to multiple antigens simultaneously. There is still no known antibody that exhibits both cytotoxic activity mediated by T cells and activation activity of T cells and other immune cells via CD137 in a cancer antigen-specific manner while avoiding harmful reactions.

[0243] Therefore, a possible way to control such an undesirable cross-linking reaction is a dual binding Fab, which is one variable (Fab) region that binds to CD3 through a part of it and binds to CD137 through a different part not involved in this binding to the first antigen (Figure 1). As shown in Figure 1, if two parts positioned proximally in one variable (Fab) region are essential for binding to their respective antigens, then the binding to CD3 inhibits the binding to CD137, while the binding to CD137 also inhibits the binding to CD3. Therefore, an improved antibody having the properties of such a dual binding Fab cannot bind to CD3 and CD137 simultaneously, and thus probably does not cause a cross-linking reaction between CD3 and CD137 (Figure 2). Also, the dual binding Fab can bind to both CD3 and CD137 simultaneously when CD3 and CD137 are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell, but it is considered that it does not bind simultaneously to these antigens expressed on different cells and does not cross-link these two cells (Figure 3). On the other hand, an antigen that binds to another variable (Fab) region (a third antigen) may cause a cross-linking reaction with CD3 and CD137 on T cells (Figure 4), or may cause a cross-linking reaction with CD137 on CD137-positive immune cells (Figure 5). Regarding this antibody, an Fc region that binds to FcγR may be used as the constant region, or an Fc region with reduced binding activity to FcγR may be used as the constant region region. By using the properties of such dual CD3 / CD137 binding Fabs, it is possible to provide, for example, a technique for damaging cancer cells expressing a cancer antigen by antibody-mediated redirection of T cells, together with the function of activating T cells, NK cells, and / or other immune cells, thereby achieving a higher anti-cancer potential.

[0244] Briefly stated, if the variable (Fab) region can be modified as a dual binding Fab so as to confer the following properties, an antibody having the actions as shown in FIG. 1 can be developed: 1. Having a binding activity to CD3; 2. Having a binding activity to CD137; and 3. Not binding to CD3 and CD137 simultaneously. The phrase "not binding to CD3 and CD137 simultaneously" also includes not cross-linking cells expressing CD3 and cells expressing CD137, or not binding to CD3 and CD137 simultaneously when each is expressed on different cells. This phrase further includes the case where the variable region can bind to both CD3 and CD137 simultaneously when CD3 and CD137 are not expressed on the cell membrane like soluble proteins, or when both are present on the same cell, but cannot bind to CD3 and CD137 simultaneously when each is expressed on different cells.

[0245] Similarly, if the variable (Fab) region can be modified as a dual binding Fab so as to confer the following properties, an antibody having the actions as shown in any of FIGS. 3, 4, and 5 can be developed: 1. Having a binding activity to CD3 on T cells; 2. Having a binding activity to CD137 on CD137-expressing cells; and 3. Not binding to CD3 and CD137 simultaneously.

[0246] [Example 2] Construction of a dual scFv library for ribosome display Using the antibody library fragment synthesized in Reference Example 3, a dual scFv library for ribosome display was constructed. The dual library was prepared as a library in which the H chain was diversified as shown in Table 38 (in Reference Example 4), while the L chain was fixed to the original sequence GLS3000 (SEQ ID NO: 1). The design of the ribosome display dual antibody library is shown in Fig. 6. To efficiently display the scFv library on ribosomes, a part of the bacteriophage λ gpD gene and the E. coli secM gene were used as spacer genes (SEQ ID NO: 2). The VL fragment of GLS3000 was assembled by PCR with its spacer gene and Gly / Ser-rich linker gene (SEQ ID NO: 3). The synthesized antibody VH library fragment was then fused by PCR amplification to the VL spacer gene at the 3' end and to the T7 promoter (SEQ ID NO: 4) with a 5' untranslated region (UTR) at the 5' end.

[0247] [Example 3] Obtaining scFv domains that bind to CD3ε and human CD137 from the dual scFv library (3-1) Obtaining the scFv domain that binds to human CD137 The scFv domain that binds to human CD137 was identified from the dual scFv library designed and constructed in Example 2. Biotin-labeled human CD137 fused to the human IgG1 Fc fragment (referred to as human CD137-Fc, SEQ ID NO: 16) was used as the antigen. The scFv ribosome display library constructed in Example 2 was used for in vitro transcription (T7 RiboMAX (trademark) Express Large scale RNA production system, P1320, Promega) to prepare an mRNA scFv library. The synthesized mRNA was purified by the RNeasy mini kit (catalog number 74104, QIAGEN). The obtained mRNA library was translated by the PUREfrex1.0 (PF001-0.25, Genefrontier) cell-free in vitro translation system with DnaK GroE Mix and DS supplement (PF003-0.5, PF004-0.5, PF005-0.5, Genefrontier). Subsequently, 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 the translation, and blocking buffer (1 pack in 200 mL milliQ of SuperBlock Dry Blend Blocking buffer in TBS (catalog number 37545, Pierce)) was also added. The panning method was performed with reference to the general panning method using magnetic beads (Nat Methods. 2007 Mar;4(3):269-79;Methods Mol Biol. 2012;805:261-86). The magnetic beads used were 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).

[0248] Specifically, 250 pmol of biotinylated antigen and 2 nmol of free human IgG Fc domain (wherein "free" means "not biotinylated") were added to the prepared ribosome display library solution, thereby contacting the library solution at 4 °C for 60 minutes. After the addition of magnetic beads blocked with SuperBlock, 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). After the addition of elution buffer (50 mM Tris-acetate, 150 mM NaCl, 50 mM EDTA, and 50 μg / mL Saccharomyces cerevisiae RNA (SIGMA)), the beads were suspended at 50 °C for 15 minutes, and immediately thereafter, the beads were separated using a magnetic stand to recover the mRNA solution. The purified mRNA library was converted to cDNA transcripts using the primer of SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with the primer of SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR with the primers of SEQ ID NOs: 149 and 150. This cycle, called panning, was repeated several times. In the second and subsequent rounds of panning, 150 - 50 pmol of biotinylated human CD137-Fc was used, and mRNA was recovered using either elution buffer (named EDTA elution campaign) or FabRICATOR (IdeS, a protease against the hinge region of IgG, GENOVIS) (named IdeS elution campaign). In that procedure, 5 μL of 10 units / μL Fabricator was added together with 95 μL of WBT buffer, the beads were suspended at 37 °C for 10 minutes, and immediately thereafter, the beads were separated using a magnetic stand to recover the mRNA solution.Next, 100 μL of elution buffer was added to the recovered mRNA and incubated at 50°C for 10 minutes.

[0249] (3-2) Binding of scFv domain to CD3ε or CD137 (scFv ELISA) To evaluate the binding of the scFv domain by ELISA, a FLAG-tag was added to the DNA libraries recovered in rounds 5 and 6 by PCR with the primers of SEQ ID NOs: 148 and 151. The resulting scFv-FLAG DNA fragments were ligated into the TOPO TA cloning kit dual promoter (Invitrogen) vector and transformed into DH5α Escherichia coli. The VH sequences from each single colony of E. coli were analyzed. Then, 5 - 7 clones with different VH sequences were picked from both the EDTA elution campaign and the IdeS elution campaign in rounds 5 and 6. Each scFv-FLAG gene was amplified from each colony with the primers of SEQ ID NOs: 148 and 151. PUREfrex1.0ss was added to each amplified scFv gene and incubated at 37°C for 2 hours. After the addition of 2% skim milk buffer, the solution containing scFv was subjected to ELISA by the following procedure: StreptaWell 96 microtiter plates (F. Hoffmann-La Roche Ltd.) were coated with biotinylated CD3ε peptide (SEQ ID 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 was available from Takara Bio Inc.) to remove unbound antigen. Then, the wells were blocked with 250 μL of 2% skim milk-TBS for 1 hour or more. After removal of the 2% skim milk-TBS, the prepared scFv solution was added to each well and the plate was left at room temperature for 1 hour so that scFv bound to the antigen contained in each well. Each well was washed with TBST and then MONOCLONAL ANTI-FLAG® M2, ANTIBODY (SIGMA, diluted 1000-fold in 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), HRP Conjugate (Invitrogen, diluted 2000-fold in TBS) was added to each well.The plate was incubated for 1 hour. After washing with TBST, TMB single solution (ZYMED Laboratories, Inc.) was added to the wells. The color reaction of the solution in each well was terminated by the addition of sulfuric acid. Then, the color development was assayed based on the absorbance at 450 nm. The results are shown in Figure 7. Most of the scFvs bound to either CD3ε or CD137, but two scFvs from the IdeS elution campaign, depicted in black in Figure 7, showed binding to both human CD137 and CD3ε. In other words, we succeeded in selecting clones showing binding activity to the second antigen (human CD137) by using the dual scFv library.

[0250] (3-3) Analysis of IgG binding to CD3ε or CD137 Eleven clones (dBBDu_001~011) were selected for further evaluation. These clones were converted to IgG (the VH and VL sequences of each clone were ligated to the constant domains of human H chain and L chain, respectively), and their binding activities to CD3ε and CD137 were evaluated. The VH fragment of each clone was amplified by PCR using primers that specifically bind to the H chain in the library. The amplified VH fragment was assembled into the CH1 gene of human IgG1 and incorporated into an animal expression plasmid. The prepared plasmid was used for expression in animal cells by the method of Reference Example 1. GLS3000 was used as the light chain, and its expression plasmid was prepared as shown in Reference Example 4-2.

[0251] The antigen binding of each molecule was tested by electrochemiluminescence (ECL method). Specifically, biotinylated CD3ε peptide or biotinylated human CD137 diluted to 18 pmol / mL with a TBS solution containing 0.1% Tween 20 called TBST, each antibody solution adjusted to 2 μg / mL, and a SULFO-TAG labeled (MESO SCALE DIAGNOSTICS, ruthenium (II) tris-bipyridine, N-hydroxysuccinimide) anti-human IgG antibody (Invitrogen #628400) adjusted to 18 pmol / mL were each added to a Nunc-Immuno™ MicroWell™ 96-well round plate (Nunc) at 25 μL / well, mixed, and then the plate was incubated at room temperature for 1 hour to form an antibody-antigen complex. A TBST solution containing 0.5% BSA was added to a streptavidin plate (MSD K.K., L15SA-1) at 150 μL / well, and the plate was incubated overnight at 4°C. After removing the blocking solution, each well was washed three times with 250 μL of the TBST solution. The antibody-antigen complex solution was added thereto at 75 μL / well, and the plate was incubated at room temperature for 1 hour so that biotin-anti-human IgG Ab bound to the streptavidin plate. After removing the antibody-antigen complex solution, each well was washed three times with the TBST solution, READ buffer (MSD K.K.) was added thereto at 150 μL / well, and then the luminescence signal of the sulfo-tag was detected using a Sector Imager 2400 (MSD K.K.).

[0252] Among those 11 clones, clone 011 (SEQ ID NO: 5) showed obvious binding to both CD3ε and human CD137, and several other clones also showed binding to both CD3ε and human CD137 (Figure 8), thus this result proves that those dual antibodies that bind to two different antigens could be obtained with this designed dual scFv library.

[0253] [Example 4] Acquisition of scFv domains that bind to CD3ε and human CD137 from a dual scFv library by double-round selection (4-1) Panning strategy for improving the efficiency of acquiring scFv domains that bind to human CD137 In Example 3, we succeeded in acquiring scFv domains that bind to CD3ε and CD137, but the acquisition efficiency was not very high. One strategy to consider for improving this is alternative panning, in which different antigens are used in different panning rounds. By this method, the selection pressure on both CD3ε and human CD137 could be applied to the dual scFv library in each different round, but not simultaneously. To solve this drawback, the inventors used double-round selection, in which two pannings are performed for one antigen in one round (one round means phage recovery from Escherichia coli ~ phage infection of Escherichia coli in phage display, and in vitro transcription ~ PCR amplification in ribosome display) (J Mol Biol. 1992 Aug 5;226(3):889-96). They used only one type of antigen in each panning round, but the inventors thought that two different antigen-specific antibodies could be more efficiently recovered by double-round selection using two different antigens in each panning procedure.

[0254] (4-2) Acquisition of scFv domains that bind to human CD137 by double-round selection The panning conditions are shown in Table 1. Campaigns 1 and 2 were alternative panning conditions, and Campaign 3 was a double-round selection condition in which double-round selection was performed in rounds 3, 5, and 7. Biotin-labeled CD3ε peptide antigen (amino acid sequence: SEQ ID NO: 6) and human CD137 fused to a biotin-labeled human IgG1 Fc fragment (named human CD137-Fc) were used as antigens. In Table 1, CD3 means panning with the biotin-labeled CD3 peptide, CD137 means panning with the biotin-labeled human CD137-Fc, and double means double-round selection.

[0255] (Table 1) TIFF0007710489000001.tif28170

[0256] 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 by an RNeasy mini kit (catalog number 74104, QIAGEN). The obtained mRNA library was translated by a PUREfrex1.0 (PF001-0.25, Genefrontier) cell-free in vitro translation system with DnaK GroE Mix and DS supplement (PF003-0.5, PF004-0.5, PF005-0.5, Genefrontier). Subsequently, 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 the translation, and blocking buffer (1 pack in 200 mL milliQ of SuperBlock Dry Blend Blocking buffer in TBS (catalog number 37545, Pierce)) was also added. The panning method was performed with reference to the general panning method using magnetic beads (Nat Methods. 2007 Mar;4(3):269-79;Methods Mol Biol. 2012;805:261-86). The magnetic beads used were 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).

[0257] Specifically, 250 pmol of biotinylated antigen and 2 nmol of free human IgG Fc domain (when the biotinylated antigen was human CD137-Fc) were added to the prepared ribosome display library solution, thereby contacting the library solution at 4°C for 60 minutes. After addition of magnetic beads blocked with SuperBlock, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed 2 or 3 times with WBT buffer (50 mM Tris-acetate, 150 mM NaCl, 50 mM Mg-acetate, 0.1% Tween). After addition of elution buffer (50 mM Tris-acetate, 150 mM NaCl, 50 mM EDTA, and 50 μg / mL budding yeast RNA (SIGMA)), the beads were suspended at 50°C for 15 minutes, and immediately thereafter the beads were separated using a magnetic stand 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 transcripts using the primer of SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with the primer of SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR with the primers of SEQ ID NO: 149 and 150. This cycle was repeated several times. In the second and subsequent rounds of panning, 150 - 50 pmol of biotinylated human CD137-Fc or 250 pmol of biotinylated CD3ε peptide was used.

[0258] In rounds 3, 5, and 7 of Campaign 3, double-round selections were performed. Specifically, 250 pmol of biotin-labeled CD3ε peptide was added to the prepared ribosome display library solution, thereby allowing the library solution to contact at 4°C for 60 minutes. After the addition of magnetic beads blocked with SuperBlock, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed 6 - 10 times (depending on the panning round) with WBT buffer. After the addition of elution buffer, the beads were suspended at 50°C for 15 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. The recovered mRNA was purified using the High Pure RNA Isolation kit (Roche). The purified mRNA library was then translated again by PUREfrex1.0 (GeneFrontier). 250 pmol or 100 pmol of biotin-labeled CD137-Fc and 2 nmol of free human IgG Fc domain were added to the prepared ribosome display library solution, thereby allowing the library solution to contact at 4°C for 60 minutes. After the addition of magnetic beads blocked with SuperBlock, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed 3 - 10 times (depending on the panning round) with WBT buffer. After the addition of elution buffer, the beads were suspended at 50°C for 15 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. The recovered mRNA was purified using the High Pure RNA Isolation kit (Roche). The purified mRNA library was converted to cDNA transcripts using the primer of SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with the primer of SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR with the primers of SEQ ID NO: 149 and 150.

[0259] (XX-2) Binding of scFv domains to CD3ε or CD137 (scFv ELISA) To evaluate the binding of the scFv domain by ELISA, a FLAG-tag was added to the DNA libraries recovered in rounds 6 and 7 by PCR with the primers of SEQ ID NOs: 149 and 151. The resulting scFv-FLAG DNA fragments were ligated into the TOPO TA cloning kit dual promoter (Invitrogen) and transformed into DH5α Escherichia coli. The VH sequences from each single colony of E. coli were analyzed. Next, several clones with different VH sequences were collected from each panning campaign in rounds 6 and 7. Each scFv-FLAG gene was amplified from each colony with the primers of SEQ ID NOs: 149 and 151. PUREfrex1.0ss was added to each amplified scFv gene and incubated at 37°C for 2 hours. After the addition of 2% skim milk buffer, the solution containing the scFv was subjected to ELISA by the following procedure: StreptaWell 96 microtiter plates (F. Hoffmann-La Roche Ltd.) were 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 was available from Takara Bio Inc.) to remove unbound antigen. The wells were then blocked with 250 μL of 2% skim milk-TBS for more than 1 hour. After removal of the 2% skim milk-TBS, the prepared scFv solution was added to each well and the plate was left at room temperature for 1 hour so that the scFv bound to the antigen contained 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), HRP Conjugate (Invitrogen, diluted 2000-fold with TBS) was added to each well. The plate was incubated for 1 hour.After washing with TBST, TMB single solution (ZYMED Laboratories, Inc.) was added to the wells. The color reaction of the solution in each well was terminated by the addition of sulfuric acid. Then, the color development was assayed based on the absorbance at 450 nm. The results are shown in Figure 9. All of the scFvs in Campaigns 1 and 2 bound to either CD3ε or CD137. On the other hand, many of the scFvs in Campaign 3 showed binding to both human CD137 and CD3ε. In other words, we succeeded in improving the efficiency of obtaining clones showing binding activity to both CD3ε and the second antigen (human CD137) by double-round selection using two different antigens in each panning round.

[0260] (4-3) Additional panning to obtain more scFv domains that bind to human CD137 by double-round selection To generate many more scFv domains that bind to human CD137 and CD3ε, additional rounds of panning were performed in Campaigns 2 and 3. In round 8, both conventional selection and double-round selection were used on the Campaign 2 round 7 output library, and double-round selection only was performed on the Campaign 3 round 7 output library. Each panning procedure was the same as in Example 4-2.

[0261] (4-4) Obtaining scFv domains that bind to human CD137 by double-round selection and IdeS elution The panning conditions are shown in Table 2. Campaigns 4 and 5 were alternative panning conditions, and Campaign 6 was a double-round selection condition in which double-round selection was performed in rounds 3, 5, and 7. Biotin-labeled CD3ε peptide antigen (amino acid sequence: SEQ ID NO: 6) and human CD137 fused to a biotin-labeled human IgG1 Fc fragment (named human CD137-Fc) were used as antigens. In Table 2, CD3 means panning with the biotin-labeled CD3 peptide, CD137 means panning with the biotin-labeled human CD137-Fc, and double means double-round selection.

[0262] (Table 2) TIFF0007710489000002.tif27168

[0263] The scFv ribosome display library constructed in Example 2 was used in in vitro transcription (T7 RiboMAX™ Express Large scale RNA production system, P1320, Promega) to prepare an mRNA scFv library. The synthesized mRNA was purified by the RNeasy mini kit (catalog number 74104, QIAGEN). The obtained mRNA library was translated by the PUREfrex1.0 (PF001-0.25, Genefrontier) cell-free in vitro translation system with DnaK GroE Mix and DS supplement (PF003-0.5, PF004-0.5, PF005-0.5, Genefrontier). Subsequently, 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 the translation, and blocking buffer (1 pack in 200 mL milliQ of SuperBlock Dry Blend Blocking buffer in TBS (catalog number 37545, Pierce)) was also added. The panning method was performed with reference to the general panning method using magnetic beads (Nat Methods. 2007 Mar;4(3):269-79;Methods Mol Biol. 2012;805:261-86). The magnetic beads used were 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).

[0264] 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, thereby contacting the library solution at 4 °C for 60 minutes. After the addition of magnetic beads blocked with SuperBlock, the antigen-scFv complex was attached to the magnetic beads at 4 °C for 15 minutes. The beads were washed 2 or 3 times with WBT buffer. After the addition of elution buffer, the beads were suspended at 50 °C for 15 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. The recovered mRNA and the mRNA were purified using the High Pure RNA Isolation kit (Roche). The purified mRNA library was converted to cDNA transcripts using the primer of SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with the primer of SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR with the primers of SEQ ID NOs: 149 and 150. This cycle was repeated several times. In the second and subsequent rounds of panning, 150 - 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 FabRICATOR (IdeS, a protease against the hinge region of IgG, GENOVIS). In that procedure, 5 μL of 10 units / μL Fabricator was added together with 95 μL of WBT buffer, the beads were suspended at 37 °C for 10 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. Then, 100 μL of elution buffer was added to the recovered mRNA and incubated at 50 °C for 10 minutes.When the antigen was the biotin-labeled CD3ε peptide, only the elution buffer was used in the same manner as in Round 1.

[0265] In rounds 3, 5, and 7 of Campaign 6, double-round selection was performed. Specifically, 250 pmol of biotin-labeled CD3ε peptide was added to the prepared ribosome display library solution, thereby contacting the library solution at 4 °C for 60 minutes. After the addition of magnetic beads blocked with SuperBlock, the antigen-scFv complex was attached to the magnetic beads at 4 °C for 15 minutes. The beads were washed 6 - 10 times (depending on the panning round) with WBT buffer. After the addition of elution buffer, the beads were suspended at 50 °C for 15 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. Ribosome (GeneFrontier) was added to the recovered mRNA, and the mRNA was purified using the High Pure RNA Isolation kit (Roche). The purified mRNA library was then translated again by PUREfrex1.0 (GeneFrontier). 250 pmol or 100 pmol of biotin-labeled CD137-Fc and 2 nmol of free human IgG Fc domain were added to the prepared ribosome display library solution, thereby contacting the library solution at 4 °C for 60 minutes. After the addition of magnetic beads blocked with SuperBlock, the antigen-scFv complex was attached to the magnetic beads at 4 °C for 15 minutes. The beads were washed 3 - 10 times (depending on the panning round) with WBT buffer. 5 μL of 10 units / μL Fabricator was added together with 95 μL of WBT buffer, the beads were suspended at 37 °C for 10 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. Then, 100 μL of elution buffer was added to the recovered mRNA and incubated at 50 °C for 10 minutes. The recovered mRNA was purified using the High Pure RNA Isolation kit (Roche).The purified mRNA library was converted to cDNA transcripts using the primer of SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with the primer of SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR with the primers of SEQ ID NOs: 149 and 150.

[0266] (4-5) Additional panning to obtain more scFv domains that bind to human CD137 by double-round selection and IdeS elution To generate many more scFv domains that bind to human CD137 and CD3ε, additional rounds of panning were performed in campaigns 4, 5, and 6 as in Example 4-3. Both conventional selection and double-round selection were used on the round 7 output libraries for both campaign 5 and campaign 6, and double-round selection was performed on the campaign 4 round 6 output library. Each panning procedure was the same as in Example 4-4.

[0267] (4-6) Binding of scFv domains to CD3ε or CD137 (scFv ELISA) To evaluate the binding of the scFv domain by ELISA, a FLAG-tag was added to the DNA libraries recovered in rounds 6 - 8 in Examples 4-3, 4, and 5 by PCR with the primers of SEQ ID NOs: 149 and 151. The resulting scFv-FLAG DNA fragments were ligated into the TOPO TA cloning kit dual promoter (Invitrogen) and transformed into DH5α Escherichia coli. The VH sequences from each single colony of E. coli were analyzed. Next, several clones with different VH sequences from each panning campaign were collected. Each scFv-FLAG gene was amplified from each colony with the primers of SEQ ID NOs: 149 and 151. PUREfrex1.0ss was added to each amplified scFv gene and incubated at 37°C for 2 hours. After the addition of 2% skim milk buffer, the solution containing scFv was subjected to ELISA by the following procedure: StreptaWell 96 microtiter plates (F. Hoffmann-La Roche Ltd.) were coated with biotinylated CD3ε peptide or biotinylated human CD137-human IgG1 Fc fusion at room temperature for 30 minutes. Each well of the plate was washed with TBST (TBS containing 0.1% Tween 20; TBS was available from Takara Bio Inc.) to remove unbound antigen. Then, the wells were blocked with 250 μL of 2% skim milk-TBS for more than 1 hour. After removal of 2% skim milk-TBS, the prepared scFv solution was added to each well and the plate was left at room temperature for 1 hour to allow the scFv to bind to the antigen contained in each well. Each well was washed with TBST and then MONOCLONAL ANTI-FLAG® M2, ANTIBODY (SIGMA, diluted 1000-fold in 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), HRP Conjugate (Invitrogen, diluted 2000-fold in TBS) was added to each well. The plate was incubated for 1 hour.After washing with TBST, TMB single solution (ZYMED Laboratories, Inc.) was added to the wells. The color reaction of the solution in each well was terminated by the addition of sulfuric acid. Then, the color development was assayed based on the absorbance at 450 nm. The results are shown in Figure 10. Many scFvs showed binding to both human CD137 and CD3ε in each panning campaign.

[0268] (4 - 7) Conversion to IgG1 of the antibody forms and preparation of each IgG1 molecule Eleven pools (shown in Table 3) were selected for further evaluation. The scFvs contained in these pools were converted to IgG (the VH and VL sequences of each clone were ligated to the constant domains of human heavy and light chains, respectively), and their binding activities to CD3ε and CD137 were evaluated. The VH fragment of each pool was amplified by PCR using primers (SEQ ID NOs: 152 and 153) that specifically bind to the heavy chains in the library. The amplified VH fragment was incorporated into an animal expression plasmid that already had the human IgG1 CH1 - Fc region. The prepared plasmid was used for expression in animal cells by the method of Reference Example 1. GLS3000 (SEQ ID NO: 1) was used as the light chain, and its expression plasmid was prepared as shown in Reference Example 4 - 2.

[0269] (Table 3) TIFF0007710489000003.tif78128

[0270] (4 - 8) Evaluation of the CD3ε and human CD137 binding activities of the obtained antibodies The prepared antibodies were subjected to ELISA to evaluate their binding ability to CD3ε and human CD137. First, a streptavidin-coated microplate (384 well, Greiner) was coated with 20 μL of TBS containing biotinylated CD3ε peptide or biotinylated human CD137-Fc at room temperature for 1 hour or more. After removing unbound biotinylated antigen from the plate by washing each well of the plate with TBST, the wells were blocked with 20 μL of blocking buffer (2% skim milk / TBS) for 1 hour or more. The blocking buffer was removed from each well. 10 μL of each mammalian cell supernatant containing IgG diluted 2-fold with 1% skim milk / TBS was added to the wells, and the plate was allowed to stand at room temperature for 1 hour to bind each IgG to the biotinylated antigen in each well. Then, each well was washed with TBST. Goat anti-human κ light chain alkaline phosphatase conjugate (BETHYL, A80-115AP) diluted with TBS was added to each well. The plate was incubated for 1 hour. After washing with TBST, the color reaction of the solution in each well to which Blue Phos Microwell Phosphatase Substrate System (KPL) was added was terminated by adding Blue Phos Stop Solution (KPL). Then, the color development was measured by absorbance at 615 nm. The measurement results are shown in FIG. 11.

[0271] Many clones showed binding to both CD3ε and human CD137, even in the IgG1 format. Then, the VH sequences from each single colony of E. coli were analyzed. In total, 19 different HCDR3 sequences were contained in these antibodies that bind to both CD3ε and human CD137, and many variants of HCDR1 or 2 sequences were present among them.

[0272] Antibodies that bind to two different antigens, CD3ε and human CD137, were obtained from a rationally designed library constructed using a CD3-binding antibody as a template as described in Reference Example 4, suggesting that double-round selection could much more efficiently obtain such antibodies. In conventional alternative panning, the selection pressure changes in each panning round, which may result in a progressive slowdown in the enrichment of ideal clones or the loss of ideal clones. By using double-round selection with two different antigens, the selection pressure in each panning round can be made uniform, so that ideal clones tend to gather smoothly and more directly.

[0273] [Example 5] Affinity Maturation of Antibody Domains That Bind to CD3ε and Human CD137 Derived from a Dual scFv Library Having a Designed Light Chain Library (5-1) Construction of a Light Chain Library with the Obtained Heavy Chain Although many antibodies that bind to both CD3ε and human CD137 were obtained in Example 4, their affinity for human CD137 was still low, so affinity maturation was performed to improve that affinity. To achieve this, the designed light chain library described in Reference Example 4 was combined with the heavy chain of one candidate antibody that binds to both CD3ε and human CD137. Among these 19 antibodies described in Example 4, the inventors selected the antibody named dBBDu_115 (SEQ ID NO: 7) as a candidate antibody for affinity maturation due to its better binding to both CD3ε and human CD137. The inventors constructed libraries of two different antibody formats, the VL-GS linker-VH scFv format and the Fab format. The design of the ribosome display dual antibody library is shown in FIG. 12. As in Example 2, a part of the bacteriophage λgpD gene and the E. coli secM gene were used as spacer genes to efficiently display the scFv or Fab library on ribosomes.

[0274] The synthesized antibody VL library fragment described in Reference Example 4 was fused by PCR amplification with the Gly / Ser-rich linker-dBBDu_115 VH-spacer gene (SEQ ID NO: 8) at the 3'-end and the T7 promoter (SEQ ID NO: 4) with a 5'-untranslated region at the 5'-end to create a VL-VH scFv format library. The synthesized antibody VL library fragment described in Reference Example 4 was fused by PCR amplification with the CL-spacer gene (SEQ ID NO: 9) at the 3'-end and the T7 promoter (SEQ ID NO: 4) with a 5'-untranslated region at the 5'-end to create a Fab format library. The VH gene fragment of dBBDu_115 was also fused by PCR amplification with the CH1 gene (SEQ ID NO: 10) at the 3'-end and the T7 promoter (SEQ ID NO: 4) with a 5'-untranslated region at the 5'-end to create a Fab format H-chain fragment.

[0275] (5-2) Obtaining Antibody Domains That Bind to Both CD3ε and Human CD137 The panning conditions are shown in Table 4. In this Table 4, double means double-round selection, and CD137Fc means conventional panning against biotin-labeled human CD137-Fc. The biotin-labeled CD3ε peptide antigen (amino acid sequence: SEQ ID NO: 6) and human CD137 fused to a biotin-labeled human IgG1 Fc fragment (named human CD137-Fc) were used as antigens.

[0276] (Table 4) TIFF0007710489000004.tif61170

[0277] The scFv ribosome display library, Fab light chain ribosome display library, and Fab heavy chain constructed in Example 5-1 were used in in vitro transcription (T7 RiboMAX™ Express Large scale RNA production system, P1320, Promega) to prepare mRNA libraries and heavy chain mRNA. The synthesized mRNA was purified by the RNeasy mini kit (catalog number 74104, QIAGEN). The obtained mRNA libraries and Fab heavy chains were translated by the PUREfrex1.0 (PF001-0.25, Genefrontier) cell-free in vitro translation system with DnaK GroE Mix and DS supplement (PF003-0.5, PF004-0.5, PF005-0.5, Genefrontier). Subsequently, WBTH buffer was added to stop the translation, and blocking buffer (2x c-block-e, Beacle) was also added. The magnetic beads used were 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).

[0278] Specifically, 60 pmol of biotinylated human CD137-Fc was added to magnetic beads at 4°C for 60 minutes, and then c-block-e (Beacle) was added to block the beads at 4°C for 60 minutes. The magnetic beads coated with this antigen and 2 nmol of free human IgG Fc domain 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 (Campaign 10) or 10 minutes (Campaigns 5, 6, and 9), and then the WBT buffer was discarded. This washing procedure was repeated 10 times with WBT buffer. After the addition of elution buffer, the beads were suspended at 50°C for 15 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. The recovered mRNA was purified using the High Pure RNA Isolation kit (Roche). The purified mRNA library was converted to cDNA transcripts using the primer of SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with the primer of SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR with the primers of SEQ ID NO: 149 and 150. This cycle called panning was repeated several times.

[0279] In the second and subsequent rounds of panning, the amount of biotinylated human CD137-Fc was changed as shown in Table 5, and the number of washes was also changed as shown in Table 6. When the antigen was biotinylated human CD137-Fc in the second and subsequent rounds in Campaigns 6, 9, and 10, mRNA was recovered using FabRICATOR (IdeS, a protease against the hinge region of IgG, GENOVIS). In that procedure, 5 μL of 10 units / μL Fabricator was added together with 95 μL of WBT buffer, the beads were suspended at 37 °C for 10 minutes, and immediately afterwards the beads were separated using a magnetic stand to recover the mRNA solution. Then, 100 μL of elution buffer was added to the recovered mRNA and incubated at 50 °C for 10 minutes.

[0280] (Table 5) Amount of biotinylated human CD137-Fc (pmol) TIFF0007710489000005.tif61170

[0281] (Table 6) Number of washes TIFF0007710489000006.tif61170

[0282] In several campaigns, double-round selection was performed. Specifically, 150 pmol of biotin-labeled CD3ε peptide was added to magnetic beads at 4 °C for 60 minutes, and then c-block-e (Beacle) was added to block the beads at 4 °C for 60 minutes. The magnetic beads coated with this antigen were added to the prepared ribosome display library solution, thereby allowing the library solution to contact at 4 °C for 60 minutes. The beads were washed 10 times with WBT buffer. After the addition of elution buffer, the beads were suspended at 50 °C for 15 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. The recovered mRNA was purified using a High Pure RNA Isolation kit (Roche). The purified mRNA library was then translated again by PUREfrex1.0 (GeneFrontier). Biotin-labeled CD137-Fc was added to magnetic beads at 4 °C for 60 minutes, and then c-block-e (Beacle) was added to block the beads at 4 °C for 60 minutes. The magnetic beads coated with this antigen and 2 nmol of free human IgG Fc domain were added to the prepared ribosome display library solution, thereby allowing the library solution to contact at 4 °C for 60 minutes. The beads were incubated with WBT buffer for 10 minutes, and then the WBT buffer was discarded. This washing procedure was repeated 10 times with WBT buffer. In Campaigns 6, 9, and 10, 5 μL of 10 units / μL Fabricator was added together with 95 μL of WBT buffer to suspend the beads at 37 °C for 10 minutes, and immediately the beads were separated using a magnetic stand to recover the mRNA solution. Then, 100 μL of elution buffer was added to the recovered mRNA and 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 to cDNA transcripts using the primer of SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with the primer of SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). The T7 promoter gene was added to the amplified DNA library by PCR with the primers of SEQ ID NOs: 149 and 150.

[0283] (5-3) Conversion to IgG1 in antibody format and preparation of each IgG1 molecule The light chain genes of the scFv or Fab domain library derived from affinity maturation panning described in Example 5-2 were converted to IgG, and their binding activities to CD3ε and CD137 were evaluated. The VL-CL fragments of the pools of Campaigns 5, 6, and 9 were amplified by PCR using primers (SEQ ID NOs: 154 and 155) that specifically bind to the L chain in the library. The amplified VL-CL fragments were incorporated into an animal expression plasmid and transformed into DH5α E. coli strain. The obtained plasmid was also used for the construction of the light chain expression vector derived from Campaign 10. The VL region gene was excised from the obtained expression vector with restriction enzymes SfiI and KpnI. The VL fragments of the pool of Campaign 10 were amplified by PCR using primers (SEQ ID NOs: 154 and 156) that specifically bind to the VL region in the library. The prepared VL fragments were introduced into the digested expression vector. The number of colonies collected is shown in Table 7. The prepared plasmid was used for expression in animal cells by the method of Reference Example 1. The dBBDu_115 heavy chain expression plasmid constructed in Example 4 was also used to express full-length IgG.

[0284] (Table 7) TIFF0007710489000007.tif32128

[0285] (5-4) Evaluation of the obtained antibodies for their CD3ε and human CD137 binding activities The prepared antibody was subjected to ELISA to evaluate its binding ability to CD3ε and human CD137. First, a streptavidin-coated microplate (384 wells, Greiner) was coated with 20 μL of TBS containing biotin-labeled CD3ε peptide, biotin-labeled human CD137-Fc, and biotin-labeled human IgG1 Fc region at room temperature for 1 hour or more. After removing unbound biotin-labeled antigen from the plate by washing each well with TBST, the wells were blocked with 20 μL of blocking buffer (2% skim milk / TBS) for 1 hour or more. The blocking buffer was removed from each well. 10 μL of each mammalian cell supernatant containing 20 μg / mL IgG diluted 2-fold with 2% skim milk / TBS was added to the wells, and the plate was allowed to stand at room temperature for 1 hour to bind each IgG to the biotin-labeled antigen in each well. Then, each well was washed with TBST. Goat anti-human κ light chain alkaline phosphatase conjugate (BETHYL, A80-115AP) diluted with TBS was added to each well. The plate was incubated for 1 hour. After washing with TBST, the color reaction of the solution in each well to which Blue Phos Microwell Phosphatase Substrate System (KPL) was added was terminated by adding Blue Phos Stop Solution (KPL). Then, the color development was measured by absorbance at 615 nm. The measurement results are shown in FIG. 13.

[0286] (5-5) Evaluation of simultaneous binding of IgG having the obtained Fab domain to CD3ε and human CD137 Five antibodies (shown in Table 8) were selected for further evaluation. These antibodies were expressed and purified according to Example 5-3 and Reference Example 1. The purified antibodies were subjected to ELISA to evaluate their simultaneous binding ability to CD3ε and human CD137.

[0287] (Table 8) TIFF0007710489000008.tif43128

[0288] First, MyOne-T1 streptavidin beads were mixed with 0.625 pmol of biotinylated human CD137-Fc or biotinylated 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 plate (Corning, 3792 black round-bottom PS plate) and incubated at room temperature for 60 minutes or more. Thereafter, 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 "not biotinylated") or TBS, and then added to the magnetic beads in each well. The plate was allowed to stand at room temperature for 1 hour to bind each IgG to the biotinylated antigen in each well. Thereafter, each well was washed with TBST. Goat anti-human κ light chain alkaline phosphatase conjugate (BETHYL, A80-115AP) diluted with TBS was added to each well. The plate was incubated for 1 hour. After washing with TBST, APS-5 (Lumigen) was added to each well. After 2 minutes, the fluorescence of each well was detected. The measurement results are shown in FIG. 14 and Table 9.

[0289] (Table 9) TIFF0007710489000009.tif66136

[0290] Inhibition of the binding to human CD137-Fc by free CD3ε peptide was observed for all tested antibodies, but not by free Fc domain. This result means that the obtained antibodies could not bind to human CD137-Fc in the presence of CD3ε peptide, in other words, these antibodies do not bind to human CD137 and CD3ε peptide simultaneously. Therefore, it was proved that we successfully obtained Fab domains that can bind to two different antigens, CD137 and CD3ε, but not simultaneously, by using the designed library and ribosome displayable round selection.

[0291] [Example 6] Obtaining Fab Domains that Bind to CD3ε and Human CD137 from a Dual Fab Phage Display Library (6-1) Construction of a Heavy Chain Phage Display Library with GLS3000 Light Chain Using the antibody library fragment synthesized in Reference Example 4, a dual Fab library for phage display was constructed. The dual library was prepared as a library in which the H chain was diversified as shown in Reference Example 4, but the L chain was fixed to the original sequence GLS3000 (SEQ ID NO: 1). The H chain library sequence derived from CE115HA000 by adding V11L / L78I mutations to the FR (framework) and further diversifying the CDRs as shown in Table 38 (in Reference Example 4) was entrusted to DNA2.0, Inc., a DNA synthesis company, to obtain an antibody library fragment (DNA fragment). The obtained antibody library fragment was inserted into a phagemid for phage display amplified by PCR. GLS3000 was selected as the L chain. The constructed phagemid for phage display was transferred into E. coli by electroporation to prepare E. coli carrying the antibody library fragment.

[0292] A phage library presenting Fab domains was produced from Escherichia coli carrying the constructed phagemid by infection with the helper phage M13KO7TC / FkpA encoding the FkpA chaperone gene, and then incubated overnight at 25 °C in the presence of 0.002% arabinose (this phage library is named the DA library) or at 20 °C in the presence of 0.02% arabinose (this phage library is named the DX library). M13KO7TC is a helper phage having an insert of a trypsin cleavage sequence between the N2 domain and the CT domain of the pIII protein on the helper phage (see International Patent Application Domestic Publication No. 2002-514413). The introduction of the insert gene into the M13KO7TC gene has already been disclosed elsewhere (see the domestic publication of International Patent Application No. WO2015046554).

[0293] (6-2) Obtaining Fab domains that bind to CD3ε and human CD137 by double-round selection Fab domains that bind to CD3ε and human CD137 were identified from the dual Fab library constructed in Example 6-1. Biotin-labeled CD3ε peptide antigen (amino acid sequence: SEQ ID NO: 6), biotin-labeled CD3ε peptide antigen through a disulfide bond linker (shown in Figure 15, called C3NP1-27; amino acid sequence: SEQ ID NO: 145, synthesized by Genscript), human CD137 fused to a biotin-labeled human IgG1 Fc fragment (named human CD137-Fc), and human CD137 fused to an SS-biotinylated 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, catalog number 21445) for human CD137 fused to a human IgG1 Fc fragment. Biotinylation was carried out according to the instruction manual.

[0294] Phages were produced from Escherichia coli carrying the constructed phagemid for phage display. 2.5 M NaCl / 10% PEG was added to the culture broth of Escherichia coli that had produced phages, 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 library solution. The panning method was carried out with reference to a general panning method using an antigen 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 streptavidin-coated beads (Dynabeads M-280 Streptavidin). Subtraction for magnetic beads and biotin-labeled human Fc was performed to exclude phages presenting antibodies that bind to the magnetic beads themselves or the human IgG1 Fc region.

[0295] Specifically, the phage solution was mixed with 250 pmol of human CD137-Fc and 4 nmol of free human IgG1 Fc domain and incubated at room temperature for 60 minutes. The magnetic beads were blocked with 2% skim milk / TBS containing free streptavidin (Roche) at room temperature for more than 60 minutes, washed three times with TBS, and then mixed with the incubated phage solution. After incubation at room temperature for 15 minutes, the beads were washed three times with TBST (TBS containing 0.1% Tween 20; TBS was 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, incubated at room temperature for 15 minutes, and immediately the beads were separated using a magnetic stand to recover the phage solution. The phage was infected into the E. coli strain through gentle agitation culture at 37 °C for 1 hour. The infected E. coli was inoculated onto a 225 mm × 225 mm plate. Next, the phage was recovered from the culture solution of the inoculated E. coli to prepare a phage library solution. In this first round of panning procedure, phages presenting antibodies that bind to human CD137 were enriched. In the second round of panning, 250 pmol of ss-human CD137-Fc was used as the biotinylated antigen, and the washing was performed three times with TBST and then twice with TBS. Elution was performed with 25 mM DTT at room temperature for 15 minutes and then digested with trypsin. In the third and sixth rounds of panning, 62.5 pmol of C3NP1-27 was used as the biotinylated antigen, and the washing was performed three times with TBST and then twice with TBS. Elution was performed with 25 mM DTT at room temperature for 15 minutes and then digested with trypsin. In the fourth, fifth, and seventh rounds of panning, 62.5 pmol of ss-human CD137-Fc was used as the biotinylated antigen, and the washing was performed three times with TBST and then twice with TBS. Elution was performed with 25 mM DTT at room temperature for 15 minutes and then digested with trypsin.

[0296] (6-3) Binding of the Fab domain presented by the phage to CD3ε or human CD137 The culture supernatant containing phage was recovered from each of the 96 single colonies of E. coli obtained by the above method according to a general method (Methods Mol. Biol. (2002) 178, 133-145). The culture supernatant containing phage was subjected to ELISA by the following procedure: Streptavidin-coated microplates (384 wells, Greiner, catalog number 781990) were coated with 10 μL of TBS containing biotinylated antigen (biotinylated CD3ε peptide or biotinylated human CD137-Fc) overnight at 4°C or for 1 hour at room temperature. Each well of the plate 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 more. After removal of TBS / 2% skim milk, the prepared culture supernatant was added to each well and the plate was left at room temperature for 1 hour so that the antibody presented by the phage bound to the antigen contained in each well. Each well was washed with TBST and then HRP / Anti M13 (GE Healthcare 27-9421-01) was added to each well. The plate was incubated for 1 hour. After washing with TBST, TMB single solution (ZYMED Laboratories, Inc.) was added to the wells. The color reaction of the solution in each well was terminated by the addition of sulfuric acid. The color development was then assayed based on the absorbance at 450 nm. The results are shown in Figure 16. As shown in Figure 16, all the clones showed binding to human CD3ε, but did not show binding to human CD137 despite performing the panning procedure against human CD137 five times. This may depend on the lower sensitivity of this phage ELISA assay on streptavidin-coated microplates, and therefore phage ELISA on streptavidin-coated beads was also performed.

[0297] (6-4) Phage-displayed Fab domain binding to human CD137 (phage bead ELISA) First, MyOne-T1 beads, magnetic beads coated with streptavidin, were washed three times with a blocking buffer containing 0.5x block Ace, 0.02% Tween, and 0.05% ProClin 300, and then blocked with this blocking buffer for 60 minutes or more at room temperature. After washing once with TBST, 0.625 pmol of ss-human CD137-Fc was added to the magnetic beads and incubated for 10 minutes or more at room temperature, and then the magnetic beads were applied to each well of a 96-well plate (Corning, 3792 black round-bottom PS plate). A phage solution presenting 12.5 μL of each Fab was added to the well together with 12.5 μL of TBS, and the plate was allowed to stand at room temperature for 30 minutes so that each Fab bound to the biotinylated antigen in each well. Thereafter, each well was washed with TBST. Anti-M13 (p8) Fab-HRP diluted with a blocking buffer containing 0.5x block Ace, 0.02% Tween, and 0.05% ProClin 300 was added to each well. The plate was incubated for 10 minutes. After washing three times with TBST, LumiPhos-HRP (Lumigen) was added to each well. Two minutes later, the fluorescence of each well was detected. The measurement results are shown in Fig. 17.

[0298] Some clones showed obvious binding to human CD137. This result indicated that some Fab domains that bind to both human CD3ε and CD137 were also obtained from this designed library by the phage display panning strategy. Still, the binding to human CD137 was still weak compared to the CD3ε peptide. The VH fragments of each human CD137-binding clone were amplified by PCR using primers (SEQ ID NOs: 157 and 158) that specifically bind to the phagemid vector, and the DNA sequences were analyzed. The results showed that all the binding clones had the same VH sequence, which meant that only one Fab clone showed binding to both human CD137 and CD3ε. To improve this, in the following experiment, double-round selection was also applied to the phage display strategy.

[0299] [Example 7] Obtaining Fab Domains that Bind to CD3ε and Human CD137 from a Dual Fab Phage Display Library by the Double-Round Selection Method (7-1) Construction of a Heavy Chain Phage Display Library with GLS3000 Light Chain The phage library presenting the Fab domain was produced from Escherichia coli carrying the constructed phagemid by infection with the helper phage M13KO7TC / FkpA encoding the FkpA chaperone (SEQ ID NO: 17), and then incubated overnight at 25°C in the presence of 0.002% arabinose (this phage library is named the DA library) or at 20°C in the presence of 0.02% arabinose (this phage library is named the DX library). M13KO7TC is a helper phage having an insert of a trypsin cleavage sequence between the N2 domain and the CT domain of the pIII protein on the helper phage (see Japanese Patent Application Publication No. 2002-514413). The introduction of the insert gene into the M13KO7TC gene has already been disclosed elsewhere (see WO2015 / 046554).

[0300] (7-2) Acquisition of Fab domains that bind to CD3ε and human CD137 by double round selection Fab domains that bind to CD3ε and human CD137 were identified from the dual Fab library constructed in Example 7-1. Biotin-labeled CD3ε peptide antigen (amino acid sequence: SEQ ID NO: 6), biotin-labeled CD3ε peptide antigen (C3NP1-27: SEQ ID NO: 145) through a disulfide bond linker, and human CD137 fused to a biotin-labeled human IgG1 Fc fragment (named human CD137-Fc) were used as antigens.

[0301] To generate many more Fab domains that bind to human CD137 and CD3ε, double round selection was also applied to phage display panning in panning round 2 and subsequent rounds. Phages were produced from Escherichia coli carrying the constructed phagemid for phage display. 2.5 M NaCl / 10% PEG was added to the culture broth of the Escherichia coli that had produced the phages, 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 library solution. The panning method was carried out with reference to a general panning method using an antigen 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 streptavidin-coated beads (Dynabeads M-280 Streptavidin). Subtraction was performed for the magnetic beads and biotin-labeled human Fc to eliminate phages presenting antibodies that bind to the magnetic beads themselves or to the human IgG1 Fc region.

[0302] Specifically, in panning round 1, magnetic beads were blocked with 2% skim milk / TBS at room temperature for 60 minutes or more and washed three times with TBS. The phage solution of the DA library or DX library was added to the blocked magnetic beads, incubated at room temperature for 60 minutes or more, and then the supernatant was collected. 500 pmol of biotin-labeled human IgG1 Fc was added to fresh magnetic beads, incubated at room temperature for 15 minutes, and then 2% skim milk / TBS was added. After blocking at room temperature for 60 minutes or more, the magnetic beads were washed three times with TBS. The collected phage solution was added to the blocked magnetic beads, incubated at room temperature for 60 minutes or more, and then the supernatant was collected. 500 pmol of biotin-labeled CD137-Fc was added to fresh magnetic beads, incubated at room temperature for 15 minutes, and then 2% skim milk / TBS was added. After blocking at room temperature for 60 minutes or more, the magnetic beads were washed three times with TBS. The collected phage solution was added to the blocked magnetic beads, 8 nmol of free human IgG1 Fc domain was also added, and then 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.), and then further washed once 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, and immediately the beads were separated using a magnetic stand to collect the phage solution. The collected phage solution was added to Escherichia coli strain ER2738 in the logarithmic growth phase (OD600: 0.4 - 0.5). The phage was infected into the Escherichia coli strain through gentle stirring culture at 37°C for 1 hour. The infected Escherichia coli was inoculated onto a 225 mm × 225 mm plate. Next, phage was recovered from the culture solution of the inoculated Escherichia coli to prepare a phage library solution.

[0303] In this panning round 1 procedure, since phages presenting antibodies that bind to human CD137 were enriched, double-round selection was performed in the nex...

Claims

Claim 1 A method for producing an antigen-binding molecule having agonist activity against CD137, which comprises an antibody variable region that can bind to a first antigen that is either CD3 or CD137 and a second antigen that is either CD3 or CD137 different from the first antigen, but does not bind to the first antigen and the second antigen simultaneously, and an antibody variable region that binds to a third antigen different from the first antigen and the second antigen, comprising: (i) (a) providing a library comprising a plurality of antigen-binding domains, wherein the library comprises an antigen-binding domain in which an amino acid maintaining binding activity to the first antigen appears, said step; (b) contacting the library provided in step (a) with the first antigen and collecting the antigen-binding domains bound to the first antigen; (c) contacting the antigen-binding domains collected in step (b) with a second antigen different from the first antigen and collecting the 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 A method for screening an antigen-binding domain that binds to a first antigen and a second antigen different from the first antigen, but does not bind to the first antigen and the second antigen simultaneously, which method does not include a step of amplifying a nucleic acid encoding the antigen-binding domains collected in step (b) between step (b) and step (c), to obtain a nucleic acid encoding an antibody variable region contained in the antigen-binding domain that binds to the first antigen and the second antigen different from the first antigen; (ii) culturing a host cell comprising a nucleic acid encoding an antibody variable region contained in an antigen-binding domain that binds to a first antigen and a second antigen different from the first antigen obtained in step (i), and a nucleic acid encoding an antibody variable region that binds to a third antigen different from the first antigen and the second antigen, to express an antigen-binding molecule comprising an antibody variable region that can bind to the first antigen and the second antigen, but does not bind to the first antigen and the second antigen simultaneously, and an antibody variable region that binds to the third antigen; (iii) recovering the antigen-binding molecule from the host cell culture; and (iv) A step of selecting an antigen-binding molecule having agonistic activity against CD137 from the antigen-binding molecules recovered in step (iv) of the engineering, comprising, a production method.

2. The method according to claim 1, wherein the third antigen is a molecule specifically expressed in cancer tissue.

3. The method according to claim 1 or 2, wherein the antigen-binding molecule has at least one characteristic selected from the group consisting of the following (1) to (3): (1) The variable region binds to the extracellular domain of CD3ε containing the amino acid sequence of SEQ ID NO: 91, (2) The antigen-binding molecule induces CD3 activation of T cells against cells expressing the molecule of the third antigen, but does not induce CD3 activation of T cells against cells expressing CD137, and (3) The antigen-binding molecule does not induce cytokine release from PBMC in the absence of cells expressing the molecule of the third antigen.

4. The method according to any one of claims 1 to 3, wherein the antigen-binding molecule further comprises an antibody Fc region.

5. The method according to claim 4, wherein the Fc region is an Fc region having a reduced binding activity to FcγR as compared with the Fc region of a natural human IgG1 antibody.

6. The antigen-binding domain is a fusion polypeptide formed by fusing the antigen-binding domain with a scaffold for cross-linking the antigen-binding domain with the nucleic acid encoding the antigen-binding domain and the antigen-binding domain, The method according to any one of claims 1 to 5.

7. The method according to claim 6, wherein the scaffold is a bacteriophage.

8. The method according to claim 6, further comprising a step of translating the nucleic acid encoding the antigen-binding domain collected in step (b) between step (b) and step (c) in step (i).

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