Antigen-binding molecule comprising altered antibody variable region binding 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 technology]

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and rarely cause adverse reactions (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-Patent Document 1) and Eur J Pharm Biopharm. (2005) 59 (3), 389-396 (Non-Patent Document 2)). Antibodies not only bind to antigens and have agonistic or antagonistic effects, but also induce effector cell-mediated cytotoxicity (also known as effector function), such as ADCC (antibody-dependent cellular cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), or CDC (complement-dependent cytotoxicity). In particular, antibodies of the IgG1 subclass exhibit effector function against cancer cells, and therefore many antibody drugs 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 (FcγR) and various complement components present on effector cells (e.g., NK cells or macrophages). In humans, the FcγR protein family has been reported to include FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isoforms, and their respective allotypes have also been reported (Immunol. Lett. (2002) 82, 57-65 (Non-Patent Document 3)). Of these isoforms, FcγRIa, FcγRIIa, and FcγRIIIa contain a domain called ITAM (immunoreceptor tyrosine-based activation motif) in their intracellular domain, which transduces activation signals. In contrast, only FcγRIIb contains a domain called ITIM (immunoreceptor tyrosine-based inhibitory motif) in its intracellular domain, which transduces inhibitory signals. All of these isoforms of FcγR are known to transmit signals by cross-linking with immune complexes, etc. (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-Patent Document 4)). In fact, when antibodies exert their effector function against cancer cells, FcγR molecules on the effector cell membrane are clustered by the Fc regions of multiple antibodies bound to the cancer cell membrane, thereby transmitting an activation signal through the effector cells. As a result, a cytocidal effect is exerted. In this regard, FcγR cross-linking is limited to effector cells located near cancer cells, indicating that immune activation 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 FcRn (a binding molecule that interacts with the Fc region of IgG) binds to each heavy chain of the antibody, one molecule at a time. Therefore, it has been reported that two FcRn molecules bind to one IgG antibody molecule. Unlike FcRn, FcγR interacts with the antibody via the hinge region and CH2 domain, and only one FcγR molecule binds to one IgG antibody molecule (J. Bio. Chem., (20001) 276, 16469-16477). It has been found that several amino acid residues in the hinge region and CH2 domain of an antibody, as well as the sugar chain attached to Asn 297 (EU numbering) in the CH2 domain, are important for the binding between FcγR and the Fc region of an 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)). Focusing on this binding site, Fc region variants with various FcγR-binding properties have been studied, and Fc region variants with higher binding activity to activating FcγRs 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 approximately 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. USA (2006) 103, 4005-4010 (Non-Patent Document 9) and WO2006 / 019447 (Patent Document 2)). This modified form has approximately 9-fold higher binding activity than the wild-type form in terms of the ratio of FcγRIIIa to FcγIIb (A / I ratio).Alternatively, Shinkawa et al. succeeded in increasing the FcγRIIIa-binding activity by approximately 100-fold by deleting the fucose in the sugar chain attached to Asn 297 (EU numbering) (J. Biol. Chem. (2003) 278, 3466-3473 (Non-Patent Document 10)). These methods can significantly improve the ADCC activity of human IgG1 compared to native human IgG1.

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

[0006] Antibodies that bind to two or more antigens with a single molecule (these antibodies are called bispecific antibodies) are being studied as molecules that inhibit multiple targets. By modifying natural IgG antibodies, it is possible to confer binding activity to two different antigens (first antigen and 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 enhance antitumor activity by crosslinking cytotoxic cells to cancer cells. Previously reported molecular forms of bispecific antibodies include molecules in which antigen-binding sites are added to the N- or C-terminus of an antibody (DVD-Ig, TCB, and scFv-IgG), molecules in which the two Fab regions of an antibody have different sequences (common L-chain bispecific antibodies and hybrid hybridomas), molecules in which one Fab region recognizes two antigens (two-in-one IgG and DutaMab), and molecules in which the CH3 domain loop serves as a separate antigen-binding site (Fcab) (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 via their Fc regions, the effector functions of the antibody are preserved.

[0007] If all antigens recognized by a bispecific antibody are specifically expressed in cancer, a bispecific antibody that binds to one of the antigens will exhibit cytotoxic activity against cancer cells, and thus is expected to have a more effective anticancer effect than conventional antibody drugs that recognize a single antigen. However, if one of the antigens recognized by the bispecific antibody is expressed in normal tissues or immune cells, cross-linking with FcγR will cause damage to normal tissues or release of cytokines (J. Immunol. (1999) Aug. 1, 163(3), 1246-52 (Non-Patent Document 15)). As a result, strong adverse reactions will be 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 antigens). Catumaxomab binds to a cancer antigen (EpCAM) and the CD3ε chain expressed on T cells, respectively, via its two Fab fragments. Catumaxomab induces T cell-mediated cytotoxicity by simultaneously binding to a cancer antigen and CD3ε, and induces NK cell- or antigen-presenting cell (e.g., macrophage)-mediated cytotoxicity by simultaneously binding to a cancer antigen and FcγR. By utilizing these two cytotoxic activities, catumaxomab has demonstrated a high therapeutic effect against malignant ascites when administered intraperitoneally, and has therefore been approved in Europe (Cancer Treat Rev. (2010) Oct 36(6), 458-67 (Non-Patent Document 16)). Furthermore, cases have been reported in which administration of catumaxomab resulted in the appearance of antibodies that react with cancer cells, demonstrating the induction of adaptive immunity (Future Oncol. (2012) Jan 8(1), 73-85 (Non-Patent Document 17)). Based on these results, such antibodies that possess both T cell-mediated cytotoxic activity and the effect mediated by cells such as NK cells or macrophages via FcγR (these antibodies are particularly referred to as trifunctional antibodies) are attracting attention because they are expected to have strong antitumor effects and induce adaptive immunity.

[0009] However, trifunctional antibodies simultaneously bind to CD3ε and FcγR even in the absence of cancer antigens. Therefore, even in the absence of cancer cells, they crosslink CD3ε-expressing T cells to FcγR-expressing cells, resulting in the production of large amounts of various cytokines. Due to this induction of cytokine production independent of cancer antigens, trifunctional antibodies are currently administered only intraperitoneally (Cancer Treat Rev. 2010 Oct 36(6), 458-67 (Non-Patent Document 16)). Systemic administration of trifunctional antibodies is extremely difficult due to the severe cytokine storm-like adverse reactions (Cancer Immunol Immunother. 2007 Sep; 56(9): 1397-406 (Non-Patent Document 18)). Bispecific antibodies of the prior art can bind to both antigens, i.e., a cancer antigen (EpCAM) as a first antigen and CD3ε as a second antigen, simultaneously while binding to FcγR. Therefore, due to their molecular structure, such adverse reactions caused by simultaneous binding to FcγR and CD3ε as a second antigen cannot be avoided. In recent years, improved antibodies have been provided that induce T cell-mediated cytotoxicity while avoiding adverse reactions by using an Fc region with reduced binding activity to FcγR ( WO2012 / 073985 ). However, due to their molecular structure, such antibodies cannot bind to cancer antigens while simultaneously acting on two immune receptors, namely CD3ε and FcγR. No antibody is known that exerts both T cell-mediated and non-T cell-mediated cytotoxicity in a cancer antigen-specific manner while avoiding adverse reactions.

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

[0011] CD137 agonist antibodies have been experimentally demonstrated to exhibit antitumor effects, primarily through the activation of CD8-positive T cells and NK cells (Houot, 2009, Blood, 114, 3431-8). T cells engineered to carry chimeric antigen receptor molecules consisting of a tumor antigen-binding domain as the extracellular domain and CD3 and CD137 signaling domains as the intracellular domain (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 nonspecific hepatotoxicity are a clinical and nonclinical problem, preventing progress in drug development (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22). It has been suggested that the main cause of side effects is antibody binding 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 their 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 event of a CD137 agonist antibody while maintaining the antitumor activity of the antibody.WO2015 / 156268 further describes that the antitumor activity can be further enhanced and these adverse events can be avoided by combining this bispecific antibody 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, no antibody has been known that exerts both T cell-mediated cytotoxicity and CD137-mediated activation of T cells and other immune cells in a cancer antigen-specific manner while avoiding adverse reactions.

[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 US A. 2004 Mar 2;101(9):2806-10 (Non-Patent Document 30); Proc Natl Acad Sci US 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 US 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)). Several reported techniques for obtaining such domains that bind to two different antigens include alternating different antigens in different panning rounds, and first obtaining a binding domain for a first antigen and then obtaining a binding domain for a second antigen from a library created by randomizing the binding domain for the first antigen. However, these strategies require a gene amplification step after the recovery of the first antigen-binding domain to amplify the recovered polynucleotides.

[0014] A phage display method called double-round selection, in which selection pressure against one antigen is applied twice consecutively without an intervening step of nucleic acid amplification, has been reported (Hawkins et al., J. Mol. Biol. 226:889-96 (1992)). However, no method is known for more efficiently collecting binding domains against two or more different antigens by applying selection pressure twice or more consecutively against two or more different antigens. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] WO2000 / 042072 [Patent Document 2] WO2006 / 019447 [Patent Document 3] WO2015 / 156268 [Patent Document 4] WO2014 / 116846 [Non-patent literature]

[0016] [Non-Patent Document 1] Nat. Biotechnol. (2005) 23, 1073-1078 [Non-licensed document 2] Eur J Pharm Biopharm. (2005) 59 (3), 389-396 [Non-licensed document 3] Immunol. Lett. (2002) 82, 57-65

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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, because an antibody having such a molecular format can simultaneously bind to three different antigens, the present inventors speculated that the trispecific antibody might simultaneously bind to CD3 and CD137, thereby cross-linking CD3ε-expressing T cells and CD137-expressing cells (e.g., T cells, B cells, NK cells, DCs, etc.). Furthermore, it has been previously reported that bispecific antibodies against CD8 and CD3ε cross-link the two and thus induce mutual cytotoxic activity between CD8-positive T cells (Wong, Clin. Immunol. Immunopathol. 1991, 58(2), 236-250). Therefore, the present inventors speculated that bispecific antibodies against a molecule expressed on T cells and CD3ε would also cross-link cells expressing that molecule with cells expressing CD3ε, thereby inducing mutual cytotoxic activity between T cells.

[0018] To obtain antigen domains that bind to two different antigens, some previously reported techniques have been used, such as alternately using different antigens in different panning rounds, and first obtaining a binding domain for a first antigen, and then obtaining a binding domain for a second antigen from a library created by randomizing the binding domain for the first antigen.However, these strategies require the steps of recovering the binding domain for the first antigen, then amplifying the recovered nucleotides encoding the binding domain for the first antigen, and further recovering and amplifying the nucleic acid of the binding domain that can also bind to the second antigen.The inventors believe that this step will result in each panning round step specifically enriching the binding domain that shows stronger binding to one of the various antigens used therein than the other antigens, compared to the binding domain that shows binding to each of the various antigens, thereby preventing the efficient recovery of desired molecules.

[0019] It is understood that in some methodologies, such as cell display, yeast display, or bacterial display, which can use FACS (fluorescence-activated cell sorting) for selection, it is possible to simultaneously apply two or more selection pressures against two or more different antigens. However, the present inventors believe that in methodologies such as phage display, ribosome display, mRNA display, or CIS display, which cannot use FACS, it is difficult to simultaneously apply two or more selection pressures against two or more different antigens. [Means for solving the problem]

[0020] The present invention provides antigen-binding domains that bind to CD3 and CD137, and methods for using the same. The present invention also provides methods for more efficiently obtaining antigen-binding domains that bind 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 simultaneously bind to a T cell receptor and CD137; and a variable region that binds to a third antigen different from a 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 that is 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 simultaneously bind to CD3 and CD137. 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 simultaneously bind to CD3 and CD137.

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

[0024] In some embodiments, the antigen-binding molecules of the present invention comprise an antibody Fc region. In further embodiments, the antigen-binding molecules of the present invention comprise an antibody Fc region that has reduced FcγR-binding activity 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 agonistic activity against CD137; (3) the antigen-binding molecule induces CD3 activation of T cells against cells expressing a molecule of a third antigen, 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 molecules of the present invention have at least one characteristic selected from the group consisting of the following (1) and (2): (1) the antigen-binding molecule does not compete with a CD137 ligand for binding to CD137; and (2) The antigen-binding molecule induces T cell cytotoxicity against cells expressing a third antigen molecule, but does not induce T cell cytotoxicity 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 Light 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 comprising at least one amino acid selected from The modified heavy chain variable domain sequence, HVR-H3, 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; Amino acids at about 100g: Gly, Tyr, Phe, or Val (Kabat numbering) The amino acid sequence comprises at least one amino acid selected from the group consisting of:

[0029] In some embodiments, the antigen-binding molecule of the present 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).

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

[0031] The present invention also provides isolated nucleic acids encoding the antigen-binding molecules of the present invention. The present invention also provides host cells containing the nucleic acids of the present invention. The present invention also provides methods for producing antibodies, comprising culturing the host cells of the present invention so that the antibodies are produced.

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

[0033] The antigen-binding molecules of the present invention may be for use as medicines, and may be for use in the treatment of various types of cancer. The antigen-binding molecules of the present invention may be used in the manufacture of a medicament. In some embodiments, the medicament is for the treatment of various types of cancer. The present invention also provides methods for treating individuals with various types of cancer. In some embodiments, the methods comprise administering to the individual an effective amount of an antigen-binding molecule of the present invention.

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

[0035] The present inventors also succeeded in developing a method for more efficiently obtaining antigen-binding domains that bind to two or more different antigens. In some embodiments, the method of screening for antigen-binding domains that bind to at least two or more 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 antigen-binding domains that bind to the first antigen; (c) contacting the antigen-binding domain collected in step (b) with a second antigen of interest and collecting the antigen-binding domain bound to the second antigen; and (d) amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; wherein the method does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain 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 the antigen-binding domain with a scaffold for bridging the antigen-binding domain with 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 the above steps (b) and (c) of the present invention is EDTA or IdeS.

[0039] In some embodiments, the method of the present invention for screening antigen-binding domains that bind to at least two or more different antigens of interest comprises: (a) providing a library comprising a plurality of antigen-binding domains; (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains 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 domain collected in step (b) with a second antigen of interest and collecting the antigen-binding domain bound to the second antigen; and (d) amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; wherein the method does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b).

[0040] In some embodiments, the method of the present invention for generating antigen-binding domains that bind to at least two or more different antigens of interest comprises: (a) providing a library comprising a plurality of antigen-binding domains; (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains that bind to the first antigen; (c) contacting the antigen-binding domain collected in step (b) with a second antigen of interest and collecting the antigen-binding domain bound to the second antigen; and (d) amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; (e) ligating the polynucleotides encoding the candidate antigen-binding domains selected in step (d) with a polynucleotide encoding a polypeptide comprising an Fc region; (f) culturing a cell into which a vector to which the polynucleotide obtained in step (d) is operably linked has been introduced; and (g) collecting antigen-binding molecules from the culture medium of the cells cultured in step (f) above. wherein the method does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b).

[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-described method.

[0043] More specifically, the present invention relates to the following: [1] an antibody variable region that can bind to CD3 and CD137, but does not simultaneously bind to CD3 and CD137; and A variable region that binds to a third antigen distinct from CD3 and CD137 An antigen-binding molecule comprising: [2] The antigen-binding molecule of [1], wherein the third antigen is a molecule that is specifically expressed in cancer tissue. [3] An antigen-binding molecule according to [1] or [2], wherein the variable region that does not simultaneously bind to CD3 and CD137 is a variable region that does not simultaneously bind to CD3 and CD137, each of which is 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 reduced FcγR-binding activity compared to the Fc region of a native human IgG1 antibody. [6] An antigen-binding molecule according to any one of [1] to [5], which 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 agonistic activity against CD137; (3) the antigen-binding molecule induces CD3 activation of T cells against cells expressing a molecule of a 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 a third antigen molecule. [7] Any of the antigen-binding molecules of [1] to [6], which 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. [8] An amino acid sequence obtained by introducing one or more amino acid alterations 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, 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 Light 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 comprising at least one amino acid selected from The modified heavy chain variable domain sequence HVR-H3 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; Amino acids at about 100g: Gly, Tyr, Phe, or Val (Kabat numbering) comprising at least one amino acid selected from An antigen-binding molecule according to 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) The antigen-binding molecule of any one of [1] to [8], comprising:

[10] A pharmaceutical composition comprising the antigen-binding molecule of any one of [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, comprising: (a) providing a library comprising a plurality of antigen-binding domains; (b) contacting the library provided in step (a) with a first antigen of interest and collecting antigen-binding domains that bind to the first antigen; (c) contacting the antigen-binding domain collected in step (b) with a second antigen of interest and collecting the antigen-binding domain bound to the second antigen; and (d) amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains. Including, The method does not include a step between steps (b) and (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b).

[12] The antigen-binding domain: A fusion polypeptide formed by fusing an antigen-binding domain with a scaffold to bridge the antigen-binding domain and the nucleic acid encoding the antigen-binding domain. This is the method of

[11] .

[13] The method of

[12] , wherein the scaffold is a bacteriophage.

[14] The method of

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

[15] The method of

[12] or

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

[0044] [Figure 1] Schematic diagram of an antibody that binds to CD3 and CD137 but does not simultaneously bind to these antigens. [Figure 2]Schematic diagram of an antibody that does not simultaneously bind to CD3 and CD137 and therefore does not cause cross-linking. On the other hand, a trifunctional antibody against CD3, CD137, and a third antigen causes cross-linking between T cells and CD137-positive cells. [Figure 3] Schematic diagram of an antibody that binds to CD3 and CD137 but does not simultaneously ligate the two cells. [Figure 4] FIG. 1 is a conceptual diagram of an antibody that crosslinks a third antigen-positive cell with a T cell that expresses CD3 and CD137. [Figure 5] FIG. 1 is a conceptual diagram of an antibody that crosslinks a third antigen-positive cell with a cell that expresses CD137. [Figure 6] FIG. 1 is a schematic diagram of the design and construction flow of a dual scFv VH ribosome display library. [Figure 7-1]

[0023] Figure 1 shows a series of graphs showing the results of ELISA of clones obtained by ribosome display against CD3 and CD137. The Y axis indicates the specificity of each clone for CD137-Fc, and the X axis indicates the specificity for CD3. Clones in black were identified as positive scFvs that showed binding to both CD137 and CD3. [Figure 7-2] Continued from Figure 7-1. [Figure 8] 1 is a graph showing the results of ECL analysis of IgG obtained by ribosome display against CD3 and CD137. The Y axis represents the response to both CD137, CD3, and the plate itself. [Figure 9-1] 1 is a series of graphs showing the results of ELISA of clones obtained by ribosome display against CD3 and CD137. The Y axis indicates the specificity of each clone for CD137-Fc, and the X axis indicates the specificity for CD3. Campaign 3 indicates ribosome display panning with double-round selection. [Figure 9-2] Continued from Figure 9-1. [Figure 9-3] Continued from Figure 9-2. [Figure 10]1 is a graph showing the results of ELISA of clones obtained by ribosome display against CD3 and CD137. The Y axis indicates the specificity of each clone for CD137-Fc, and the X axis indicates the specificity for CD3. [Figure 11] 1 is a graph showing the results of ELISA of IgG obtained by ribosome display against CD3 and CD137. The Y axis indicates the specificity of each clone for CD137-Fc, and the X axis indicates the specificity for CD3. [Figure 12] FIG. 1 is a schematic diagram of the design of a dual scFv VL ribosome display library and a dual Fab VL ribosome display library. [Figure 13] 1 is a graph showing the results of ELISA of IgG obtained by ribosome display affinity maturation against CD3 and CD137. The Y axis indicates the specificity of each clone for CD137-Fc, and the X axis indicates the specificity for CD3. [Figure 14] Graph showing the results of competitive ELISA of IgG obtained by ribosome display affinity maturation against CD3 and CD137. The Y axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. Excess human CD3 or human Fc was used as a competitor. [Figure 15] 1 shows the design of C3NP1-27, a CD3ε peptide antigen, which is biotinylated through a disulfide bond linker. [Figure 16] Graph showing the results of phage ELISA of clones obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for CD137-Fc, and the X axis indicates the specificity for CD3. [Figure 17]16 is a graph showing the results of phage ELISA of clones obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for CD137-Fc in bead ELISA, and the X axis indicates the specificity for CD3 in the same plate ELISA as in FIG. [Figure 18] 1 shows comparative data between the amino acid sequence of human CD137 and that of cynomolgus monkey CD137. [Figure 19] Graph showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for cynomolgus monkey CD137-Fc, and the X axis indicates the specificity for human CD137. [Figure 20] Graph showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y-axis indicates specificity for CD3e. [Figure 21] Graph showing the results of competitive ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. Excess human CD3 or human Fc was used as a competitor. [Figure 22A] This graph shows the results of phage ELISA of the phage display panning output pools for CD3 and CD137. The Y axis indicates specificity for human CD137. The X axis indicates the panning output pools, with "initial" indicating the pool before phage display panning, and R1 to R6 indicating the panning output pools after rounds 1 to 6 of phage display panning, respectively. [Figure 22B]Graph showing the results of phage ELISA of phage display panning output pools for CD3 and CD137. The Y axis indicates specificity for cynomolgus monkey CD137. The X axis indicates panning output pools, with "initial" indicating the pool before phage display panning, and R1 to R6 indicating the panning output pools after rounds 1 to 6 of phage display panning, respectively. [Figure 22C] Graph showing the results of phage ELISA of the phage display panning output pools for CD3 and CD137. The Y axis indicates specificity for CD3. The X axis indicates the panning output pools, where "initial" indicates the pool before phage display panning, and R1 to R6 indicate the panning output pools after rounds 1 to 6 of phage display panning, respectively. [Figure 23-1] 1 is a series of graphs showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for human CD137-Fc, and the X axis indicates the specificity for human CD137 or CD3. [Figure 23-2] Continuation of Figure 23-1. [Figure 23-3] Continuation of Figure 23-2. [Figure 24] 1 is a series of graphs showing the results of ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis indicates the specificity of each clone for human CD137-Fc, and the X axis indicates the specificity for human CD137 or CD3. [Figure 25] Graph showing the results of competitive ELISA of IgG obtained by phage display against CD3 and CD137. The Y axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. An excess amount of human CD3 was used as a competitor. [Figure 26]Graph showing the results of ELISA of IgG obtained by phage display against CD3 and CD137 to identify the epitope domain of each clone. The Y axis represents the ELISA response to each domain of human CD137. [Figure 27] 1 is a series of graphs showing the results of ELISA of IgG obtained by phage display affinity maturation against CD3 and CD137. The Y axis indicates the specificity of each clone for human CD137-Fc, and the X axis indicates the specificity for human CD137 or CD3. [Figure 28-1]

[0023] Figure 1 is a series of graphs showing the results of competitive ELISA of phage-displayed IgG against CD3 and CD137. The Y-axis represents the ELISA response to biotin-human CD137-Fc or biotin-human Fc. An excess of human CD3 was used as a competitor. [Figure 28-2] Continuation of Figure 28-1. [Figure 28-3] Continuation of Figure 28-2. [Figure 28-4] Continuation of Figure 28-3. [Figure 28-5] Continued from Figure 28-4. [Figure 29A] 1 shows the mechanism of IL-6 secretion from activated B cells mediated by anti-human GPC3 / Dual-Fab antibody. [Figure 29B] 10 shows graphs showing the results of evaluating the CD137-mediated agonistic activity of various anti-human GPC3 / Dual-Fab antibodies based on the level of IL-6 production secreted by activated B cells. Ctrl indicates a negative control human IgG1 antibody. [Figure 30A] 1 shows the mechanism of luciferase expression in activated Jurkat T cells mediated by anti-human GPC3 / Dual-Fab antibody. [Figure 30B] 1 shows a graph showing the results of evaluating the CD3-mediated agonist activity of various anti-human GPC3 / Dual-Fab antibodies based on the level of luciferase production expressed in activated Jurkat T cells. Ctrl indicates a negative control human IgG1 antibody. [Figure 31]

[0023] Figure 1 is a series of graphs showing the results of assessing cytokine (IL-2, IFN-γ, and TNF-α) release from human PBMC-derived T cells in the presence of each immobilized antibody. The Y-axis represents the concentration of each secreted cytokine, and the X-axis represents the concentration of immobilized antibody. A control anti-CD137 antibody (B), a control anti-CD3 antibody (CE115), a negative control antibody (Ctrl), and one of the dual antibodies (L183L072) were used for the assay. [Figure 32] This is a series of graphs showing the results of assessing T cell-dependent cytotoxicity (TDCC) against GPC3-positive target cells (SK-pca60 and SK-pca13a) using each bispecific antibody. The Y axis represents the cytostatic (CGI) ratio, and the X axis represents the concentration of each bispecific antibody. Anti-GPC3 / Dual bispecific antibody (GC33 / H183L072), negative control / Dual bispecific antibody (Ctrl / H183L072), anti-GPC3 / anti-CD137 bispecific antibody (GC33 / B), and negative control / anti-CD137 bispecific antibody (Ctrl / B) were used for this assay. Five times the amount of effector (E) cells was added to tumor (T) cells (ET5). [Figure 33] 1 shows the design and construction procedure for trispecific antibodies (mAb AB). [Figure 34] The naming rules for the prepared trispecific antibodies are shown. [Figure 35]

[0023] Figure 1 is a series of graphs showing the results of Biacore analysis of simultaneous binding of GPC3 / CD137xCD3 trispecific antibody and anti-GPC3 / dual-Fab antibody. The Y-axis represents the binding response to each antigen. First, human CD3 (hCD3) was used as the analyte, followed by hCD3 (shown as a dashed line) or a mixture of human CD137 (hCD137) and hCD3 (shown as a solid line). [Figure 36]35A and 35B are a series of sensor graphs showing the results of FACS analysis of each antibody against CD137-positive CHO cells or Jurkat cells. Figures 35(a) and (c) show the results of binding to human CD137-positive CHO cells, and Figures 35(b) and (d) show the results for parental CHO cells. In Figures 35(a) and (b), the solid line shows the results for the anti-GPC3 / dual antibody (GC33 / H183L072), and the solid area shows the results for the control antibody (Ctrl). In Figures 35(c) and (d), the solid line, dark gray area, and light gray area show the results for the GPC3 / CD137xCtrl trispecific antibody, GPC3 / CD137xCD3 trispecific antibody, and Ctrl / CtrlxCD3 trispecific antibody, respectively. Figures 35(e) and (f) show the results of binding to Jurkat CD3-positive cells. In Figure 35(e), the solid line and filled areas indicate the results for the anti-GPC3 / dual antibody (GC33 / H183L072) and the control antibody (Ctrl), respectively. In Figure 35(f), the solid line, dark gray filled areas, and light gray filled areas indicate the results for the GPC3 / CtrlxCD3 trispecific antibody, GPC3 / CD137xCD3 trispecific antibody, and Ctrl / CD137xCtrl trispecific antibody, respectively. [Figure 37] Graphs show the results of evaluating the CD3-mediated agonistic activity of various antibodies against GPC3-positive target cells SK-pca60 by measuring the level of luciferase production expressed in activated Jurkat T cells. Six types of trispecific antibodies, an anti-GPC3 / Dual-Fab antibody (GPC3 / H183L072), and a control / Dual-Fab antibody (Ctrl / H183L072) were used for this assay. The X-axis indicates the concentration of each antibody used. [Figure 38]Graphs show the results of evaluating the CD3-mediated agonistic activity of various antibodies against human CD137-positive CHO cells and parental CHO cells by measuring the level of luciferase production expressed in activated Jurkat T cells. Six trispecific antibodies, an anti-GPC3 / Dual-Fab antibody (GPC3 / H183L072), and a control / Dual-Fab antibody (Ctrl / H183L072) were used for this assay. The X-axis indicates the concentration of each antibody used. [Figure 39]

[0023] Figure 1 is a series of graphs showing the results of assessing cytokine (IL-2, IFN-γ, and TNF-α) release from human PBMCs in the presence of each soluble antibody. The Y-axis represents the concentration of each secreted cytokine, and the X-axis represents the concentration of the antibody used. Ctrl / CD137xCD3 trispecific antibody and control / Dual-Fab antibody (Ctrl / H183L072) were used for this assay. [Figure 40] 1 is a graph showing the results of cell ELISA of CE115 against CD3e. [Figure 41] FIG. 1 shows the molecular form of EGFR_ERY22_CE115. [Figure 42] 1 is a graph showing the results of TDCC (SK-pca13a) of EGFR_ERY22_CE115. [Figure 43] 1 is an exemplary sensorgram of an antibody with a binding ratio of less than 0.8, where the vertical axis represents RU value (response) and the horizontal axis represents time. DETAILED DESCRIPTION OF THE INVENTION

[0045] Description of Aspects 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 simultaneously bind to CD3 and CD137, 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 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 simultaneously bind to a T cell receptor and CD137, and a variable region that binds to a third antigen different from a T cell receptor and CD137. 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 but does not simultaneously bind to CD3 and CD137, and a variable region that binds to a molecule specifically expressed in cancer tissues.

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

[0047] In some embodiments, the antigen-binding molecules of the present invention can activate T cells and induce the cytotoxic activity of T cells against target cells through their agonistic activity against CD3, and can enhance the activation, survival, and differentiation of T cells into memory T cells through their costimulatory agonistic activity against CD137 and CD3. On the other hand, the antigen-binding molecules of the present invention do not simultaneously bind to CD3 and CD137, thereby avoiding the adverse effects caused by cross-linking of CD137 and CD3.

[0048] In some embodiments, the antigen-binding molecules of the present invention can also activate immune cells expressing CD137 and enhance immune responses to target cells through agonistic activity against CD137.

[0049] In the present invention, the term "antibody variable region" generally refers to a region containing a domain composed of four framework regions (FR) and three flanking complementarity-determining regions (CDRs), and also includes subsequences thereof, as long as the subsequence has the activity of binding to part or all of an antigen. Regions containing an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH) are particularly preferred. The antibody variable regions of the present invention may have any sequence and may be derived from any antibody, such as mouse, rat, rabbit, goat, or camel antibodies, or humanized antibodies obtained by humanizing any of these non-human antibodies, or human antibodies. "Humanized antibodies," also known as reshaped human antibodies, are obtained by grafting the complementarity-determining regions (CDRs) of an antibody derived from a non-human mammal, such as a mouse antibody, onto the CDRs of a human antibody. Methods for identifying CDRs are known in the art (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institutes of Health, Bethesda, Md.; and Chothia et al., Nature (1989) 342: 877). General recombinant techniques for this purpose are also known in the art (see European Patent Application Publication No. EP 125023 and WO 96 / 02576).

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

[0051] In the present invention, one amino acid modification may be used alone, or multiple amino acid modifications may be used in combination. When multiple amino acid modifications are used in combination, the number of modifications to be combined is not particularly limited and can be appropriately set within the scope that allows the object of the invention to be achieved. The number of modifications to be combined is, for example, 2 to 30, preferably 2 to 25, 2 to 22, 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 2 to 3. The multiple amino acid modifications to be combined may be made only to the heavy or light chain variable domain of the antibody, or may be distributed appropriately among both the heavy and light chain variable domains.

[0052] One or more amino acid residues in the variable region are permissible for modification as long as antigen-binding activity is maintained. When amino acids in the variable region are modified, the resulting variable region preferably maintains the binding activity of the corresponding unmodified antibody, and preferably has a binding activity that is at least 50%, more preferably at least 80%, and even more preferably at least 100% higher than that before modification, although the variable region of the present invention is not limited thereto. The binding activity may be increased by the amino acid modification, for example, by 2-fold, 5-fold, or 10-fold compared to the binding activity before modification.

[0053] Examples of preferred regions for amino acid modification include solvent-exposed regions and loops in the variable region. CDR1, CDR2, CDR3, FR3, and loops are particularly preferred. Specifically, positions 31-35, 50-65, 71-74, and 95-102 (Kabat numbering) in the H-chain variable domain, and positions 24-34, 50-56, and 89-97 (Kabat numbering) in the L-chain variable domain are preferred. Positions 31, 52a-61, 71-74, and 97-101 (Kabat numbering) in the H-chain variable domain, and positions 24-34, 51-56, and 89-96 (Kabat numbering) in the L-chain variable domain are more preferred. Furthermore, amino acids that enhance antigen-binding activity may be introduced during amino acid modification.

[0054] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain whose sequences ("complementarity determining regions" or "CDRs") are hypervariable and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include the following: (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) A combination of (a), (b), and / or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0055] In the present invention, a "loop" means a region containing residues that are 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 is used interchangeably with amino acid mutation, and can be used with the same meaning.

[0056] Substitution of an amino acid residue is carried out, for example, by replacing it with another amino acid residue for the purpose of altering any of the following (a) to (c): (a) the polypeptide backbone structure of a region having a sheet or helix 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 common side chain properties: (1) hydrophobic residues: norleucine, Met, Ala, Val, Leu, and Ile; (2) neutral hydrophilic residues: Cys, Ser, Thr, Asn, and Gln; (3) acidic residues: Asp and Glu; (4) basic residues: His, Lys, and Arg; (5) residues that affect chain orientation: Gly and Pro; and (6) aromatic residues: Trp, Tyr, and Phe.

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

[0058] Modifications of amino acid residues also include the selection of variable regions capable of binding to CD3 and CD137 but incapable of simultaneously binding to these antigens from those obtained by randomly modifying amino acids in the variable regions of antibodies that bind to CD3 or CD137, where the modifications do not abolish antigen-binding; and modifications by inserting into the above-mentioned regions a peptide previously known to have binding activity against the desired antigen.

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

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

[0061] Furthermore, for example, amino acid modifications that change the antigen-binding activity depending on the concentration of a target tissue-specific compound may be added to such an antibody variable region that binds to a third antigen (WO2013 / 180200).

[0062] The variable region may be further modified to, for example, enhance avidity, improve specificity, lower pI, confer pH-dependent antigen binding properties, improve thermostability of the bond, improve solubility, improve stability against chemical modification, reduce glycosylation-induced heterogeneity, avoid T cell epitopes identified by in silico prediction or by using in vitro T cell-based assays to reduce immunogenicity, or introduce T cell epitopes to activate regulatory T cells (mAbs 3:243-247, 2011).

[0063] Whether an antibody variable region of the present invention is "capable of binding to CD3 and CD137" can be determined by methods known in the art. This can be determined, for example, by electrochemiluminescence (ECL) (BMC Research Notes 2011, 4:281). Specifically, for example, a region of a biotin-labeled test antigen-binding molecule capable of binding to CD3 and CD137, such as a small antibody composed of the 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 to a streptavidin-immobilized plate. During this procedure, the biotin-labeled test antigen-binding molecule binds to the streptavidin on the plate. Light is generated from the sulfo-tag, and the luminescence signal is detected using a Sector Imager 600 or 2400 (MSD KK), etc., thereby confirming the binding of the above-mentioned region of the test antigen-binding molecule to CD3 or CD137. Alternatively, the assay may be performed by ELISA, FACS (fluorescence activated cell sorting), ALPHAScreen (amplified luminescence proximity homogeneous assay screen), BIACORE method based on the surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

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

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

[0066] ALPHAScreen is implemented using ALPHA technology, which uses two types of beads (donor and acceptor), based on the following principle: luminescence signals are detected only when a biological interaction between a molecule bound to a donor bead and a molecule bound to an acceptor bead brings these two beads into close proximity. A photosensitizer in the donor bead, excited by a laser, converts ambient oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, thereby triggering a chemiluminescent reaction in the bead, ultimately resulting in the emission of light. If there is no interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead will not reach the acceptor bead. Therefore, the chemiluminescent reaction will not occur.

[0067] One of the substances (ligands) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to induce total internal reflection at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (SPR signal) is formed in a portion of the reflected light. The other substance (analyte) whose interaction is to be observed is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, causing a change in the refractive index of the solvent on the sensor chip surface. This change in refractive index causes a shift in the position of the SPR signal (conversely, when the bound molecule dissociates, the signal returns to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the ordinate, and displays the time-dependent change in mass as assay data (sensorgram). The amount of analyte bound to the ligand captured on the sensor chip surface (the amount of change in response on the sensorgram before and after analyte interaction) can be determined from the sensorgram. However, because the amount of binding also depends on the amount of ligand, comparisons must be performed under conditions using substantially the same amount of ligand. Kinetics, i.e., the association rate constant (ka) and dissociation rate constant (kd), can be determined from the curve of the sensorgram, while affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also suitable for use in the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

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

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

[0070] In one aspect, while one antigen (e.g., CD3) is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD3 can be determined in the presence of another antigen (e.g., CD137) by a method known in the prior art (i.e., ELISA, ECL, etc.). In another aspect, while CD137 is immobilized, the attenuation of the binding signal of the antigen-binding molecule to CD137 can also be determined in the presence of CD3. When either one of the above two aspects is performed, if the binding signal is attenuated by at least 50%, preferably 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, or even more preferably 95% or more, it is determined that the antigen-binding molecule of the present invention does not simultaneously bind to CD3 and CD137 (see Examples 5-5, 7-5, 8-9, 9-4). In some embodiments, the concentration of the antigen injected as the analyte is at least 1-fold, 2-fold, 5-fold, 10-fold, 30-fold, 50-fold, or 100-fold higher than the concentration of the other antigen that is immobilized. In a preferred mode, the concentration of the antigen injected as analyte is 100 times higher than the concentration of the other antigen to be immobilized, and binding is inhibited by at least 80%. In one embodiment, the ratio of the KD value for the CD3 (analyte)-binding activity of the antigen-binding molecule to the KD value for the CD137 (immobilized)-binding activity of the antigen-binding molecule (KD(CD3) / KD(CD137)) is calculated, and a CD3 (analyte) concentration that is 10-fold, 50-fold, 100-fold, or 200-fold higher than the CD137 (immobilized) concentration by this KD value ratio (KD(CD3) / KD(CD137)) can be used for the above measurement. (For example, if the KD value ratio is 0.1, a concentration 1-fold, 5-fold, 10-fold, or 20-fold higher can be selected. Furthermore, if 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 a sulfo-tag (Ru complex), and unlabeled CD137 are prepared. If the test antigen-binding molecule can bind to both CD3 and CD137 but does not simultaneously bind to both CD3 and CD137, the mixture of the test antigen-binding molecule and labeled CD3 is added to a streptavidin-immobilized plate, and the luminescence signal of the sulfo-tag is detected in the absence of unlabeled CD137 by subsequent light emission. In contrast, the luminescence signal decreases in the presence of unlabeled CD137. The decrease in the luminescence signal can be quantified to determine the relative binding activity. This analysis can be similarly performed using labeled CD137 and unlabeled CD3.

[0072] In ALPHAScreen, a test antigen-binding molecule interacts with CD3 in the absence of competing CD137, generating a signal at 520-620 nm. Untagged CD137 competes with CD3 for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or similar techniques is known in the art. For example, CD3 can be tagged with GST by any suitable method, including fusing a polynucleotide encoding CD3 in frame with a polynucleotide encoding GST; expressing the resulting fusion gene in cells carrying a vector capable of expressing it; and then purifying it using a glutathione column. The resulting signal is preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego), which is fitted with a one-site competition model based on nonlinear regression analysis. This analysis can be performed similarly using tagged CD137 and untagged CD3. Alternatively, a method using fluorescence resonance energy transfer (FRET) may be used. FRET is a phenomenon in which excitation energy is directly transferred between two closely spaced fluorescent molecules due to electronic resonance. When FRET occurs, the excitation energy of the donor (a fluorescent molecule in an excited state) is transferred to the acceptor (another fluorescent molecule located near the donor), causing the fluorescence emitted from the donor to be quenched (more precisely, the fluorescence lifetime is shortened), and instead, fluorescence is emitted from the acceptor. This phenomenon can be used to analyze whether an antibody simultaneously binds to CD3 and CD137. For example, when CD3 containing a fluorescent donor and CD137 containing a fluorescent acceptor simultaneously bind to a test antigen-binding molecule, the fluorescence of the donor is quenched, while fluorescence is emitted from the acceptor. Therefore, a change in fluorescence wavelength is observed. Such an antibody is confirmed to simultaneously bind to CD3 and CD137. On the other hand, if mixing CD3, CD137, and the test antigen-binding molecule does not change the fluorescence wavelength of the fluorescent donor bound to CD3, the test antigen-binding molecule can be considered to be 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 glutathione S-transferase (GST)-tagged CD3 is bound to acceptor beads. The test antigen-binding molecule interacts with CD3 in the absence of a competing second antigen, generating a signal at 520-620 nm. The untagged second antigen competes with CD3 for interaction with the test antigen-binding molecule. The resulting decrease in fluorescence is quantified, thereby determining relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or similar is known in the art. For example, CD3 can be tagged with GST by any suitable method, including fusing a polynucleotide encoding CD3 in frame with a polynucleotide encoding GST; expressing the resulting fusion gene in cells carrying a vector capable of expressing it; and then purifying it using a glutathione column. The resulting signals are preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) fitted to a one-site competition model based on nonlinear regression analysis.

[0074] Tagging is not limited to GST tagging, and may be performed with any tag, including, but not limited to, histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, etc. Binding of the test antigen-binding molecule to the donor beads is not limited to binding using 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] In addition, when CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or when both are present on the same cell, the ability of the variable region to simultaneously bind to CD3 and CD137, but not to CD3 and CD137 expressed on different cells, can also be assayed by methods known in the art. Specifically, a test antigen-binding molecule that has been confirmed to be positive in ECL-ELISA for detecting simultaneous binding to CD3 and CD137 is also mixed with cells expressing CD3 and cells expressing CD137. Unless the antigen-binding molecule and these cells simultaneously bind to each other, the test antigen-binding molecule can be shown not to simultaneously bind to CD3 and CD137 expressed on different cells. This assay can be performed, for example, by cell-based ECL-ELISA. CD3-expressing cells are immobilized on a plate in advance. After the test antigen-binding molecule is bound to it, CD137-expressing cells are added to the plate. Different antigens expressed only on CD137-expressing cells are detected using sulfo-tagged antibodies against these antigens. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a signal is observed. If the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed. Alternatively, this assay can be carried out by the ALPHAScreen method. Test antigen-binding molecule is mixed with the cells expressing CD3 bound to donor beads and the cells expressing CD137 bound to acceptor beads. When the antigen-binding molecule simultaneously binds to the two antigens expressed on the two cells, respectively, a signal is observed. When the antigen-binding molecule does not simultaneously bind to these antigens, no signal is observed. Alternatively, this assay can be performed using Octet interaction analysis. First, cells expressing peptide-tagged CD3 are bound to a biosensor that recognizes the peptide tag. CD137-expressing cells and a test antigen-binding molecule are placed in a well and analyzed for interaction. If the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively, a large wavelength shift is observed due to the binding of the test antigen-binding molecule and the CD137-expressing cells to the biosensor. If the antigen-binding molecule does not simultaneously bind to these antigens, a small wavelength shift is observed due to the binding of only the test antigen-binding molecule to the biosensor.

[0076] Instead of these binding activity-based methods, biological activity-based assays can be performed. For example, CD3-expressing cells and CD137-expressing cells are mixed and cultured with a test antigen-binding molecule. When the antigen-binding molecule simultaneously binds to the two antigens, the two antigens expressed on the two cells are mutually activated via the test antigen-binding molecule. Therefore, changes in activation signals, such as increases in the phosphorylation levels downstream of each antigen, can be detected. Alternatively, cytokine production is induced as a result of activation. Therefore, the amount of cytokine produced can be measured, thereby confirming simultaneous binding to the two cells. Alternatively, cytotoxic activity against CD137-expressing cells can be induced as a result of activation. Alternatively, reporter gene expression can be induced by a promoter activated downstream of the CD137 or CD3 signaling pathway as a result of activation. Therefore, the cytotoxic activity or the amount of reporter protein produced can be measured, thereby confirming simultaneous binding to the two cells.

[0077] In the present invention, the term "Fc region" refers to a region of an antibody molecule comprising a hinge or a portion thereof, and a fragment consisting of the CH2 and CH3 domains. The Fc region of an IgG class refers, for example, but is not limited to, the region from cysteine ​​226 (EU numbering (also referred to herein as the EU index)) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus. The Fc region can be obtained, for example, by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, followed by re-elution of the fraction adsorbed to a protein A or protein G column. There is no particular limitation on the type of protease, so long as it can digest a full-length antibody to form Fab or F(ab')2 under appropriately selected enzyme reaction conditions (e.g., pH). Examples include pepsin and papain.

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

[0079] In some embodiments, the "antigen-binding molecule" of the present invention is not particularly limited to molecules comprising an "antibody variable region." In certain embodiments, antigen-binding molecules other than antibodies comprising a variable region can bind to two different antigens, and for example, affibodies, etc., may be obtained by methods commonly 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 US A. 2011 Aug 23;108(34):14067-72).

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

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

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

[0083] The Fc region of the present invention is particularly preferably an Fc region with reduced binding activity to an Fcγ receptor. Here, Fcγ receptor (also referred to herein as FcγR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, or IgG4, and refers to any member of a protein family substantially encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); and any unidentified 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 FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).

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

[0085] The reduced binding activity to Fcγ receptors 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 based on the ALPHA technology, which uses two types of beads (donor and acceptor), and is based on the following principle: a luminescent signal is detected only when a molecule bound to a donor bead and a molecule bound to an acceptor bead are brought into close proximity due to a biological interaction between these two beads. A photosensitizer in the donor bead, excited by a laser, converts ambient oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, thereby triggering a chemiluminescent reaction in the bead, ultimately resulting in the emission of light. If there is no interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead will not reach the acceptor bead. Therefore, the chemiluminescent reaction will not occur.

[0086] For example, a biotin-labeled test antigen-binding molecule is bound to donor beads, while an Fcγ receptor tagged with glutathione S-transferase (GST) is bound to acceptor beads. In the absence of a competing antigen-binding molecule with a mutant Fc region, the antigen-binding molecule with a wild-type Fc region interacts with the Fcγ receptor and generates a signal at 520-620 nm. The antigen-binding molecule with an untagged mutant Fc region competes with the antigen-binding molecule with the wild-type Fc region for interaction with the Fcγ receptor. The resulting decrease in fluorescence is quantified, allowing relative binding affinity to be determined. Biotinylation of antigen-binding molecules (e.g., antibodies) using sulfo-NHS-biotin or similar is known in the art. For example, an Fcγ receptor can be tagged with GST by an appropriate method, including fusing a polynucleotide encoding an Fcγ receptor with a polynucleotide encoding GST in frame, expressing the resulting fusion gene using a cell carrying a vector capable of expressing it, and then purifying it using a glutathione column. The resulting signal is preferably analyzed using, for example, the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego) fitted to a one-site competition model based on nonlinear regression analysis.

[0087] One of the substances (ligands) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to cause total internal reflection at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (SPR signal) is formed in a portion of the reflected light. The other substance (analyte) whose interaction is to be observed is injected onto the surface of the sensor chip. When the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, when the bound molecule dissociates, the signal returns to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the ordinate, and displays the time-dependent change in mass as assay data (sensorgram). Kinetics, i.e., the association rate constant (ka) and dissociation rate constant (kd), can be determined from the curve of the sensorgram, while affinity (KD) can be determined from the ratio of these constants. Inhibition assays are also suitable for use in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

[0088] As used herein, "decreased Fcγ receptor-binding activity" means that a test antigen-binding molecule exhibits, based on the above-described analytical method, a binding activity that is, 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, and particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the binding activity of a control antigen-binding molecule comprising an Fc region. An antigen-binding molecule having the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be used as a control antigen-binding molecule, as appropriate. The structure of the Fc region is set forth in SEQ ID NO: 94 (RefSeq Accession No. AAC82527.1 with an A added to the N-terminus), SEQ ID NO: 95 (RefSeq Accession No. AAB59393.1 with an A added to the N-terminus), SEQ ID NO: 96 (RefSeq Accession No. CAA27268.1 with an A added to the N-terminus), or SEQ ID NO: 97 (RefSeq Accession No. AAB59394.1 with an A added to the N-terminus). When an antigen-binding molecule having a variant of an Fc region of an antibody of a particular isotype is used as a test substance, the effect of mutations in the variant on Fcγ receptor-binding activity is tested using this antigen-binding molecule having an Fc region of this particular isotype as a control. Antigen-binding molecules having an Fc region variant confirmed to have reduced Fcγ receptor-binding activity are appropriately prepared.

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

[0090] The following substitution groups of constituent amino acids, defined according to EU numbering (numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution; and the single-letter amino acid code 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 IgG2 antibody Fc region, such as those described above, can also be used appropriately.

[0091] The following substitution groups of constituent amino acids, defined according to EU numbering (numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution; and the single-letter amino acid code 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 an IgG3 antibody Fc region, such as those described above, can also be used appropriately.

[0092] The following substitution groups of constituent amino acids, defined according to EU numbering (numbers represent the positions of amino acid residues defined according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution; and the single-letter amino acid code 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 an IgG4 antibody Fc region, such as those described above, can also be used appropriately.

[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 any of the following amino acids at positions 233, 234, 235, 236, 237, 327, 330, and 331, as defined according to EU numbering, with amino acids at the corresponding positions according to EU numbering in the Fc region of the counterpart IgG2 or IgG4.

[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 one or more of the following amino acids: 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. Particularly preferred are antigen-binding molecules having an Fc region in which one or more of the amino acids at positions 234, 235, and 297 are substituted with alanine.

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

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

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

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

[0099] The antibody can also be prepared in which one to three pairs of amino acid residues are selected from pairs of amino acid residues (1) to (3) in a second H chain CH3 domain that is different from the first H chain CH3 domain, so that they correspond to pairs of amino acid residues (1) to (3) in the first H chain CH3 domain that have the same charge but have the opposite charge to the corresponding amino acid residues in the first H chain CH3 domain.

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

[0101] In the above-described antibodies, each of the "charged amino acid residues" is preferably selected from, for example, amino acid residues included in either of the following groups (a) and (b): (a) glutamic acid (E) and aspartic acid (D); and (b) Lysine (K), arginine (R), and histidine (H).

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

[0103] In a preferred embodiment, the antibody may have a first H chain CH3 domain and a second H chain CH3 domain cross-linked by a disulfide bond. The amino acid residues to be modified according to the present invention are not limited to those in the antibody variable region or constant region described above. Those skilled in the art can identify the amino acid residues that constitute the interface of a polypeptide variant or heteromultimer by homology modeling using commercially available software, and can modify the amino acid residues at those positions to control the association.

[0104] The assembly of the multispecific antibodies of the present invention can also be achieved by alternative techniques known in the art. An amino acid side chain present in the variable domain of one antibody heavy chain is replaced with a larger side chain (knob), and its partner amino acid side chain present in the variable domain of the other heavy chain is replaced with a smaller side chain (hole). The knob can be positioned in the hole to ensure efficient assembly of polypeptides of Fc domains with different amino acid sequences (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 portion of the CH3 of one antibody H chain is converted to its corresponding IgA-derived sequence, and the complementary portion of the CH3 of the other antibody H chain is converted to its corresponding IgA-derived sequence. The resulting chain-exchange engineered domain CH3 can be used to efficiently associate polypeptides with different sequences through complementary CH3 association (Protein Engineering Design & Selection, 23; 195-202, 2010). This technique, known in the art, can also be used to efficiently form the desired multispecific antibodies.

[0106] Alternatively, multispecific antibodies can be formed by, for example, antibody preparation techniques using antibody CH1-CL association and VH-VL association as described in WO2011 / 028952, techniques for preparing bispecific antibodies using separately prepared monoclonal antibodies (Fab arm exchange) as described in WO2008 / 119353 and WO2011 / 131746, techniques for controlling the association between antibody heavy chain CH3 domains as described in WO2012 / 058768 and WO2013 / 063702, techniques for preparing bispecific antibodies composed of two types of light chains and one type of heavy chain as described in WO2012 / 023053, or techniques for preparing bispecific antibodies using two bacterial cell lines each expressing antibody half molecules 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 assembly techniques, CrossMab technology (Scaefer et al., Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192), a known heterologous light chain assembly technique in which a light chain forming a variable region binding to a first epitope and a light chain forming a variable region binding to a second epitope are assembled with a heavy chain forming a variable region binding to a first epitope and a heavy chain forming a variable region binding to a second epitope, respectively, can also be used to prepare the multispecific or multiparatopic antigen-binding molecules provided by the present invention. An example of a technique for preparing bispecific antibodies using separately prepared monoclonal antibodies includes a method comprising subjecting a monoclonal antibody in which specific amino acids have been substituted in the heavy chain CH3 domain to reducing conditions to promote antibody heterodimerization and obtain the desired bispecific antibody. Examples of preferred amino acid substitution sites for this method include residues at EU numbering positions 392 and 397 in the CH3 domain.Furthermore, bispecific antibodies can also be prepared by using antibodies in which one to three pairs of amino acid residues selected from the following pairs of amino acid residues (1) to (3) in a first H-chain CH3 domain have the same electric charge: (1) the amino acid residues at positions 356 and 439 (EU numbering) in the H-chain CH3 domain; (2) the amino acid residues at positions 357 and 370 (EU numbering) in the H-chain CH3 domain; and (3) the amino acid residues at positions 399 and 409 (EU numbering) in the H-chain CH3 domain. Bispecific antibodies can also be prepared by using antibodies in which one to three pairs of amino acid residues are selected from pairs of amino acid residues (1) to (3) in a second H-chain CH3 domain that is different from the first H-chain CH3 domain so that they correspond to pairs of amino acid residues (1) to (3) in the first H-chain CH3 domain that have the same electric charge but have the opposite electric charge to the corresponding amino acid residues in the first H-chain CH3 domain.

[0107] Even if the desired multispecific antibody cannot be efficiently formed, the multispecific antibody of the present invention can be obtained by separating and purifying the desired multispecific antibody from the produced antibodies. For example, a previously reported method involves introducing amino acid substitutions into the variable domains of two types of H chains to impart a difference in isoelectric point, so that two types of homodimers and the desired heterodimerized antibody can be separately purified by ion exchange chromatography (WO2007114325). A method for purifying heterodimerized antibodies consisting of a mouse IgG2a H chain capable of binding to Protein A and a rat IgG2b H chain that cannot bind to Protein A has previously been reported (WO98050431 and WO95033844). Alternatively, the amino acid residues at positions 435 and 436 (EU numbering), which constitute the Protein A-binding site of IgG, can be substituted with amino acids such as Tyr and His, which provide different Protein A binding strengths, and the resulting H chains are used to alter the interaction of each H chain with Protein A. As a result, only the heterodimerized antibody can be efficiently purified by use of a Protein A column.

[0108] Multiple techniques, for example, two or more, may be used in combination. These techniques may also be applied separately to the two H chains to be associated. The antigen-binding molecules of the present invention may be prepared based on such modified forms, but may also be prepared as antigen-binding molecules having the same amino acid sequence.

[0109] Amino acid sequence alterations can be accomplished by a variety of methods known in the art. Examples of these methods that may be performed include site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer, W, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456). 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 may be used, but are not limited to these methods.

[0110] The "antigen-binding molecule" of the present invention may be an antibody fragment that contains both the heavy and light chains constituting the "antibody variable region" of the present invention in a single polypeptide chain, but lacks the constant region. Such an antibody fragment 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 WO93 / 11161). These polypeptide chains are connected via a linker that is so short, for example, about 5 residues, that the light chain variable domain (VL) and the heavy chain variable domain (VH) on the same polypeptide chain cannot pair with each other. Because of this short linker, the VL and VH encoded on the same polypeptide chain cannot form a single-chain Fv, but instead dimerize with the VH and VL, respectively, on separate polypeptide chains 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 VL and two VH, linked via a linker such as a peptide linker (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VH and VL may be derived from different monoclonal antibodies. Preferred examples include bispecific sc(Fv)2, which recognizes two different 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 configuration of the antigen-binding domain that constitutes the sc(Fv)2 described herein include antibodies in which two VHs and two VLs are arranged 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 configuration and may be in any order. Examples also include the following configuration: [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, a person skilled in the art can appropriately prepare a desired sc(Fv)2 in order to prepare the antigen-binding molecules disclosed herein.

[0115] The antigen-binding molecules of the present invention may be conjugated to a carrier polymer such as PEG or an organic compound such as an anticancer agent. Furthermore, a glycosylation sequence can be inserted to suitably add a sugar chain to the antigen-binding molecules of the present invention to produce a desired effect.

[0116] For example, any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (e.g., a linker disclosed in Protein Engineering, 9 (3), 299-305, 1996) can be used as a linker for linking antibody variable domains. In the present invention, peptide linkers are preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The length is preferably 5 amino acids or more (the upper limit is not particularly limited, but is usually 30 amino acids or less, preferably 20 amino acids or less), and is particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, all of these peptide linkers used may have the same length or 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-Gly (SEQ ID NO: 169), (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 164)), and (Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 165))n, (where 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] The synthetic compound linker (chemical crosslinker) is a crosslinker 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-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), or bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available. Three linkers are usually required to link four antibody variable domains, and all of the linkers used may be the same or different linkers.

[0119] F(ab')2 comprises two light chains and two heavy chains containing constant regions (part of the CH1 domain and CH2 domain) such that interchain disulfide bonds are formed between the two heavy chains. The F(ab')2 that constitutes the polypeptide complexes disclosed herein can be preferably obtained, for example, by partially digesting a full-length monoclonal antibody containing the desired antigen-binding domain with a protease such as pepsin, followed by removal of the Fc fragment adsorbed to a Protein A column. There is no particular limitation on the protease used, as long as it is capable of digesting the full-length antibody to form F(ab')2 under appropriately selected enzyme reaction conditions (e.g., pH). Examples include pepsin and ficin.

[0120] In addition to the amino acid modifications described above, the antigen-binding molecules of the present invention may further contain additional modifications. The additional modifications may be selected from, for example, amino acid substitutions, deletions, and modifications, and combinations thereof. For example, the antigen-binding molecules of the present invention can be further modified arbitrarily without substantially changing the intended function of the molecule. Such mutations can be made, for example, by conservative substitution of amino acid residues. Alternatively, modifications that alter the intended function of the antigen-binding molecules of the present invention may also be made, as long as the altered function is within the scope of the present invention.

[0121] The amino acid sequence modification according to the present invention also includes post-translational modification. Specifically, post-translational modification can refer to the addition or deletion of a sugar chain. For example, an antigen-binding molecule of the present invention having an IgG1-type constant region can have a sugar chain-modified amino acid residue at EU numbering position 297. The sugar chain structure used 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: mammalian antibody-producing cells; 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 antibody-encoding DNA. On the other hand, the antibodies of the present invention also include antibodies that are not glycosylated at that position. Antibodies with constant regions that are not glycosylated can be obtained by expressing genes encoding these antibodies in prokaryotic cells such as E. coli.

[0122] More specifically, the additional modification according to the present invention may be, for example, the addition of sialic acid to a 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 may be made to improve binding activity to FcRn (J Immunol. 2006 Jan 1;176(1):346-56; J Biol Chem. 2006 Aug 18;281(33):23514-24; Int Immunol. 2006 Dec;18(12):1759-69; Nat Biotechnol. 2010 Feb;28(2):157-9; WO2006 / 019447; WO2006 / 053301; and WO2009 / 086320), or to improve antibody heterogeneity or stability ((WO2009 / 041613)).

[0124] In the present invention, the term "antibody" is used in the broadest sense and includes any antibody, such as a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, an antibody variant, an antibody fragment, a multispecific antibody (e.g., a bispecific antibody), a chimeric antibody, and a humanized antibody, so long as it exhibits 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 antibody origins include, but are not 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 can be produced by hybridoma method (Kohler and Milstein, Nature 256:495 (1975)) or recombinant method (U.S. Patent No. 4,816,567). Alternatively, monoclonal antibodies can be isolated from phage display antibody libraries (Clackson et al., Nature 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991)). Monoclonal antibodies can also be isolated from single B cell clones (N. Biotechnol. 28(5): 253-457 (2011)).

[0127] Humanized antibodies are also called reshaped human antibodies. Specifically, for example, humanized antibodies consisting of human antibodies grafted with CDRs from non-human animal (e.g., mouse) antibodies are known in the art. General genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, for example, overlap extension PCR is known in the art as a method for grafting CDRs from mouse antibodies onto human FRs.

[0128] A vector for expressing a humanized antibody can be prepared by inserting DNA encoding an antibody variable domain, each containing three linked CDRs and four FRs, and DNA encoding a human antibody constant domain into an expression vector so that the variable domain DNA is fused in frame with the constant domain DNA. These vectors with inserts are introduced into a host to establish recombinant cells. The recombinant cells are then cultured to express the DNA encoding the humanized antibody, and the humanized antibody is produced in culture (see European Patent Publication No. EP 239400 and International Publication No. WO1996 / 002576).

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

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

[0131] In addition, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, human antibody V regions are expressed as single-chain antibodies (scFvs) on the surface of phages using phage display methods. Phages expressing antigen-binding scFvs can be selected. The genes of the selected phages can be analyzed to determine the DNA sequence encoding the V region of the antigen-binding human antibody. After determining the DNA sequence of the antigen-binding scFv, the V region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is then transfected into the preferred expression cells listed above for the expression of genes 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, techniques using cell-free translation systems, techniques displaying antigen-binding molecules on the surface of cells or viruses, and emulsion-based techniques are known as techniques for obtaining human antibodies by panning using human antibody libraries. For example, ribosome display, which involves forming a complex between mRNA and the translated protein via ribosomes by removing a stop codon, cDNA or mRNA display, which involves covalently binding the translated protein to a gene sequence using a compound such as puromycin, or CIS display, which involves forming a complex between a gene and the translated protein using a nucleic acid-binding protein, can be used as a cell-free translation system-based technique. Phage display, as well as E. coli display, Gram-positive bacteria display, yeast display, mammalian cell display, and virus display can be used as techniques for displaying antigen-binding molecules on the surface of cells or viruses. For example, in vitro virus display, which uses genes and translation-related molecules encapsulated in an emulsion, can be used as an emulsion-based technique. These methods are known in the art (Nat Biotechnol. 2000 Dec; 18 (12): 1287-92; Nucleic Acids Res. 2006; 34 (19): e127; Proc Natl Acad Sci US A. 2004 Mar 2; 101 (9): 2806-10; Proc Natl Acad Sci US A. 2004 Jun 22; 101 (25): 9193-8; Protein Eng Des Sel. 2008 Apr; 21 (4): 247-55; Proc Natl Acad Sci US A. 2000 Sep 26; 97 (20): 10701-5; MAbs. 2010 Sep-Oct; 2 (5): 508-18; and Methods Mol Biol. 2012;911:183-98).

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

[0134] As used herein, the term "third antigen" is not particularly limited and may be any antigen. Examples of antigens include 17-IA, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, 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 beta, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, apoA1, apo-serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptide A, atrial natriuretic peptide B, atrial natriuretic peptide C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis anthracis) protective antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, β-2-microglobulin, β-lactamase, bFGF, BID, Bik, BIM, BLC, BL -CAM, BLK, B lymphocyte stimulating factor (BlyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR,BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, bombesin, bone-derived neurotrophic factor, bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte cell adhesion molecule, C10, C1 inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, cadherin-3, calcitonin, cAMP, carbonic anhydrase-IX, carcinoembryonic antigen (CEA), cancer-associated antigen, cardiotrophin-1, cathepsin A, cathepsin B, cathepsin C / DP PI, 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 / MPI F-1, CCL24 / eotaxin-2, CCL25 / TECK, CCL26 / eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CCL3 / M1P-1-α, CCL3Ll / LD-78-β, CCL4 / MIP-l-β, CCL5 / RANTES, 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, C D11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, C D27L, 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 / Gr o-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, endosialin, endothelin receptor, endotoxin, enkephalinase, eNOS, Eot,Eotaxin, eotaxin-2, eotaxini, EpCAM, ephrin B2 / EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO), erythropoietin receptor, E-selectin, ET-1, Exodus-2, RSV F protein, F10, F11, F12, F13, F5, F9, factor Ia, factor IX, factor Xa, factor VII, factor VIII, factor VIIIc, Fas, FcαR, FcεRI, FcγIIb, FcγRI, FcγRII a, 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, glutamate carboxypeptidase II, glycoprotein hormone receptor, glycoprotein IIb / IIIa (GP) IIb / IIIa), glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO / MGSA, growth hormone-releasing factor, GRO-β, GRO-γ, H. pylori,Hapten (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 proteins such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue-type plasminogen activator (t-PA), huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-α, IFN-β, IFN-γ, IgA, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptor, IL-11, IL-11 receptor, IL-12, IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-1 6, IL-16 receptor, IL-17, IL-17 receptor, IL-18(IGIF), IL-18 receptor, IL-1α, IL-1β, IL-1 receptor, IL-2, IL-2 receptor, IL-20, IL-20 receptor, IL-21, IL-21 receptor, IL-23, IL-23 receptor, IL-2 receptor, IL-3, IL-3 receptor, IL-31, IL-31 receptor, IL-3 receptor, IL-4, IL-4 receptor, IL-5, IL-5 receptor, IL-6, IL-6 receptor, IL-7 , IL-7 receptor, IL-8, IL-8 receptor, IL-9, IL-9 receptor, immunoglobulin immune complex, immunoglobulin, INF-α, INF-α receptor, INF-β, INF-β receptor, INF-γ, INF-γ receptor, type I IFN,Type I IFN receptor, influenza, inhibin, inhibin α, inhibin β, iNOS, insulin, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein, integrin, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α-V / β-3, integrin α-V / β-6, integrin α4 / β7, integrin α5 / β1, integrin α5 / β3, integrin α5 / β6, integrin ασ (αV), integrin αθ, integrin β1, integrin β2, integrin β3 (GPIIb-IIIa), IP-10, I-TA C, JE, kallikrein, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, kallistatin, 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, islet amyloid polypeptide), LAP (TGF-1), latency-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-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotactin, lymphotoxin β receptor, lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 aa), MDC (69 aa), megsin, Mer,MET tyrosine kinase receptor family, 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-attracting protein, monocyte colony inhibitory factor, mouse gonadotropin-related peptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Müllerian inhibitory substance, Mug, MuSK, myelin-associated glycoprotein, myeloid progenitor 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, neurturin, NGF-β, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, hepatitis C virus nonstructural 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, osteoinductive factor, osteopontin, OX40L, OX40R, oxidized LDL p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator inhibitor-1, platelet growth factor, plgR, PLP, polyglycol chains of various sizes (e.g., PEG-20, PEG-30, PEG40), PP14, prekallikrein,Prion protein, procalcitonin, , programmed cell death protein 1, proinsulin, prolactin, proprotein convertase PC9, prorelaxin, prostate-specific membrane antigen (PSMA), protein A, protein C, protein D, protein S, protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, rheumatoid factor, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF / KL, SCGF, sclerostin, SDF-1, SDF1α, SDF1β, SERINE, serum amyloid P, serum albumin, sFRP-3, Shh, Shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor 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 lymphoprotein (Thymic stromallymphoprotein) and activator Ck-1 activates thyroid hormone (TSH). Commercial Tie, TIMP, TIQ. Built-in bathrooms, bathrooms, snowflakes Liquids TMEFF2, Tmpo, TMPRSS2, TNF complex I, TNF complex II, TNF-α TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1 Apo-2 / DR4)、TNFRSF10B(TRAIL R2 DR5 / KILLER / TRICK-2A / TRICK-B)、TNFRSF10C(TRAIL R3 DcR1 / LIT / TRID)、TNFRSF10D(TRAIL R4 DcR2 / TRUNDD)、TNFRSF11A(RANK ODF R / TRANCE R)、TNFRSF11B(OPG OCIF / TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF12A、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHO ATAR / HveA / LIGHT R / TR2、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY CROWN / TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF Rl CD120a / p55-60, TNFRSF1B (TNF RII CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3). Apo-3 / LARD / TR-3 / TRAMP / WSL-1); Apo-1 / APT1 / CD95), TNFRSF6B(DcR3 M68 / TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9(4-1 BB CD137 / ILA), TNFRST23(DcTRAIL R1 TNFRH1), TNFSF10 (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 / DIF / TNFSF2), TNFSF1B (TNF-b LTa / TNFSF1), TNFSF3 (LTb TNFSF / p33), TNFSF4 (OX40 ligand gp34 / TXGP1), TNFSF5 (CD40 ligand CD154 / gp39 / HIGM1 / IMD3 / TRAP), TNFSF6 (Fas ligand Apo-1 ligand / APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1 BB ligand CD137 ligand), TNF-α, TNF-β, TNIL-I, toxic metabolites, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGFs), such as TGF-α and TGF-β, transmembrane glycoprotein NMB, transthyretin, TRF, Trk, TROP-2, trophoblast glycoprotein, TSG, TSLP, tumor necrosis factor (TNF), tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, 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, VL These include A, 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.

[0135] Specific examples of molecules specifically expressed on T cells include CD3 and T cell receptors. CD3 is particularly preferred. For example, in the case of human CD3, the site in CD3 to which the antigenic molecule of the present invention binds may be any epitope present in the sequence of the γ, δ, or ε chain that constitutes 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, δ, and ε chains that constitute CD3 are shown in SEQ ID NOs: 170 (NM_000073.2), 172 (NM_000732.4), and 174 (NM_000733.3), and their polypeptide sequences 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 not systemically expressed on cells or organs. The third antigen is preferably, for example, a tumor cell-specific antigen, and also includes antigens expressed in association with the malignant transformation of cells and abnormal sugar chains that appear on cell surfaces or protein molecules during the malignant transformation of cells. Specific examples include ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1 / 4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate-specific antigen (PSA), melanoma-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), and Burkitt's lymphoma antigen 38.13, CD19, human B lymphoma antigen CD20, CD33, melanoma-specific antigens (e.g., ganglioside GD2, ganglioside GD3, ganglioside GM2, and ganglioside GM3), tumor-specific transplantation antigens (TSTA), T antigens, virus-induced tumor antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), colon CEA, oncofetal antigen α-fetoprotein (e.g., oncofetal trophoblast glycoprotein 5T4 and oncofetal bladder tumor antigen), differentiation antigens (e.g., human Lung cancer antigens L6 and L20), fibrosarcoma antigen, human T-cell leukemia-associated antigen Gp37, neoglycoproteins, 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 preimplantation embryos or gastric cancer, I found in mammary epithelium, M18, M39 found in bone marrow cells, SSEA-1, VEP8, VEP9, Myl, VIM-D5 found in colorectal cancer, D156-22 found in colorectal cancer, TRA-1-85 (blood type 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 embryonal carcinoma cells, TL5 (blood type A), EGF receptor found in A431 cells, E1 series found in pancreatic cancer (blood type B), FC10.2 found in embryonal carcinoma cells, gastric cancer antigen, CO-514 (blood type Lea) found in adenocarcinoma, NS-10 found in adenocarcinoma, CO-43 (blood type Leb), G49 found in the EGF receptor of A431 cells, MH2 (blood type ALeb / Ley) found in colon cancer, 19.9 found in colon cancer, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2, GD3, D1 found in embryonal carcinoma cells.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8, SSEA-3 and SSEA-4 found in 4-cell to 8-cell embryos, cutaneous T-cell lymphoma-associated antigen, MART-1 antigen, sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, paraneoplastic-associated brain-testis cancer antigen (tumor neural antigen MA2 and paraneoplastic neural antigen), neuro-oncological abdominal antigen 2 (NOVA2), blood cell cancer antigen gene 520, tumor-associated antigen CO-029, tumor These include tumor-associated antigens MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b, MAGE-X2, cancer-testis antigen (NY-EOS-1), YKL-40, and any fragments of these polypeptides, as well as modified structures thereof (such as the aforementioned modified phosphate groups and sugar chains), EpCAM, EREG, CA19-9, CA15-3, sialyl SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A.

[0137] The term "CD137," as used herein, also referred to as 4-1BB, is a member of the tumor necrosis factor (TNF) receptor family. Examples of factors belonging to the TNF superfamily or TNF receptor superfamily include CD137, CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL.

[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 comprising the amino acid sequence of SEQ ID NO: 91; (2) the antigen-binding molecule has agonistic activity against CD137; (3) the antigen-binding molecule induces CD3 activation of T cells against cells expressing a molecule of a third antigen, 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 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 comprising the amino acid sequence of SEQ ID NO: 91; (2) the antigen-binding molecule has agonistic activity against CD137; (3) the antigen-binding molecule induces T cell cytotoxicity against cells expressing a third antigen molecule, but does not induce T cell activation against cells expressing CD137; and (4) The antigen-binding molecule does not induce cytokine release from PBMCs in the absence of cells expressing a molecule of the third antigen. In some embodiments, the antigen-binding molecules of the present invention have at least one characteristic selected from the group consisting of the following (1) and (2): (1) the antigen-binding molecule does not compete with a CD137 ligand for binding to CD137; and (2) The antigen-binding molecule induces T cell cytotoxicity against cells expressing a third antigen molecule, but does not induce T cell cytotoxicity against cells expressing CD137.

[0140] In one aspect, a "CD137 agonist antibody" or an "antigen-binding molecule having agonist activity against CD137" of the present invention refers to an antibody or antigen-binding molecule that, when added to cells, tissues, or organisms expressing CD137, activates at least about 5%, specifically at least about 10%, or more specifically at least about 15% of CD137-expressing cells, where 0% activation is the background level (e.g., IL6 secretion) of non-activated cells expressing CD137. In various embodiments, CD137 agonist antibodies for use as pharmaceutical compositions of the present invention can activate cellular 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, when added to cells, tissues, or organisms expressing CD137, activates at least about 5%, specifically at least about 10%, or more specifically at least about 15% of cells expressing CD137, where 100% activation is the level of activation achieved by an equimolar amount of binding partner under physiological conditions. In various embodiments, CD137 agonist antibodies for use as pharmaceutical compositions of the present invention can activate cellular 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, a "binding partner," as used herein, is a molecule known to bind to CD137 and induce activation of cells expressing CD137. In a further embodiment, examples of binding partners include urelumab (CAS Registry Number 934823-49-1) and its variants described in WO2005 / 035584A1, utomilumab (CAS Registry Number 1417318-27-4) and its variants described in WO2012 / 032433A1, and various known CD137 agonist antibodies. In certain embodiments, examples of binding partners include CD137 ligands. In a further embodiment, the activation of CD137-expressing cells by anti-CD137 agonist antibodies 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 embodiment, 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 as a binding partner at 30 ug / mL for measurement (see, e.g., Example 10-2 herein).

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

[0142] In a non-limiting embodiment, CD137 agonist activity can be confirmed using B cells known to express CD137 on their surface. In a non-limiting embodiment, the HDLM-2 B cell line can be used as the B cells. Because IL-6 expression is induced as a result of CD137 activation, CD137 agonist activity can be assessed by the amount of human interleukin-6 (IL-6) produced. In this assessment, the amount of IL-6 is used to assess the increase in IL-6 expression from non-activated B cells as a 0% background level, thereby determining the percentage of CD137 agonist activity of the molecule being evaluated.

[0143] In some embodiments, the antigen-binding molecules of the present invention induce CD3 activation of T cells against cells expressing a third antigen, but not against cells expressing CD137. Whether an 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 CD3 activation of 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 reporter gene expression or the activity of the reporter gene product as an indicator of T cell activation. 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 an antigen-binding molecule, detection of reporter gene expression or reporter gene product activity in a dose-dependent manner of the antigen-binding molecule indicates that the antigen-binding molecule induces T cell activation against cells expressing a third antigen. Similarly, whether an antigen-binding molecule does not induce CD3 activation of T cells against 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 CD3 activation of T cells as described above. If reporter gene expression or reporter gene product activity is absent, below the detection limit, or below that of a negative control 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, it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137.In one aspect, 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 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%, it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137, where 100% activation is the level of activation achieved by an antigen-binding molecule that simultaneously binds to CD3 and CD137. In one aspect, 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 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%, it is determined that the antigen-binding molecule does not induce T cell activation against cells expressing CD137, where 100% activation is the level of activation achieved by the same antigen-binding molecule against cells expressing a molecule of a third antigen.

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

[0145] 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.

[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 a heavy chain variable region and the amino acid sequence of SEQ ID NO: 99 as a light chain variable region; [2] An antibody comprising the amino acid sequence of SEQ ID NO: 100 as a heavy chain variable region and the amino acid sequence of SEQ ID NO: 101 as a light chain variable region; [3] An antibody comprising the amino acid sequence of SEQ ID NO: 102 as a heavy chain variable region and the amino acid sequence of SEQ ID NO: 103 as a light chain variable region; [4] An antibody comprising the amino acid sequence of SEQ ID NO: 104 as a heavy chain variable region and the amino acid sequence of SEQ ID NO: 105 as a light chain variable region; [5] An antibody comprising the amino acid sequence of SEQ ID NO: 106 as a heavy chain variable region and the amino acid sequence of SEQ ID NO: 107 as a light chain variable region; [6] An antibody comprising the amino acid sequence of SEQ ID NO: 108 as a heavy chain variable region and the amino acid sequence of SEQ ID NO: 109 as a light chain variable region; [7] Any one of the antibodies of [1] to [6], which comprises the amino acid sequence of SEQ ID NO: 110 as a 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 a light chain constant region; and [8] An antibody having activity equivalent to the activity of any one of the antibodies of [1] to [7]; and [9] An antibody that binds to the same epitope as any one of the antibodies in [1] to [7].

[0147] In the antibody [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 [1] to [7].

[0148] Whether a test antibody shares a common epitope with a particular antibody can be evaluated based on the competition between the two antibodies for the same epitope. Competition between antibodies can be detected by cross-blocking assays, etc. For example, competitive ELISA assays are preferred cross-blocking assays. Specifically, in cross-blocking assays, 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. In other words, the higher the affinity of the test antibody for the same epitope, the lower the amount of the anti-CD137 antibody of the present invention bound to the wells coated with CD137 protein, and the higher the amount of the test antibody bound to the wells coated with CD137 protein.

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

[0150] Furthermore, if 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 wells can be measured by using a labeled antibody that recognizes the constant region of that antibody. Alternatively, if the antibodies are derived from the same species but belong to different classes, the amount of antibody bound to the wells can be measured using antibodies that distinguish between the individual classes.

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

[0152] In another embodiment, the ability of a test antibody to competitively or cross-competitively bind to another antibody can be determined by one of skill in the art using standard binding assays such as BIAcore analysis or flow cytometry, as known in the art.

[0153] Methods for determining spatial conformation of epitopes 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 a test antibody shares a common epitope with a CD137 ligand can also be evaluated based on the competition between the test antibody and the CD137 ligand for the same epitope. Competition between an antibody and a CD137 ligand can be detected by the above-mentioned cross-blocking assay or the like. In another embodiment, the ability of a test antibody to competitively or cross-competitively bind to a CD137 ligand can be appropriately determined by a person skilled in the art using standard binding assays known in the art, such as BIAcore analysis or flow cytometry.

[0155] In some embodiments, convenient examples of the antigen-binding molecules of the present invention include antigen-binding molecules that bind to the same epitope of human CD137 as that bound by an antibody selected from the group consisting of: In human CD137 protein an antibody recognizing a region including the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 81); An antibody recognizing a region containing the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (SEQ ID NO: 76); An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC (SEQ ID NO: 79), and An antibody that recognizes a region containing the sequence LQDPCSNCPAGTFCDNNRNQIC (sequence number: 74).

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

[0157] "Epitope" refers to 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 an 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 glycan, the epitope can be specified by its specific glycan structure.

[0158] A linear epitope is one whose primary amino acid sequence contains the epitope being recognized. Such a linear epitope typically contains at least 3, and most commonly at least 5, e.g., about 8-10 or 6-20 amino acids in its specific sequence.

[0159] In contrast to linear epitopes, "conformational epitopes" are epitopes in which the primary amino acid sequence containing the epitope is not the sole determinant of the recognized epitope (e.g., the primary amino acid sequence of a conformational epitope is not necessarily recognized by the antibody that defines the epitope). Conformational epitopes may contain a greater number of amino acids than linear epitopes. 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 conformational structure of an epitope include, but are not limited to, 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. Methods for evaluating the binding of an epitope in a target antigen by another binding domain can also be performed as appropriate according to the following examples.

[0161] For example, whether a test antigen-binding molecule containing an antigen-binding domain against a cancer-specific antigen recognizes a linear epitope in an antigen molecule can be confirmed, for example, 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. The test antigen-binding molecule containing the antigen-binding domain against a cancer-specific antigen is then evaluated for its binding activity to the linear peptide containing the amino acid sequence forming the extracellular domain. For example, the binding activity of the antigen-binding molecule to the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen. 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-mentioned 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 binds strongly to cancer-specific antigen-expressing cells upon contact, but does not substantially bind to an immobilized linear peptide containing an amino acid sequence that forms the extracellular domain of the cancer-specific antigen. As used herein, "does not substantially bind" means that the binding activity to antigen-expressing cells is 80% or less, generally 50% or less, preferably 30% or less, and particularly preferably 15% or less, compared to the binding activity to antigen-expressing cells in ELISA or fluorescence-activated cell sorting (FACS) using 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 to antigen-expressing cells can be quantitatively evaluated by comparing the signal levels generated by the enzymatic reaction. Specifically, the test antigen-binding molecule is added to an ELISA plate on which antigen-expressing cells are immobilized. The cell-bound test antigen-binding molecule is then detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, when using FACS, a dilution series of the test antigen-binding molecule can be prepared, and the antigen-binding titer to the antigen-expressing cells can be determined, and the binding activity of the test antigen-binding molecule to the antigen-expressing cells can be compared.

[0164] The binding of a test antigen-binding molecule to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices: FACSCanto(TM) II FACSAria(trademark) FACSArray™ FACSVantage(TM) SE FACSCalibur™ (all trade 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 trade names of Beckman Coulter).

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

[0166] Whether a test antigen-binding molecule comprising an antigen-binding domain of the present invention shares a common epitope with another antigen-binding molecule can be evaluated based on the competition between the two molecules for the same epitope.Competition between antigen-binding molecules can be detected by cross-blocking assays, etc.For example, competitive ELISA assays are preferred cross-blocking assays.

[0167] Specifically, in cross-blocking assays, the antigens coated on the wells of microtiter plates are pre-incubated in the presence or absence of candidate competing antigen-binding molecules, and then test antigen-binding molecules are added thereto. The amount of test antigen-binding molecules bound to the antigen in the wells is indirectly correlated with the binding ability of the candidate competing antigen-binding molecules that compete for binding to the same epitope. That is, the higher the affinity of the competing antigen-binding molecules for the same epitope, the lower the binding activity of the test antigen-binding molecules to the wells coated with antigen.

[0168] The amount of test antigen-binding molecules bound to the wells via 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 conjugates and appropriate substrates. In particular, cross-blocking assays using enzyme labels such as peroxidase are called "competitive ELISA assays." Antigen-binding molecules can also be labeled with other labeling substances that allow detection or measurement. Specifically, radiolabels, fluorescent labels, etc. are known. When a candidate competitor antigen-binding molecule is able to block binding of a test antigen-binding molecule comprising an antigen-binding domain by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity in a control experiment performed in the absence of the competitor antigen-binding molecule, the test antigen-binding molecule is determined to substantially bind to the same epitope as the competitor antigen-binding molecule, or to compete for binding to the same epitope.

[0169] When the structure of the epitope bound by a test antigen-binding molecule comprising an antigen-binding domain of the present invention 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 toward a peptide prepared by introducing amino acid mutations into the peptide that forms the epitope.

[0170] For example, the binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide containing a mutation can be measured by comparison in the ELISA format described above. In addition to ELISA, the binding activity of the test antigen-binding molecule and the control antigen-binding molecule 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 a mutant peptide to a column in the form of a GST-fusion peptide are known.

[0171] Alternatively, if the identified epitope is a conformational epitope, whether the test and control antigen-binding molecules share a common epitope can be assessed using the following method. First, cells expressing the antigen targeted by the antigen-binding domain and cells expressing the antigen bearing the mutated epitope are prepared. These cells are suspended in an appropriate buffer, such as PBS, to prepare a cell suspension, to which the test and control antigen-binding molecules are added. The cell suspension is then washed appropriately with buffer, and an FITC-labeled antibody capable of recognizing the test and control antigen-binding molecules is added. The fluorescence intensity and number of cells stained with the labeled antibody are determined using a FACSCalibur (BD). The test and control antigen-binding molecules are appropriately diluted with an appropriate buffer and used at the desired concentration. For example, they may be used at a concentration ranging from 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 labeled antibody bound to the cells. That is, the binding activity of the test and control antigen-binding molecules, represented by the amount of bound labeled antibody, can be measured by determining the geometric mean value.

[0172] 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 modified 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 Light 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) is selected from The modified heavy chain variable domain sequence, HVR-H3, 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; Amino acid Gly, Tyr, Phe, or Val at the 100th position (Kabat numbering) The amino acid sequence comprises at least one amino acid selected from the group consisting of:

[0173] In some embodiments, the antigen-binding molecule of the present 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 commonly known to those skilled in the art. For example, antibodies can be prepared by the methods shown below, but methods for preparing the antibodies of the present invention are not limited thereto. Many combinations of host cells and expression vectors are known in the art for preparing antibodies by transferring an isolated gene encoding the polypeptide into an appropriate host. 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 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 embryonic kidney cell line with sheared adenovirus (Ad) 5 DNA), PER.C6 cells (human embryonic 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 E. coli (mAbs 2012 Mar-Apr; 4(2): 217-225) or yeast (WO2000023579). Antibodies prepared using E. coli are non-glycosylated, whereas antibodies prepared using yeast are glycosylated.

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

[0176] Alternatively, DNA encoding a protein in which one or more amino acid residues in an antibody variable domain prepared by methods known in the art against a specific antigen have been substituted with a different amino acid of interest may be designed in advance and chemically synthesized to obtain DNA encoding a heavy chain in which one or more amino acid residues in the variable domain have been substituted with a different amino acid of interest. The amino acid substitution site and type are not particularly limited. Examples of preferred regions for amino acid modification include solvent-exposed regions and loops in the variable domain. CDR1, CDR2, CDR3, FR3, and loops are particularly preferred. Specifically, positions 31-35, 50-65, 71-74, and 95-102 (Kabat numbering) in the H-chain variable domain and positions 24-34, 50-56, and 89-97 (Kabat numbering) in the L-chain variable domain are preferred. More preferred are positions 31, 52a to 61, 71 to 74, and 97 to 101, according to the Kabat numbering, in the H-chain variable domain, and positions 24 to 34, 51 to 56, and 89 to 96, according to the Kabat numbering, in the L-chain variable domain. Amino acid alterations are not limited to substitutions, but may also be deletions, additions, insertions, or modifications, or combinations thereof.

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

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

[0179] The DNA encoding the antibody of interest is incorporated into an expression vector so that it is expressed under the control of an expression control region, such as an enhancer and a promoter.The resulting expression vector is then transformed into a host cell to express the antibody.In this case, a suitable 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 excising cDNA.

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

[0182] The vector may contain a signal sequence for polypeptide secretion. In the case of production in the periplasm of E. coli, the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4397, the entire contents of which are incorporated herein by reference) 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 expression vectors for E. coli, examples of vectors for producing the polypeptides of the present invention include mammalian expression vectors (e.g., pcDNA3 (Invitrogen Corp.), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322, the entire contents of which are incorporated herein by reference), pEF, and pCDM8), insect cell-derived expression vectors (e.g., "Bac-to-BAC Baculovirus Expression System" (GIBCO BRL), and pBacPAK8), plant-derived expression vectors (e.g., pMH1 and pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derived expression vectors (e.g., "Pichia Expression Kit" (Invitrogen Corp.), pNV11, and SP-Q01), and Bacillus subtilis (Bacillus Examples of suitable expression vectors include those derived from L. subtilis (e.g., pPL608 and pKTH50).

[0184] For expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, or HEK293 cells, the vector must have a promoter necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, the entire contents of which are incorporated herein by reference), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, the entire contents of which are incorporated herein by reference), the CAG promoter (Gene. (1991) 108, 193, the entire contents of which are incorporated herein by reference), or the CMV promoter. More preferably, the vector has a gene for screening transformed cells (e.g., a drug resistance gene that can serve as a marker for drugs (e.g., neomycin, G418, etc.)). Examples of vectors with such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13. Additionally, the EBNA1 protein may be coexpressed to increase gene copy number using a vector carrying the replication origin OriP (Biotechnol Bioeng. 2001 Oct 20;75(2):197-203; and Biotechnol Bioeng. 2005 Sep 20;91(6):670-7).

[0185] An exemplary method for stably expressing a gene and increasing its copy number in cells includes transforming CHO cells deficient in a nucleic acid synthesis pathway with a vector (e.g., pCHOI) carrying a complementing DHFR gene and using methotrexate (MTX) for gene amplification. An exemplary method for transiently expressing a gene includes transforming COS cells carrying the SV40 T antigen gene on their chromosome with a vector (e.g., pcD) carrying an SV40 origin of replication. Origins of replication derived from polyoma virus, adenovirus, bovine papilloma virus (BPV), and the like can also be used. To increase gene copy number in a host cell system, the expression vector can contain a selection marker such as the aminoglycoside phosphotransferase (APH) gene, the thymidine kinase (TK) gene, the Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, or the dihydrofolate reductase (dhfr) gene.

[0186] Antibodies can be recovered, for example, by culturing the transformed cells and then isolating the antibody from the transformed cells or the culture medium. Antibodies can be isolated and purified by an appropriate combination of methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, C1q, FcRn, protein A, and protein G columns, affinity chromatography, ion exchange chromatography, and gel filtration chromatography.

[0187] The above-mentioned 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 the technique of suppressing unintended association between H chains by introducing charge repulsion (WO2006 / 106905), can be applied to methods for efficiently preparing multispecific antibodies.

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

[0189] Examples may include a method of making comprising the steps of: (i) preparing a library of antigen-binding molecules each of which has at least one amino acid altered in its antibody variable region that binds to CD3 or CD137, wherein the at least one amino acid in the altered variable region differs from each other; (ii) selecting, from the prepared library, antigen-binding molecules comprising variable regions that have binding activity to CD3 and CD137 but do not simultaneously bind to CD3 and CD137; (iii) culturing host cells containing 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 does not simultaneously bind 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 method of preparation, step (ii) may be an optional step of: (v) selecting, from the prepared library, antigen-binding molecules comprising variable regions that have binding activity to CD3 and CD137 but do not simultaneously bind to CD3 and CD137, each of which is expressed on different cells.

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

[0192] The amino acids to be modified are selected, for example, from amino acids in the variable region of an antibody that binds to CD3 or CD137, the modification of which does not abolish binding to the antigen.

[0193] In the present invention, one amino acid modification may be used alone, or multiple amino acid modifications may be used in combination. When multiple amino acid modifications are used in combination, the number of modifications to be combined is not particularly limited, and may be, for example, 2 to 30, preferably 2 to 25, 2 to 22, 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 2 to 3. The multiple amino acid modifications to be combined may be made only to the heavy or light chain variable domain of the antibody, or may be distributed appropriately among both the heavy and light chain variable domains.

[0194] Examples of preferred regions for amino acid modification include solvent-exposed regions and loops in the variable region. CDR1, CDR2, CDR3, FR3, and loops are particularly preferred. Specifically, positions 31-35, 50-65, 71-74, and 95-102 (Kabat numbering) in the H-chain variable domain, and positions 24-34, 50-56, and 89-97 (Kabat numbering) in the L-chain variable domain are preferred. Positions 31, 52a-61, 71-74, and 97-101 (Kabat numbering) in the H-chain variable domain, and positions 24-34, 51-56, and 89-96 (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 of the antibody variable region that binds to CD3 or CD137, and insertion of a peptide previously known to have binding activity to CD3 or CD137 into the above-mentioned regions. The antigen-binding molecules of the present invention can be obtained by selecting, from antigen-binding molecules modified in this way, a variable region that can bind to CD3 and CD137 but cannot simultaneously bind to these antigens.

[0196] Whether the variable region is capable of binding to CD3 and CD137 but is unable to simultaneously bind to these antigens, and further whether the variable region is capable of simultaneously binding to both CD3 and CD137 when either CD3 or CD137 is present on a cell and the other antigen is present alone, when both antigens are present alone, or when both antigens are present on the same cell, but is unable to simultaneously bind to these antigens each expressed on different cells, can also be confirmed using the methods described above.

[0197] The present inventors also succeeded in developing a method for more efficiently obtaining antigen-binding domains that bind to two or more different antigens. In some embodiments, the method of screening for antigen-binding domains that bind to at least two or more 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 antigen-binding domains that bind to the first antigen; (c) contacting the antigen-binding domain collected in step (b) with a second antigen of interest and collecting the antigen-binding domain bound to the second antigen; and (d) amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; wherein the method does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b). In the above-described methods, the number of steps of contacting the antigen-binding domain with the antigen is not particularly limited. In some embodiments, when the number of antigens of interest is two or more, the screening method of the present invention may include three or more contacting steps. In further embodiments, the screening method of the present invention may include two or more steps of contacting the antigen-binding domain with 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 consecutively two or more times, or may be contacted first with one antigen once or more times and then with another antigen 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 the antigen, the method does not include a step of amplifying the nucleic acid encoding the collected antigen-binding domain between any two consecutive contacting steps.

[0198] 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 the antigen binding domain with a scaffold and crosslinking the antigen binding domain with 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 in steps (b) and (c) above using an elution solution that is an acid solution, a base solution, DTT, or IdeS. In some embodiments, the elution solution used in the above steps (b) and (c) of the present invention is EDTA or IdeS.

[0201] In some embodiments, the method of screening for antigen-binding domains that bind to at least two or more 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 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 domain collected in step (b) with a second antigen of interest and collecting the antigen-binding domain bound to the second antigen; and (d) amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; wherein the method does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b).

[0202] In some embodiments, the method for generating antigen-binding domains that bind to at least two or more 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 antigen-binding domains that bind to the first antigen; (c) contacting the antigen-binding domain collected in step (b) with a second antigen of interest and collecting the antigen-binding domain bound to the second antigen; and (d) amplifying the genes encoding the antigen-binding domains collected in step (c) and identifying candidate antigen-binding domains; (e) ligating the polynucleotides encoding the candidate antigen-binding domains selected in step (d) with a polynucleotide encoding a polypeptide comprising an Fc region; (f) culturing a cell into which a vector to which the polynucleotide obtained in step (d) is operably linked has been introduced; and (g) collecting antigen-binding molecules from the culture medium of the cells cultured in step (f) above. wherein the method does not include a step between step (b) and step (c) of amplifying the nucleic acid encoding the antigen-binding domain collected in step (b).

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

[0204] In one aspect, the screening method of the present invention makes it possible to more efficiently obtain antigen-binding domains that bind to at least two or more different antigens of interest.

[0205] As used herein, the term "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 a plurality of fusion polypeptides comprising antigen-binding molecules contained in the library have different sequences, and are antigen-binding molecules or fusion polypeptides comprising antigen-binding molecules that do not have a single sequence. In some embodiments, the library of the present invention is a design library. In a further embodiment, the design library is the design library disclosed in WO2016 / 076345.

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

[0207] In one embodiment, the antigen-binding molecule of the present invention can be an ScFv, a Fab fragment, F(ab)2, or F(ab')2. In another embodiment, the present invention provides a library consisting essentially of a plurality of fusion polypeptides with different sequences, each comprising any of these antigen-binding molecules and a heterologous polypeptide. Specifically, the present invention provides a library consisting essentially of a plurality of fusion polypeptides with different sequences, each comprising any of these antigen-binding molecules fused to at least a portion of a viral coat protein selected from the group consisting of viral coat proteins pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, and variants thereof. The antigen-binding molecules of the present invention may further comprise a dimerization domain. In one embodiment, the dimerization domain can be located between the variable region domain of the heavy or light chain of the antibody and at least a portion of the viral coat protein. This dimerization domain can include at least one dimerization sequence and / or a sequence containing one or more cysteine ​​residues. The dimerization domain can preferably be linked to the C-terminus of the heavy chain variable domain or the constant domain. The dimerization domain can assume various structures depending on whether the antibody variable domain is prepared as a fusion polypeptide component with a viral coat protein component (there is no amber stop codon after the dimerization domain) or whether the antibody variable domain is prepared primarily without a viral coat protein component (e.g., there is an amber stop codon after the dimerization domain). When the antibody variable domain is prepared primarily as a fusion polypeptide with a viral coat protein component, bivalent display is achieved by one or more disulfide bonds and / or a single dimerization sequence.

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

[0209] As used herein, "phage display" refers to a technique in which variant polypeptides are displayed as fusion proteins with at least a portion of a coat protein on the particle surface of phage, e.g., filamentous phage. Phage display is useful because it allows large libraries of randomized protein variants to be rapidly and efficiently screened for sequences that bind to target antigens with high affinity. Display of peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties. Polyvalent phage display methods have been used to display small random peptides and small proteins through fusion with 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 protein or peptide library to gene III or a portion thereof so that each phage particle displays 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 lower avidity effects than polyvalent phage and are therefore screened based on intrinsic ligand affinity using phagemid vectors, 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 containing a bacterial origin of replication, such as ColE1, and a copy of the intergenic region of a bacteriophage. Phagemids derived from any bacteriophage known in the art, such as filamentous or lambdoid bacteriophages, can be used as appropriate. Plasmids usually also contain a selectable marker for antibiotic resistance. DNA fragments cloned into these vectors can be propagated as plasmids. When cells harboring these vectors possess all the genes necessary for phage particle production, the replication pattern of the plasmid shifts to rolling circle replication, forming copies of a single plasmid DNA strand and packaging phage particles. Phagemids can form infectious or non-infectious phage particles. This term includes phagemids containing a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene by gene fusion, such that the heterologous polypeptide is displayed on the surface of the phage particle.

[0211] The term "phage vector" refers to a double-stranded replicative bacteriophage that contains a heterologous gene and is capable of replication. The phage vector has a phage origin of replication that allows phage replication and phage particle formation. The phage is preferably a filamentous bacteriophage, such as M13, f1, fd, or Pf3 phage or its derivatives, or a lambda-type phage, such as lambda, 21, phi80, phi81, 82, 424, 434, or any other phage or its derivatives.

[0212] The term "coat protein" refers to a protein, at least a portion of which is present on the surface of a virus particle. From a functional standpoint, a coat protein is any protein that binds to a virus particle during virus assembly in a host cell and remains associated with it until the virus infects other host cells. A coat protein may be a larger or smaller coat protein. Smaller coat proteins are typically coat proteins present in the virus capsid, preferably at least about 5, more preferably at least about 7, and even more preferably at least about 10 or more protein copies per virion. Larger coat proteins may be present in tens, hundreds, or thousands of copies per virion. Examples of larger coat proteins include the filamentous phage p8 protein.

[0213] "Ribosome display," as used herein, refers to a technique by which variant polypeptides are displayed 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 ribosomal stall sequence, such as E. coli secM (J. Mol. Biol. 2007 Sep14;372(2):513-24), or does not have a 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 are fused to the variant polypeptide and encode peptides that allow the variant polypeptide to pass through the ribosomal tunnel after translation and enable the variant polypeptide to express its function. Any in vitro translation system can be used for ribosome display, such as the E. coli S30 system, the PURE system, the rabbit reticulocyte lysate system, or the wheat germ cell-free translation system.

[0214] The term "oligonucleotide" refers to a short single-stranded or double-stranded polydeoxynucleotide that is chemically synthesized by methods known in the art (for example, phosphotriester, phosphite, or phosphoramidite chemistry using solid-phase methods, such as those described in EP266032; or 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 polymerase chain reaction and other autoprimer methods described below, as well as oligonucleotide synthesis on solid support.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 the nucleic acid sequence complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, possible nucleic acid sequences can be 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 acids" refers to an experimental procedure that increases the moles of nucleic acids. In a non-limiting embodiment, nucleic acids include single-stranded RNA (ssRNA), double-stranded DNA (dsDNA), or single-stranded DNA (ssDNA). In a non-limiting embodiment, PCR (polymerase chain reaction) is a commonly used method for amplifying nucleic acids, but any method capable of amplifying nucleic acids can be used. Alternatively, nucleic acids can be amplified in host cells by introducing a nucleic acid vector into the host cell. In a non-limiting embodiment, electroporation, heat shock, infection with a phage or virus carrying the vector, or chemical reagents can be used to introduce nucleic acids into cells. Alternatively, transcription of DNA or reverse transcription of mRNA followed by transcription thereof can also amplify nucleic acids. In a non-limiting embodiment, introduction of a phagemid vector into E. coli is commonly used to amplify nucleic acids encoding binding domains, but PCR can also be used in phage display technology. In ribosome display, cDNA display, mRNA display, and CIS display, PCR or transcription is commonly used to amplify nucleic acids.

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

[0217] The term "scaffold" in "fusion polypeptide formed by fusing an antigen-binding domain with a scaffold" refers to a molecule that bridges the antigen-binding domain with the nucleic acid encoding the antigen-binding domain. Non-limiting examples of scaffolds that can be used in each display methodology include phage coat proteins in phage display, ribosomes in ribosome display, puromycin in mRNA or cDNA display, RepA protein in CIS display, viral coat proteins in viral display, mammalian cell membrane-anchored proteins in mammalian cell display, yeast cell membrane-anchored proteins in yeast display, and bacterial cell membrane-anchored proteins in bacterial or E. coli display.

[0218] In the present invention, the term "one or more amino acids" is not limited to a specific number of amino acids, but 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 display of fusion polypeptides, the fusion polypeptides of the variable regions of antigen-binding molecules can be displayed 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. 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. Display of multivalent forms is preferred, perhaps in part because the displayed multivalent forms usually have multiple antigen-binding sites, which allows for the identification of low-affinity clones and / or allows for more efficient selection of rare clones during the selection process.

[0220] Methods for displaying fusion polypeptides containing antibody fragments on the surface of bacteriophage are known in the art and are described, for example, in WO1992001047 and herein. Other related methods are described in WO1992020791, WO1993006213, WO1993011236, and WO1993019172. Those skilled in the art can use these methods as appropriate. Other publications (H.R. Hoogenboom & G. Winter (1992) J. Mol. Biol. 227, 381-388, WO1993006213, and WO1993011236) disclose the identification of antibodies against various antigens displayed on the surface of phage using artificially rearranged variable region gene repertoires.

[0221] When a vector is constructed for display in the form of an scFv, the vector contains nucleic acid sequences encoding the light chain variable domain and 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 a nucleic acid sequence encoding a 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 via a nucleic acid sequence encoding a peptide linker. The peptide linker generally contains approximately 5 to 15 amino acids. Optionally, for example, an additional sequence encoding a tag 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, the nucleic acid sequence encoding the heavy chain variable domain of the antigen-binding molecule, or both.

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

[0223] Regarding the introduction of vectors into host cells, the vectors constructed as described above are introduced into host cells for amplification and / or expression. The vectors can be introduced into host cells by transformation methods known in the art, including electroporation, calcium phosphate precipitation, etc. If the vector is an infectious particle such as a virus, the vector itself will invade the host cell. The fusion protein is displayed on the surface of the phage particle by transfecting the host cell with a replicable expression vector having an insert of a polynucleotide encoding the fusion protein and producing phage particles by methods known in the art.

[0224] Replicable expression vectors can be introduced into host cells by various methods. In a non-limiting embodiment, the vector can be introduced into cells by electroporation, as described in WO2000106717. Cells are cultured in standard culture medium at 37°C, optionally for approximately 6 to 48 hours (or until the OD at 600 nm reaches 0.6 to 0.8). The culture medium is then 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)). The suspension is then centrifuged again, and the supernatant is removed. The resulting cell pellet is resuspended, for example, in 5 to 20% V / V diluted glycerol. Following removal of the supernatant, the suspension is again centrifuged 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 with water or diluted glycerin.

[0225] An example of a preferred recipient cell includes the E. coli strain SS320, which is capable of responding to electroporation (Sidhu et al., Methods Enzymol. (2000) 328, 333-363). E. coli strain SS320 was prepared by coupling MC1061 cells with XL1-BLUE cells under conditions sufficient to transfer the fertility episome (F' plasmid) or XL1-BLUE into the MC1061 cells. E. coli strain SS320 has been deposited with the ATCC (10801 University Boulevard, Manassas, Virginia) under 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 as commercially available products (TG1, CJ236, CSH18, DHF', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18, etc.).

[0226] The use of higher DNA concentrations (approximately 10-fold) in electroporation improves transformation frequency and increases the amount of DNA that transforms host cells. The use of higher cell densities also improves efficiency (approximately 10-fold). Increasing the amount of DNA transferred can result in libraries with greater diversity and a greater number of independent clones with different sequences. Transformed cells are usually selected based on the presence or absence of growth on antibiotic-containing media.

[0227] The present invention further provides nucleic acids encoding the antigen-binding molecules of the present invention. The nucleic acids 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 depending on the host cell to receive the vector. For example, any of the vectors described above 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 host cells described above can be used.

[0230] The present invention also provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the present invention can be formulated according to methods known in the art by adding a pharmaceutically acceptable carrier to the antigen-binding molecules of the present invention. For example, the pharmaceutical compositions can be used in the form of parenteral injection of a sterile solution or suspension with water or any other pharmaceutically acceptable solution. For example, pharmaceutical compositions can be formulated by mixing the antigen-binding molecules with an appropriate combination of pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., in a unit dosage form required for commonly accepted pharmaceutical practice. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, saccharides, carboxymethyl cellulose, corn starch, and inorganic salts. The amount of the active ingredient in such preparations is determined so that an appropriate dosage within the indicated range can be achieved.

[0231] Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, using vehicles such as distilled water for injection.Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose and other auxiliary agents (for example, D-sorbitol, D-mannose, D-mannitol and sodium chloride).These solutions can be used in combination with suitable solubilizers, such as alcohol (particularly ethanol) or polyalcohols (for example, propylene glycol and polyethylene glycol), or nonionic surfactants, such as polysorbate 80 (trademark) or HCO-50.

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

[0233] The administration method can be selected appropriately depending on the patient's age and symptoms. The dose of a pharmaceutical composition containing a polypeptide or a polynucleotide encoding the polypeptide can be selected, for example, within the range of 0.0001 to 1,000 mg per kg of body weight per dose. Alternatively, the dose can be selected, for example, within the range of 0.001 to 100,000 mg per patient, but is not necessarily limited to these values. The dose and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can select the dose and method appropriately.

[0234] The present invention also provides methods for treating cancer, which comprise a step of administering the antigen-binding molecule of the present invention; the antigen-binding molecule of the present invention for use in treating cancer; use of the antigen-binding molecule of the present invention in producing a cancer therapeutic agent; and a process for producing a cancer therapeutic agent, which comprises a step of using the antigen-binding molecule of the present invention.

[0235] As used herein, the three-letter amino acid codes and corresponding one-letter codes 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] It should be understood by those skilled in the art that any one or any combination of two or more of the aspects described herein is also included in the present invention, unless a technical contradiction arises based on the common general knowledge of those skilled in the art.

[0237] All references cited herein are incorporated by reference in their entirety.

[0238] The present invention is further illustrated with reference to the following examples, which, however, are not intended to be limiting. [Example]

[0239] [Example 1] Concept of an engineered immunoglobulin variable (Fab) region that binds to CD3 and CD137 but does not simultaneously bind to CD3 and CD137 T cells play an important role in tumor immunity and are known to be activated by two signals: 1) T cell receptor (TCR) binding to antigenic peptides presented by major histocompatibility complex (MHC) class I molecules and activation of the TCR; and 2) costimulatory molecules on the surface of T cells binding to ligands on antigen-presenting cells and activation of the costimulatory molecules. Furthermore, 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 been demonstrated to exhibit antitumor effects, primarily through the activation of CD8-positive T cells and NK cells (Houot, 2009, Blood, 114, 3431-8). It has also been shown that T cells engineered with chimeric antigen receptor molecules (CAR-T cells) consisting of a tumor antigen-binding domain as the extracellular domain and CD3 and CD137 signaling domains as the intracellular domain 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 nonspecific hepatotoxicity are a clinical and nonclinical problem, preventing the development of these drugs (Dubrot, Cancer Immunol. Immunother., 2010, 28, 512-22). It has been suggested that the main cause of side effects is related to antibody binding to Fcγ receptors via the antibody constant region (Schabowsky, Vaccine, 2009, 28, 512-22). Furthermore, it has been reported that agonistic antibodies targeting receptors belonging to the TNF receptor superfamily require antibody cross-linking by Fcγ receptor-expressing cells (FcγRII-expressing cells) to exert their agonistic 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 agonistic activity and a binding domain for a tumor-specific antigen can exert CD137 agonistic activity and activate immune cells only in the presence of cells expressing the tumor-specific antigen, thereby avoiding the hepatotoxic adverse events of CD137 agonistic antibodies while maintaining the antitumor activity of the antibody. WO2015 / 156268 further describes that the antitumor 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 with three binding domains for CD137, CD3, and a tumor-specific antigen (EGFR) has also been reported (WO2014 / 116846). However, because this molecule simultaneously binds to CD3ε and CD137, it is expected to crosslink CD3ε-expressing T cells with CD137-expressing cells (T cells, B cells, NK cells, DCs, etc.) even in the absence of tumor-specific antigen-expressing cells (Figure 2). In fact, a bispecific antibody against CD3 and CD8 has been reported to crosslink CD8-positive T cells and induce cytotoxic activity between them (Wong, Clin. Immunol. Immunopathol. 1991, 58(2), 236-250). Therefore, crosslinking CD3 with antigens expressed on T cells is expected to induce crosslinking of T cells and result in mutual killing of T cells.

[0241] Catumaxomab is a bispecific antibody that recognizes a protein expressed on T cells and a protein expressed on cancer cells (cancer antigens). Its two Fab fragments bind to a cancer antigen (EpCAM) and the CD3ε chain expressed on T cells, respectively. Even in the absence of cancer antigens, it simultaneously binds to CD3ε and FcγR, thereby crosslinking CD3ε-expressing T cells to FcγR-expressing cells and producing large amounts of various cytokines, even in the absence of cancer cells. Due to this induction of various cytokines independent of cancer antigens, 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, systemic administration of trifunctional antibodies is extremely difficult due to the severe cytokine storm-like adverse reactions (Cancer Immunol Immunother. 2007 Sep; 56(9): 1397-406 (Non-Patent Document 18)).

[0242] On the other hand, conventional multispecific antibodies bind to multiple antigens simultaneously. Depending on the combination of antigens, simultaneous binding to multiple antigens may not be desirable. No antibody has yet been discovered that exerts both T cell-mediated cytotoxicity and CD137-mediated activation of T cells and other immune cells in a cancer antigen-specific manner while avoiding adverse reactions.

[0243] Therefore, a potential method for controlling such undesired cross-linking reactions is a dual-binding Fab, which is a single variable (Fab) region that binds to CD3 through one portion and to CD137 through a different portion that is not involved in the binding to the first antigen (Figure 1). As shown in Figure 1, if the two proximal portions of a single variable (Fab) region are essential for binding to each antigen, binding to CD3 inhibits binding to CD137, while binding to CD137 also inhibits binding to CD3. Therefore, an improved antibody with such dual-binding Fab properties cannot simultaneously bind to CD3 and CD137 and therefore likely does not cause cross-linking reactions between CD3 and CD137 (Figure 2). Furthermore, dual-binding Fabs can simultaneously bind to both CD3 and CD137 when CD3 and CD137 are not expressed on the cell membrane as soluble proteins or when both are present on the same cell. However, they do not simultaneously bind to these antigens expressed on different cells, and are therefore unlikely to crosslink these two cells (Figure 3). On the other hand, an antigen (third antigen) bound to another variable (Fab) region may crosslink CD3 and CD137 on T cells (Figure 4) or CD137 on CD137-positive immune cells (Figure 5). For this antibody, an Fc region that binds to FcγR may be used as the constant region, or an Fc region with reduced FcγR binding activity may be used as the constant region. The use of such dual CD3 / CD137 binding Fab properties can provide, for example, a technique for damaging cancer cells expressing cancer antigens by antibody-mediated redirection of T cells, together with the function of activating T cells, NK cells, and / or other immune cells, thereby achieving higher anti-cancer potential.

[0244] In brief, if the variable (Fab) region can be modified to have the following properties as a dual-binding Fab, an antibody with the functions shown in Figure 1 can be developed: 1.Has binding activity to CD3; 2. Has binding activity to CD137; and 3. It does not bind to CD3 and CD137 simultaneously. The phrase "does not simultaneously bind to CD3 and CD137" also includes not cross-linking cells expressing CD3 with cells expressing CD137, or not simultaneously binding to CD3 and CD137, each expressed on different cells. This phrase also includes cases where the variable region can simultaneously bind to both CD3 and CD137 when CD3 and CD137 are not expressed on the cell membrane as soluble proteins, or when both are present on the same cell, but cannot simultaneously bind to CD3 and CD137, each expressed on different cells.

[0245] Similarly, if the variable (Fab) region can be improved as a dual-binding Fab so as to confer the following properties, it will be possible to develop antibodies having the effects shown in any of Figures 3, 4, and 5: 1. Has binding activity to CD3 on T cells; 2. Has binding activity to CD137 on CD137-expressing cells; and 3. It does not bind to CD3 and CD137 simultaneously.

[0246] [Example 2] Construction of a dual scFv library for ribosome display A dual scFv library for ribosome display was constructed using the antibody library fragments synthesized in Reference Example 3. The dual library was prepared as a library in which the H chain was diversified as shown in Table 38 (in Reference Example 4), but 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 Figure 6. To efficiently display the scFv library on ribosomes, the bacteriophage λgpD gene and a portion of the E. coli secM gene were used as spacer genes (SEQ ID NO: 2). The VL fragment of GLS3000 was assembled with the spacer gene and a Gly / Ser-rich linker gene by PCR (SEQ ID NO: 3). The synthesized antibody VH library fragments were then fused to the VL spacer gene at the 3' end and to a T7 promoter (SEQ ID NO: 4) with a 5' untranslated region (UTR) at the 5' end by PCR amplification.

[0247] [Example 3] Isolation of scFv domains that bind to CD3ε and human CD137 from a dual scFv library (3-1) Isolation of scFv domains that bind to human CD137 An 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 a 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™ Express Large-scale RNA production system, P1320, Promega) to prepare an mRNA scFv library. The synthesized mRNA was purified using an RNeasy mini kit (Cat. No. 74104, QIAGEN). The resulting mRNA library was translated using a PUREfrex1.0 (PF001-0.25, Genefrontier) cell-free in vitro translation system with DnaK GroE Mix and DS supplements (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 translation, and blocking buffer (one pack of SuperBlock Dry Blend Blocking buffer in TBS (Cat. No. 37545, Pierce) in a 200 mL milliQ tube) was also added. Panning was performed according to the general panning method with 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-coated or FG NeutrAvidin beads) or streptavidin-coated beads (Dynabeads M-280 Streptavidin or Dynabeads MyOne Streptavidin). T1 beads).

[0248] Specifically, 250 pmol of biotin-labeled antigen and 2 nmol of free human IgG Fc domain ("free" here means "not biotin-labeled") were added to the prepared ribosome display library solution, allowing the library solution to contact with the library solution at 4°C for 60 minutes. After adding SuperBlock-blocked magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed three times with WBT buffer (50 mM Tris-acetate, 150 mM NaCl, 50 mM Mg-acetate, 0.1% Tween). After adding elution buffer (50 mM Tris-acetate, 150 mM NaCl, 50 mM EDTA, and 50 μg / mL S. cerevisiae RNA (SIGMA)), the beads were suspended at 50°C for 15 minutes, and then immediately separated using a magnetic stand to recover the mRNA solution. The purified mRNA library was converted to cDNA reverse transcriptase using primer SEQ ID NO:147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), then amplified by PCR using primer SEQ ID NO:148 and KOD-FX polymerase (TOYOBO). A T7 promoter gene was added to the amplified DNA library by PCR using primers SEQ ID NO:149 and 150. This cycle, called panning, was repeated several times. In the second and subsequent rounds of panning, mRNA was recovered using either 150-50 pmol of biotin-labeled human CD137-Fc and either elution buffer (referred to as the EDTA elution campaign) or FabRICATOR (IdeS, a protease against the hinge region of IgG, GENOVIS) (referred to as the IdeS elution campaign). In this procedure, 5 uL of 10 units / μL Fabricator was added together with 95 uL of WBT buffer, and the beads were suspended at 37°C for 10 minutes, and then immediately 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.

[0249] (3-2) Binding of scFv domains to CD3ε or CD137 (scFv ELISA) To evaluate the binding of the scFv domains by ELISA, FLAG tags were added to the DNA libraries recovered in rounds 5 and 6 by PCR using primers SEQ ID NOs: 148 and 151. The resulting scFv-FLAG DNA fragments were ligated into the TOPO TA cloning kit dual promoter vector (Invitrogen) and transformed into DH5α E. coli. The VH sequences from each single E. coli colony were analyzed. Five to seven clones with distinct VH sequences were then collected 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 using primers SEQ ID NOs: 148 and 151. PUREfrex 1.0 ss was added to each amplified scFv gene and incubated at 37°C for 2 hours. After adding 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 (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. The wells were then blocked with 250 μL of 2% skim milk-TBS for at least 1 hour. After removing 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 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 1:1000 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 1:2000 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 adding sulfuric acid. Color development was then assayed based on 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, clones exhibiting binding activity to the second antigen (human CD137) were successfully selected by using a 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 into IgG (the VH and VL sequences of each clone were linked to the constant domains of human H and L chains, 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 inserted into an animal expression plasmid. The prepared plasmid was used for expression in animal cells according to 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] Antigen binding of each molecule was tested by electrochemiluminescence (ECL) assay. Specifically, biotinylated CD3ε peptide or biotinylated human CD137 diluted to 18 pmol / mL in TBS solution containing 0.1% Tween 20 (TBST), each antibody solution adjusted to 2 μg / mL, and SULFO-TAG-labeled (MESO SCALE DIAGNOSTICS, ruthenium(II) tris-bipyridine, N-hydroxysuccinimide) anti-human IgG antibody (Invitrogen #628400) adjusted to 18 pmol / mL were added to a Nunc-Immuno™ MicroWell™ 96-well round plate (Nunc) at 25 μL / well, mixed, and then incubated at room temperature for 1 hour to allow antibody-antigen complex formation. TBST solution containing 0.5% BSA was added to a streptavidin plate (MSD KK, 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 TBST solution. Antibody-antigen complex solution was added to it at 75 μL / well, and the plate was incubated at room temperature for 1 hour to allow the biotin-anti-human IgG Ab to bind to the streptavidin plate. After removing the antibody-antigen complex solution, each well was washed three times with TBST solution, and READ buffer (MSD KK) was added to it at 150 μL / well. The luminescent signal of the sulfo-tag was then detected using a Sector Imager 2400 (MSD KK).

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

[0253] [Example 4] Obtaining scFv domains that bind to CD3ε and human CD137 from a dual scFv library through double-round selection (4-1) Panning strategy to improve the efficiency of obtaining scFv domains that bind to human CD137 In Example 3, we successfully isolated scFv domains binding to CD3ε and CD137, but the isolation efficiency was not very high. One strategy worth considering to improve this is alternative panning, in which different antigens are used in different panning rounds. This method allows selective pressure against both CD3ε and human CD137 to be applied to the dual scFv library in different rounds, but not simultaneously. To address this drawback, we used double-round selection, which has been reported to perform two pannings against one antigen in one round (one round refers to the recovery of phage from E. coli and infection of E. coli in phage display, and in vitro transcription and PCR amplification in ribosome display) (J Mol Biol. 1992 Aug 5;226(3):889-96). Although they used only one type of antigen in each panning round, we believe that double-round selection, in which two different antigens are used in each panning procedure, can more efficiently isolate antibodies specific to two different antigens.

[0254] (4-2) Double-round selection to obtain scFv domains that bind to human CD137 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 biotin-labeled human CD137 fused to human IgG1 Fc fragment (designated human CD137-Fc) were used as antigens. In Table 1, CD3 refers to panning with biotin-labeled CD3 peptide, CD137 refers to panning with biotin-labeled human CD137-Fc, and double refers to double-round selection.

[0255] (Table 1) TIFF2025143451000002.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 using an RNeasy mini kit (Cat. No. 74104, QIAGEN). The resulting mRNA library was translated using a PUREfrex1.0 (PF001-0.25, Genefrontier) cell-free in vitro translation system with DnaK GroE Mix and DS supplements (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 translation, and blocking buffer (one pack of SuperBlock Dry Blend Blocking buffer in TBS (Cat. No. 37545, Pierce) in a 200 mL milliQ tube) was also added. Panning was performed according to the general panning method with 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-coated or FG NeutrAvidin beads) or streptavidin-coated beads (Dynabeads M-280 Streptavidin or Dynabeads MyOne Streptavidin). T1 beads).

[0257] 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, allowing the library solution to contact with the library solution at 4°C for 60 minutes. After adding SuperBlock-blocked magnetic beads, the antigen-scFv complex was attached to the magnetic beads for 15 minutes at 4°C. The beads were washed two or three times with WBT buffer (50 mM Tris-acetate, 150 mM NaCl, 50 mM Mg-acetate, 0.1% Tween). After adding 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 then immediately 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 reverse transcriptase using primer SEQ ID NO:147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), then amplified by PCR using primer SEQ ID NO:148 and KOD-FX polymerase (TOYOBO). A T7 promoter gene was added to the amplified DNA library by PCR using primers SEQ ID NO: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.

[0258] Double-round selection was performed in rounds 3, 5, and 7 of Campaign 3. Specifically, 250 pmol of biotin-labeled CD3ε peptide was added to the prepared ribosome display library solution, allowing the library solution to contact with the library solution at 4°C for 60 minutes. After the addition of SuperBlock-blocked magnetic beads, the antigen-scFv complex was allowed to attach to the magnetic beads for 15 minutes at 4°C. 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 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 using PUREfrex 1.0 (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, allowing the library solution to contact with the library solution at 4°C for 60 minutes. After adding SuperBlock-blocked magnetic beads, the antigen-scFv complexes were allowed to attach to the magnetic beads for 15 minutes at 4°C. The beads were washed 3 to 10 times (depending on the panning round) with WBT buffer. After adding elution buffer, the beads were suspended at 50°C for 15 minutes and immediately 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 reverse transcriptase using primer SEQ ID NO:147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR using primer SEQ ID NO:148 and KOD-FX polymerase (Toyobo). A T7 promoter gene was added to the amplified DNA library by PCR with primers SEQ ID NOs:149 and 150.

[0259] (XX-2) Binding of scFv domains to CD3ε or CD137 (scFv ELISA) To evaluate the binding of the scFv domains by ELISA, FLAG tags were added to the DNA libraries recovered in rounds 6 and 7 by PCR using 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 E. coli. The VH sequences from each single colony of E. coli were analyzed. Several clones with different VH sequences were then collected from each panning campaign in rounds 6 and 7. Each scFv-FLAG gene was amplified from each colony using 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 adding 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 at least 1 hour. After removing 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 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 1:1000 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 1:2000 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 adding sulfuric acid. Color development was then assayed based on absorbance at 450 nm. The results are shown in Figure 9. All scFvs in campaigns 1 and 2 bound to either CD3ε or CD137. On the other hand, many scFvs in campaign 3 showed binding to both human CD137 and CD3ε. In other words, the efficiency of obtaining clones exhibiting binding activity to both CD3ε and a second antigen (human CD137) was successfully improved by double-round selection, in which two different antigens were used in each panning round.

[0260] (4-3) Double-round selection for additional panning to obtain more scFv domains that bind to human CD137 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 and double-round selections were used on the campaign 2 round 7 output library, and only double-round selection 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 through double-round selection and IdeS elution 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 biotin-labeled human CD137 fused to human IgG1 Fc fragment (designated human CD137-Fc) were used as antigens. In Table 2, CD3 refers to panning with biotin-labeled CD3 peptide, CD137 refers to panning with biotin-labeled human CD137-Fc, and double refers to double-round selection.

[0262] (Table 2) TIFF2025143451000003.tif27168

[0263] The scFv ribosome display library constructed in Example 2 was used for in vitro transcription (T7 RiboMAX™ Express Large-scale RNA production system, P1320, Promega) to prepare an mRNA scFv library. The synthesized mRNA was purified using an RNeasy mini kit (Cat. No. 74104, QIAGEN). The resulting mRNA library was translated using a PUREfrex1.0 (PF001-0.25, Genefrontier) cell-free in vitro translation system with DnaK GroE Mix and DS supplements (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 translation, and blocking buffer (one pack of SuperBlock Dry Blend Blocking buffer in TBS (Cat. No. 37545, Pierce) in a 200 mL milliQ tube) was also added. Panning was performed according to the general panning method with 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-coated 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, allowing the library solution to contact with the library solution at 4°C for 60 minutes. After adding SuperBlock-blocked magnetic beads, the antigen-scFv complex was attached to the magnetic beads at 4°C for 15 minutes. The beads were washed two or three times with WBT buffer. After adding elution buffer, the beads were suspended at 50°C for 15 minutes and immediately separated using a magnetic stand to recover the mRNA solution. The recovered mRNA and its mRNA were purified using the High Pure RNA Isolation kit (Roche). The purified mRNA library was converted to cDNA reverse transcriptase using primer SEQ ID NO:147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), then amplified by PCR using primer SEQ ID NO:148 and KOD-FX polymerase (TOYOBO). A T7 promoter gene was added to the amplified DNA library by PCR using primers SEQ ID NO: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 this procedure, 5 μL of 10 units / μL Fabricator was added together with 95 μL of WBT buffer, and the beads were suspended at 37°C for 10 minutes. The beads were then immediately separated using a magnetic stand to recover the mRNA solution. 100 μL of elution buffer was then added to the recovered mRNA and incubated at 50°C for 10 minutes.When the antigen was biotinylated CD3ε peptide, elution buffer alone was used as in round 1.

[0265] Double-round selection was performed in rounds 3, 5, and 7 of Campaign 6. Specifically, 250 pmol of biotin-labeled CD3ε peptide was added to the prepared ribosome display library solution, allowing the library solution to contact with the library solution at 4°C for 60 minutes. After the addition of SuperBlock-blocked magnetic beads, the antigen-scFv complexes were attached to the magnetic beads for 15 minutes at 4°C. 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 separated using a magnetic stand to recover the mRNA solution. Ribosomes (GeneFrontier) were 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 using PUREfrex 1.0 (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, allowing the library solution to contact with the library solution for 60 minutes at 4°C. After the addition of SuperBlock-blocked magnetic beads, the antigen-scFv complexes were allowed to attach to the magnetic beads for 15 minutes at 4°C. The beads were washed 3–10 times (depending on the panning round) with WBT buffer. 5 μL of 10 units / μL Fabricator was added along with 95 μL of WBT buffer, and the beads were suspended at 37°C for 10 minutes. The beads were immediately separated using a magnetic stand to recover the mRNA solution. 100 μL of elution buffer was then 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 reverse transcriptase using primer SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with primer SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). A T7 promoter gene was added to the amplified DNA library by PCR with primers SEQ ID NO: 149 and 150.

[0266] (4-5) Double-round selection and IdeS elution followed by additional panning to obtain more scFv domains that bind to human CD137 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 and double-round selections were used on the round 7 output libraries of both campaigns 5 and 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 domains by ELISA, FLAG tags were added to the DNA libraries recovered in rounds 6 to 8 in Examples 4-3, 4, and 5 by PCR using primers set forth in 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 E. coli. The VH sequences from each single E. coli colony were analyzed. Several clones with different VH sequences were then collected from each panning campaign. Each scFv-FLAG gene was amplified from each colony using primers set forth in SEQ ID NOs: 149 and 151. PUREfrex 1.0 ss was added to each amplified scFv gene and incubated at 37°C for 2 hours. After adding 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-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. The wells were then blocked with 250 μL of 2% skim milk-TBS for at least 1 hour. After removing 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 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 1:1000 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 1:2000 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 adding sulfuric acid. The color development was then 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 of antibody format to IgG1 and preparation of individual IgG1 molecules Eleven pools (shown in Table 3) were selected for further evaluation. The scFvs contained in these pools were converted into IgG (the VH and VL sequences of each clone were linked to the constant domains of human H and L chains, respectively), and their binding activities to CD3ε and CD137 were evaluated. The VH fragments of each pool were amplified by PCR using primers (SEQ ID NOs: 152 and 153) that specifically bind to the H chain in the library. The amplified VH fragments were incorporated into an animal expression plasmid already containing a human IgG1 CH1-Fc region. The prepared plasmid was used for expression in animal cells according to 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) TIFF2025143451000004.tif78128

[0270] (4-8) Evaluation of the CD3ε and human CD137 binding activity 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 biotin-labeled CD3ε peptide or biotin-labeled human CD137-Fc for at least 1 hour at room temperature. After washing each well with TBST to remove unbound biotin-labeled antigen, the wells were blocked with 20 μL of blocking buffer (2% skim milk / TBS) for at least 1 hour. The blocking buffer was then removed from each well. Ten μL of IgG-containing mammalian cell supernatants, diluted two-fold with 1% skim milk / TBS, were added to each well, and the plate was left to stand at room temperature for 1 hour to allow the IgG to bind to the biotin-labeled antigen in each well. Then, each well was washed with TBST. Goat anti-human kappa light chain alkaline phosphatase conjugate (BETHYL, A80-115AP) diluted in 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 containing Blue Phos Microwell Phosphatase Substrate System (KPL) was terminated by adding Blue Phos Stop Solution (KPL). The color development was then measured by absorbance at 615 nm. The measurement results are shown in Figure 11.

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

[0272] This suggests that antibodies binding 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, and that double-round selection could significantly improve the efficiency of obtaining such antibodies. In conventional alternative panning, the selection pressure changes in each panning round, which can lead to slower and slower concentration of ideal clones or even loss of ideal clones. By using double-round selection with two different antigens, the selection pressure in each panning round can be made uniform, which tends to result in smoother and more direct concentration of ideal clones.

[0273] [Example 5] Affinity maturation of antibody domains binding to CD3ε and human CD137 derived from a dual scFv library with a designed light chain library (5-1) Construction of a light chain library with the obtained heavy chains Although many antibodies that bind to both CD3ε and human CD137 were obtained in Example 4, their affinity for human CD137 was still low, and therefore affinity maturation was performed to improve their 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, we selected an antibody designated 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 present inventors constructed two different antibody format libraries: a VL-GS linker-VH scFv format and a Fab format. The design of the ribosome display dual antibody library is shown in Figure 12. As in Example 2, a portion 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 fragments described in Reference Example 4 were fused at the 3' end to a Gly / Ser-rich linker-dBBDu_115 VH-spacer gene (SEQ ID NO: 8) and at the 5' end to a T7 promoter with a 5' untranslated region (SEQ ID NO: 4) by PCR amplification to create a VL-VH scFv format library. The synthesized antibody VL library fragments described in Reference Example 4 were fused to a CL-spacer gene (SEQ ID NO: 9) at the 3' end and to a T7 promoter (SEQ ID NO: 4) with a 5' untranslated region at the 5' end by PCR amplification to create a Fab format library. The VH gene fragments of dBBDu_115 were also fused to a CH1 gene (SEQ ID NO: 10) at the 3' end and to a T7 promoter (SEQ ID NO: 4) with a 5' untranslated region at the 5' end by PCR amplification to create Fab format H chain fragments.

[0275] (5-2) Isolation of antibody domains that bind to both CD3ε and human CD137 The panning conditions are shown in Table 4. In Table 4, "double" means double-round selection, and "CD137Fc" means conventional panning against biotin-labeled human CD137-Fc. Biotin-labeled CD3ε peptide antigen (amino acid sequence: SEQ ID NO: 6) and biotin-labeled human CD137 fused to human IgG1 Fc fragment (designated human CD137-Fc) were used as antigens.

[0276] (Table 4) TIFF2025143451000005.tif61170

[0277] The scFv ribosome display library, Fab light chain ribosome display library, and Fab heavy chain constructed in Example 5-1 were used for in vitro transcription (T7 RiboMAX™ Express Large-scale RNA production system, P1320, Promega) to prepare an mRNA library and heavy chain mRNA. The synthesized mRNA was purified using an RNeasy mini kit (Cat. No. 74104, QIAGEN). The resulting mRNA library and Fab heavy chain were translated using a PUREfrex1.0 (PF001-0.25, Genefrontier) cell-free in vitro translation system with DnaK GroE Mix and DS supplements (PF003-0.5, PF004-0.5, PF005-0.5, Genefrontier). Subsequently, WBTH buffer was added to stop 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-coated or FG NeutrAvidin beads) or streptavidin-coated beads (Dynabeads M-280 Streptavidin or Dynabeads MyOne Streptavidin T1 beads).

[0278] Specifically, 60 pmol of biotin-labeled human CD137-Fc was added to magnetic beads for 60 minutes at 4°C, followed by the addition of c-block-e (Beacle) to block the beads for 60 minutes at 4°C. The antigen-coated magnetic beads and 2 nmol of free human IgG Fc domain were added to the prepared ribosome display library solution, allowing them to contact the library solution for 75 minutes at 4°C. The beads were incubated with WBT buffer for 2 minutes (Campaign 10) or 10 minutes (Campaigns 5, 6, and 9), after which 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 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 reverse transcriptase using primer SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR using primer SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). A T7 promoter gene was added to the amplified DNA library by PCR using primers 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 biotin-labeled human CD137-Fc was varied as shown in Table 5, and the number of washes was also varied as shown in Table 6. When the antigen was biotin-labeled human CD137-Fc in the second and subsequent rounds of campaigns 6, 9, and 10, mRNA was recovered using FabRICATOR (IdeS, a protease against the hinge region of IgG, GENOVIS). In this procedure, 5 μL of 10 units / μL Fabricator was added together with 95 μL of WBT buffer, and the beads were suspended at 37°C for 10 minutes. The beads were then immediately 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 biotin-labeled human CD137-Fc (pmol) TIFF2025143451000006.tif61170

[0281] Table 6: Number of washes TIFF2025143451000007.tif61170

[0282] In some campaigns, double-round selection was performed. Specifically, 150 pmol of biotin-labeled CD3ε peptide was added to magnetic beads for 60 minutes at 4°C, followed by the addition of c-block-e (Beacle) to block the beads for 60 minutes at 4°C. The antigen-coated magnetic beads were added to the prepared ribosome display library solution, allowing them to contact the library solution for 60 minutes at 4°C. 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 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 using PUREfrex 1.0 (GeneFrontier). Biotin-labeled CD137-Fc was added to the magnetic beads for 60 minutes at 4°C, followed by the addition of c-block-e (Beacle) to block the beads for 60 minutes at 4°C. The antigen-coated magnetic beads and 2 nmol of free human IgG Fc domain were added to the prepared ribosome display library solution, allowing them to contact the library solution for 60 minutes at 4°C. The beads were incubated with WBT buffer for 10 minutes, and the WBT buffer was then 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 along with 95 μL of WBT buffer to suspend the beads for 10 minutes at 37°C. The beads were then immediately separated using a magnetic stand to recover the mRNA solution. 100 μL of elution buffer was then 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 reverse transcriptase using primer SEQ ID NO: 147 and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific), and then amplified by PCR with primer SEQ ID NO: 148 and KOD-FX polymerase (TOYOBO). A T7 promoter gene was added to the amplified DNA library by PCR with primers SEQ ID NO: 149 and 150.

[0283] (5-3) Conversion of antibody format to IgG1 and preparation of individual IgG1 molecules The light chain genes of the scFv or Fab domain libraries derived from the affinity maturation panning described in Example 5-2 were converted to IgG, and their binding activity to CD3ε and CD137 was evaluated. VL-CL fragments from pools from campaigns 5, 6, and 9 were amplified by PCR using primers (SEQ ID NOs: 154 and 155) that specifically bind to the L chains in the libraries. The amplified VL-CL fragments were incorporated into animal expression plasmids and transformed into the DH5α E. coli strain. The resulting plasmids were also used to construct light chain expression vectors from campaign 10. The VL region genes were removed from the resulting expression vectors with restriction enzymes SfiI and KpnI. VL fragments from pools from 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 vectors. The number of collected colonies is shown in Table 7. The prepared plasmids were used for expression in animal cells according to the method described in 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) TIFF2025143451000008.tif32128

[0285] (5-4) Evaluation of the CD3ε and human CD137 binding activity 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 biotin-labeled CD3ε peptide, biotin-labeled human CD137-Fc, and biotin-labeled human IgG1 Fc region for at least 1 hour at room temperature. After washing each well of the plate with TBST to remove unbound biotin-labeled antigen, the wells were blocked with 20 μL of blocking buffer (2% skim milk / TBS) for at least 1 hour. The blocking buffer was then removed from each well. 10 μL of mammalian cell supernatant containing 20 μg / mL IgG, diluted 2-fold with 2% skim milk / TBS, was added to each well, and the plate was left to stand at room temperature for 1 hour to allow each IgG to bind to the biotin-labeled antigen in each well. Then, each well was washed with TBST. Goat anti-human kappa light chain alkaline phosphatase conjugate (BETHYL, A80-115AP) diluted in 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 containing Blue Phos Microwell Phosphatase Substrate System (KPL) was terminated by adding Blue Phos Stop Solution (KPL). The color development was then measured by absorbance at 615 nm. The measurement results are shown in Figure 13.

[0286] (5-5) Evaluation of simultaneous binding of IgG with the isolated 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) TIFF2025143451000009.tif43128

[0288] First, MyOne-T1 streptavidin beads were mixed with 0.625 pmol of biotin-labeled human CD137-Fc or biotin-labeled human Fc and incubated at room temperature for 10 minutes. Then, 2% skim milk / TBS was added to block the magnetic beads. The mixture 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. The magnetic beads were then 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 left to stand at room temperature for 1 hour to allow each IgG to bind to the biotinylated antigen in each well. Each well was then washed with TBST. Goat anti-human kappa light chain alkaline phosphatase conjugate (BETHYL, A80-115AP) diluted in 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, fluorescence in each well was detected. The measurement results are shown in Figure 14 and Table 9.

[0289] (Table 9) TIFF2025143451000010.tif66136

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

[0291] [Example 6] Isolation of 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 A dual Fab library for phage display was constructed using the antibody library fragments synthesized in Reference Example 4. The dual library was prepared with the heavy chain diversified as shown in Reference Example 4, while the light chain was fixed to the original sequence GLS3000 (SEQ ID NO: 1). The heavy chain library sequence derived from CE115HA000 by adding V11L / L78I mutations to the framework and further diversifying the CDRs as shown in Table 38 (Reference Example 4) was entrusted to DNA2.0, Inc., a DNA synthesis company, to obtain antibody library fragments (DNA fragments). The resulting antibody library fragments were inserted into phagemids amplified by PCR for phage display. GLS3000 was selected as the light chain. The constructed phagemids for phage display were transformed into E. coli by electroporation to generate E. coli carrying the antibody library fragments.

[0292] A phage library displaying Fab domains was produced from E. coli harboring the constructed phagemid by infection with helper phage M13KO7TC / FkpA, encoding the FkpA chaperone gene, and then incubated overnight in the presence of 0.002% arabinose at 25°C (this phage library is designated the DA library) or 0.02% arabinose at 20°C (this phage library is designated the DX library). M13KO7TC is a helper phage with a trypsin cleavage sequence inserted between the N2 and CT domains of the pill protein on the helper phage (see International Patent Application Publication No. 2002-514413). Introduction of an insert gene into the M13KO7TC gene has been previously described (see International Patent Application Publication No. WO2015046554).

[0293] (6-2) Double-round selection to obtain Fab domains that bind to CD3ε and human CD137 Fab domains binding 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), CD3ε peptide antigen biotin-labeled via a disulfide bond linker (Figure 15, designated C3NP1-27; amino acid sequence: SEQ ID NO: 145, synthesized by Genscript), human CD137 fused to a biotin-labeled human IgG1 Fc fragment (designated human CD137-Fc), and human CD137 fused to a SS-biotinylated human IgG1 Fc fragment (designated ss-human CD137-Fc) were used as antigens. ss-human CD137-Fc was prepared by using an EZ-Link Sulfo-NHS-SS-Biotinylation Kit (PIERCE, catalog no. 21445) on human CD137 fused to a human IgG1 Fc fragment. Biotinylation was carried out according to the manufacturer's instructions.

[0294] Phages were produced from E. coli carrying the constructed phage display phagemid. 2.5 M NaCl / 10% PEG was added to the culture medium of the phage-producing E. coli, 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. Panning was performed using a standard panning method using antigens immobilized on magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1-2), 191-203; Biotechnol. Prog. (2002) 18 (2) 212-20; and Mol. Cell Proteomics (2003) 2 (2), 61-9). The magnetic beads used were NeutrAvidin-coated (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated (Dynabeads M-280 Streptavidin) beads. Subtraction of magnetic beads and biotin-labeled human Fc was performed to exclude phage displaying antibodies that bound 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 for 60 minutes at room temperature. Magnetic beads were blocked with 2% skim milk / TBS containing free streptavidin (Roche) for at least 60 minutes at room temperature, washed three times with TBS, and then mixed with the incubated phage solution. After 15 minutes of incubation at room temperature, the beads were washed three times with TBST (TBS containing 0.1% Tween 20; TBS was available from Takara Bio Inc.), and then washed twice with 1 mL of TBS. Five μL of 100 mg / mL trypsin and 495 μL of TBS were added, followed by 15 minutes of incubation at room temperature. The beads were then immediately separated using a magnetic stand to recover the phage solution. The phage was then used to infect the E. coli strain by culturing the strain at 37°C for 1 hour with gentle agitation. The infected E. coli was inoculated onto a 225 mm x 225 mm plate, and then phages were collected from the culture medium of the inoculated E. coli to prepare a phage library solution. In this panning round 1 procedure, phage displaying antibodies that bind to human CD137 were enriched. In the second round of panning, 250 pmol of ss-human CD137-Fc was used as a biotin-labeled antigen, and the phage were washed three times with TBST and then twice with TBS. Elution was performed with 25 mM DTT at room temperature for 15 minutes, followed by digestion with trypsin. In the third and sixth rounds of panning, 62.5 pmol of C3NP1-27 was used as a biotin-labeled antigen, washed three times with TBST and then twice with TBS, eluted 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 biotin-labeled antigen, washed three times with TBST and then twice with TBS, eluted with 25 mM DTT for 15 minutes at room temperature, and then digested with trypsin.

[0296] (6-3) Binding of phage-displayed Fab domains to CD3ε or human CD137 Phage-containing culture supernatants were collected from each of the 96 single colonies of E. coli obtained by the above method according to a standard method (Methods Mol. Biol. (2002) 178, 133-145). The phage-containing culture supernatants were subjected to ELISA by the following procedure: streptavidin-coated microplates (384 wells, Greiner, catalog number 781990) were coated with 10 μL of TBS containing biotin-labeled antigen (biotin-labeled CD3ε peptide or biotin-labeled human CD137-Fc) overnight at 4°C or at room temperature for 1 hour. 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 at least 1 hour. After removing the TBS / 2% skim milk, the prepared culture supernatant was added to each well, and the plate was left at room temperature for 1 hour to allow the phage-displayed antibodies to bind to the antigens 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 clones showed binding to human CD3ε but not to human CD137 despite five rounds of panning against human CD137, which may be due to the lower sensitivity of this phage ELISA analysis on streptavidin-coated microplates; therefore, phage ELISA on streptavidin-coated beads was also performed.

[0297] (6-4) Binding of phage-displayed Fab domains to human CD137 (phage bead ELISA) First, streptavidin-coated magnetic beads, MyOne-T1 beads, were washed three times with a blocking buffer containing 0.5x Block Ace, 0.02% Tween, and 0.05% ProClin 300, and then blocked with this blocking buffer for at least 60 minutes at room temperature. After washing once with TBST, 0.625 pmol of ss-human CD137-Fc was added to the magnetic beads and incubated at room temperature for at least 10 minutes. The magnetic beads were then applied to each well of a 96-well plate (Corning, 3792 black round-bottom PS plate). 12.5 μL of each Fab-displaying phage solution was added to each well along with 12.5 μL of TBS, and the plate was left to stand at room temperature for 30 minutes to allow each Fab to bind to the biotin-labeled antigen in each well. Each well was then washed with TBST. Anti-M13(p8) Fab-HRP diluted in 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. After 2 minutes, fluorescence in each well was detected. The measurement results are shown in Figure 17.

[0298] Some clones showed clear binding to human CD137. This result indicated that some Fab domains binding to both human CD3ε and CD137 were also obtained from this designed library using the phage display panning strategy. Nevertheless, the binding to human CD137 was still weaker compared to the CD3ε peptide. The VH fragment of each human CD137-binding clone was amplified by PCR using primers (SEQ ID NOs: 157 and 158) that specifically bind to the phagemid vector, and the DNA sequence was analyzed. The results showed that all of 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 next 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 using a double-round selection method (7-1) Construction of a heavy chain phage display library with GLS3000 light chain A phage library displaying Fab domains was produced from E. coli harboring the constructed phagemid by infection with helper phage M13KO7TC / FkpA encoding the FkpA chaperone (SEQ ID NO: 17), followed by overnight incubation in the presence of 0.002% arabinose at 25°C (this phage library is designated the DA library) or in the presence of 0.02% arabinose at 20°C (this phage library is designated the DX library). M13KO7TC is a helper phage carrying a trypsin cleavage sequence inserted between the N2 and CT domains of the pill protein on the helper phage (see Japanese Patent Application Publication No. 2002-514413). Introduction of an insert gene into the M13KO7TC gene has been previously described elsewhere (see WO2015 / 046554).

[0300] (7-2) Double-round selection to obtain Fab domains that bind to CD3ε and human CD137 Fab domains binding 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) via a disulfide bond linker, and biotin-labeled human CD137 fused to a human IgG1 Fc fragment (designated 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 E. coli carrying the constructed phage display phagemid. 2.5 M NaCl / 10% PEG was added to the culture medium of the phage-producing E. coli, 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. Panning was performed using a standard panning method using antigens immobilized on magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9; J. Immunol. Methods. (2001) 247 (1-2), 191-203; Biotechnol. Prog. (2002) 18 (2) 212-20; and Mol. Cell Proteomics (2003) 2 (2), 61-9). The magnetic beads used were NeutrAvidin-coated (Sera-Mag SpeedBeads NeutrAvidin-coated) or streptavidin-coated (Dynabeads M-280 Streptavidin) beads. Subtraction of magnetic beads and biotin-labeled human Fc was performed to exclude phage displaying antibodies that bound to the magnetic beads themselves or 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 or DX library was added to the blocked magnetic beads and incubated at room temperature for 60 minutes or more, and the supernatant was then collected. 500 pmol of biotin-labeled human IgG1 Fc was added to new magnetic beads and 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 and 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 new magnetic beads and 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 recovered phage solution was added to the blocked magnetic beads, and 8 nmol of free human IgG1 Fc domain was also added, followed by incubation 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 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 separated using a magnetic stand to recover the phage solution. The recovered phage solution was added to E. coli strain ER2738 in logarithmic growth phase (OD600: 0.4-0.5). The strain was incubated at 37°C for 1 hour with gentle agitation to allow the phage to infect the E. coli strain. The infected E. coli was inoculated into a 225 mm x 225 mm plate. Next, phages were collected from the culture medium of the inoculated E. coli to prepare a phage library solution.

[0303] Since phages displaying antibodies that bind to human CD137 were enriched in this panning round 1 procedure, a double-round selection was performed to recover phages displaying antibodies that bind to both CD3ε and human CD137 from the next round of the panning procedure.

[0304] Specifically, in panning round 2, the 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 was added to the blocked magnetic beads and incubated at room temperature for 60 minutes or more, and the supernatant was then collected. 500 pmol of biotin-labeled human IgG1 Fc was added to new magnetic beads and 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 and 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 new magnetic beads and 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 recovered phage solution was added to the blocked magnetic beads and then incubated at room temperature for 60 minutes. The beads were washed three times with TBST (TBS containing 0.1% Tween 20; TBS was available from Takara Bio Inc.), and then washed twice with 1 mL of TBS. Antibody-displaying phages were recovered using FabRICATOR (IdeS, a protease against the hinge region of IgG, GENOVIS) (referred to as the IdeS elution campaign). In this procedure, 20 μL of 10 units / μL Fabricator was added together with 80 μL of TBS buffer, and the beads were suspended at 37°C for 30 minutes. Immediately after, the beads were separated using a magnetic stand to recover the phage solution.

[0305] Because the first cycle of this panning procedure enriched phage displaying antibodies that bound to human CD137, we then proceeded to a second-cycle panning procedure to recover phage displaying antibodies that also bound to CD3ε before phage infection and amplification. 500 pmol of biotin-labeled CD3ε was added to new magnetic beads and incubated at room temperature for 15 minutes, followed by the addition of 2% skim milk / TBS. After blocking at room temperature for at least 60 minutes, the magnetic beads were washed three times with TBS. The recovered phage solution, 50 μL of TBS, and 250 μL of 8% BSA blocking buffer were added to the blocked magnetic beads and then incubated at 37°C for 30 minutes, room temperature for 60 minutes, overnight at 4°C, and then room temperature for 60 minutes to transfer the antibody-displaying phage from human CD137 to CD3ε. The beads were washed three times with TBST (TBS containing 0.1% Tween 20; TBS was available from Takara Bio Inc.) and then twice with 1 mL of TBS. The beads were suspended in 0.5 mL of 1 mg / mL trypsin at room temperature for 15 minutes, and then immediately separated using a magnetic stand to recover the phage solution. The phage recovered from the trypsinized phage solution was added to E. coli strain ER2738 in logarithmic growth phase (OD600: 0.4-0.7). The strain was cultured at 37°C for 1 hour with gentle agitation to infect the phage. The infected E. coli was inoculated onto a 225 mm x 225 mm plate. Next, phage were collected from the inoculated E. coli culture to recover the phage library solution.

[0306] In the third and fourth rounds of panning, the number of washes was increased to five with TBST and then two with TBS. In the second cycle of double-round selection, the C3NP1-27 antigen was used instead of the biotin-labeled CD3ε peptide antigen, and elution was performed with DTT solution to cleave the disulfide bond between the CD3ε peptide and biotin. Specifically, after two washes with TBS, 500 μL of 25 mM DTT solution was added and the beads were suspended at room temperature for 15 minutes. The beads were then immediately separated using a magnetic stand to recover the phage solution. 0.5 mL of 1 mg / mL trypsin was added to the recovered phage solution and incubated at room temperature for 15 minutes.

[0307] (7-3) Binding of IgGs with the isolated Fab domains to human CD137 and cynomolgus monkey CD137 From each panning output pool of the DA and DX libraries in rounds 3 and 4, 96 clones were collected and their VH gene sequences were analyzed. Twenty-nine VH sequences were obtained, all of which were converted to IgG format. The VH fragments of each clone were amplified by PCR using primers (SEQ ID NOs: 157 and 158) that specifically bind to the phagemid vector. The amplified VH fragments were incorporated into an animal expression plasmid already containing a human IgG1 CH1-Fc region. The prepared plasmid was used for expression in animal cells according to 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.

[0308] The prepared antibodies were subjected to ELISA to evaluate their binding ability to human CD137 (SEQ ID NO: 146) and cynomolgus monkey (called cynomolgus monkey) CD137 (SEQ ID NO: 18). Figure 18 shows the differences i...

Claims

[Claim 1] The invention described in the present specification.

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