RU2812875C1 - Cytotoxicity-inducing therapeutic agent - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody that specifically binds to CD3 epsilon has been proposed. A nucleic acid encoding an antibody, including said nucleic acid, an expression vector and a cell, and a method for producing the antibody are also proposed.

EFFECT: invention can be used to obtain drugs intended for treatment or prevention of various types of cancer.

7 cl, 15 dwg, 14 tbl, 4 ex

Description

Field of technology to which the invention relates

The present invention relates to multispecific antigen binding molecules, their uses and the like.

Prerequisites for creating the invention

Antibodies have attracted attention as therapeutic agents due to their high stability in plasma and low incidence of adverse reactions (Non-Patent Documents 1 and 2). Antibodies are known to induce not only antigen-binding activity, agonist action, and antagonist action, but also effector-mediated cytotoxic activities (also called effector functions), such as antibody-mediated cell-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement dependent cytotoxicity (CDC), and have antitumor effects against cancer cells (Non-Patent Document 3). ADCC is effector cell-mediated cytotoxicity against antibody-loaded target cancer cells through binding of the Fc region of the antibody to the Fc receptor present on effector cells such as NK cells and macrophages. The complement complex binds to the complement-binding site present in the antibody structure. CDC is harmful to cells as a result of cellular destruction, in which the influx of water and ions into cells is increased due to the formation of pores on the cell membrane of antibody-loaded cells with the help of complement components present in the complex. A number of therapeutic antibodies with established very high antitumor activities have been developed as pharmaceutical agents for the treatment of cancer (Non-Patent Document 4); and although it has been established that the known therapeutic antibodies have very high effects, the treatment outcome achieved with the use of these antibodies is still unsatisfactory.

For an antibody to mediate ADCC, ADCP and CDC, the presence of an Fc region in the antibody and the presence of a receptor for the antibody (FcγR) on effector cells such as NK cells and macrophages and various complement components available for binding are required. In humans, FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb isoforms have been described as the FcγR protein family, and corresponding allotypes have also been described (Non-Patent Document 5). Among these isoforms, FcγRIa, FcγRIIa and FcγRIIIa carry an immunoreceptor tyrosine-based activating motif (ITAM) in the intracellular domain and transmit activation signals. On the other hand, only FcγRIIb carries a domain known as the receptor tyrosine inhibitory motif (ITIM) in the intracellular domain and transmits inhibitory signals. It is known that each of the FcγRs signals through cross-linking by immune complexes and the like. (Non-Patent Document 6). When antibodies actually exhibit effector function on cancer cells, FcγRs on the membrane of the effector cell form clusters on the Fc regions of several antibodies bound on the membrane of the cancer cell, and activation signals are transmitted by the effector cells. The result is a cytocidal effect, but since FcγRs are only cross-linked on effector cells that are present in the vicinity of the cancer cells at that time, it is found that immune activation occurs locally in the cancer cells (Non-Patent Document 7).

Naturally occurring immunoglobulins bind to antigens in their variable regions and bind to receptors such as FcγR, FcRn, FcαR and FcεR, and complements in their constant regions. FcRn is one of the binding molecules that interact with the Fc region of IgG, and it is found that since each of the antibody heavy chains binds to one FcRn molecule, two FcRn molecules bind to one IgG antibody molecule. However, unlike FcRn et al., FcγR interacts with the antibody hinge region and CH2 domain, and only one FcγR molecule binds to one IgG-type antibody molecule (Non-Patent Document 8). In addition, the canonical naturally occurring IgG antibody recognizes and binds a single epitope through its variable region (Fab); therefore, it can only bind to one antigen. On the other hand, many types of proteins are known to be associated with cancer and inflammation and therefore crosstalk between proteins may occur. For example, it is known that several inflammatory cytokines (TNF, IL1 and IL6) may be involved in immunological diseases (Non-Patent Document 9). In addition, it is known that one of the mechanisms by which cancer acquires drug resistance is the activation of other receptors (Non-Patent Document 10). In such cases, canonical antibodies that recognize a single epitope cannot inhibit multiple proteins.

Antibodies (bispecific antibodies), one molecule of which binds to two or more types of antigens, have been studied as molecules that inhibit multiple targets. It is possible to obtain binding activities with two different antigens (a first antigen and a second antigen) by modifying naturally occurring IgG-type antibodies (Non-Patent Document 11). Thus, it is possible not only to neutralize two or more types of antigens with a single molecule, but also to enhance antitumor activity through cross-talk between cells that have cytotoxic activity and cancer cells. To date, the molecular formats of a bispecific antibody have been described as a molecule containing an antigen-binding site added to the N- or C-terminus of the antibody (DVD-Ig and scFv-IgG), a molecule having different sequences of the two Fab regions of the antibody (containing a common L- bispecific antibody chain and hybrid hybridoma), a molecule in which one Fab region recognizes two antigens (IgG two-in-one), and a molecule having a CH3 loop region as a new antigen-binding site (Fcab) (Non-Patent Documents 12 and 13) . Because all bispecific antibodies interact at their Fc regions with the FcγR, the effector functions of the antibody are preserved. Thus, the bispecific antibody binds to any antigen that it recognizes and simultaneously binds to FcγR and mediates ADCC activity against cells expressing the antigen.

If all of the antigens recognized by a bispecific antibody are specifically expressed in cancer, then the bispecific antibody is cytotoxic to cancer cells when it binds to any of the antigens. Thus, compared to a canonical pharmaceutical antibody that recognizes a single antigen, this antibody can be expected to have a more effective antitumor effect. However, when any one of the antigens recognized by the bispecific antibody is expressed in healthy tissues or cells expressed on immunocytes, damage to healthy tissues or release of cytokines due to cross-linking with FcγR occurs (Non-Patent Document 14). As a result, strong adverse reactions are induced.

A T cell redirecting antibody that exploits cytotoxicity by activating T cells as effector cells, which is the mechanism of its antitumor action, has been known since the 1980s (Non-Patent Documents 15, 16 and 17). Unlike antibodies that use ADCC by activating NK cells or macrophages as effector cells, which is the mechanism of their antitumor action, a T cell redirecting antibody is an antibody against any one of the subunits that make up the T cell receptor complex ( TCR) on T cells, and is a specific bispecific antibody that contains an antibody that binds to the CD3 epsilon chain and an antibody that binds to an antigen on the target cancer cell. T cells come into contact with cancer cells through the simultaneous binding of the CD3 epsilon chain and cancer antigen by a T cell redirecting antibody. This results in antitumor effects against cancer cells due to T cell-mediated cytotoxic activity.

Catumaxomab, a known T cell redirecting antibody, binds via two Fabs to cancer antigen (EpCAM) and to the CD3ε (CD3 epsilon) chain expressed on T cells. Catumaxomab induces T cell-mediated cytotoxic activity by simultaneously binding to cancer antigen and CD3ε, and induces cytotoxic activity mediated by antigen-presenting cells such as NK cells and macrophages by simultaneously binding to cancer antigen and FcγR. Due to these two types of cytotoxic activity, catumaxomab has a high therapeutic effect against malignant ascites when administered intraperitoneally and therefore its use is approved in Europe (Non-Patent Document 18). In addition, there are cases where the administration of catumaxomab reportedly leads to the formation of antibodies reactive against cancer cells, which clearly demonstrates the induction of acquired immunity (Non-Patent Document 19). Given this result, antibodies that have both T cell-mediated cytotoxic activity and FcγR-mediated actions of cells such as NK cells or macrophages (these antibodies are referred to as trifunctional antibodies) have attracted attention, since they can be expected to have a strong antitumor effect and induce acquired immunity.

However, trifunctional antibodies bind simultaneously to CD3ε and FcγR even in the absence of cancer antigen and therefore cross-link CD3ε-expressing T cells with FcγR-expressing cells even in the absence of cancer cells in the environment, resulting in the production of large quantities of various cytokines. This cancer antigen-independent induction of the production of various cytokines currently limits the use of trifunctional antibodies by the intraperitoneal route (Non-Patent Document 20). Trifunctional antibodies are very difficult to administer systemically due to severe cytokine storm-like adverse reactions. In fact, a phase I clinical trial found that the maximum tolerated dose of systemically administered catumaxomab to patients with non-small cell lung cancer was a very low dose of 5 μg/body, and that administration of a higher dose caused serious adverse reactions (Non-Patent Document 21) .

Thus, bispecific antibodies created using conventional techniques can bind to both antigens, with the first antigen being a cancer antigen (EpCAM) and the second antigen being CD3ε, while at the same time they bind to FcγR; and therefore, given their molecular structure, it is impossible to avoid unwanted reactions caused by simultaneous binding to FcγR and the second antigen CD3ε.

However, unlike catumaxomab, BiTE (bispecific T-cell activator (conductor)) does not contain an Fcγ receptor binding site and therefore does not cross-link with receptors that are expressed on T cells and cells such as NK cells and macrophages, in a cancer antigen-dependent manner. Thus, it was demonstrated that BiTE does not cause the cancer antigen-independent induction of cytokines that was found with catumaxomab. However, since BiTE is a modified small molecule antibody molecule without an Fc region, the problem is that its half-life in the blood after administration to a patient is significantly shorter than that of IgG-type antibodies that are commonly used as therapeutic antibodies. In fact, according to published data, the blood half-life of BiTE when administered in vivo is approximately several hours (Non-Patent Documents 22 and 23). In clinical trials, blinatumomab was administered by continuous intravenous infusion using a minipump. This method of administration is not only extremely inconvenient for patients, but also carries the potential risk of medical complications due to device malfunction or the like. Therefore, this method of administration cannot be considered to be desirable.

In recent years, the use of an Fc region with a reduced ability to bind to FcγR has allowed the potent antitumor activity of BiTE to be retained and the very good safety profile without inducing a “cytokine storm” in a cancer antigen-dependent manner, and has enabled the creation of new polypeptide structures with long half-lives in blood (patent document 1).

On the other hand, when a conventionally generated bispecific antibody is expressed, two types of H chains and two types of L chains are expressed, which can result in ten combinations. Of these, only one of the resulting combinations has binding specificity of interest. Thus, to obtain a bispecific antibody of interest, one antibody of interest must be isolated from ten types of antibodies, which is very inefficient and complex.

As a solution to this problem, a method has been described that consists of preferentially secreting IgG with a heterodimeric combination of H chains, for example, a combination of an H chain against antigen A and an H chain against antigen B, by introducing amino acid substitutions into the CH3 region of the H chain IgG (patent documents 2, 3, 4, 5, 6, 7 and non-patent documents 24 and 25). As such methods, methods based on physical disturbance are described, i.e. “protrusion” and “hollow”, and a method based on the repulsion of electric charges.

To obtain a molecule of interest with increased potency, methods based on the use of L chains that can bind to two different antigens, even though the L chains have the same amino acid sequence, have been described (Patent Documents 8 and 9). However, the affinity for the antigen can be significantly reduced when using common L chains, and it is difficult to find common L chains that retain the affinity for the antigen.

List of listed documents

[Patent documents]

[Patent Document 1] WO 2012/073985.

[Patent Document 2] WO 96/27011.

[Patent Document 3] WO 2006/106905.

[Patent Document 4] WO 2007/147901

[Patent Document 5] WO 2009/089004.

[Patent Document 6] WO 2010/129304.

[Patent Document 7] WO 2013/065708.

[Patent Document 8] WO 98/050431.

[Patent Document 9] WO 2006/109592.

[Non-patent documents]

[Non-Patent Document 1] Nat. Biotechnol. 23, 2005, pp. 1073-1078.

[Non-Patent Document 2] Eur J Pharm Biopharm. 59 (3), 2005, pp. 389-396.

[Non-Patent Document 3] Drug Des Devel Ther 3, 2009, pp. 7-16.

[Non-Patent Document 4] Clin Cancer Res. 16 (1), 2010, pp. 11-20.

[Non-Patent Document 5] Immunol. Lett. 82, 2002, pp. 57-65.

[Non-Patent Document 6] Nat. Rev. Immunol. 8, 2008, pp. 34-47.

[Non-Patent Document 7] Ann. Rev. Immunol. 6, 1988, pp. 251-281.

[Non-Patent Document 8] J. Bio. Chem., 276, 2001, pp. 16469-16477.

[Non-Patent Document 9] Nat. Biotech., 28, 2011, pp. 502-510.

[Non-Patent Document 10] Endocr Relat Cancer 13, 2006, ss. 45-51.

[Non-Patent Document 11] MAbs. March 1, 2012, 4(2).

[Non-Patent Document 12] Nat. Rev. 10, 2010, ss. 301-316.

[Non-Patent Document 13] Peds 23(4), 2010, ss. 289-297.

[Non-Patent Document 14] J. Immunol. 163(3), 1 August 1999, ss. 1246-1252.

[Non-Patent Document 15] Nature 314 (6012), 1985, ss. 628-631.

[Non-Patent Document 16] Int J Cancer 41 (4), 1988, ss. 609-615.

[Non-Patent Document 17] Proc Natl Acad Sci USA 83 (5), 1986, ss. 1453-1457.

[Non-Patent Document 18] Cancer Treat Rev. 36(6), October 2010, pp. 458-467.

[Non-Patent Document 19] Future Oncol. 8(1), January 2012, pp. 73-85.

[Non-Patent Document 20] Cancer Immunol Immunother. 56(9), 2007, pp. 1397-1406.

[Non-Patent Document 21] Cancer Immunol Immunother. 56 (10), 2007, pp. 1637-1644.

[Non-Patent Document 22] Cancer Immunol Immunother. 55(5), 2006, pp. 503-514.

[Non-Patent Document 23] Cancer Immunol Immunother. 58(1), 2009, pp. 95-109.

[Non-Patent Document 24] Protein Engineering, vol. 9, 1996, pp. 617-621.

[Non-Patent Document 25] Nature Biotechnology, vol. 16, 1998, pp. 677-681.

Summary of the invention

Objectives underlying the present invention

The present invention has been made taking into account the above circumstances. The purpose of the present invention was to create multispecific antigen binding molecules that have the ability to bring T cells into contact with target cancer cells and that can be used to treat cancer by exploiting the cytotoxic activity of T cells against cancer target tissues that contain glypican expressing tissues. 3 cells, and are molecular formats that can be produced with high efficiency; develop methods for producing antigen-binding molecules; and pharmaceutical compositions that contain antigen-binding molecules as active ingredients.

Means for solving these problems

The present invention describes an L chain shared with a domain that contains the variable region of a glypican 3 binding antibody and a domain that contains the variable region of an antibody that binds to a T cell receptor complex, wherein the common L chain has the ability to increase affinity to both antigens. This allows the production of molecular forms with high efficiency, and additionally new multispecific antigen-binding molecules have been described that retain the strong antitumor activity characteristic of T-cell redirecting antibodies such as BiTE and have very good safety characteristics without inducing a “cytokine storm” depending on from cancer antigen and also have a long half-life in the blood. In addition, the present invention has discovered that multispecific antigen binding molecules that contain common L chains target glypican 3 expressing cancer cells and cause cell damage. Based on this discovery, the present invention has found that the multispecific antigen binding molecules of the present invention cause damage to cancer tissues that contain glypican 3 expressing cancer cells.

More specifically, the present invention provides:

[1] A multispecific antigen-binding molecule that contains:

(1) a domain containing an antibody variable region that has binding activity for glypican 3,

(2) a domain containing an antibody variable region that has binding activity for the T cell receptor complex, and

(3) a domain containing an Fc region with reduced binding activity towards the Fcγ receptor,

wherein the L chain variable regions contained in the variable region specified in subparagraph (1) and in the variable region specified in subparagraph (2) have a common amino acid sequence; wherein the multispecific antigen binding molecule has cytotoxic activity equivalent to or greater than that of the bispecific antibody GPC3_ERY22_rCE115, which contains a glypican 3 binding domain containing SEQ ID NOs: 47 and 48, and a T cell receptor complex binding domain containing SEQ ID NO: 49 and 50.

[2] The multispecific antigen-binding molecule according to claim [1], the cytotoxic activity of which is a T cell-dependent cytotoxic activity.

[3] The multispecific antigen-binding molecule of claim [1] or claim [2], wherein the T-cell receptor complex binding activity is a T-cell receptor binding activity.

[4] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[3], wherein the T cell receptor complex binding activity is the CD3ε chain binding activity.

[5] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[4], in which the variable region of the antibody specified in subparagraph (1) in paragraph [1] is an antibody variable region that contains any one of the combinations of CDR1, CDR2 and CDR3 of the H chain selected from specified in subparagraphs (a1)-(a5) below, or a functionally equivalent antibody variable region:

(a1) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 40;

(a2) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 197;

(a3) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 206;

(a4) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 211; And

(a5) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 215.

[6] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[4], in which the variable region of the antibody specified in subparagraph (2) in paragraph [1] is an antibody variable region that contains any one of the combinations of the amino acid sequences CDR1, CDR2 and CDR3 of the H chain, selected from those indicated below in subparagraphs (b1) - (b15), or a functionally equivalent variable region of the antibody:

(b1) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 52;

(b2) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 103;

(b3) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 122;

(b4) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 128;

(b5) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 129;

(b6) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 132;

(b7) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 142;

(b8) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 144;

(b9) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 164;

(b10) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 168;

(b11) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 421;

(b12) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 424;

(b13) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 426;

(b14) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 429; And

(b15) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 430.

[7] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[4], in which the variable regions of the antibody specified in subparagraphs (1) and (2) in paragraph [1] are variable regions of the antibody that contain any one of the combinations of CDR1, CDR2 and CDR3 H- a chain selected from those specified in subparagraphs (c1) to (c19) below, or functionally equivalent antibody variable regions:

(c1) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 40; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 52;

(c2) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 40; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 421;

(c3) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 40; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 426;

(c4) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 40; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 429;

(c5) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 40; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 430;

(c6) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 197; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 128;

(c7) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 206; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 142;

(c8) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 206; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 144;

(c9) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 206; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 164;

(c10) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 206; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 168;

(c11) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 142;

(c12) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 144;

(c13) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 164;

(c14) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 168;

(c15) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103;

(c16) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 122;

(c17) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 129;

(c18) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 132; And

(c19) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; and CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 424.

[8] Multispecific antigen-binding molecule according to one of paragraphs. [5]-[7], in which CDR1, CDR2 and CDR3 are CDR1, CDR2 and CDR3 regions defined based on Cabot numbering.

[9] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[4], in which the variable region of the antibody specified in subparagraph (1) in paragraph [1] is an antibody variable region that contains any one of the H chain variable regions selected from those specified in the subparagraphs below (a1)-(a5), or functionally equivalent antibody variable region:

(a1) an H chain variable region which has the amino acid sequence of SEQ ID NO: 40;

(a2) an H chain variable region which has the amino acid sequence of SEQ ID NO: 197;

(a3) an H chain variable region which has the amino acid sequence of SEQ ID NO: 206;

(a4) an H chain variable region which has the amino acid sequence of SEQ ID NO: 211; And

(a5) an H chain variable region which has the amino acid sequence of SEQ ID NO: 215.

[10] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[4], in which the variable region of the antibody specified in subparagraph (2) in paragraph [1] is an antibody variable region that contains any one of the H chain variable regions selected from those specified in the subparagraphs below (b1)-(b15), or functionally equivalent antibody variable region:

(b1) an H chain variable region which has the amino acid sequence of SEQ ID NO: 52;

(b2) an H chain variable region which has the amino acid sequence of SEQ ID NO: 103;

(b3) an H chain variable region which has the amino acid sequence of SEQ ID NO: 122;

(b4) an H chain variable region which has the amino acid sequence of SEQ ID NO: 128;

(b5) an H chain variable region which has the amino acid sequence of SEQ ID NO: 129;

(b6) an H chain variable region which has the amino acid sequence of SEQ ID NO: 132;

(b7) an H chain variable region which has the amino acid sequence of SEQ ID NO: 142;

(b8) an H chain variable region which has the amino acid sequence of SEQ ID NO: 144;

(b9) an H chain variable region which has the amino acid sequence of SEQ ID NO: 164;

(b10) an H chain variable region which has the amino acid sequence of SEQ ID NO: 168;

(b11) an H chain variable region which has the amino acid sequence of SEQ ID NO: 421;

(b12) an H chain variable region which has the amino acid sequence of SEQ ID NO: 424;

(b13) an H chain variable region which has the amino acid sequence of SEQ ID NO: 426;

(b14) an H chain variable region which has the amino acid sequence of SEQ ID NO: 429; And

(b15) an H chain variable region which has the amino acid sequence of SEQ ID NO: 430.

[11] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[4], in which the antibody variable regions specified in subparagraphs (1) and (2) in paragraph [1] are antibody variable regions that contain any one of the combination of H chain variable regions selected from those listed below in subparagraphs (c1) - (c19), or functionally equivalent variable regions of the antibody:

(c1) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 40; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 52;

(c2) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 40; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 421;

(c3) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 40; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 426;

(c4) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 40; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 429;

(c5) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 40; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 430;

(c6) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 197; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 128;

(c7) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 206; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 142;

(c8) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 206; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 144;

(c9) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 206; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 164;

(b10) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 206; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 168;

(c11) an H chain variable region which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 211; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 142;

(b12) an H chain variable region which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 211; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 144;

(c13) an H chain variable region which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 211; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 164;

(c14) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 211; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 168;

(c15) an H chain variable region which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 215; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 103;

(c16) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 215; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 122;

(c17) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 215; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 129;

(c18) an H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 215; and an H chain variable region, which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 132; And

(c19) an H chain variable region which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 215; and an H chain variable region which is contained in the antibody variable region specified in subparagraph (2) in paragraph [1], which has the amino acid sequence of SEQ ID NO: 424.

[12] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[11], in which the common L-chain specified in paragraph [1] is a common L-chain that contains any one of the combinations of CDR1, CDR2 and CDR3 selected from those specified below in subparagraphs (d1 )-(g11), or the functional equivalent of the general L-chain:

(d1) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 53;

(d2) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 223;

(d3) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 299;

(d4) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 301;

(d5) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 302;

(d6) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 304;

(d7) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 306;

(d8) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDRS regions contained in SEQ ID NO: 307;

(d9) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 309;

(d10) CDR1, CDR2 and CDR3, identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 310; And

(g11) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 319.

[13] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[11], in which the variable region of the L-chain specified in paragraph [1] is the variable region of any one of the amino acid sequences of the L-chain selected from those indicated below in subparagraphs (d1)-(d11) :

(d1) an L chain containing the amino acid sequence of SEQ ID NO: 53;

(d2) an L chain containing the amino acid sequence of SEQ ID NO: 223;

(d3) an L chain containing the amino acid sequence of SEQ ID NO: 299;

(d4) an L chain containing the amino acid sequence of SEQ ID NO: 301;

(d5) an L chain containing the amino acid sequence of SEQ ID NO: 302;

(d6) an L chain containing the amino acid sequence of SEQ ID NO: 304;

(d7) an L chain containing the amino acid sequence of SEQ ID NO: 306;

(d8) an L chain containing the amino acid sequence of SEQ ID NO: 307;

(d9) an L chain containing the amino acid sequence of SEQ ID NO: 309;

(d10) L chain containing the amino acid sequence SEQ ID NO: 310; And

(d11) L chain containing the amino acid sequence of SEQ ID NO: 319.

[14] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[4], in which the variable regions of the antibody specified in subparagraphs (1) and (2) in paragraph [1] and the variable region of the common L chain are variable regions of the antibody containing any one of the combinations of CDR1 , CDR2 and CDR3 of the H chain and CDR1, CDR2 and CDR3 of the L chain, selected from those listed below in subparagraphs (e1)-(e25), or functionally equivalent variable regions of the antibody:

(e1) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 197; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 128; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 53;

(e2) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 197; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 128; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 299;

(e3) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 197; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 128; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 310;

(e4) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 197; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 128; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 319;

(e5) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 206; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 142; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 223;

(e6) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 206; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 144; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 223;

(e7) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 206; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 164; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 223;

(e8) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 206; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 168; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 223;

(e9) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 142; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 223;

(e10) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 142; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 299;

(e11) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 144; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 223;

(e12) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 164; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of SEQ ID NO: 223;

(e13) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 211; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 168; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 223;

(e14) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 53;

(e15) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 299;

(e16) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 301;

(e17) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 302;

(e18) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 304;

(e19) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 306;

(e20) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 307;

(e21) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 103; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 309;

(e22) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 122; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 53;

(e23) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 129; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 53;

(e24) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 132; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 53; And

(e25) CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 215; CDR1, CDR2 and CDR3, which are contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 424; and CDR1, CDR2 and CDR3, which are contained in the variable region of the general L chain of the antibody, and are identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 53.

[15] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[4], in which the variable regions of the antibody specified in subparagraphs (1) and (2) in paragraph [1], and the variable region of the common L chain are variable regions of the antibody containing any one of the combinations of variable regions selected from those specified in subparagraphs (e1)-(e26) below, or functionally equivalent antibody variable regions:

(e1) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 197; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 128; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 53;

(e2) an H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 197; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 128; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 299;

(e3) an H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 197; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 128; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 310;

(e4) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 197; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 128; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 319;

(e5) an H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 206; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 142; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 223;

(e6) the H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 206; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 144; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 223;

(e7) the H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 206; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 164; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 223;

(e8) the H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 206; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 168; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 223;

(e9) the H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 211; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 142; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 223;

(e10) the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 211; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 142; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 299;

(e11) an H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 211; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 144; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 223;

(e12) the H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 211; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 164; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 223;

(e13) the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 211; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 168; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 223;

(e14) an H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 103; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 53;

(e15) the H chain variable region, which is contained in the antibody variable region specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 103; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 299;

(e16) the H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 103; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 301;

(e17) the H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 103; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 302;

(e18) the H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 103; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 304;

(e19) the H chain variable region, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 103; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 306;

(e20) the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 103; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 307;

(e21) the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 103; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 309;

(e22) the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 122; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 53;

(e23) the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 129; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 53;

(e24) the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 132; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 53;

(e25) the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (1) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 215; the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph [1], and is identical to the amino acid sequence of SEQ ID NO: 424; and a variable region of the general L chain of the antibody identical to the amino acid sequence of the variable region contained in SEQ ID NO: 53; And

(e26) a multispecific antigen binding molecule that binds to an epitope overlapping with each of the epitopes of glypican 3 and the T cell receptor complex that binds the multispecific antigen binding molecule specified in one of subparagraphs (e1) to (e25), and which has a common L-chain.

[16] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[15], in which the Fc region specified in subparagraph (3) in paragraph [1] is an Fc region with an amino acid mutation of any of the amino acid sequences forming the Fc region SEQ ID NO: 23-26 (IgG1-IgG4).

[17] The multispecific antigen-binding molecule according to claim [16], wherein the Fc region specified in sub-clause (3) in clause [1] is an Fc region with a mutation of at least one amino acid selected from the amino acids in the following amino acid positions indicated according to EU numbering:

position 220, position 226, position 229, position 231, position 232, position 233, position 234, position 235, position 236, position 237, position 238, position 239, position 240, position 264, position 265, position 266, position 267 , position 269, position 270, position 295, position 296, position 297, position 298, position 299, position 300, position 325, position 327, position 328, position 329, position 330, position 331 and position 332.

[18] The multispecific antigen-binding molecule according to paragraph [16], wherein the Fc region specified in subparagraph (3) in paragraph [1] is an Fc region that contains at least one amino acid selected from the following amino acids, according to EU numbering:

Arg at amino acid position 234, Ala or Arg at amino acid position 235, Lys at amino acid position 239, and Ala at amino acid position 297.

[19] Multispecific antigen-binding molecule according to one of paragraphs. [16]-[18], in which the Fc region specified in subparagraph (3) in paragraph [1] additionally contains an amino acid mutation that enhances the formation of a heterodimeric Fc region.

[20] The multispecific antigen-binding molecule according to claim [19], wherein the heterodimeric Fc region is the following combination of (g1) or (g2) amino acid sequences:

(g1) a combination of an amino acid sequence identical to the constant region of the Fc region which contains the amino acid sequence of SEQ ID NO: 57, and an amino acid sequence identical to the constant region of the Fc region which contains the amino acid sequence of SEQ ID NO: 58; And

(g2) a combination of an amino acid sequence identical to the constant region of the Fc region which contains the amino acid sequence of SEQ ID NO: 60 or 62, and an amino acid sequence identical to the constant region of the Fc region which contains the amino acid sequence of SEQ ID NO: 61.

[21] Multispecific antigen-binding molecule according to one of paragraphs. [1]-[20], where the multispecific antigen binding molecule is a bispecific antibody.

[22] Bispecific antibody according to one of the following subparagraphs (h1)-(h25):

(h1) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 424, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 53;

(h2) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 103, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 53;

(33) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: 215 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 103, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 299;

(h4) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: 215 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 103, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 301;

(h5) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 103, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 302;

(36) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 103, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 304;

(h7) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: 215 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 103, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 306;

(38) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 103, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 307;

(39) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: 215 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 103, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 309;

(h10) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: 215 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 122, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 53;

(h11) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: 215 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 129 and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 53;

(312) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 215, and a constant region that has the amino acid sequence of SEQ ID NO: 215 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 132, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 53;

(313) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 197, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 128, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 299;

(314) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 197, and a constant region that has the amino acid sequence of SEQ ID NO: 197 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 128, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 310;

(315) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 197, and a constant region that has the amino acid sequence of SEQ ID NO: 197 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 128, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 319;

(316) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 197, and a constant region that has the amino acid sequence of SEQ ID NO: 197 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 128, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 53;

(317) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 211, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 142, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 299;

(318) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 211, and a constant region that has the amino acid sequence of SEQ ID NO: 211 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 142, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 223;

(319) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 211, and a constant region that has the amino acid sequence of SEQ ID NO: 211 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 144, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 223;

(320) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 206, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 144, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 223;

(321) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 206, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 142, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 223;

(322) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 206, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 164, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 223;

(323) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 206, and a constant region that has the amino acid sequence of SEQ ID NO: 206 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 168, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 223;

(324) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 211, and a constant region that has the amino acid sequence of SEQ ID NO: : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 164, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 223; And

(325) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 211, and a constant region that has the amino acid sequence of SEQ ID NO: 211 : 61; An antibody H chain that has binding activity for a T cell receptor complex, comprising an antibody H chain variable region that has the amino acid sequence of SEQ ID NO: 168, and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain having the amino acid sequence of SEQ ID NO: 223.

[23] Nucleic acid that encodes a multispecific antigen-binding molecule according to one of the paragraphs. [1]-[20] or a bispecific antibody according to paragraph [21] or paragraph [22].

[24] A vector into which the nucleic acid according to paragraph [23] is introduced.

[25] A cell containing a nucleic acid according to paragraph [23] or a vector according to paragraph [24].

[26] A method for producing a multispecific antigen-binding molecule according to one of paragraphs. [1]-[20] or a bispecific antibody according to item [21] or item [22], which consists in culturing the cell according to item [25].

[27] A multispecific antigen-binding molecule or bispecific antibody obtained/obtained by the method according to claim [26].

[28] Pharmaceutical composition containing a multispecific antigen-binding molecule according to one of paragraphs. [1]-[20] or a bispecific antibody according to paragraph [21] or paragraph [22] and a pharmaceutically acceptable carrier.

[29] The pharmaceutical composition according to claim [28], which induces cytotoxicity.

[30] The pharmaceutical composition according to claim [29], wherein the cytotoxicity is T-cell dependent cytotoxicity.

[31] The pharmaceutical composition according to claim [28], which is intended for administration to a patient in need of it, a multispecific antigen-binding molecule according to one of claims. [1]-[20] or a bispecific antibody according to paragraph [21] or paragraph [22].

The present invention also provides a kit for use in the method of the present invention, which contains a multispecific antigen binding molecule of the present invention or a multispecific antigen binding molecule produced by the method of the present invention. The present invention also relates to the use of a multispecific antigen binding molecule of the present invention or a multispecific antigen binding molecule obtained by the method of the present invention for the preparation of a pharmaceutical composition for promoting cytotoxic activity. The present invention also relates to a multispecific antigen binding molecule of the present invention or a multispecific antigen binding molecule produced by the method of the present invention for use in the method of the present invention. As used herein, multispecific antigen binding molecules include the bispecific antibodies of the present invention.

In addition, the present invention relates to a multispecific antigen binding molecule that contains the following domains:

(1) a domain containing an antibody variable region that has binding activity for glypican 3,

(2) a domain containing an antibody variable region that has binding activity for the T cell receptor complex;

wherein the L chain variable regions included in the variable regions specified in subparagraphs (1) and (2) have a common amino acid sequence. The present invention also relates to the domain referred to in subparagraph (1), which more specifically is a domain containing variable regions of the heavy chain and/or light chain of an antibody that have binding activity for glypican 3, and which is part of a multispecific antigen binding molecule . The present invention also relates to the domain referred to in subparagraph (2), which is more specifically a domain containing an antibody variable region that has binding activity for a T cell receptor complex, and which is part of a multispecific antigen binding molecule. The components of the domains specified in subparagraphs (1) and (2) may include those specified above in paragraphs. [1]-[22]. The multispecific antigen binding molecule may be a bispecific antibody. In addition, the multispecific antigen binding molecule may contain a domain including an Fc region and the Fc region may have reduced Fcγ receptor binding activity. Components of the domain containing the Fc region may include those specified in paragraphs above. [1]-[22]. The present invention relates to a nucleic acid encoding a multispecific antigen binding molecule or domains, a vector into which the nucleic acid is introduced, a cell that contains the nucleic acid or vector, a method for producing a multispecific antigen binding molecule that involves culturing cells, and a multispecific antigen binding molecule or domains that contain an antibody variable region that has binding activity for glypican 3 or binding activity for the T-cell receptor complex obtained by the method. In addition, the present invention relates to a pharmaceutical composition containing a multispecific antigen-binding molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition can induce cell damage, the cell damage can be due to T cell-dependent cellular cytotoxicity, and the composition can be administered to a patient who requires the use of a multispecific antigen binding molecule.

The present invention also relates to a multispecific antigen-binding molecule that binds to epitopes that overlap and/or compete with epitopes of both glypican 3 and the T-cell receptor complex that are bound by the multispecific antigen-binding molecule specified in one of subparagraphs (e1) to (e25) above. in paragraph [14], a multispecific antigen-binding molecule that binds to epitopes that overlap and/or compete with epitopes of both glypican 3 and the T-cell receptor complex bound by the multispecific antigen-binding molecule specified above in one of subparagraphs (e1)-( e25) in section [15].

Regarding the two Fc regions described above in subparagraphs (g1) and (g2) in paragraph [20], the first Fc region can be included in the H chain of an antibody, which has binding activity against glypican 3, and the second Fc region the region may be included in the H chain of an antibody that has binding activity for the T cell receptor complex; or the first Fc region may be included in an antibody H chain that has binding activity for the T cell receptor complex, and the second Fc region can be included in an antibody H chain that has binding activity for glypican 3.

Effects of the invention

The present invention provides novel multispecific antigen-binding molecules having molecular forms that can be produced with high efficiency, which retain the strong antitumor activity characteristic of BiTE and very good safety characteristics, resulting in the absence of induction of a cancer antigen-independent “cytokine storm” and etc., and have a long half-life in the blood. Pharmaceutical compositions that activate cytotoxic activity, containing as an active substance the multispecific antigen-binding molecule proposed in the present invention, specifically affect cancer tissues that contain glypican 3-expressing cancer cells, causing cell damage, and can be used to treat or prevent various types of cancer. The invention provides the required treatment, which not only has a high level of safety, but also reduces physical stress on patients and is very convenient for patients.

Brief description of drawings

The drawings show:

Fig. 1 schematic diagram a: ERY22 and b: ERY27;

in fig. 2 is a graph illustrating the cytotoxic activity of GPC3_ERY22_rCE115 and GPC3_ERY27_hCE115 when used as an NCI-H446 target cell. Filled diamonds ( ) and filled triangles ( ) indicates the cytotoxic activity of GPC3_ERY22_rCE115 and GPC3_ERY27_hCE115, respectively;

in fig. 3 is a graph illustrating the cytotoxic activity of GPC3_ERY22_rCE115 and GPC3_ERY27_hCE115 when used as a PC-10 target cell. Filled diamonds ( ) and filled triangles ( ) indicates the cytotoxic activity of GPC3_ERY22_rCE115 and GPC3_ERY22_rCE115, respectively;

in fig. 4 is a graph illustrating the cytotoxic activity of the optimized antibodies when used as a target cell NCI-H446;

in fig. 5 is a graph illustrating the cytotoxic activity of the optimized antibodies when used as a target cell NCI-H446;

in fig. 6 is a graph illustrating the cytotoxic activity of the optimized antibodies when used as a target cell NCI-H446;

in fig. 7 is a graph illustrating the cytotoxic activity of the optimized antibodies when used as a target cell NCI-H446;

in fig. 8 is a graph illustrating the cytotoxic activity of the optimized antibodies when used as a target cell NCI-H446;

in fig. 9 is a graph illustrating the cytotoxic activity of the optimized antibodies when used as a target cell NCI-H446;

in fig. 10 - data on the in vivo antitumor activity of optimized antibodies when used as a target cell PC-10;

in fig. 11 - data on the in vivo antitumor activity of optimized antibodies when used as a target cell NCI-H446;

in fig. 12 shows the relationship between the amino acid residues forming the Fc regions of IgG1, IgG2, IgG3 and IgG4 and the Cabot EU numbering system (indicated in the context of this description as the EU index);

in fig. 13-1 - sequences of heavy chain variable regions and their numbering according to Cabot et al.;

in fig. 13-2 - sequences of heavy chain variable regions and their numbering according to Cabot et al.;

in fig. 14 sequences of light chain variable regions and their numbering according to Cabot et al.

Embodiments of the Invention

The following definitions are provided to facilitate understanding of the present invention.

Antibody

As used herein, “antibody” refers to a naturally occurring immunoglobulin or an immunoglobulin produced in whole or in part by synthesis. Antibodies can be isolated from naturally occurring sources, such as naturally occurring plasma and serum, or from culture supernatants of antibody-producing hybridomas. Alternatively, antibodies can be partially or fully synthesized using techniques such as genetic recombination. Preferred antibodies are, for example, those belonging to an immunoglobulin isotype or subclass thereof. Known human immunoglobulins include antibodies of the following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM. Of these isotypes, the antibodies of the invention include IgG1, IgG2, IgG3 and IgG4.

Methods for preparing antibodies with the desired binding activity are known to those skilled in the art. The following is an example that describes a method for producing an antibody (anti-GPC3 antibody) that binds to glypican-3 (hereinafter referred to as GPC3), which belongs to the family of GPI-anchored receptors (Int J Cancer. 103(4), 2003 , pp. 455-465). According to the example described below, antibodies that bind to the T-cell receptor complex can also be produced.

Antibodies to GPC3 can be obtained as polyclonal or monoclonal antibodies using known methods. Antibodies to GPC3 are preferably obtained as monoclonal antibodies derived from mammals. These mammalian-derived antibodies include antibodies produced from hybridomas or host cells transformed with an expression vector that carries the antibody gene using genetic engineering techniques.

Hybridomas producing monoclonal antibodies can be obtained using known methods, for example, those described below. In particular, mammals are immunized using conventional immunization methods using the GPC3 protein as the sensitizing antigen. The resulting immune cells are fused with known parent cells using conventional fusion techniques. Anti-GPC3 antibody-producing hybridomas can then be selected by screening for monoclonal antibody-producing cells using conventional screening methods.

In particular, monoclonal antibodies are produced according to the method described below. First, the GPC3 gene, the nucleotide sequence of which is presented in RefSeq accession number NM_001164617.1 (SEQ ID NO: 1), can be expressed to obtain the GPC3 protein, the sequence of which is presented in RefSeq accession number NP_001158089.1 (SEQ ID NO: 2), which can be used as a sensitizing antigen to produce an antibody. To do this, the gene sequence encoding GPC3 is inserted into a known expression vector and the corresponding host cells are transformed with this vector. The desired human GPC3 protein is purified from host cells or culture supernatants using known methods. For example, to obtain soluble GPC3 from culture supernatants, the amino acid at positions 564-580, which forms the hydrophobic region corresponding to the GPI-anchoring sequence used to anchor GPC3 to the cell membrane, is removed by deleting the GPC3 polypeptide sequence SEQ ID NO: 2, and then the resulting protein is expressed in place of the GPC3 protein having SEQ ID NO: 2. Alternatively, purified naturally occurring GPC3 protein can be used as the sensitizing antigen.

Purified GPC3 protein can be used as a sensitizing antigen for immunization of mammals. Partial GPC3 peptides can also be used as sensitizing antigens. In this case, partial peptides can be obtained by chemical synthesis based on the amino acid sequence of human GPC3. In addition, they can also be obtained by inserting part of the GPC3 gene into an expression vector and expressing it therein. In addition, they can be obtained by cleaving the GPC3 protein with a protease, however, a particular embodiment of the invention does not impose restrictions on the length and region of the GPC3 peptide used as a partial peptide. As the preferred region, any sequence of the amino acid sequence that corresponds to the amino acids at positions 524-563, or more preferably, any sequence of the amino acid sequence that corresponds to the amino acids at positions 537-563 in the amino acid sequence SEQ ID NO: 2 can be selected. any sequence from an amino acid sequence region not containing an amino acid sequence that corresponds to amino acids at positions 550-663 in amino acid sequence SEQ ID NO: 2. Preferably, any sequence corresponding to positions 544-553 may be selected, and more preferably, any sequence from amino acid sequence sequence corresponding to positions 546-551 in the amino acid sequence SEQ ID NO: 2. The number of amino acids forming a peptide that can be used as a sensitizing antigen is preferably at least five or more, or preferably, for example, six or more, or seven or more. More specifically, a peptide consisting of 8-50 residues, more preferably 10-30 residues, can be used as the sensitizing antigen.

Alternatively, a fusion protein obtained by fusing the desired partial polypeptide or GPC3 protein peptide with another polypeptide can be used as the sensitizing antigen. For example, antibody Fc fragments and peptide tags are preferably used to produce fusion proteins for use as sensitizing antigens. Expression vectors for these fusion proteins can be constructed by in-frame fusion of genes encoding two or more of the desired polypeptide fragments and insertion of the fusion gene into the expression vector described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook J. et al., Molecular Cloning, 2nd ed. 1989, 9.47-9.58, Cold Spring Harbor Lab. Press). Methods for preparing GPC3 for use as a sensitizing antigen and methods for immunization using GPC3 are specifically described in WO 2003/000883, WO 2004/022754 and WO 2006/006693.

There is no specific limitation regarding the mammals to be immunized with the sensitizing antigen. However, it is preferable to select mammals based on their compatibility with the parent cells used for cell fusion. In general, rodents such as mice, rats, as well as hamsters, rabbits, and monkeys are preferably used.

The above animals are immunized with the sensitizing antigen using known methods. Common methods for immunizing mammals are, for example, intraperitoneal or subcutaneous injection of a sensitizing antigen. In particular, the sensitizing antigen can be suitably diluted in PBS (phosphate buffered saline), saline or the like. If necessary, a conventional adjuvant, such as Freund's complete adjuvant, is mixed with the antigen and the mixture is emulsified. The sensitizing antigen is then administered to the mammal several times at 4-21 day intervals. When immunizing with a sensitizing antigen, appropriate vehicles can be used. In particular, when a low molecular weight partial peptide is used as the sensitizing antigen, it is sometimes necessary to combine the peptide representing the sensitizing antigen with a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.

Alternatively, hybridomas that produce the desired antibody can be prepared by DNA immunization as described below. DNA immunization is an immunization method that provides immunostimulation through the expression of a sensitizing antigen in the immunized animal through the introduction of a DNA vector designed to cause expression of a gene encoding the antigenic protein in the animal. Compared to conventional immunization methods, in which the animals to be immunized are injected with a protein antigen, DNA immunization appears to have the following advantages:

- immunostimulation can be carried out while maintaining the structure of a membrane protein such as GPC3; And

- there is no need to purify the antigen for immunization.

To obtain the monoclonal antibody of the present invention using DNA immunization, DNA expressing the GPC3 protein is first administered to the animal to be immunized. DNA encoding GPC3 can be synthesized using known methods such as PCR. The resulting DNA is inserted into an appropriate expression vector and then it is administered to the animal to be immunized. Preferably, expression vectors used for this purpose include, for example, commercially available expression vectors such as pcDNA3.1. Vectors can be introduced into the body using conventional methods. For example, DNA immunization is carried out using a gene gun to introduce gold particles coated with an expression vector into the body cells of the animal to be immunized. Antibodies recognizing GPC3 can also be obtained by methods described in WO 2003/104453. After the above-described immunization of a mammal, an increased titer of GPC3-binding antibody is confirmed in its serum. After this, immune cells are obtained from the mammal's body and then used for cell fusion. In particular, splenocytes are preferably used as immune cells.

A mammalian myeloma cell is used as a cell to be fused with the above immunocyte. The myeloma cells preferably contain an acceptable selection marker for screening. A selection marker imparts characteristics to cells that ensure their survival (or death) under specific culture conditions. Hypoxanthine guanine phosphoribosyltransferase deficiency (abbreviated hereinafter as HGPRT deficiency) and thymidine kinase deficiency (abbreviated hereinafter as TK deficiency) are known as selection markers. Cells deficient in HGPRT or TK are sensitive to hypoxanthine-aminopterin-thymidine (abbreviated hereinafter as HAT-sensitivity). Cells with HAT sensitivity cannot synthesize DNA in the HAT selection medium and die as a result. However, when the cells are fused with healthy cells, they can continue to synthesize DNA using the healthy cell's "recycling" pathway, and as a result they can grow even in a HAT selection environment.

HGPRT-deficient and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (abbreviated hereinafter as 8AG) or 5'-bromodeoxyuridine, respectively. Healthy cells are destroyed because they incorporate these pyrimidine analogues into their DNA. Moreover, cells deficient in these enzymes can survive in the selection environment, since they cannot include these pyrimidine analogues. In addition, resistance to G418 is a selection marker, which is provided by the neomycin resistance gene, which confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogues). Various types of myeloma cells are known that can be used for cell fusion.

For example, the following cells can be preferably used as myeloma cells:

P3(P3x63Ag8.653) (J. Immunol. 123 (4), 1979, pp. 1548-1550);

P3x63Ag8U.l (Current Topics in Microbiology and Immunology 81, 1978, pp. 1-7);

NS-1 (C. Eur. J. Immunol. 6 (7), 1976, cc. 511-519);

MPC-11 (Cell 8 (3), 1976, pp. 405-415);

SP2/0 (Nature 276 (5685), 1978, pp. 269-270);

FO (J. Immunol. Methods 35 (1-2), 1980, cc. 1-21);

S194/5.XX0.BU.1 (J. Exp. Med. 148 (1), 1978, cc. 313-323);

R210 (Nature 277 (5692), 1979, pp. 131-133), etc.

Cellular fusion of immunocytes and myeloma cells is typically carried out using known methods, for example, the method described by Kohler and Milstein et al. (Methods Enzymol. 73, 1981, cc. 3-46).

More specifically, cell fusion can be carried out, for example, in a conventional culture medium in the presence of a cell fusion enhancing agent. Cell fusion enhancing agents include, for example, polyethylene glycol (PEG) and Sendai virus (hemagglutinating mouse virus of Japan) (HVJ). If necessary, an auxiliary substance such as dimethyl sulfoxide is also added to increase the efficiency of fusion.

The ratio of immunocytes to myeloma cells can be determined at the discretion of the investigator, preferably, for example, one myeloma cell for every 1-10 immunocytes. Culture media used for cell fusions include, for example, media suitable for growing myeloma cell lines, such as RPMI1640 medium and MEM medium, as well as other conventional culture media used for this type of cell culture. In addition, serum supplements such as fetal calf serum (FCS) can preferably be added to the culture medium.

For cell fusion, the above immune cells and myeloma cells taken in predetermined quantities are mixed well in the above culture medium. A PEG solution (eg, having an average molecular weight of about 1000 to 6000) preheated to about 37° C. is then added at a concentration of typically 30 to 60% (w/v). The mixture is gently stirred until the desired fused cells (hybridoma) are obtained. The above culture medium is then gradually added to the cells and centrifuged again to remove the supernatant. In this way, it is possible to remove cell fusion agents that are undesirable for hybridoma growth.

The hybridomas thus obtained can be selected by cultivation using a conventional selection medium, for example, HAT medium (culture medium containing hypoxanthine, aminopterin and thymidine). Cells other than the required hybridomas (unfused cells) can be destroyed by subsequent cultivation in the above HAT medium for a sufficient period of time. Typically, the period ranges from several days to several weeks. Hybridomas producing the desired antibody are then screened and individually cloned using conventional serial dilution methods.

The hybridromas thus obtained can be selected using a selection medium based on the selection marker carried by the myeloma cells used for cell fusion. For example, cells with HGPRT or TK deficiency can be selected by culturing using HAT medium (a culture medium containing hypoxanthine, aminopterin and thymidine). In particular, when HAT-sensitive myeloma cells are used for cell fusion, cells that are characterized by successful fusion with healthy cells can selectively multiply in the HAT environment. Cells other than the required hybridomas (unfused cells) can be killed by culturing in the above HAT medium for a sufficient period of time. In particular, the desired hybridomas can be selected by culturing, typically over a period of several days to several weeks. Hybridomas producing the desired antibody are then screened and individually cloned using conventional serial dilution methods.

The desired antibodies can preferably be selected and individually cloned using screening methods based on a known antigen/antibody reaction. For example, a GPC3-binding monoclonal antibody can bind to GPC3 expressed on the cell surface. These monoclonal antibodies can be screened using fluorescence excitation cell separation (FACS). FACS is a system that evaluates the binding of an antibody to a cell surface by analyzing cells in contact with a fluorescent antibody using a laser beam and by assessing the fluorescence emitted by individual cells.

To screen using FACS for hybridomas that produce the monoclonal antibody of the present invention, GPC3-expressing cells are first prepared. The cells that are preferably used for screening are mammalian cells that are forced to express GPC3. As a control, the ability of the antibody to bind to cell surface GPC3 can be selectively determined using untransformed mammalian host cells. In particular, hybridomas that produce a monoclonal antibody to GPC3 can be isolated by selecting for hybridomas that produce an antibody that binds to cells overexpressing GPC3 but not to host cells.

Alternatively, the ability of an antibody to bind to immobilized GPC3-expressing cells can be assessed based on the ELISA principle. For example, GPC3-expressing cells are immobilized onto the wells of an ELISA plate. Hybridoma culture supernatants are brought into contact with immobilized cells in the wells and antibodies that bind to the immobilized cells are detected. When the monoclonal antibodies are of murine origin, the cell-bound antibodies can be detected using an anti-mouse immunoglobulin antibody. Hybridomas producing the desired antibody that has antigen-binding activity are selected by the screening described above and can be cloned by serial dilution or similar methods.

The hybridomas obtained in this way, producing monoclonal antibodies, can be subcultured into a conventional culture medium and stored in liquid nitrogen for a long period of time.

The above hybridomas are cultured using a conventional method and the desired monoclonal antibodies can be obtained from the culture supernatants. Alternatively, hybridomas are introduced and grown into suitable mammals, and monoclonal antibodies are obtained from ascites. The first method is suitable for obtaining antibodies with a high degree of purity.

Preferably, antibodies encoded by antibody genes that are cloned from antibody-producing cells, such as the hybridomas mentioned above, can also be used. The cloned antibody gene is inserted into an appropriate vector and introduced into a host to express the antibody encoded by the gene. Methods for isolating antibody genes, inserting genes into vectors, and transforming host cells have been developed previously (see, for example, Vandamme et al., Eur. J. Biochem. 192(3), 1990, pp. 767-775). Methods for producing recombinant antibodies are also known and are described below.

For example, cDNA encoding the variable region (V region) of an anti-GPC3 antibody is prepared from hybridoma cells expressing an anti-GPC3 antibody. For this purpose, total RNA is first extracted from the hybridomas. Methods that are used to extract mRNA from cells include, for example:

- ultracentrifugation method in the presence of guanidine (Biochemistry 18(24), 1979, cc. 5294-5299) and

- AGPC method (Anal. Biochem. 162(1), 1987, pp. 156-159).

Extracted mRNA can be purified using an mRNA purification kit (GE Healthcare Bioscience) or similar kit. Alternatively, commercially available kits for extracting mRNA directly from cells, such as the QuickPrep mRNA purification kit (GE Healthcare Bioscience), can also be used. mRNA can be obtained from hybridomas using such kits. cDNAs encoding the V region antibodies can be synthesized from the resulting mRNAs using reverse transcriptase. cDNA can be synthesized using a first-strand cDNA synthesis kit containing AMV reverse transcriptase (Reverse Transcriptase First-strand cDNA Synthesis Kit) (from Seikagaku Co.) or the like. In addition, the SMART RACE cDNA amplification kit (Clontech) and the PCR-based 5'-RACE (Rapid Amplification of cDNA Ends) method (Proc. Natl. Acad. Sci. USA 85(23)) can be used to synthesize and amplify cDNA. ), 1988, pp. 8998-9002; Nucleic Acids Res. 17(8), 1989, pp. 2919-2932). In this cDNA synthesis process, the corresponding restriction enzyme sites described below can be introduced at both ends of the cDNA.

The cDNA fragment of interest is purified from the resulting PCR product and then inserted by ligation into a DNA vector. In this way, a recombinant vector is created and introduced into E. coli or a similar host. After colony selection, the desired recombinant vector can be prepared from colony-forming E. coli. It is then determined whether the recombinant vector has the cDNA nucleotide sequence of interest using a known method such as the dideoxy method.

The 5'-RACE method, which uses primers to amplify the variable region gene, is typically used to isolate the gene encoding the variable region. First, a cDNA library used for 5'-RACE is constructed using RNA extracted from hybridoma cells as a template. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize a 5'RACE cDNA library.

The antibody gene is amplified by PCR using the resulting 5'-RACE cDNA library as a template. Primers for amplifying a mouse antibody gene can be designed based on known antibody gene sequences. The nucleotide sequences of the primers vary depending on the immunoglobulin subclass. Thus, it is preferable to pre-subclassify using a readily available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

In particular, for example, to isolate the genes encoding murine IgG, primers are used that provide amplification of the genes encoding the heavy chains γ1, γ2a, γ2b and γ3 and the light chains κ and λ. In general, a primer whose hybridization site (“annealing”) to the constant region is located adjacent to the variable region is used as a 3'-end primer for amplification of the IgG variable region gene. In this case, a primer attached to a set of 5' RACE cDNA library constructs is used as a 5'-terminal primer.

PCR products amplified in this way are used to reconstruct immunoglobulins, consisting of a combination of heavy and light chains. The desired antibody can be selected using the GPC3 binding activity of the reconstituted immunoglobulin as an indicator. For example, when the goal is to isolate an antibody to GPC3, it is more preferable that the binding of the antibody to GPC3 is specific. You can screen for GPC3-binding antibodies, for example, using the following steps:

(1) contacting a GPC3-expressing cell with an antibody containing a V region that is encoded by cDNA isolated from the hybridoma;

(2) detect the binding of the antibody to the GPC3-expressing cell; And

(3) selecting an antibody that binds to the GPC3-expressing cell.

Methods for determining antibody binding to GPC3-expressing cells are known. In particular, antibody binding to GPC3-expressing cells can be determined using the techniques described above, such as FACS. Immobilized samples of GPC3-expressing cells can be used to assess antibody binding activity.

Preferred antibody screening methods that use binding activity as an indicator also include panning methods based on the use of phage vectors. Screening methods using phage vectors are advantageous when antibody genes are isolated from heavy chain and light chain subclass libraries from a population of cells expressing polyclonal antibodies. The genes encoding the heavy chain and light chain variable regions can be linked using a suitable linker sequence to form a single chain Fv (scFv). Phages presenting scFv on their surface can be produced by inserting the gene encoding scFv into a phage vector. Phages are brought into contact with the antigen of interest. DNA encoding the scFv that has the binding activity of interest can then be isolated by collecting phages bound to the antigen. This process can be repeated as necessary to enrich scFvs that have binding activity of interest.

After the cDNA encoding the V region of the anti-GPC3 antibody of interest is isolated, the cDNA is digested with restriction enzymes that recognize restriction sites introduced into both ends of the cDNA. Preferred restriction enzymes recognize and cleave a nucleotide sequence that occurs at low frequency in the nucleotide sequence of an antibody gene. In addition, to insert the single-copy cleavage fragment in the correct orientation, it is preferable to introduce into the vector of interest a restriction site for the enzyme that forms the sticky end. The anti-GPC3 antibody V coding region cDNA is digested according to the method described above and inserted into a suitable expression vector to create an antibody expression vector. When the gene encoding the constant region (C region) of an antibody and the gene encoding the above V region are fused in frame, a chimeric antibody is obtained. As used herein, “chimeric antibody” means that the constant region and the variable region differ in origin. Thus, in addition to mouse/human heterochimeric antibodies, the chimeric antibodies proposed in the present invention also include human/human allochimeric antibodies. A chimeric antibody expression vector can be created by inserting the above V region gene into an expression vector that already contains a constant region. In particular, for example, the sequence recognized by a restriction enzyme that excises the above V region gene should preferably be placed in the 5' region of an expression vector carrying DNA that encodes the constant region (C region) of the desired antibody. A chimeric antibody expression vector is created by in-frame fusion of two genes digested by the same combination of restriction enzymes.

To obtain a monoclonal antibody to GPC3, the antibody genes are inserted into an expression vector so that gene expression occurs under the control of an expression regulatory region. The expression regulatory region for antibody expression includes, for example, enhancers and promoters. In addition, an appropriate signal sequence can be attached to the amino terminus so that the antibody is secreted from the cells to the outside. In the examples described below, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) is used as a signal sequence. Along with it, other suitable signal sequences can be attached. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence and the resulting polypeptide is secreted out of the cells as a mature polypeptide. Suitable host cells are then transformed with the expression vector to produce recombinant cells expressing DNA that encodes an anti-GPC3 antibody.

DNAs that encode the antibody heavy chain (H chain) and the antibody light chain (L chain) are separately inserted into different expression vectors to express the antibody gene. An antibody molecule having H and L chains can be expressed by contransfecting the same host cell with vectors into which the H chain and L chain genes are respectively inserted. Alternatively, host cells can be transformed with a single expression vector into which DNA encoding the H and L chains is inserted (see WO 94/11523).

Various combinations of host cells/expression vectors are known for producing antibodies by introducing isolated antibody genes into appropriate hosts. All of these expression systems can be used to isolate antigen binding domains, including variable regions, of the antibodies of the present invention. Suitable eukaryotic cells that are used as host cells include animal cells, plant cells and fungal cells. In particular, animal cells are, for example, the following cells:

(1) mammalian cells: CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero or the like;

(2) amphibian cells: clawed frog (Xenopus) oocytes or the like. And

(3) insect cells: sf9, sf21, Tn5 or the like.

In addition, as a plant cell, an antibody gene expression system is known that uses cells obtained from members of the genus Nicotiana, for example, Nicotiana tabacum. Cultured callus cells can be used to transform plant cells.

In addition, the following cells can be used as fungal cells:

yeast: Saccharomyces genus, for example Saccharomyces cerevisiae, and Pichia genus, for example Pichia pastoris, and

filamentous fungi: of the Aspergillus genus, for example, Aspergillus niger.

In addition, antibody gene expression systems are known that use prokaryotic cells. For example, bacterial cells can be used according to the present invention, i.e. E. coli cells, Bacillus subtilis cells, etc. Expression vectors that carry the antibody genes of interest are introduced into these cells by transfection. The transfected cells are cultured in vitro, and the desired antibody can be obtained from a culture of transformed cells.

In addition to the above host cells, transgenic animals can also be used to produce a recombinant antibody. This means that the antibody can be obtained from an animal into which the gene encoding the antibody of interest has been introduced. For example, an antibody gene can be generated as a fusion gene by insertion in frame into a gene that encodes a protein specifically produced in milk. For example, goat β-casein can be used as a protein secreted into milk. DNA fragments containing a fusion gene that includes the antibody gene are injected into a goat embryo and the embryo is then introduced into a female goat. The desired antibodies can be obtained as a protein fused to milk protein from the milk of a transgenic goat born from the embryo recipient goat (or its offspring). In addition, to increase the volume of milk containing the desired antibody that is produced by the transgenic goat, hormones can be administered to the transgenic goat if necessary (Ebert K. M. et al., Bio/Technology 12 (7), 1994, pp. 699-702) .

When the antigen binding molecule provided herein is administered to a human, the domain of the antigen binding molecule may be a domain derived from an antibody produced by genetic recombination that has been artificially modified to reduce heterologous antigenicity in humans and other animals. These antibodies obtained by genetic recombination include, for example, humanized antibodies. These modified antibodies can be produced using known methods.

The antibody variable region used to obtain the antigen binding molecule domain that includes the variable region of the antibody provided herein typically consists of three hypervariable regions (CDRs) separated by four framework regions (FRs). The CDR is a region that largely determines the binding specificity of an antibody. CDR amino acid sequences are characterized by a high degree of variability. On the other hand, the amino acid sequences that form the FR often exhibit high identity even among antibodies with different binding specificities. Thus, generally, by transplanting a CDR, the binding specificity of a particular antibody can be introduced into another antibody.

A humanized antibody is also called a reshaped human antibody. In particular, humanized antibodies obtained by transplanting the CDR of an antibody from a non-human animal, such as a mouse antibody, into a human antibody and the like are known. There are also generally known genetic engineering techniques designed to produce humanized antibodies. In particular, for example, PCR with overlapping primers is a known method for transplanting the CDR of a mouse antibody into a human FR. When performing PCR with overlapping primers, the nucleotide sequence that encodes the CDR of the mouse antibody to be transplanted is added to the primers designed to synthesize the FR of the human antibody. Primers for each of the four FRs are prepared. It is generally accepted that when a murine CDR is transplanted into a human FR, it is advisable to select a human FR that has a high level of identity with the murine FR to maintain CDR function. Thus, it is generally preferred to use a human FR that contains an amino acid sequence having a high level of identity with the amino acid sequence of the FR that is adjacent to the murine CDR to be transplanted.

The nucleotide sequences to be ligated are designed so that they are connected to each other in reading frame. Human FRs are synthesized individually using appropriate primers. The result is products in which DNA encoding a mouse CDR is fused to DNA encoding individual FRs. The nucleotide sequences encoding the murine CDR of each product are designed to overlap each other. A complementary strand synthesis reaction is then carried out to anneal the overlapping CDR regions of the products synthesized using the human antibody gene as a template. Using this reaction, human FRs are inserted by ligation through mouse CDR sequences.

The full-length V region gene, into which three CDRs and four FRs are ultimately ligated, is amplified using 5' or 3' end hybridizing primers that are added with sequences recognized by suitable restriction enzymes. An expression vector for a humanized antibody can be prepared by inserting the DNA obtained according to the method described above and the DNA that encodes the C region of the human antibody into the expression vector so as to ligate them in frame. After transfecting a host with a recombinant vector to generate recombinant cells, the recombinant cells are cultured and DNA encoding the humanized antibody is expressed to produce a humanized antibody in cell culture (see European Patent Publication EP 239400 and International Patent Application Publication WO 1996/002576).

By qualitatively or quantitatively assessing and measuring the antigen binding activity of a humanized antibody prepared according to the method described above, human antibody FRs that allow the CDR to form a preferred antigen binding site when ligated using the CDR can be selected. The amino acid residues in the FR can be changed as needed so that the CDRs of the reshaped human antibody form a suitable antigen binding site. For example, amino acid sequence mutations can be introduced into the FR using the PCR method used to transplant a murine CDR into a human FR. More specifically, partial nucleotide sequence mutations can be introduced into primers that hybridize to FR. Nucleotide sequence mutations are introduced into FRs synthesized using the indicated primers. Mutant FR sequences having the desired characteristics can be selected by measuring and evaluating the antigen binding activity of the amino acid substitution mutant antibody using the method mentioned above (Sato K. et al., Cancer Res. 53, 1993, pp. 851-856 ).

Alternatively, the desired human antibodies can be produced by immunizing transgenic animals having the full range of human antibody genes (see WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO 1996/034096; WO 1996 /033735), using DNA immunization.

In addition, techniques for producing human antibodies by panning using libraries of human antibodies are known. For example, the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of a phage using a phage display technique. Phages that express a scFv that binds to an antigen can be selected. The DNA sequence encoding the V region of a human antibody that binds the antigen can be determined by analyzing the genes of selected phages. The DNA sequence of the scFv that binds to the antigen is determined. An expression vector is prepared by fusing the in-frame V region sequence to the C region sequence of the desired human antibody and then inserting it into a suitable expression vector. The expression vector is introduced into cells suitable for the above expression. A human antibody can be produced by expressing a gene encoding a human antibody in cells. Such methods have already been described (see WO 1992/001047; WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO 1995/001438; WO 1995/015388).

Domain containing an antibody variable region that has binding activity for glypican 3 (GPC3)

As used herein, the phrase “domain comprising an antibody variable region that has binding activity for glypican 3 (GPC3)” refers to a region of an antibody that contains a region that specifically binds to the GPC3 protein described above or to all or a portion of a partial peptide protein GPC3, and is also complementary to it. Antibody variable region-containing domains can be derived from the variable domains of one or more antibodies. Preferably, the antibody variable region containing domains comprise the light chain and heavy chain variable regions (VL and VH) of the antibody. Suitable examples of such domains containing antibody variable regions are “single chain Fv (scFv)”, “single chain antibody”, “Fv”, “single chain Fv2 (scFv2)”, “Fab”, “F(ab')2”, etc. .d.

Domain containing the variable region of an antibody that has binding activity for the T cell receptor complex

As used herein, the phrase “domain comprising an antibody variable region that has binding activity for a T cell receptor complex” refers to a region of an antibody that contains a region that specifically binds to all or a portion of the T cell receptor complex, and is also complementary to it. The T cell receptor complex may be the T cell receptor itself or an adapter molecule that is included in the T cell receptor complex along with the T cell receptor. A suitable adapter molecule is CD3.

Domain containing the variable region of an antibody that has T cell receptor binding activity

As used herein, the phrase “domain comprising an antibody variable region that has T cell receptor binding activity” refers to the T cell receptor binding region of an antibody obtained by including a region that specifically binds to all or a region of T -cellular receptor and is also complementary to it.

The region of the T cell receptor to which the domain of the present invention binds may be a variable region or a constant region, but an epitope present in the constant region is preferred. Examples of constant region sequences include the T cell receptor α chain, which has RefSeq accession no. CAA26636.1 (SEQ ID NO: 4), T-cell receptor β-chain, which has RefSeq accession No. C25777 (SEQ ID NO: 5), T-cell receptor γ1 chain, which has RefSeq accession No. A26659 (SEQ ID NO: 6), T cell receptor γ2 chain, which has RefSeq accession No. AAB63312.1 (SEQ ID NO: 7), and T cell receptor δ chain, which has RefSeq accession No. AAA61033.1 (SEQ ID NO : 8).

Domain containing the variable region of an antibody that has CD3 binding activity

As used herein, the phrase “a domain comprising an antibody variable region that has CD3 binding activity” refers to a CD3 binding region of an antibody that is obtained by including a region that specifically binds to all or a region of CD3 and that is also complementary thereto .

Preferably, the domain comprises the light chain and heavy chain variable regions (VL and VH) of the anti-CD3 antibody. Acceptable examples of such a domain are “single chain Fv (scFv)”, “single chain antibody”, “Fv”, “single chain Fv2 (scFv2)”, “Fab”, “F(ab')2”, etc.

The inventive domain containing an antibody variable region that has CD3 binding activity can be any epitope binding domain as long as the epitope is present in the γ chain, δ chain or ε chain sequence forming human CD3. Preferably, the present invention may use a domain comprising an anti-CD3 antibody light chain variable region (VL) and an anti-CD3 antibody heavy chain variable region (VH) that bind to an epitope present in the extracellular region of the ε chain of the human CD3 complex. In addition to the anti-CD3 antibody light chain variable region (VL) and the anti-CD3 antibody heavy chain variable region (VH), which are described in the Examples section, known CD3-binding domains that contain the anti-CD3 antibody light chain variable region (VL) , and the heavy chain variable region (VH) of the anti-CD3 antibody and derived from the OKT3 antibody (Proc. Natl. Acad. Sci. USA 77, 1980, cc. 4914-4917) can be used as these domains. Accordingly, an antibody variable region-containing domain derived from an anti-CD3 antibody that has the desired properties, generated by immunizing the desired animal using the method described above with the γ chain, δ chain or ε chain forming human CD3, can be used. Human antibodies and suitably humanized antibodies can be used, respectively, as an anti-CD3 antibody to generate an antibody variable region-containing domain that has CD3-binding activity. Regarding the structure of the γ chain, δ chain or ε chain forming human CD3, their polynucleotide sequences are shown in SEQ ID NO: 9 (NM_000073.2), 10 (NM_000732.4) and 11 (NM_000733.3), and their polypeptide sequences are presented in SEQ ID NO: 12 (NP 000064.1), 13 (NP 000723.1) and 14 (NP 000724.1) (RefSeq accession numbers are indicated in parentheses).

The antibody variable region domains in the antigen binding molecules of the present invention may bind to the same epitope. As used herein, the same epitope may be present in a protein that contains the amino acid sequence of SEQ ID NO: 2 or 14. Alternatively, antibody variable region containing domains in the antigen binding molecules of the present invention may bind to different epitopes respectively. As used herein, various epitopes may be present in a protein that contains the amino acid sequence of SEQ ID NO: 2 or 14.

Specificity

The term "specificity" means that one of the molecules involved in specific binding does not have any significant ability to bind to molecules other than one or more binding partners of the molecules. In addition, the concept is also used when the domain containing the variable region of the antibody is specific for a particular epitope of several epitopes included in the antigen. When the epitope to which the variable region-containing domain binds is included in several different antigens, antigen-binding molecules containing the variable region-containing domain can bind to different antigens that contain the epitope.

Epitope

"Epitope" means an antigenic determinant in an antigen and refers to the antigenic site to which the domain of an antigen binding molecule, including the variable region of an antibody, as described herein, binds. For example, an epitope can be characterized based on its structure. Alternatively, an epitope can be characterized based on the antigen binding activity of the antigen binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be determined by the amino acid residues that form the epitope. Alternatively, when the epitope is a sugar chain, the epitope can be determined by its specific sugar chain structure.

A linear epitope is an epitope that contains an epitope whose primary amino acid sequence is recognized. Said linear epitope typically contains at least three and most often at least five, for example about 8 to 10 or 6 to 20 amino acid residues in a particular sequence.

In contrast to a linear epitope, a “conformational epitope” is an epitope in which the primary amino acid sequence forming the epitope is not the sole determinant of the epitope being recognized (e.g., it is not necessary that the primary amino acid sequence of the conformational epitope be recognized by an epitope-specific antibody). Conformational epitopes may contain a larger number of amino acid residues compared to linear epitopes. A conformational epitope recognition antibody recognizes the three-dimensional structure of a peptide or protein. For example, when a protein molecule is folded into a three-dimensional structure, the amino acids and/or polypeptide backbones that form the conformational epitope are aligned and the epitope becomes recognizable to the antibody. Methods for determining epitope conformations include, but are not limited to, for example, x-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, ed. Morris, vol. 66, 1996).

An example of a method for assessing the binding of an epitope by a test antigen binding molecule containing a domain that contains an antibody variable region having GPC3 binding activity is described below, and a method for confirming binding to an epitope by a test antigen binding molecule containing a domain that contains an antibody variable region having binding activity in relation to the T-cell receptor complex, can also be carried out according to the examples below.

For example, to confirm that a test antigen binding molecule containing a domain that contains an antibody variable region having GPC3 binding activity recognizes a linear epitope on the GPC3 molecule, the method described below can be used. For the above purpose, a linear peptide is synthesized containing the amino acid sequence forming the extracellular domain of GPC3. The peptide can be synthesized chemically. Alternatively, it can be produced by genetic engineering techniques using a region in the GPC3 cDNA that encodes an amino acid sequence corresponding to the extracellular domain. Binding activity is then assessed between a linear peptide that contains an amino acid sequence forming an extracellular domain and a test antigen binding molecule containing a domain that contains an antibody variable region having GPC3 binding activity. For example, an immobilized linear peptide can be used as an antigen in an ELISA to evaluate the binding activity of an antigen binding molecule to the peptide. Alternatively, binding activity for a linear peptide can be assessed by the level of inhibition by the linear peptide of binding of an antigen-binding molecule to GPC3-expressing cells. These assays can demonstrate the binding activity of antigen-binding molecules to a linear peptide.

In addition, the following method can be used to confirm that a test antigen binding molecule containing a domain that contains an antibody variable region having GPC3 binding activity recognizes the three-dimensional structure of an epitope. For the above purpose, GPC3 expressing cells are prepared. A test antigen-binding molecule containing a domain that contains an antibody variable region having GPC3-binding activity is considered to recognize a conformational epitope if it binds strongly to GPC3-expressing cells upon contact but binds little to an immobilized linear peptide that contains amino acid sequence forming the extracellular domain of GPC3. As used herein, “slightly bound” means that the binding activity is 80% or less, typically 50% or less, preferably 30% or less, and most preferably 15% or less compared to the binding activity to cells expressing human GPC3.

Methods for assaying the binding activity of a test antigen-binding molecule containing a domain that binds a GPC3 antigen to GPC3-expressing cells include, for example, those described in: Antibodies: A Laboratory Manual (ed. Harlow, David Lane, Cold Spring Harbor Laboratory, 1988, pp. 359-420). In particular, the assessment can be carried out based on the ELISA principle or the fluorescence excitation-based cell separation (FACS) method using GPC3-expressing cells as the antigen.

In the ELISA format, the binding activity of a test antigen-binding molecule containing a domain that binds a GPC3 antigen to GPC3-expressing cells can be quantified by comparing the levels of signals generated during the enzymatic reaction. Specifically, the antigen-binding molecule to be tested is added to an ELISA plate on which GPC3-expressing cells are immobilized. The test antigen binding molecule bound to the cells is then detected using an enzyme-labeled antibody that recognizes the test antigen binding molecule. Alternatively, when FACS is used, serial dilutions of the test antigen-binding molecule are prepared and the titer of antibody binding to GPC3-expressing cells can be determined by comparing binding activity to GPC3-expressing cells.

The binding of a test antigen-binding molecule to an antigen that is expressed on the surface of cells suspended in a buffer or similar medium can be detected using a flow cytometer. Known flow cytometers include, for example, the following devices:

FACS Canto™ II,

FACSAria™

FACSArray™

FACSVantage™ SE

FACSCalibur™ (all trademarks of BD Biosciences),

EPICS ALTRA HyPerSort,

Cytomics FC 500,

EPICS XL-MCL ADC EPICS XL ADC,

Cell Lab Quanta/Cell Lab Quanta SC (all trademarks of Beckman Coulter).

Preferred methods for assaying the binding activity of a test antigen binding molecule containing a GPC3 antigen binding domain to an antigen include, for example, the following method. GPC3-expressing cells are first exposed to the antigen-binding molecule to be tested and then stained with an FITC-labeled secondary antibody that recognizes the polypeptide complex. The antigen binding molecule to be tested is suitably diluted with a suitable buffer to obtain the complex at the required concentration. For example, the complex can be used at a concentration ranging from 10 μg/ml to 10 ng/ml. The fluorescence intensity and cell number are then determined using a FACSCalibur (BD). The fluorescence intensity determined using assays based on the CELL QUEST program (BD), namely, expressed as geometric means, reflects the level of binding of the antibody to cells. This means that the binding activity of the test antigen binding molecule, which is characterized by the amount of bound test antigen binding molecule, can be assessed by determining the geometric mean value.

Whether a test antigen binding molecule that contains a GPC3 antigen binding domain shares an epitope with another antigen binding molecule can be assessed by competition between the two complexes for the same epitope. Competition between antigen-binding molecules can be determined by cross-blockade assay or similar assay. For example, a preferred cross-blockade assay is a competitive ELISA assay.

Specifically, in a cross-blockade assay, GPC3 protein immobilized in the wells of a microtiter plate is preincubated with or without a candidate antigen binding molecule and then the antigen binding molecule to be tested is added. The amount of GPC3 protein-bound test antigen-binding molecule in the wells is inversely correlated with the binding activity of the candidate antigen-binding molecule that competes for binding to the same epitope. This means that the higher the affinity of a candidate antigen binding molecule for the same epitope, the lower the binding activity of the test antigen binding molecule to GPC3 protein-sensitized wells.

The amount of test antigen-binding molecule bound via the GPC3 protein to the wells can be easily determined by pre-labeling the antigen-binding molecule. For example, a biotin-labeled antigen-binding molecule is assessed using an avidin/peroxidase conjugate and an appropriate substrate. In particular, a cross-blockade assay that uses enzymes such as peroxidase as labels is called a “competition ELISA.” The antigen-binding molecule can also be labeled with other labeling substances that can be detected or assessed. In particular, radioactive labels, fluorescent labels, etc. are known.

When the antigen binding molecule considered as a candidate competitor can block the binding of a test antigen binding molecule that contains a GPC3 antigen binding domain by at least 20%, preferably by at least 20-50%, and preferably by at least 50% Compared with the binding activity determined in a control experiment, which is carried out in the absence of the antigen-binding molecule considered as a competitor, the tested antigen-binding molecule is considered to have a strong ability to bind to the same epitope to which the antigen-binding molecule considered as a competitor binds. competitor, or it competes for binding to the same epitope.

If the structure of the epitope associated with a test antigen-binding molecule that contains a GPC3 antigen-binding domain has already been identified, then the binding activities of the two antigen-binding molecules with the peptide can be compared to determine whether the test and control antigen-binding molecules have the same epitope. , obtained by introducing amino acid mutations into the peptide that forms the epitope.

To evaluate the above binding activities, for example, compare the binding activities of test and control antigen binding molecules to a linear peptide into which a mutation has been introduced using the above ELISA format. In addition to the ELISA method, binding activity for a mutant peptide bound to a column can be determined by passing test and control antigen binding molecule through the column and then assessing the amount of antigen binding molecule eluted into the elution solution. Methods are known for adsorbing a mutant peptide onto a column, for example in the form of a GST fusion peptide.

Alternatively, when the identified epitope is a conformational epitope, the following method can be used to decide whether the test and control antigen-binding molecules share a common epitope. First, GPC3-expressing cells and GPC3-expressing cells with a mutation introduced into the epitope are prepared. The test and control molecules are added to a cell suspension prepared by suspending the cells in a suitable buffer such as PBS. The cell suspensions are then appropriately washed with buffer and an FITC-labeled antibody that recognizes the test and control antigen-binding molecules is added. The fluorescence intensity and number of cells stained with the labeled antibody were determined using a FACSCalibur (BD). The test and control polypeptide complexes are appropriately diluted with a suitable buffer and used at the required concentration. For example, they can be used at concentrations ranging from 10 μg/ml to 10 ng/ml. Fluorescence intensity is determined by analysis using the CELL QUEST program (BD) to determine the geometric mean value, which reflects the amount of labeled antibody bound to the cells. This means that the binding activities of test and control antigen-binding molecules, which are characterized by the amount of labeled antibody bound, can be determined by assessing the geometric mean.

When implementing the method described above to determine whether an antigen binding molecule exhibits “little binding to cells that express mutant GPC3,” the following method can be used, for example. First, test and control antigen-binding molecules bound to cells that express mutant GPC3 are stained with a labeled antibody. The fluorescence intensity in the cells is then determined. When a flow cytometer is used to evaluate fluorescence, the fluorescence intensity can be analyzed using the CELL QUEST program. Based on the geometric mean values with and without the antigen-binding molecule, a comparison value (ΔGeo-Mean), which characterizes the degree of increase in fluorescence intensity resulting from binding to the antigen-binding molecule, can be calculated according to the formula below.

ΔGeo-Mean = Geo-Mean (in the presence of antigen-binding molecule)/Geo-Mean (in the absence of antigen-binding molecule)

The geometric mean used for comparison (the ΔGeo-Mean value for the mutant GPC3 molecule), determined using the above analysis, which reflects the amount of the test antigen-binding molecule associated with cells that express the mutant GPC3, is compared with the relative AGeo-Mean value, which reflects the amount of test antigen-binding molecule bound to GPC3-expressing cells. In this case, the concentrations of the test antigen binding molecule used to determine the relative ΔGeo-Mean values for GPC3 expressing cells and mutant GPC3 expressing cells are most preferably adjusted to be the same or substantially the same. An antigen binding molecule confirmed to recognize an epitope in GPC3 is used as a control antigen binding molecule.

If the reference ΔGeO-Mean of the test antigen binding molecule against cells expressing mutant GPC3 is lower than the reference ΔGeO-Mean value of the test antigen binding molecule against cells expressing GPC3 by at least 80%, preferably 50%, more preferably 30% and most preferably 15%, the antigen-binding molecule tested is considered to “bind slightly to cells that express mutant GPC3.” The formula for determining the Geo-Mean value is described in the CELL QUEST software manual (BD biosciences). When the relative values are found to be substantially equivalent by comparison, the test and control antigen-binding molecules can be considered to have the same epitope.

Variable fragment (Fv)

As used herein, the term “variable fragment (Fv)” refers to the minimal unit of an antibody-derived antigen binding domain that consists of a pair comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In 1988, Skerra and Pluckthun discovered that homogeneous and active antibodies could be produced from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science 240(4855), 1988, pp. 1038-1041). In Fv obtained from the periplasmic fraction, VH associates with VL so as to bind antigen.

According to the present description, the Fv preferably includes, for example, a pair of Fvs that are an antigen binding molecule or the like containing:

(1) a bivalent antigen binding domain, which is a bivalent scFv in which one monovalent scFv of a bivalent scFv is linked to one polypeptide forming an Fc domain through the heavy chain of an Fv fragment forming a CD3 binding domain, and another monovalent scFv is linked to another a polypeptide forming an Fc domain via a light chain of an Fv fragment forming a CD3 binding domain;

(2) a domain containing an Fc domain that has no Fcγ receptor binding activity and that is derived from the amino acids forming the Fc domain of IgG1, IgG2a, IgG3, or IgG4, and

(3) at least one monovalent CD3 binding domain,

where the light chain and the heavy chain of the Fv fragments, as a result of association, form a CD3-binding domain that can bind to the CD3 antigen.

scFv, single chain antibody and sc(Fv)2

As used herein, the terms "scFv", "single chain antibody" and "sc(Fv)2" all refer to an antibody fragment in the form of a single polypeptide chain that contains variable regions derived from the heavy and light chains, but does not contain a constant region. Typically, a single chain antibody also contains a polypeptide linker between the VH and VL domains to form the desired structure that is likely to mediate antigen binding. The single-chain antibody is described in detail by Pluckthun in: “The Pharmacology of Monoclonal Antibodies”, vol. 113, ed. Rosenburg and Moore, Springer-Verlag, New York, 1994, cc. 269-315" (see also international patent publication WO 1988/001649; US No. 4946778 and 5260203). In a specific embodiment of the invention, the single chain antibody may be bispecific and/or humanized.

scFv is an antigen binding domain in which the VH and VL forming the Fv are linked to each other by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. 85(16), 1988, cc. 5879-5883). VH and VL can be held in close proximity by a peptide linker.

sc(Fv)2 is a single chain antibody in which the four variable regions of two VL and two VH are linked by linkers such as peptide linkers to form a single chain (J Immunol. Methods 231(1-2), 1999, pp. 177-189 ). Two VH and two VL can be obtained from various monoclonal antibodies. Said sc(Fv)2 preferably includes, for example, a bispecific sc(Fv)2 recognizing two epitopes that are present in one antigen, as described in Journal of Immunology 152(11), 1994, cc. 5368-5374. sc(Fv)2 can be prepared by methods known to those skilled in the art. For example, sc(Fv)2 can be produced by linking the scFv with a linker, such as a peptide linker.

As used herein, the form of the antigen binding domain forming sc(Fv)2 refers to an antibody in which two VH units and two VL units are organized in the following order: VH, VL, VH and VL ([VH]-linker-[VL]- linker-[VH]-linker-[VL]), starting from the N-terminus of the single-chain polypeptide. The arrangement order of the two VH units and two VL units is not limited to the above form, and they can be arranged in any order. An example of the order of arrangement in various forms is given below. [VL]-linker-[VH]-linker-[VH]-linker-[VL], [VH]-linker-[VL]-linker-[VL]-linker-[VH], [VH]-linker- [VH]-linker-[VL]-linker-[VL], [VL]-linker-[VL]-linker-[VH]-linker-[VH], [VL]-linker-[VH]-linker- [VL]-linker-[VH].

The molecular form of sc(Fv)2 is also described in detail in WO 2006/132352. Thus, according to these descriptions, those skilled in the art can obtain the desired sc(Fv)2 intended for the creation of antigen-binding molecules presented in the present description.

In addition, the antigen binding molecules of the present invention can be conjugated to a carrier polymer such as PEG or an organic compound such as an anticancer agent. Alternatively, additional sugar chain sequence is preferably inserted into polypeptide complexes in order for the sugar chain to provide the desired effect.

Linkers for binding antibody variable regions include arbitrary peptide linkers that can be introduced by genetic engineering, synthetic linkers and linkers described, for example, in: Protein Engineering, 9(3), 1996, cc. 299-305. However, for the purposes of the present invention, peptide linkers are most preferred. The length of the peptide linkers is not specifically limited and may be selected by those skilled in the art depending on the application at hand. The preferred length is five or more amino acids (with an upper limit of, but not limited to, typically up to 30 amino acids or less, preferably 20 amino acids or less), and most preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, their length may be the same or different.

For example, specified peptide linkers include:

Ser,

Gly⋅Ser,

Gly⋅Gly⋅Ser,

Ser⋅Gly⋅Gly,

Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 15),

Ser⋅Gly⋅Gly⋅Gly (SEQ ID NO: 16),

Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 17),

Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 18),

Gly⋅Gly⋅Gly⋅Gly⋅Gly Ser (SEQ ID NO: 19),

Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 20),

Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 21),

Ser⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 22),

(Gly⋅Gly⋅Gly⋅Gly⋅Ser (SEQ ID NO: 17))n,

(Ser⋅Gly⋅Gly⋅Gly⋅Gly (SEQ ID NO: 18))n,

where n denotes an integer of 1 or more. Those skilled in the art can select the length or sequence of peptide linkers depending on the task at hand.

As a rule, synthetic linkers (chemical cross-linking agents) are used for cross-linking, they are, for example:

N-Hydroxysuccinimide (NHS),

disuccinimidyl suberate (DSS),

bis(sulfosuccinimidyl)suberate (BS ³ ),

dithiobis(succinimidyl propionate) (DSP),

dithiobis(sulfosuccinimidyl propionate) (DTSSP),

ethylene glycolbis(succinimidyl succinate) (EGS),

ethylene glycolbis(sulfosuccinimidyl succinate) (sulfo-EGS),

disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),

bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES) and

bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These cross-linking agents are commercially available.

Typically, three linkers are required to join together the four variable regions of an antibody. The linkers used may be of the same type or of different types.

Fab, F(ab')2 and Fab'

A "Fab" consists of one light chain and a CH1 domain and a variable region of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bridges with the heavy chain of another molecule.

"F(ab')2" or "Fab" is produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and it refers to the antibody fragment produced by cleavage of the immunoglobulin (monoclonal antibody) near the disulfide bridges present between the hinge regions in each of the two H chains. For example, papain cleaves IgG in front of the disulfide bridges present between the hinge regions in each of the two H chains, producing two homologous antibody fragments in which the L chain containing VL (variable region of the L chain) and CL (constant region of the L chain) -chains) are linked to a fragment of the H-chain containing VH (variable region of the H-chain) and CHγ1 (γ1-region in the constant region of the H-chain), through a disulfide bridge in their C-terminal regions. Each of these two homologous antibody fragments is designated Fab'.

“F(ab')2” consists of two light chains and two heavy chains containing a CH1 domain and part of CH2 domains in the constant region, as a result of which disulfide bridges are formed between the two heavy chains. F(ab')2 forming the antigen binding molecule presented herein can preferably be prepared as follows. A complete monoclonal antibody or similar antibody containing the desired antigen-binding domain is partially cleaved with a protease such as pepsin; and the Fc fragments are removed by adsorption onto a protein A column. There is no limitation on a particular protease as long as it has the ability to selectively cleave the complete antibody to form F(ab')2 under reaction conditions appropriate to the enzyme, such as pH. Said proteases are, for example, pepsin and ficin.

Fc domain

The Fc domain that forms the antigen binding molecule provided herein can preferably be prepared as follows. An antibody, such as a monoclonal antibody, is partially cleaved with a protease such as pepsin. The resulting fragment is then adsorbed onto a Protein A or Protein G column and eluted with an appropriate elution buffer. There is no limitation on a particular protease as long as it has the ability to selectively cleave antibodies, such as monoclonal antibodies, under reaction conditions, such as pH, appropriate for the enzyme. Said proteases are, for example, pepsin and ficin.

Antigen-binding molecules provided herein contain an Fc domain with reduced Fcγ receptor binding ability, which include amino acids forming the Fc domain of IgG1, IgG2, IgG3 or IgG4.

The isotype of an antibody is determined based on the structure of the constant region. The constant regions of the IgG1, IgG2, IgG3, and IgG4 isotypes are designated Cγ1, Cγ2, Cγ3, and Cγ4, respectively. The amino acid sequences of the Fc domain of the polypeptides that form human Cγ1, Cγ2, Cγ3 and Cγ4 are exemplified in SEQ ID NOs: 23, 24, 25 and 26, respectively. The relationship between the amino acid residues forming each amino acid sequence and the Cabot EU numbering (referred to herein as the EU index) is shown in FIG. 12.

The term "Fc domain" refers to the region outside F(ab')2, which contains two light chains and two heavy chains containing part of the constant region, which includes the CH1 domain and the region between the CH1 and CH2 domains, which allows the formation of disulfide bridges between two heavy chains. The Fc domain forming the antigen binding molecule presented herein can preferably be prepared as follows. A monoclonal antibody in the form of IgG1, IgG2, IgG3 or IgG4 or a similar antibody is partially digested with a protease such as pepsin, followed by elution of the fraction adsorbed on the protein A column. There is no limitation on a particular protease as long as it has the ability to selectively digest the entire antibody with the formation of F(ab')2 under reaction conditions appropriate for a given enzyme, such as pH value. Said proteases are, for example, pepsin and ficin.

Fcγ receptor

The term "Fcγ receptor" refers to a receptor having the ability to bind to the Fc domain of monoclonal antibodies in the form of IgG1, IgG2, IgG3 or IgG4, and it includes all members belonging to the family of proteins that are encoded primarily by the Fcγ receptor gene. In humans, the family includes FcγRI (CD64), including the FcγRIa, FcγRIb, and FcγRIc isoforms; FcγRII (CD32), including isoforms FcγRIIa (including allotype H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including the FcγRIIIa isoform (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); and all unidentified human FcγRs, FcγR isoforms, and their allotypes. However, the Fcγ receptor is not limited to these examples. FcγRs include, but are not limited to, FcγRs derived from human, mouse, rat, rabbit and monkey antibodies. FcγR can be obtained from any organism. Murine FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as all unidentified mouse FcγRs, FcγR isoforms, and allotypes thereof. Said preferred Fcγ receptors include, for example, human FcγI (CD64), FcγIIA (CD32), FcγIIB (CD32), FcγIIIA (CD16) and/or FcγIIIB (CD 16). The polynucleotide sequence and amino acid sequence of FcγI are presented in SEQ ID NO: 27 (NM 000566.3) and 28 (NP 000557.1), respectively; the polynucleotide sequence and amino acid sequence of FcγIIA are presented in SEQ ID NO: 29 (BC020823.1) and 30 (AAH20823.1), respectively; the polynucleotide sequence and amino acid sequence of FcγIIB are presented in SEQ ID NO: 31 (BC146678.1) and 32 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of FcγIIIA are presented in SEQ ID NO: 33 (BC033678.1) and 34 (AAH33678.1), respectively, and the polynucleotide sequence and amino acid sequence of FcγIIIB are presented in SEQ ID NO: 35 (BC128562.1) and 36 (AAI28563. 1) respectively (the registration number of each sequence in the RefSeq database is indicated in parentheses). Whether the Fcγ receptor has the ability to bind to the Fc domain of a monoclonal antibody IgG1, IgG2, IgG3 or IgG4 can be assessed using the ALPHA Screen® assay ( Amplified Luminescent P roximity H omogeneous Assay )), BIACORE method based on surface plasmon resonance (SPR), etc. (Proc. Natl. Acad. Sci. USA 103(11), 2006, cc. 4005-4010), in addition to the FACS and ELISA formats described above.

The term “Fc ligand” or “effector ligand” refers to a molecule, and preferably a polypeptide, that binds to the Fc domain of an antibody to form an Fc/Fc ligand complex. The molecule can be obtained from any organism. Binding of Fc ligand to Fc preferentially induces one or more effector functions. These ligands include, but are not limited to, Fc receptors, FcγR, FcαR, FcεR, FcRn, C1q and C3, MBL, mannose receptor, Staphylococcus protein A, Staphylococcus protein G, and viral FcγRs. Fc ligands also include Fc receptor homologues (FcRH) (Davis et al., Immunological Reviews 190, 2002, pp. 123-136), which are a family of Fc receptors homologous to FcγRs. Fc ligands also include unidentified molecules that bind to Fc.

Fcγ receptor binding activity

Impaired binding activity of the Fc domain to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA and/or FcγIIIB can be assessed using the FACS and ELISA formats described above, as well as the ALPHA Screen assay (homogeneous proximity-enhanced luminescence assay, BIACORE method based on surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA 103(11), 2006, cc. 4005-4010).

ALPHA Screen analysis is carried out using ALPHA technology, which is based on the principle described below, using two types of granules. The luminescent signal becomes detectable only when there is a biological interaction between molecules bound to the donor beads and molecules bound to the acceptor beads, and when both beads are in close proximity to each other. The photosensitizer in the donor granules, excited by a laser beam, converts oxygen into excited singlet oxygen. When singlet oxygen diffuses from donor granules and reaches acceptor granules localized in close proximity, a chemiluminescent reaction is induced in the acceptor granules. This reaction ultimately results in the emission of light. If the molecules associated with the donor beads do not interact with the acceptor beads, then the singlet oxygen that is produced by the donor beads does not reach the acceptor beads and the chemiluminescent reaction does not occur.

For example, a biotin-labeled antigen-binding molecule is immobilized on donor beads, and a glutathione β-transferase (GST)-labeled Fcγ receptor is immobilized on acceptor beads. In the absence of an antigen-binding molecule containing a competing mutant Fc domain, the Fcγ receptor interacts with an antigen-binding molecule containing a wild-type Fc domain, resulting in a signal with a wavelength of 520 to 620 nm. An antigen-binding molecule containing an untagged mutant Fc domain competes with an antigen-binding molecule containing a wild-type Fc domain for interaction with the Fcγ receptor. Relative binding affinity can be assessed by quantifying the decrease in fluorescence due to competition. Methods for biotinylation of antigen-binding molecules such as antibodies using sulfo-NHS-biotin or similar agents are known. Suitable methods for introducing a GST tag into the Fcγ receptor include methods that involve in-frame fusion of polypeptides encoding Fcγ and GST, expression of the fusion gene using cells into which a vector carrying the gene has been introduced, and subsequent purification using a glutathione-containing column. . The induced signal can preferably be analyzed, for example, by fitting a single-site competition model based on nonlinear regression analysis using a program such as GRAPHPAD PRISM (GraphPad; San Diego).

One of the substances intended to study their interaction is immobilized as a ligand on a thin layer of gold on a sensor chip. When light penetrates the back surface of the sensor chip such that there is total reflection at the interface between the thin gold layer and the glass, the intensity of the reflected light at a particular site is partially reduced (SPR signal). Another substance, intended to study its interaction, is injected as an analyte onto the surface of the sensor chip. When an analyte binds to a ligand, the mass of the immobilized ligand molecule increases. This changes the refractive index of the solvent on the surface of the sensor chip. A change in refractive index causes a positive shift in the SPR signal (and conversely, dissociation shifts the signal back to its original position). In the Biacore system, the level of the shift described above (i.e., the change in mass on the surface of the sensor chip) is plotted along the vertical axis, and thus a graph of mass change versus time (sensogram) is obtained as quantitative data. Kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from sensorgrams presented in the form of curves, and affinity (KD) is determined as the ratio of these two constants. BIACORE methods are preferably used for inhibition assays. Examples of such inhibition assays are described in Proc. Natl. Acad. Sci. USA 103(11), 2006, cc. 4005-4010.

As used herein, “reduced Fcy receptor binding activity” means, for example, that using the assay described above, the competitive activity of the antigen binding molecule being tested is 50% or less, preferably 45% or less, 40% or less, 35% or less , 30% or less, 20% or less or 15% or less and most 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% less than the competitive activity of the control antigen binding molecule.

Antigen binding molecules containing the Fc domain of a monoclonal antibody as IgG1, IgG2, IgG3 or IgG4 can be used as control antigen binding molecules, respectively. The structures of the Fc domain are presented in SEQ ID NO: 37 (A added to the N-terminus of the sequence presented in RefSeq under accession number AAC82527.1), SEQ ID NO: 38 (A added to the N-terminus of the sequence presented in RefSeq under accession number number AAB59393.1), SEQ ID NO: 25 (A added to the N-terminus of the sequence presented in RefSeq under accession number CAA27268.1) and SEQ ID NO: 39 (A added to the N-terminus of the sequence presented in RefSeq under accession number number AAV59394.1). In addition, when an antigen binding molecule containing a mutant Fc domain of an antibody of a particular isotype is used as a test substance, the effect of the mutant mutation on Fcγ receptor binding activity is assessed using an antigen binding molecule containing an Fc domain of the same isotype as a control. As described above, antigen binding molecules are accordingly prepared containing a mutant Fc domain with truly reduced Fcγ receptor binding activity.

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

In particular, preferred antigen binding molecules are those that contain an Fc domain with an amino acid substitution at position 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266 , 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, or 332 (EU numbering) in the amino acids forming the Fc domain of an antibody of a particular isotype. The invention is not limited to the isotype of the antibody from which the Fc domain is derived, and a corresponding Fc domain derived from a monoclonal antibody in the form of IgG1, IgG2, IgG3 or IgG4 can be used. Preferably, an Fc domain derived from antibodies in the form of IgG1 should be used.

Preferred antigen binding molecules are, for example, molecules that contain an Fc domain containing one of the following substitutions, the position of which is determined according to EU numbering (each number indicates the position of an amino acid residue according to EU numbering; and a single letter amino acid symbol that appears before the number , denotes the amino acid residue before its substitution, and the single-letter amino acid symbol located after the number denotes the amino acid residue after the substitution), among the amino acids forming the Fc domain of the antibody in the form of IgG1:

(a) L234F, L235E, P331S;

(b) C226S, C229S, P238S;

(c) C226S, C229S;

(d) C226S, C229S, E233P, L234V, L235A;

(e) L234A, L235A or L235R, N297A;

(f) L235A or L235R, S239K, N297A,

as well as molecules that contain an Fc domain with a deletion of amino acids at positions 231-238.

In addition, preferred antigen-binding molecules are also molecules that contain an Fc domain containing one of the following substitutions, the position of which is determined according to EU numbering, among the amino acids that form the Fc domain of the IgG2 antibody:

(g) H268Q, V309L, A330S and P331S;

(h) V234A;

(i) G237A;

(k) V234A and G237A;

(l) A235E and G237A;

(m) V234A, A235E and G237A.

Each number indicates the position of an amino acid residue according to EU numbering; and the single-letter amino acid symbol that precedes the number indicates the amino acid residue before the substitution, and the single-letter amino acid symbol that follows the number denotes the amino acid residue after the substitution.

In addition, preferred antigen-binding molecules are those that contain an Fc domain containing one of the following substitutions, as determined by EU numbering, among the amino acids that form the Fc domain of an IgG3 antibody:

(n) F241A;

(o) D265A;

(n) V264A.

Each number indicates the position of an amino acid residue according to EU numbering; and the single-letter amino acid symbol that precedes the number indicates the amino acid residue before the substitution, and the single-letter amino acid symbol that follows the number denotes the amino acid residue after the substitution.

In addition, preferred antigen-binding molecules are those that contain an Fc domain containing one of the following substitutions, the position of which is determined according to EU numbering, among the amino acids that form the Fc domain of an IgG4 antibody:

(p) L235A, G237A and E318A;

(c) L235E;

(t) F234A and L235A.

Each number indicates the position of an amino acid residue according to EU numbering; and the single-letter amino acid symbol that precedes the number indicates the amino acid residue before the substitution, and the single-letter amino acid symbol that follows the number denotes the amino acid residue after the substitution.

Other preferred antigen binding molecules are those containing an Fc domain in which any amino acid at position 233, 234, 235, 236, 237, 327, 330 or 331 (EU numbering) among the amino acids forming the Fc domain of an IgG1 antibody , is replaced by an amino acid that is in the corresponding position according to the EU numbering in the corresponding IgG2 or IgG4.

Preferred antigen binding molecules are also molecules containing an Fc domain in which one or more amino acids at positions 234, 235 and 297 (EU numbering) among the amino acids forming the Fc domain of an IgG1 antibody are replaced by other amino acids . The invention is not limited to the type of amino acid after replacement; however, most preferred are antigen binding molecules containing an Fc domain in which one or more amino acids at positions 234, 235 and 297 are replaced by alanine.

Preferred antigen binding molecules are also those containing an Fc domain in which the amino acid at position 265 (EU numbering) among the amino acids forming the Fc domain of an IgG1 antibody is replaced by another amino acid. The invention is not limited to the type of amino acid after replacement; however, the most preferred are antigen binding molecules containing an Fc domain in which the amino acid at position 265 is replaced by alanine.

Multispecific antigen binding molecule

Examples of preferred embodiments of the "multispecific antigen binding molecule" of the present invention include multispecific antibodies. When an Fc region with reduced Fcγ receptor binding activity is used as an Fc region of a multispecific antibody, an Fc region derived from a multispecific antibody can accordingly be used. Bispecific antibodies are most preferred as the multispecific antibodies of the present invention. In this case, a bispecific antibody is an antibody having two different specificities. Bispecific IgG-type antibodies can be secreted from a hybrid hybridoma (quadroma), obtained by fusing two types of hybridomas that produce an antibody in the form of IgG (Milstein S. et al., Nature 305, 1983, pp. 537-540).

In addition, bispecific IgG antibodies are secreted through the introduction of a total of four genes into cells, i.e. genes of L-chains and H-chains forming the two types of IgG of interest, and their co-expression. However, there are theoretically ten combinations of IgG H chains and L chains that can be produced by such methods. However, it is difficult to purify IgG consisting of the desired combination of H chains and L chains from ten types of IgG. In addition, the theoretical amount of secreted IgG with the required combination is also greatly reduced, so large-scale cultivation is required. This further increases the cost of production.

Thus, for the multispecific antigen-binding molecules of the present invention, techniques can be used that enhance the association between H chains and between L and H chains having the desired combinations.

For example, techniques for inhibiting unwanted H chain association by introducing electrostatic repulsion at the interface of the second constant region or third constant region of an antibody H chain (CH2 or CH3) can be used for multispecific antibody association (WO 2006/106905).

When implementing the method of suppressing unwanted H-chain association by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues in contact at the interface with another constant region of the H-chain include regions corresponding to residues located according to EU numbering at positions 356 , 439, 357, 370, 399 and 409 in the CH3 region.

More specifically, examples include an antibody containing two types of H chain CH3 regions, in which 1 to 3 pairs of amino acid residues in the CH3 region of the first H chain are selected from the pairs of amino acid residues specified in subparagraphs (1) to (3) below ), carry the same type of charge: (1) amino acid residues in the CH3 region of the H chain, which are located, according to EU numbering, at positions 356 and 439; (2) amino acid residues in the CH3 region of the H chain, which are located according to EU numbering at positions 357 and 370; and (3) amino acid residues in the CH3 region of the H chain, which are located according to EU numbering at positions 399 and 409.

In addition, the antibody may be an antibody in which pairs of amino acid residues in the CH3 region of the second H chain, which is different from the above-mentioned CH3 region of the first H chain, are selected from the above amino acid pairs in subparagraphs (1) to (3). residues in which 1-3 pairs of amino acid residues that correspond to the pairs of amino acid residues above in subparagraphs (1)-(3), carrying the same type of charge as that indicated above for the CH3 region of the first H-chain, carry opposite charges relative to corresponding amino acid residues in the above CH3 region of the first H chain.

The corresponding amino acid residues indicated in subparagraphs (1)-(3) above are located close to each other when associated. Those skilled in the art can identify sites for a desired H chain CH3 region or H chain constant region that correspond to the above residues specified in subparagraphs (1) to (3) through homology modeling and the like using the available sales program, and the amino acid residues of these sites can be subject to modifications if necessary.

In the above antibodies, the “charged amino acid residues” are preferably selected, for example, from amino acid residues that are included in any one of the following groups (a) and (b):

(a) glutamic acid (E) and aspartic acid (D) and

(b) lysine (K), arginine (R) and histidine (H).

With respect to the above antibodies, "having the same type of charge" means, for example, that two or more amino acid residues are all amino acid residues included in any one of the above groups (a) and (b). The term “have an opposite charge” means, for example, that when at least one of two or more amino acid residues is an amino acid residue included in any one of the above groups (a) and (b), the remaining residues must be amino acid residues residues included in another group.

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

According to the present invention, the amino acid residues to be changed are not limited to the amino acid residues of the constant region or variable region of the antibody described above. For polypeptide mutants or heteromultimers, those skilled in the art can identify the amino acid residues that form the interface through homology modeling and the like using a commercially available program, and the amino acid residues of these sites can be modified as necessary to control the association .

Other known techniques can also be used to associate the multispecific antibodies of the present invention. Polypeptides with different amino acids in the Fc region can be efficiently associated with each other by replacing the side chain of an amino acid present in one of the variable regions of the antibody H chain with a larger side chain ("overhang") and replacing the side chain of the amino acid in the corresponding variable region another H-chain onto a smaller side chain (the "valley") so that the "ridge" fits into the "valley" (WO 1996/027011; Ridgway J.B. et al., Protein Engineering 9, 1996, pp. 617-621 ; Merchant A.M. et al., Nature Biotechnology 16, 1998, pp. 677-681 and US 20130336973).

In addition, other known techniques can also be used to associate the multispecific antibodies proposed in the present invention. Association of polypeptides having different sequences can be efficiently induced by complementary association of CH3 regions using an engineered strand exchange CH3 domain obtained by changing the CH3 portion of one of the antibody H chains to the corresponding IgA-derived sequence and introducing into the complementary CH3 portion in the other H chain corresponding to the IgA-derived sequence (Protein Engineering Design & Selection, 23, 2010, pp. 195-202). This known technique can also be used to efficiently obtain multispecific antibodies of interest.

In addition, the following methods can be used to obtain multispecific antibodies: methods for obtaining antibodies based on the association of CH1 and CL antibodies and the association of VH and VL, which are described in WO 2011/028952, WO 2014/018572 and in Nat Biotechnol. 32(2), February 2014, pp. 191-198; methods for producing bispecific antibodies using a combination of separately obtained monoclonal antibodies (Fab arm exchange, FAE), described in WO 2008/119353 and WO 2011/131746; methods for regulating the association between the CH3 domain of antibody heavy chains, described in WO 2012/058768 and WO 2013/063702; methods for producing bispecific antibodies consisting of two types of light chains and one type of heavy chain described in WO 2012/023053; methods for producing bispecific antibodies using two strains of bacterial cells that individually express one of antibody chains containing one H chain and one L chain, described by Christoph et al., Nature Biotechnology vol. 31, 2013, pp. 753-758).

Alternatively, even if the multispecific antibody of interest cannot be produced efficiently, the multispecific antibody of the present invention can be obtained by separating the multispecific antibody of interest from the resulting antibodies and purifying it. For example, a method is described that allows for the purification of two types of homomeric forms and a heteromeric antibody of interest using ion exchange chromatography by identifying differences in isoelectric points resulting from the introduction of amino acid substitutions into the variable regions of two types of H chains (WO 2007/114325). To date, a method has been described for the purification of heteromeric forms that uses Protein A to purify a heterodimeric antibody containing a mouse IgG2a H chain that binds to Protein A and a rat IgG2b H chain that does not bind to Protein A ( WO 98/050431 and WO 95/033844). In addition, the heterodimerized antibody itself can be efficiently purified using a Protein A column by altering the interaction between each of the H chains and Protein A by using H chains that have amino acid residues in the IgG-Protein A binding site at positions 435 and 436 (EU numbering) are replaced by amino acid residues that provide a different binding affinity for Protein A, such as Tyr, His, or the like, or using H chains with different affinities for Protein A, which are prepared according to the method specified in Example 5 for comparison, to change the interaction of each of the H chains with protein A, and then use a column with protein A.

Alternatively, a common L chain that can provide the ability to bind to multiple different H chains can be prepared and used as the common L chain of a multispecific antibody. To achieve efficient expression of multispecific IgG, genes for the common L chain and multiple different H chains can be introduced into cells and the IgG expressed (Nature Biotechnology, 16, 1998, pp. 677-681). The method of selecting a common L chain, which has a strong ability to bind to any different H chains, can also be used to select a common H chain (WO 2004/065611).

In addition, an Fc region characterized by improved C-terminal heterogeneity can be used as the Fc region of the present invention. More specifically, Fc regions are proposed that lack glycine at position 446 and lysine at position 447 (EU numbering) in the amino acid sequences of two polypeptides forming an Fc region that is derived from IgG1, IgG2, IgG3 or IgG4.

Several, for example two or more, of these techniques can be used in combination with each other. Moreover, these techniques can be suitably and separately applied to the two H chains to be associated. In addition, these techniques can be used in combination with the Fc region described above, which has reduced Fcγ receptor binding activity. Moreover, the antigen binding molecule of the present invention may be a molecule derived separately from the antigen binding molecule modified as described above such that it has the same amino acid sequence.

A suitable multispecific antigen binding molecule of the present invention comprises

(1) a domain containing an antibody variable region that has binding activity for glypican 3;

(2) a domain containing an antibody variable region that has binding activity for the T cell receptor complex; And

(3) a domain containing the specified Fc region with a reduced ability to bind to the Fcy receptor, its structure is not limited to this.

According to the present invention, each of the above domains can be cross-linked directly using peptide bonds. For example, when using F(ab')2 as a domain containing a variable region of the antibody specified in subparagraphs (1) and (2), and the specified Fc regions as a domain containing an Fc region with reduced ability to bind to Fcγ- receptor specified in subparagraph (3), then polypeptides formed by crosslinking the variable region-containing domains specified in subparagraphs (1) and (2) and the Fc region-containing domain specified in subparagraph (3) using peptide bonds, can form an antibody structure. Such antibodies can be obtained by purification from the above hybridoma culture medium, as well as by purifying antibodies from the culture medium of the desired host cells that stably carry polynucleotides encoding the polypeptides that form the antibody.

Examples of the preferred antibody H chain variable region of the present invention contained in the antibody variable region with glypican 3 binding activity include the antibody H chain variable regions that are shown in Table 1 or the antibody H chain variable regions that have CDR sequences whose amino acid sequences CDR1, CDR2 and CDR3 are the same as the amino acid sequences CDR1, CDR2 and CDR3 contained in the H chain variable regions that are listed in Table 1, or the antibody H chain variable regions that are functionally equivalent the above variable regions.

Examples of the preferred antibody variable region that has T-cell receptor complex binding activity of the present invention include antibody variable regions that have T-cell receptor binding activity. Of the T cell receptors, CD3 is the preferred one, and CD3ε is the most preferred. Examples of the antibody H chain variable region contained in these antibody variable regions include the antibody H chain variable regions which are shown in Table 2, or the antibody H chain variable regions which have CDR sequences whose amino acid sequences are CDR1, CDR2 and CDR3 are the same as the amino acid sequences CDR1, CDR2 and CDR3 contained in the H chain variable regions that are listed in Table 2, or the antibody H chain variable regions that are functionally equivalent to the above variable regions.

The relationship between the amino acid residues of the CDR regions forming the amino acid sequence of the antibody H chain and the Cabot EU numbering is presented in FIG. 13.

Regarding the antibody L chain variable regions contained in the antibody variable region that has glypican 3 binding activity and the antibody variable region that has T cell receptor complex binding activity of the present invention, it is preferable to obtain total L -a chain that can impart the binding activity of an H chain that has binding activity for glypican 3, and the binding activity of an H chain that has binding activity for the T cell receptor complex, and use it as a variable region of the common L- chains of a multispecific antigen-binding molecule.

Examples of the general L chain variable region that can be used according to the present invention include the L chain variable regions listed in Table 3, antibody L chain variable regions that have CDR sequences whose amino acid sequences CDR1, CDR2 and CDR3 are the same that the amino acid sequences CDR1, CDR2 and CDR3 contained in the antibody L chain variable regions shown in Table 3, and the antibody L chain variable regions that are functionally equivalent to the above variable regions.

The relationship between the amino acid residues of the CDR regions forming the amino acid sequence of the antibody L chain and the Cabot EU numbering is presented in FIG. 14.

In the present invention, the phrase "functionally equivalent" means that the antigen binding affinities are equivalent, or alternatively means that the cytotoxic activity on glypican 3 expressing cells or tissues containing such cells is equivalent when used as a multispecific antigen binding molecule. Binding affinity and cytotoxic activity can be measured using the method specified in the present description. The cells used to measure the cytotoxic activity may be the desired GPC3-expressing cells or the desired tissue containing the cells, and, for example, human cancer cell lines and PC-10 or NCI-H446, which express GPC3, can be used. With respect to antibody constant regions, the phrase may imply that the binding activity for the Fcγ receptor is equivalent.

For example, the phrase “an antibody H chain variable region functionally equivalent to the antibody H chain variable region specified herein (i.e., the original H chain variable region)” means that the region has the same binding affinity when it is combined with a variable region of the L chain of an antibody as defined herein which pairs with the parent H chain, or alternatively means that the region has the same cytotoxic activity against glypican 3 expressing cells or tissues containing said cells, when used to produce a multispecific antigen-binding molecule. In addition, the phrase “an antibody L chain variable region functionally equivalent to the antibody L chain variable region specified herein (i.e., the original L chain variable region)” means that the region has the same binding affinity as when combined with an antibody H chain variable region as defined herein that pairs with the parent L chain, or alternatively means that the region has the same cytotoxic activity against glypican 3 expressing cells or tissues containing said cells , when used to produce a multispecific antigen-binding molecule.

The term "equivalent" does not necessarily imply the same level of activity, and the activity may be increased. Specifically, the antigen binding affinity includes, for example, the case in which the coefficient (KD value/KD value of the parent antibody) obtained by comparison with the binding affinity of the variable region of the antibody that serves as a control (KD value of the parent antibody) is 1.5 or less. The KD value/KD value of the parent antibody is preferably 1.3 or less, more preferably 1.2 or less, 1.1 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0. 7 or less, 0.6 or less, or 0.5 or less. There is no lower limit, examples include factors of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 or 10 ^-6 . More specifically, according to the present invention, the KD value/KD value of the parent antibody is preferably 10 ^-6 to 1.5×10 ^-0 , more preferably 10 ^-6 to 10 ^-1 , even more preferably 10 ^-6 to 10 ^{- 2} and even more preferably from 10 ^-6 to 10 ^-3 . Regarding cytotoxic activity, examples include the case in which the coefficient (cell growth inhibition rate/cell growth inhibition rate of the parent antibody) obtained by comparison with the cell growth inhibition rate of a multispecific antigen binding molecule that serves as a control (cell growth inhibition rate of the parent antibody) , is 0.7 or more. The concentration of the added multispecific antigen binding molecule can be determined accordingly, but is preferably, for example, 0.01, 0.05, 0.1, 0.5 or 1 nM; and preferably measurements are carried out at a concentration of 0.05 or 0.1 nM. The cell growth inhibition rate/cell growth inhibition rate of the parent antibody is preferably 0.8 or more, more preferably 0.9 or more, 1.0 or more, 1.2 or more, 1.5 or more, 2 or more, 3 or more, 5 or more, 10 or more, or 20 or more. There is no upper limit; the coefficient can be 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ .

In addition, regarding cytotoxic activity, examples include the case in which the coefficient (concentration causing 50% cell growth inhibition/concentration of parent antibody causing 50% cell growth inhibition) obtained by comparison with the concentration of the parent multispecific antigen binding molecule causing cell growth inhibition 50% growth (the concentration of the parent antibody that causes 50% inhibition of cell growth) is 1.5 or less. The concentration causing 50% cell growth inhibition means the concentration of the multispecific antigen binding molecule required to reduce the rate of cell proliferation by half compared to a variant in which the multispecific antigen binding molecule is not added. The ratio "concentration causing 50% cell growth inhibition/concentration of parent antibody causing 50% cell growth inhibition" is preferably 1.3 or less, more preferably 1.2 or less, 1.1 or less, 1.0 or less than, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less. There is no lower limit, examples include factors of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 or 10 ^-6 . In particular, the coefficient is preferably from 10 ^-6 to 1.5×10 ^-0 , more preferably from 10 ^-6 to 10 ^-1 , even more preferably from 10 ^-6 to 10 ^-2 and even more preferably from 10 ^-6 to 10 ^-3 .

For a domain containing an antibody variable region that has GPC3 binding activity, the KD value indicative of binding to GPC3 (eg, human GPC3) may be, for example, 5×10 ^-9 M or less, preferably 4×10 ^-9 M or less, for example, 3×10 ^-9 M or less, 2×10 ^-9 M or less, 1×10 ^-9 M or less, 8×10 ^-10 M or less, 5×10 ^-10 M or less, 4 ×10 ^-10 M or less, 3×10 ^-10 M or less, 2×10 ^-10 M or less, 1×10 ^-10 M or less, 8×10 ^-11 M or less, 5×10 ^-11 M or less, 4×10 ^-11 M or less, 3×10 ^-11 M or less, 2×10 ^-11 M or less, 1×10 ^-11 M or less, 8×10 ^-12 M or less, 5× 10 ^-12 M or less, 4×10 ^-12 M or less, 3×10 ^-12 M or less, 2×10 ^-12 M or less, 1×10 ^-12 M or less, 8×10 ^-13 M or less, 5×10 ^-13 M or less, 4×10 ^-13 M or less, 3×10 ^-13 M or less, 2×10 ^-13 M or less, or 1×10 ^-13 M or less.

With respect to a domain containing a variable region that has binding activity for a T cell receptor complex, the KD value for binding to a human T cell receptor complex, such as human T cell receptor or more specifically, for example, human CD3ε, may be , for example, 2×10 ^-7 M or less, preferably 1.5×10 ^-7 M or less, for example 1.4×10 ^-7 M or less, 1.3×10 ^-7 M or less, 1, 2×10 ^-7 M or less, 1×10 ^-7 M or less, 3×10 ^-8 M or less, 2×10 ^-8 M or less, 1×10 ^-8 M or less, 8×10 ^-9 M or less, 5×10 ^-9 M or less, 4×10 ^-9 M or less, 3×10 ^-9 M or less, 2×10 ^-9 M or less, 1×10 ^-9 M or less, 8 ×10 ^-10 M or less, 5×10 ^-10 M or less, 4×10 ^-10 M or less, 3×10 ^-10 M or less, 2×10 ^-10 M or less, 1×10 ^-10 M or less, 8×10 ^-11 M or less, 5×10 ^-11 M or less, 4×10 ^-11 M or less, 3×10 ^-11 M or less, 2×10 ^-11 M or less, 1× 10 ^-11 M or less, 8×10 ^-12 M or less, 5×10 ^-12 M or less, 4×10 ^-12 M or less, 3×10 ^-12 M or less, 2×10 ^-12 M or less or 1×10 ^-12 M or less.

The multispecific antigen binding molecules of the present invention preferably have KD values for binding to human GPC3 and the human T cell receptor complex (e.g., human CD3ε chain) that are 5 x 10 ^-9 M or less and 2 x 10 ^-7 M or less, respectively, and more preferably 1×10 ^-9 M or less and 5×10 ^-8 M or less, respectively.

In the present invention, antibody variable regions that are “functionally equivalent” are not limited to specific regions as long as they are antibody H chain and/or L chain variable regions that satisfy the above requirements. Examples of such antibody variable regions include regions obtained by introducing substitution, deletion, addition and/or insertion of one or more amino acids (for example, 1, 2, 3, 4, 5 or 10 amino acids) into the amino acid sequences of the variable regions as set forth above in tables 1-3. A method well known to those skilled in the art for introducing one or more amino acid substitutions, deletions, additions and/or insertions into an amino acid sequence is a method for introducing mutations into proteins. For example, those skilled in the art can produce variable regions that are functional equivalents of antibody variable regions having the above functions by introducing appropriate mutations into the amino acid sequences using methods such as site-directed mutagenesis (Hashimoto-Gotoh T., Mizuno T., Ogasahara Y. and Nakagawa M., An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 1995, pp. 271-275; Zoller M. J. and Smith M., Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 1983, pp. 468-500; Kramer W. Drutsa V., Jansen H. W., Kramer W., Pflugfelder M. and Fritz H. J., The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W. and Fritz, H. J., Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 1987, pp. 350-367; and Kunkel T. A., Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad. Sci. USA. 82, 1985, cc. 488-492).

When an amino acid residue is changed, the amino acid is preferably replaced by mutation with other amino acid(s) retaining the characteristics of the amino acid side chains. Examples of amino acids with these side chain characteristics are: hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S and T), amino acids containing aliphatic side chains (G, A, V, L, I and P), amino acids with hydroxyl group-containing side chains (S, T and Y), amino acids with side chains containing a sulfur atom ( C and M), amino acids with carboxylic acid and amide side chains (D, N, E and Q), amino acids with basic side chains (R, K and H) and amino acids with aromatic side chains (H, F, Y and W ) (in parentheses are the designations of amino acids in accordance with the one-letter code). Amino acid substitutions within each of these groups are designated conservative substitutions. It is well known that a polypeptide containing a modified amino acid sequence in which one or more amino acid residues in the specified amino acid sequence has been deleted, added and/or replaced with other amino acids can retain the original biological activity (Mark D.F. et al., Proc. Natl. Acad. Sci. USA 81, 1984, pp. 5662-5666; Zoller M. J. and Smith M., Nucleic Acids Res. 10, 1982, pp. 6487-6500; Wang A. et al., Science 224, 1984, 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl. Acad. Sci. USA) 79, 1982, cc. 6409-6413). The variable regions of the present invention containing the specified amino acid modifications have amino acid sequences that are at least 70% identical, more preferably at least 75%, even more preferably at least 80%, even more preferably at least at least 85%, even more preferably at least 95%, of the amino acid sequences of the CDR sequences, FR variable region sequences or complete variable regions before modification. As used herein, sequence identity is defined as the percentage of residues identical to residues in the original H chain variable region or L chain variable region amino acid sequence determined after sequence alignment and introduction of appropriate gaps to maximize sequence identity if desired. The identity of amino acid sequences can be determined using the method described below.

In addition, a “functionally equivalent antibody variable region” can be obtained, for example, from nucleic acids that hybridize under stringent conditions with nucleic acids that contain a nucleotide sequence encoding the amino acid sequence of the variable region specified in Tables 1-3 above. Stringent hybridization conditions used to isolate a nucleic acid that hybridizes under stringent conditions to a nucleic acid that contains a nucleotide sequence encoding a variable region amino acid sequence include, for example, the following conditions: 6 M urea, 0.4% SDS, 0.5× SSC and 37°C or hybridization conditions substantially equivalent to those indicated. Isolation of nucleic acids with the highest possible homology can be expected when more stringent conditions are used, for example the following conditions: 6 M urea, 0.4% SDS, 0.1 x SSC and 42°C. Wash conditions after hybridization are, for example, a wash using 0.5×SSC (1×SSC is 0.15M NaCl and 0.015M sodium citrate, pH 7.0) and 0.1% SDS at 60°C, more preferably a wash with 0.2xSSC and 0.1% SDS at 60°C, even more preferably a wash with 0.2xSSC and 0.1% SDS at 62°C, even more preferably a wash with 0. 2xSSC and 0.1% SDS at 65°C and even more preferably a wash using 0.1xSSC and 0.1% SDS at 65°C. The sequences of the isolated nucleic acids can be determined by known methods, which are described below. The overall nucleotide sequence homology of the isolated nucleic acid is at least 50% or greater, preferably 70% or greater, and most preferably 90% or greater (e.g., 95%, 96%, 97%, 98%, 99% or greater) when assessed degree of sequence identity.

Nucleic acids that hybridize under stringent conditions to a nucleic acid containing a nucleotide sequence that encodes the amino acid sequence of the variable region can also be isolated using, instead of the hybridization-based methods described above, gene amplification methods such as polymerase chain reaction (PCR), in which uses primers synthesized on the basis of nucleotide sequence information that encodes the amino acid sequence of the variable region.

The identity of a single nucleotide sequence or amino acid sequence can be determined using the BLAST algorithm described in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90, 1993, cc. 5873-5877). Based on this algorithm, the BLASTN and BLASTX programs were developed (Altschul et al., J. Mol. Biol. 215, 1990, pp. 403-410). For BLASTN-based nucleotide sequence analysis, parameters are set such as score = 100 and word length = 12. On the other hand, parameters used for BLAST-based amino acid sequence analysis include, for example, score = 50 and word length=3. The default settings apply on a per-program basis when using the BLAST and Gapped BLAST programs. Specific techniques for these assays are known in the art (see National Center for Biotechnology Information (NCBI) website, Basic Local Alignment Search Tool (BLAST); http://www.ncbi.nlm.nih.gov).

The combination of an antibody variable region having glypican 3 binding activity and an antibody variable region having T-cell receptor complex binding activity included in the multispecific antigen binding molecule of the present invention is not limited to a particular combination as long as it has the above types of activity. However, according to the present invention, the cytotoxic activity of the multispecific antigen binding molecule is preferably equivalent to or greater than that of the bispecific antibody GPC3_ERY22_rCE115 described in Example 3. As used herein, "equivalent" does not necessarily imply the same degree of activity as above, and the activity may be higher. The molecule is equivalent to GPC3_ERY22_rCE115, for example, when the coefficient (cell growth inhibition rate/cell growth inhibition rate (GPC3_ERY22_rCE115)) relative to the cell growth inhibition rate of GPC3_ERY22_rCE115 (cell growth inhibition rate (GPC3_ERY22_rCE115)) is 0.7 or more, preferably 0.8 or more than, 0.9 or more, 1.0 or more, 1.2 or more, 1.5 or more, 2 or more, 3 or more, 5 or more, 10 or more, or 20 or more. While there is no upper limit, the score could be, for example, 10, ¹⁰² , ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ or ¹⁰⁶ . The concentration of the added multispecific antigen binding molecule can be determined accordingly, but is preferably, for example, 0.01, 0.05, 0.1, 0.5 or 1 nM; and preferably measurements are carried out at a concentration of 0.05 or 0.1 nM.

In addition, examples include the case where the coefficient "concentration causing 50% cell growth inhibition/concentration (GPC3_ERY22_rCE115) causing 50% cell growth inhibition" obtained by comparing with the concentration ( _{GPC3_ERY22_rCE115} ) causing cell growth inhibition 50% (the concentration of (GPC3_ERY22_rCE115) causing 50% inhibition of cell growth) is 1.5 or less. The ratio "concentration causing 50% cell growth inhibition/concentration (GPC3_ERY22_rCE115) causing 50% cell growth inhibition" is preferably 1.3 or less, more preferably 1.2 or less, 1.1 or less, 1.0 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, or 0.5 or less. There is no lower limit, examples include factors of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 or 10 ^-6 . In particular, the coefficient is preferably from 10 ^-6 to 1.5×10 ^-0 , more preferably from 10 ^-6 to 10 ^-1 even more preferably from 10 ^-6 to 10 ^-2 and even more preferably from 10 ^-6 to 10 ^-3 .

Preferred specific KD values for binding to human GPC3 and the human T-cell receptor complex (eg, the human CD3ε chain) are also as stated above. Appropriate GPC3-expressing cells or appropriate tissue containing said cells may be used, and, for example, human cancer cell lines PC-10 or NCI-H446, which express GPC3, may be used.

Examples of said combination of an antibody variable region having binding activity for glypican 3 and an antibody variable region having binding activity for the T-cell receptor complex include combinations of antibody H chain variable regions that are shown in Table 4, combinations of H variable regions -antibody chains that have CDR sequences whose amino acid sequences CDR1, CDR2 and CDR3 are the same as the amino acid sequences CDR1, CDR2 and CDR3 that are included in the variable regions of the antibody H chain presented in Table 4, and combinations of variable regions Antibody H chains functionally equivalent to the indicated variable regions. As used herein, "functionally equivalent" has the meaning set forth above.

Preferred common L chain for such combinations of an antibody variable region having glypican 3 binding activity and an antibody variable region having T cell receptor complex binding activity includes, for example, L0000, L0011, L0201, L0203, L0204, L0206, L0208, L0209, L0211, L0212, L0222 and a common L chain that has CDR sequences (amino acid sequences CDR1, CDR2 and CDR3) identical to the amino acid sequences CDR1, CDR2 and CDR3 listed above for the common L chain. Specific combinations include, for example, combinations of antibody H chain variable regions and a common L chain, which are presented in Table 5, combinations of antibody variable regions that have CDR sequences (amino acid sequences CDR1, CDR2 and CDR3) identical to the amino acid sequences CDR1, CDR2 and CDR3, which are included in the common L chain variable regions presented in Table 5, and combinations of antibody H chain variable regions and the common L chain that are functionally equivalent to these variable regions. As used herein, "functionally equivalent" has the meaning set forth above.

There is no particular limitation on the Fc region included in the multispecific antigen binding molecule of the invention if it is an Fc region with reduced Fcγ receptor binding activity, but examples of the preferred Fc region of the present invention include a combination an E22Hh Fc region portion and an E22Hk Fc region portion, a combination of an E2702GsKsc Fc region portion and an E2704sEpsc Fc region portion, and a combination of an E2702sKsc Fc region portion and an E2704sEpsc Fc region portion.

Examples of the preferred multispecific antigen binding molecule of the present invention include bispecific antibodies that contain an antibody variable region having glypican 3 binding activity and an antibody variable region having CD3ε binding activity. More preferably, the cytotoxic activity is the same as or greater than that of the bispecific antibody GPC3_ERY22_rCE115. Examples of these bispecific antibodies include bispecific antibodies containing the H and L chains listed in Table 13, and bispecific antibodies that bind to an epitope overlapping with the epitope that binds to the above antibodies and that contain an Fc region with reduced binding activity in relation to the Fcy receptor.

Whether an antibody recognizes an epitope that overlaps with an epitope recognized by another antibody can be determined based on competition between the two antibodies for the epitope. Competition between antibodies can be assessed using competition binding assays such as enzyme-linked immunosorbent assay (ELISA), fluorescence resonance energy transfer (FRET) and fluorometric microvolume analysis technology (FMAT (registered trademark)). The amount of antibody bound to an antigen indirectly correlates with the binding ability of a competing candidate antibody (test antibody) that competitively binds to the overlapping epitope. In other words, if the amount or affinity of a test antibody for an overlapping epitope increases, then the amount of antibody bound to the antigen decreases and the amount of antigen bound by the test antibody increases. Specifically, an appropriately labeled antibody and the antibody to be studied are added simultaneously to an antigen, and the resulting antibody that binds is detected by the label. The amount of antibody bound to an antigen can be easily determined using a pre-labeled antibody. The label is not limited to a specific label, and the labeling method is selected depending on the analytical technique used. In particular, the labeling method includes introducing a fluorescent label, a radioactive label, an enzyme label, and the like.

For example, a fluorescently labeled antibody and an unlabeled antibody or test antibody are simultaneously added to beads on which GPC3 or CD3ε is immobilized, and the labeled antibody is detected using fluorometric microvolume analysis technology.

As used herein, “an antibody that binds to an overlapping epitope” means a test antibody that can reduce the amount of bound labeled antibody by at least 50% at a concentration that is typically 100-fold higher, preferably 80-fold higher, more preferably 50 times higher, even more preferably 30 times higher, and even more preferably 10 times higher than the concentration at which unlabeled antibody reduces by 50% the amount of bound labeled antibody (IC ₅₀ ).

Multispecific antigen-binding molecules, which have antigen-binding antibody sites binding to epitopes that overlap with the epitopes bound by the above antibodies, can have very high cytotoxic activity.

The multispecific antigen binding molecules of the present invention are produced using the same technology as described above for the production of recombinant antibodies.

The present invention also relates to polynucleotides encoding the antigen binding molecules of the present invention, and they can be inserted into the desired expression vectors. Suitable host cells can be transformed with expression vectors to produce cells that express antigen binding molecules. Antigen binding molecules encoded by polynucleotides can be produced by culturing cells that express the antigen binding molecules and collecting expression products from culture supernatants. Thus, the present invention relates to vectors that contain a polynucleotide encoding the antigen-binding molecule proposed in the present invention, cells that carry the vector, and methods for producing antigen-binding molecules, which involve culturing the cells and collecting antigen-binding molecules from the supernatants culture. They can be produced using the same technologies described above for the production of recombinant antibodies.

Pharmaceutical compositions

Another object of the present invention is pharmaceutical compositions containing as an active substance a multispecific antigen-binding molecule, which contains: (1) a domain containing an antibody variable region having binding activity against glypican 3, (2) a domain containing an antibody variable region having binding activity activity towards the T cell receptor complex, and (3) a domain containing an Fc region with reduced binding activity towards the Fcγ receptor. In addition, the present invention relates to pharmaceutical compositions that induce cell damage containing an antigen-binding molecule as an active ingredient. The pharmaceutical compositions of the present invention, which induce the desired cellular damage, primarily T cell-dependent cellular cytotoxicity, are preferably administered to an individual who suffers from a disease in which these activities are required for prevention or treatment, or to an individual who is likely to relapse. diseases.

In addition, according to the present invention, cytotoxicity-inducing agents and cell growth inhibitory agents and anticancer agents which contain as an active substance a multispecific antigen-binding molecule which contains:

(1) a domain containing an antibody variable region having binding activity for glypican 3,

(2) a domain containing an antibody variable region having binding activity for the T cell receptor complex, and

(3) a domain containing an Fc region with reduced binding activity towards the Fcγ receptor,

can be used in a method for inducing cell damage which comprises the step of administering an antigen binding molecule to an individual, or can use the antigen binding molecule to prepare a cytotoxicity inducing agent and a cell growth inhibitory agent.

In the present invention, the phrase “contains as an active ingredient a multispecific antigen-binding molecule that contains: (1) a domain containing an antibody variable region having binding activity for glypican 3, (2) a domain containing an antibody variable region having binding activity for T-cell receptor complex, and (3) a domain containing an Fc region with reduced Fcγ receptor binding activity" means that the composition contains an antigen-binding molecule as the main active ingredient; however, there are no specific restrictions on the relative content of antigen-binding molecule.

If desired, the multispecific antigen-binding molecules of the present invention can be encapsulated in microcapsules (microcapsules made from hydroxymethylcellulose, gelatin, poly(methyl methacrylate), etc.) and can be included in components of colloidal systems intended for the administration of drugs (liposomes , albumin microspheres, microemulsions, nanoparticles and nanocapsules) (for example, see Remington's Pharmaceutical Science 16th ed., ed. Oslo, 1980)). In addition, methods for preparing sustained release agents are well known and can be used for the multispecific antigen binding molecules of the present invention (J. Biomed. Mater. Res. 15, 1981, pp. 267-277; Chemtech. 12 , 1982, pp. 98-105; US No. 3773719; published European patents (EP) EP 58481 and EP 133988; Biopolymers 22, 1983, pp. 547-556).

The pharmaceutical compositions of the present invention or the cytotoxicity-inducing agents and cell growth-inhibiting agents can be administered orally or parenterally to a patient, and parenteral administration is preferred. Specified examples of administration methods include injection, nasal administration, transpulmonary administration, and transdermal administration. Examples of administration by injection include, for example, intravenous injection, intramuscular injection, intraperitoneal injection and subcutaneous injection. The pharmaceutical composition of the present invention or the cytotoxicity-inducing agent and the cell growth-inhibiting agent can be administered topically or systemically by injection. The method of administration can be selected depending on the patient's age and symptoms. The dose may be selected, for example, from the following range: 0.0001 to 1000 mg per kg body weight per administration. Alternatively, the dose can be selected, for example, from the following range: 0.001 to 100,000 mg/patient. However, the dosages of the pharmaceutical composition of the present invention or the cytotoxicity-inducing agent and the cell growth-inhibiting agent are not limited to these dosages.

The pharmaceutical compositions of the present invention, or cytotoxicity-inducing agents and cell growth inhibitory agents, can be prepared using conventional methods (for example, see Remington's Pharmaceutical Science, latest ed., Mark Publishing Company, Easton, U.S.A.), and they may also contain pharmaceutically acceptable carriers and additives. Examples thereof include, but are not limited to, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicizing agents, binders, disintegrants, lubricants, flow agents and flavoring agents, and others may be used. common media. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carmellose, sodium carmellose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain triglyceride, polyoxyethylenated hydrogenated castor oil 60, sucrose, carboxymethylcellulose a, corn starch, inorganic salt, etc.

The present invention also provides methods for damaging glypican 3 antigen-expressing cells or tumor tissues containing antigen-expressing cells, or methods for inhibiting the growth of such cells or tumor tissues by contacting glypican 3 antigen-expressing cells with a multispecific antigen-binding molecule, proposed in the present invention, which binds to the antigen. The multispecific antigen-binding molecule that binds to an antigen is described above as the antigen-binding molecule of the present invention that binds to an antigen, which is contained in the cytotoxicity-inducing agents and cell growth-inhibiting agents of the present invention. The cells to which the multispecific antigen binding molecule of the present invention binds, which binds to an antigen, is not limited to specific cells as long as those cells express the antigen.

According to the present invention, “contacting” can be accomplished, for example, by adding a multispecific antigen-binding molecule of the present invention that binds an antigen to the culture medium of antigen-expressing GPC3 cells that are cultured in vitro. In this case, the added antigen binding molecule can be used in a suitable form such as a solution or a solid obtained by lyophilization or the like. When added as an aqueous solution, it may be an aqueous solution that contains only the multispecific antigen binding molecule of the present invention, or it may also be a solution containing, for example, the above surfactants, excipients, dyes, flavors, preservatives , stabilizers, buffers, suspending agents, isotonicizing agents, binders, disintegrants, lubricants, flow agents and flavoring agents. There are no special restrictions on the concentration of the added molecule; however, the final concentration in the culture medium is preferably 1 pg/ml to 1 g/ml, more preferably 1 ng/ml to 1 mg/ml, and even more preferably 1 μg/ml to 1 mg/ml.

Moreover, according to another embodiment of the present invention, “contacting” is also carried out by treating non-human animals into which GPC3 antigen-expressing cells have been transplanted and animals in which the antigen is expressed endogenously. The method of administration may be oral or parenteral, and parenteral administration is preferred. Specified examples of administration methods include injection, nasal administration, transpulmonary administration, and transdermal administration. Examples of administration by injection include, for example, intravenous injection, intramuscular injection, intraperitoneal injection and subcutaneous injection. The pharmaceutical composition of the present invention or the cytotoxicity-inducing agent and cell growth inhibitory agent can be administered topically or systemically, for example, by injection. The method of administration can be selected depending on the age of the experimental animal and symptoms. When administered as an aqueous solution, an aqueous solution may be used containing only the multispecific antigen binding molecule of the present invention, or a solution may also be used containing the above surfactants, excipients, dyes, flavors, preservatives, stabilizers, buffers, suspending agents , isotonicizing agents, binders, disintegrants, lubricants, flow agents and flavoring agents, etc. The dose can be selected from the following range: 0.0001 to 1000 mg per kg body weight per administration.

Alternatively, the dose can be selected, for example, from the following range: 0.001 to 100,000 mg/patient. However, the administered amount of the multispecific antigen-binding molecule of the present invention is not limited to these doses.

The method described below is preferably used as a method for assessing or measuring cell damage induced in cells expressing glypican 3 as an antigen, which is bound by a domain that carries an antibody variable region having binding activity for glypican 3, which is part of an antigen binding molecule that is the result of contact of cells with the multispecific antigen-binding molecule proposed in the present invention. Examples of a method for assessing or measuring cytotoxic activity in vitro include methods for measuring the activity of cytotoxic T cells or the like. Whether the multispecific antigen binding molecule of the present invention has T cell cytotoxicity can be determined using known methods (see, for example, Current protocols in Immunology, Chapter 7. Immunological studies in humans, edited by John E, Coligan, et al. ., ed., John Wiley & Sons, Inc., 1993). When measuring activity, an antigen binding molecule that binds to an antigen other than glypican 3, which is an antigen that is not expressed on the cells used for evaluation, can be used as a control that is assayed in the same manner as the multispecific antigen binding molecule provided in the present invention, and the activity can be determined by assessing whether the multispecific antigen binding molecule of the present invention has greater cytotoxic activity than the control antigen binding molecule.

To evaluate or measure cytotoxic activity in vivo, for example, cells expressing glypican 3 as an antigen are transplanted intradermally or subcutaneously into a non-human experimental animal, and then the test antigen-binding molecule is administered intravenously or intraperitoneally every day or at intervals of several days starting on the day transplantation or the day after it. Cytotoxic activity can be determined by measuring tumor size daily, detecting differences in tumor size changes. Similar to the method used for in vitro assessment, an antigen binding molecule of the present invention may be considered to have cytotoxic activity if, upon administration of a control antigen binding molecule, tumors in the group administered the antigen binding molecule of the present invention are found to be significantly larger in size. smaller than the size of tumors in the control antigen-binding molecule group

As a method for assessing or measuring the inhibitory effect on the proliferation of cells expressing glypican 3 as an antigen, the method of measuring the uptake of isotope-labeled thymidine into cells or the MTT method can be used. As a method for assessing or measuring the inhibitory effect on cell proliferation in vivo, a method similar to that described above for assessing or measuring cytotoxic activity in vivo can be used.

The present invention also provides kits that can be used in the methods of the present invention that contain a multispecific antigen binding molecule of the present invention or a multispecific antigen binding molecule created by the production method of the present invention. Alternatively, the kits may be packaged together with additional pharmaceutically acceptable carrier, diluent, and instructions for use.

The present invention also relates to a multispecific antigen binding molecule of the present invention or a multispecific antigen binding molecule prepared by the method of the present invention for use in the method of the present invention.

The present invention also relates to molecules having GPC3-binding activity that contain a domain containing the variable region of an antibody that has GPC3-binding activity, the multispecific antigen-binding molecule of the present invention. In addition, the present invention provides a molecule that has GPC3-binding activity, which contains the variable regions of the H and L chains of the antibody, respectively, containing three CDRs of the H and L chains (a total of six CDRs) that are included in the molecule. The present invention also provides molecules having T cell receptor complex binding activity that comprise a domain containing an antibody variable region that has T cell receptor complex binding activity, the multispecific antigen binding molecule of the present invention. In addition, the present invention provides a molecule that has binding activity for the T cell receptor complex, which contains the variable regions of the H and L chains of an antibody, respectively, containing three CDRs of the H and L chains (a total of six CDRs), which enter the molecule. These molecules may be antibodies or polypeptides containing antigen-binding fragments of an antibody. The present invention also relates to antibodies that bind to epitopes that overlap or compete with the specified molecules or polypeptides that contain their antigen-binding fragments. Suitable examples of such polypeptides that contain antigen binding antibody fragments are scFv, single chain antibody, Fv, single chain Fv2 (scFv2), Fab and F(ab')2. In addition, these molecules do not necessarily need to be multispecific (bispecific), but can bind only to either GPC3 or the T cell receptor complex (eg, CD3ε chain).

These molecules include a molecule that contains a domain containing an antibody variable region that has GPC3-binding activity, a multispecific antigen-binding molecule, which is described in detail in the Examples section herein (which contains H chain variable regions that have GPC3-binding activity). activity and common L chain variable region), a molecule that contains a domain containing an antibody variable region that has binding activity for the T-cell receptor complex, a multispecific antigen-binding molecule, the detailed description of which is provided in the Examples section herein ( which contains variable regions of the H chain with binding activity towards the T-cell receptor complex and a variable region of the common L chain), as well as a molecule with the ability to bind to the same antigenic protein (GPC3 or T-cell receptor complex), which contains three CDRs of each of the H and L chains (six CDRs in total) that are included in the above molecule.

These molecules have CDRs that are the same as the CDRs of the multispecific antigen binding molecule of the present invention; and therefore are expected to bind to an epitope overlapping that of the multispecific antigen binding molecule of the present invention. Thus, these molecules can compete with the multispecific antigen binding molecules of the present invention when co-present with the multispecific antigen binding molecules of the present invention. Thus, these molecules can be used, for example, as regulatory agents to inhibit various activities (such as antigen binding activity, cytotoxic activity and antitumor activity) of the multispecific antigen binding molecules proposed in the present invention. In addition, the molecule may be pre-bound to a target protein (GPC3 or T-cell receptor complex), and upon addition of the multispecific antigen-binding molecule of the present invention, molecules that dissociate due to competition can be detected. In this use, the molecule is useful as an agent for assessing the binding of the multispecific antigen binding molecule of the present invention to a target protein. According to the present invention, the molecules can be labeled with appropriate fluorescent substances or the like. Alternatively, these molecules can be used to screen for new antibodies that bind to epitopes overlapping with epitopes that are bound by the multispecific antigen binding molecules of the present invention. As stated above, the molecule can be pre-bound to a target protein (GPC3 or T-cell receptor complex) and then the test antibody is added, whereby if dissociation of the bound molecules occurs, then the test antibody is a candidate antibody , which binds to an epitope overlapping with the epitope to which the multispecific antigen binding molecule of the present invention binds. This allows efficient screening of new multispecific antigen-binding molecules.

The combinations exemplified herein as combinations of each CDR of the multispecific antigen binding molecules of the present invention can be used directly as specific combinations of the H chain and L chain variable region CDRs of these molecules. The antigen affinity of these molecules (KD values) is preferably characterized by, but is not limited to, the values exemplified by the KD of the multispecific antigen binding molecule of the present invention.

The present invention also relates to nucleic acids encoding these molecules, vectors containing nucleic acids, cells containing nucleic acids or vectors, methods for producing molecules by culturing cells and molecules obtained using these methods.

All prior art documents cited herein are incorporated herein by reference. Examples

The present invention is described below with reference to specific examples, which do not limit the scope of the invention.

Example 1.

Preparation of GPC3_ERY22_rCE115 and measurement of cytotoxic activity

(1-1) Obtaining GPC3_ERY22_rCE115

A molecule in which one of the Fabs was replaced with a CD3 epsilon binding domain was prepared using IgG against cancer antigen (GPC3) as the main structure. In this case, the IgG Fc region that was used as the main structure was a silent Fc with weakened affinity for FcgR (Fcγ (Fc-gamma) receptor). Anti-GPC3 antibody H0000 (SEQ ID NO: 40)/GL4 (SEQ ID NO: 41) was used as the GPC3 binding domain. Anti-CD3 antibody rCE115H/rCE115L (SEQ ID NO: 42/SEQ ID NO: 43) was used as the GPC3 binding domain.

The G1d construct, obtained by removing Gly and Lys at the C-terminus of IgG1, was used as the constant region of the antibody H chain and was used in combination with H0000/GL4 and rCE115H/rCE115L. When the constant region of an antibody H chain was designated as H1, the sequence corresponding to the antibody H chain carrying H0000 in the variable region was designated as H0000-H1. In the present description, the amino acid change is designated, for example, as D356K. The first alphabetic character (corresponding to D in D356K) is the single-letter code for the amino acid residue before modification, the following number (corresponding to 356 in D356K) indicates the position of the modification according to EU numbering, and the last alphabetical character (corresponding to K in D356K) is the single-letter code amino acid residue after modification. G1dh (SEQ ID NO: 44), created by removing Gly and Lys at the C-terminus of IgG1, ERY22_Hk (SEQ ID NO: 45), created by introducing mutations L234A/L235A/Y349C/T366W into G1dh, and ERY22_Hh (SEQ ID NO : 46), created by introducing mutations L234A/L235A/D356C/T366S/L368A/Y407V in G1dh, were obtained according to the method described in Example 1 given for reference. Mutations L234A and L235A were introduced into the corresponding H chains to weaken the affinity for FcgR ( Fcγ receptor), and the mutations Y349C/T366W and D356C/T366S/L368A/Y407V were introduced to efficiently form heteromers of each H chain to produce heterodimeric antibodies containing two types of H chains.

The heterodimeric antibody GPC3_ERY22_rCE115, obtained by replacing the VH and VL domains of an anti-GPC3 Fab, was generated according to the method described in Reference Example 1 (Fig. 1a).

A series of expression vectors with an integrated polynucleotide encoding each of the constructs GL4-ERY22_Hk (SEQ ID NO: 47), H0000-ERY22_L (SEQ ID NO: 48), rCE115H-ERY22_Hh (SEQ ID NO: 49) and rCE115L-k0 (SEQ ID NO: 50) were obtained using methods well known to those skilled in the art, such as PCR-based methods using added primers that had corresponding sequences similar to those used in the method described above.

The following combination of expression vectors was introduced into FreeStyle 293-F cells to transiently express each target molecule.

Target molecule: GPC3_ERY22_rCE115

Polypeptides encoded by polynucleotides inserted into expression vectors: GL4-ERY22_Hk, H0000-ERY22_L, rCE115H-ERY22_Hh, rCE115L-k0.

(1-2) Cleaning GPC3_ERY22_rCE115

The resulting culture supernatant was added to a column with anti-FLAG M2 antibody (Sigma) and then the column was washed and then eluted using FLAG peptide (Sigma) at a concentration of 0.1 mg/ml. Fractions containing the molecule of interest were added to a HisTrap HP column (GE Healthcare) and the column was then washed and then eluted using an imidazole concentration gradient. The fraction containing the molecule of interest was concentrated using an ultrafiltration method and the concentrate was then applied to a column packed with Superdex 200 (GE Healthcare) and each of the purified molecules of interest was collected only as monomer fractions from the eluted solution.

(1-3) Measurement of cytotoxic activity of GPC3_ERY22_rCE115 using human peripheral blood mononuclear cells

The in vitro cytotoxic activity of GPC3_ERY22_rCE115 was assessed.

(1-3-1) Preparation of a solution of human peripheral blood mononuclear cells (PBMC)

Samples of 50 ml of peripheral blood from healthy volunteers (adults) were obtained using syringes pre-filled with 100 μl (1000 units/ml) of heparin solution (Novo-Heparin 5000 units per injection; Novo Nordisk). This peripheral blood was diluted twofold with PBS(-), divided into four aliquots and added to Leucosep lymphocyte separation tubes (catalog no. 227290; Greiner bio-one), into which 15 ml of Ficoll-Pak PLUS were previously added and centrifuged. After centrifugation of the separation tubes (2150 rpm, 10 min, room temperature), mononuclear cell fractions were collected. Cells in the mononuclear cell fraction were washed once using Dulbecco's modified Eagle's medium (SIGMA) containing 10% FBS (hereinafter referred to as 10% FBS/D-MEM) and then the cell density was adjusted to 4 × 10 ⁶ cells/ml using 10%FBS/D-MEM. The cell suspension thus obtained was used as a solution of human PBMCs in subsequent experiments.

(1-3-2) Cytotoxic activity assay

Cytotoxic activity was assessed based on the degree of cell growth inhibition using the xCELLigence Real Time Cell Analyzer (Roche Diagnostics). The target cells used were the human cancer cell line NCI-H446 or the human cancer cell line PC-10 expressing GPC3. NCI-H446 or PC-10 cells were separated from the plates and plated into an E-Plate 96 (Roche Diagnostics) in 100 μl/well aliquots, adjusted to 1 x 10 ⁴ , and viable cell analysis was started using the analyzer. cells in real time xCELLigence. The next day, the plate was removed from the xCELLigence Real-Time Cell Analyzer and 50 μl of each corresponding antibody produced was added to the plate at various concentrations (0.004, 0.04, 0.4 and 4 nM). After reacting for 15 min at room temperature, 50 μl of human PBMC solution (2 x 10 ⁵ cells/well) prepared according to the method described in section (1-2) was added and the cells were again placed into the xCELLigence real-time cell analyzer and Viable cell analysis was started. The reaction was carried out in an atmosphere of 5% nitrogen dioxide gas at 37°C and the degree of cell growth inhibition (%) was determined using the formula below using the cell index value determined 72 hours after the addition of human PBMCs. The cell index value used for calculation was standardized in such a way that the cell index value determined immediately before the addition of the antibody was taken as 1.

Degree of cell growth inhibition (%) = (A-B)×100/(A-1)

A denotes the average cellular index value in wells without the addition of antibody (target cells and human PBMCs only), and B denotes the average cellular index value in each well. The measurement was carried out in triplicate.

When peripheral blood mononuclear cells (PBMCs) derived from human blood were used as effector cells for the cytotoxicity assay of GPC3_ERY22_rCE115, very strong activity was detected (Fig. 2).

Example 2: Humanization of the H chain of an anti-CD3 antibody. , and "sharing" of a common L-chain

(2-1) Generation of hCE115HA, a humanized antibody H chain variable region

The variable region of the H chain of the anti-CD3 antibody rCE115 (SEQ ID NO: 42) was humanized. CDR and FR were determined according to Cabot (Cabot numbering).

First, the human FR sequence was selected by comparing the human antibody variable region sequences in the database with the rat variable region sequence. The database used was IMGT (http://www.imgt.org/) and NCBI GenBank (http://www.ncbi.nlm.nih.gov/genbank/). A humanized H chain variable region sequence was created by ligating the H chain variable region CDR sequence to a selected human FR sequence. In this way, a humanized H chain variable region sequence, hCE115HL (SEQ ID NO: 51) was obtained.

The amino acid residue at position 93 according to Cabot numbering is Ala in the selected human FR3 H chain sequence, but is Arg in the rCE115 variable region sequence. Using a database of rat and human germline sequences (IMGT database (http://www.imgt.org/)), only a few sequences were found that contain Arg at this site. It is known that the amino acid residue at position 94 according to Cabot numbering is involved in stabilizing the structure of the antibody by forming the upper core (Ewert et al., Methods 34(2) October 2004, pp. 184-199). Based on this information, a new humanized H chain variable region sequence was created in which the amino acid residues at Cabot positions 93 and 94 in H chain FR3 were replaced with residues present in the rCE115 variable region sequence. In this way, the humanized H chain variable region sequence of hCE115HA (SEQ ID NO: 52) was obtained.

(2-2) Generating a common L chain L0000 for anti-CD3 antibody rCE115 and anti-GPC3 antibody

FR/CDR rearrangement of the L chain variable region rCE115L (SEQ ID NO: 43) of the anti-CD3 antibody rCE115 and the L chain variable region GL4 (SEQ ID NO: 41) of the anti-GPC3 antibody were performed.

The FR sequence of GL4 was selected as the L chain FR sequence. L chain CDR2 was the same in rCE115L and GL4. The L chain CDR1 was selected from the GL4 CDR sequences and the L chain CDR3 was selected from the rCE115L CDR sequences, respectively. In addition, the L chain CDR3 was recreated by replacing the Asp amino acid residue at Cabot position 94 of the selected L chain CDR3 with the Val residue present in GL4.

A humanized L chain variable region sequence was created by stitching together the selected FRs and CDRs described above. The result was a humanized L chain variable region sequence, L0000 (SEQ ID NO: 53).

(2-3) Affinity assessment for human GPC3

Binding activity towards human GPC3 was assessed using GL4 (SEQ ID NO: 41) and L0000 (SEQ ID NO: 53) as L chain variable regions. To accomplish this, a single-arm molecular form of the antibody was used, carrying a single Fab heterodimerized in the Fc region of human IgG1 using knobs-into-hole technology. H0000 (SEQ ID NO: 40) was used as the H chain variable region of the anti-GPC3 antibody.

The affinity and binding constants of the anti-GPC3 antibody to the antigen were measured using a surface plasmon resonance-based multicycle kinetics method using a Biacore™ - T200 device (GE Healthcare Japan). HBS-EP+ (GE Healthcare Japan) was used as the running buffer, and an amine coupling kit (GE Healthcare Japan) was used to covalently couple protein A/G to the CM5 chip (carboxymethyldextran-coated chip). Each GPC3 antibody was prepared such that approximately 100 RU was captured by the A/G protein. Human GPC3 used as an analyte was prepared at concentrations of 8, 16, 32, 64 and 128 nM using HBS-EP+. When performing measurements, protein A/G was first allowed to bind to the antibody solution, and then the human GPC3 solution was injected at a flow rate of 30 μl/min for 3 min to allow the reaction to occur. The solution was then replaced with HBS-EP+ buffer and the dissociation phase was assessed for 15 min. After completion of the dissociation phase assessment, the sensor chip was regenerated by washing with 10 mM Gly-HCl at pH 1.5. Measurement was carried out similarly at 0 concentration by allowing protein A/G to "capture" in the antibody solution, injecting HBS-EP+ for 3 min to allow the reaction to occur, and then replacing the solution with HBS-EP+ to assess the dissociation phase for 15 min. . After completion of the dissociation phase assessment, the sensor chip was regenerated by washing with 10 mM Gly-HCl at pH 1.5. Only Biacore analysis software was used for data analysis, Biacore T200 Evaluation software, version 1.0 was used to perform kinetic analysis to calculate the binding reaction rate constant (ka), dissociation reaction rate constant (kd) and rate constant ratio based on the obtained sensorgrams. The results are presented in Table 6.

(2-4) Assessment of affinity for human CD3

Binding activity against human CD3 was assessed using hCE115HA (SEQ ID NO: 52) as the H chain variable region and L0000 (SEQ ID NO: 53) as the L chain variable region. To accomplish this, a single-arm molecular form of the antibody was used, carrying a single Fab heterodimerized in the Fc region of human IgG1 using knobs-into-hole technology.

The affinity and binding constants of the anti-CD3 antibody to the antigen were measured using a single-cycle kinetics method based on surface plasmon resonance using a Biacore™ T200 device (GE Healthcare Japan). HBS-EP+ (GE Healthcare Japan) was used as the running buffer, and an amine coupling kit (GE Healthcare Japan) was used to covalently couple human CD3 to the CM5 chip (carboxymethyldextran-coated chip). The anti-CD3 antibody used as an analyte was prepared at concentrations of 5 and 20 μg/ml using HBS-EP+. Measurements were performed by first injecting each of the 5 and 20 μg/ml anti-CD3 antibody solutions over 3 min at a flow rate of 20 μl/min, allowing the reaction to proceed. The solution was then replaced with HBS-EP+ and the dissociation phase was assessed for 3 minutes. After completion of the dissociation phase assessment, the sensor chip was regenerated by washing with 10 mM Gly-HCl at pH 1.5. Only Biacore analysis software was used for data analysis, Biacore T200 Evaluation software, version 1.0 was used to perform kinetic analysis to calculate the binding reaction rate constant (ka), dissociation reaction rate constant (kd) and rate constant ratio based on the obtained sensorgrams. The results are presented in Table 7.

(2-5) Obtaining GPC3_ERY27_hCE115

Anti-cancer antigen IgG4 (GPC3) was used as the backbone to produce the ERY27 molecule (FIG. 16) in which the H chain variable region of one of the Fabs was replaced with a CD3 epsilon binding domain and the L chain was common to both Fabs. In this case, the IgG4 Fc that was used as the main structure was a silent Fc with weakened affinity for FcgR (Fcγ-(Fc-gamma) receptor). H0000 (SEQ ID NO: 40) was used as the H chain variable region of the GPC3 binding domain, and hCEHSHA (SEQ ID NO: 52) was used as the H chain variable region of the CD3 binding domain. L0000 (SEQ ID NO: 53) was used as the L chain variable region. The D356K and K439E mutations were introduced into the respective H chains to efficiently form heteromers of each H chain to produce heterodimeric antibodies containing two types of H chains (WO 2006/106905). H435R is a modification that disrupts protein A binding and was introduced to efficiently separate heteromer and homomer (WO 2011/078332).

A series of expression vectors with an integrated polynucleotide encoding each of the f H0000-ERY27_HK (SEQ ID NO: 54), hCE115HA-ERY27_HE (SEQ ID NO: 55) and L0000-k0 (SEQ ID NO: 56) constructs were prepared by well-known methods.

The following combination of expression vectors was introduced into FreeStyle 293-F cells to transiently express each target molecule.

Target molecule: GPC3_ERY27_hCE115

Polypeptides encoded by polynucleotides inserted into expression vectors: H0000-ERY27_HK, hCE115HA-ERY27_HE and L0000-k0

(2-6) Cleaning GPC3_ERY27_hCE115

Each molecule of interest was purified using the method described in Example 1-2.

(2-7) Measurement of cytotoxic activity using human peripheral blood mononuclear cells

(2-7-1) Preparation of a solution of human peripheral blood mononuclear cells (PBMC)

The solution was prepared according to the method described in example 1-3-1.

(2-7-2) Evaluation of cytotoxic activity

Cytotoxic activity was assessed according to the method described in example 1-3-2.

When human blood-derived PBMCs were used as effector cells to evaluate the cytotoxicity of GPC3_ERY27_hCE115, a decrease in activity was found as a result of humanization of the rCE115 H chain and sharing of the common L chain (Fig. 2).

Example 3. Preparation and evaluation of humanized variants of bispecific antibodies to improve various properties

The T cell dependent cytotoxic activity (TDCC activity) of a bispecific humanized anti-CD3 chain (CD3 epsilon) and anti-GPC3 antibody prepared according to the method described in Example 2, GPC3_ERY27_hCE115 (SEQ ID NOs: 54, 55 and 56), was found to be lower than the T cell dependent cytotoxic activity of GPC3_ERY22_rCE115 (SEQ ID NOs: 47, 48, 49 and 50). This may result from decreased affinity for GPC3 and the CD3ε chain as a result of humanization and sharing of the common L chain. For antigens such as GPC3 and CD3ε chains, which have independent sequences from each other, there is currently no data on humanized bispecific antibodies with increased T-cell dependent cytotoxic activity and improved affinity for both antigens when using a common antibody L chain . Thus, it appears difficult to obtain humanized antibodies with dual specificity having drug efficacy equivalent to or greater than that of the GPC3_ERY22_rCE115 antibody.

Taking into account these circumstances, when creating the invention, modified humanized bispecific antibodies with modified affinity for human GPC3 and the human CD3ε chain were obtained using methods known to specialists in this field, which included extensive replacement of amino acid residues encoded by the antibody gene, obtaining antibody variants against both antigens, namely human GPC3 and human CD3ε chain, performing various screening assessments. In addition, similar methods were used to obtain modified humanized bispecific antibodies with modified physicochemical properties. Additionally, by combining amino acid residue substitutions with efficacy in modifying affinity and physicochemical properties, optimized bispecific antibodies with TDCC activity equivalent to or greater than the T cell-dependent cellular cytotoxicity of GPC3_ERY22_rCE115 prior to humanization were obtained.

Introduction of point mutations, expression and purification of antibodies, assessment of antigen affinity and determination of T cell-dependent cellular cytotoxicity when optimizing humanized bispecific antibodies were carried out according to methods similar to those described in Examples 1 and 2. CDR and FR were determined according to Cabot (Cabot numbering).

Depending on the target, the following constructs were used as constant regions of the antibody H chain (numbers correspond to EU numbering): E22Hh (SEQ ID NO: 57), which was obtained by introducing mutations L234A/L235A/N297A/D356C/T366S/L368A/Y407V /G446-deletion/K447-deletion in human IgG1; E22Hk (SEQ ID NO: 58), which was obtained by introducing mutations L234A/L235A/N297A/Y349C/T366W/G446-deletion/K447-deletion, and a Ser-Ser insertion mutation immediately before position 118 in human IgG1; G1dh was generated by introducing the D356C/T366S/L368A/Y407V/G446-deletion/K447-deletion mutations into human IgG1; none-Hi-Kn010G3 was generated by introducing the 118-215 deletion and C220S/Y349C/T366W/H435R mutations into human IgG1; E2702GsKsc (SEQ ID NO: 60) was generated by introducing the L235R/S239K/N297A/E356K/R409K/H435R/L445P/G446-deletion/K447-deletion mutations into human IgG4; E2704sEpsc (SEQ ID NO: 61) was generated by introducing the K196Q/L235R/S239K/N297A/R409K/K439E/L445P/G446-deletion/K447-deletion mutations into human IgG4; and E2702sKsc (SEQ ID NO: 62) were produced by introducing the L235R/S239K/N297A/E356K/R409K/L445P/G446-deletion/K447-deletion mutations into human IgG4. In addition, human κ chain (kappa chain) k0 (SEQ ID NO: 63) and E22L (SEQ ID NO: 432) were produced by introducing mutations R108A/T109S into the human κ chain used as L chain constant regions antibodies.

A mutation leading to the replacement of Asp with Cys at position 356 according to EU numbering, a mutation leading to the replacement of The with Ser at position 366 according to EU numbering, a mutation leading to the replacement of Leu with Ala at position 368 according to EU numbering, mutation, leading to the replacement of Tyr with Val at position 407 according to the EU numbering, a mutation leading to the replacement of Tyr with Cys at position 349 according to the EU numbering, a mutation leading to the replacement of Thr with Trp at position 366 according to the EU numbering, and a mutation leading to to the Ser-Ser insertion immediately before position 118 according to EU numbering, are mutations designed to efficiently form heterodimeric molecules of each H chain to create heteromeric antibodies. Similarly, the mutation resulting in the replacement of Glu by Lys at position 356 according to the EU numbering, and the mutation resulting in the replacement of Lys by Glu at position 439 according to the EU numbering, are also mutations designed to efficiently form heterodimeric molecules of each H- chains when creating heteromeric antibodies. They are expected to increase the efficiency of production of bispecific antibodies.

A mutation leading to the replacement of Leu with Ala at position 234 according to the EU numbering, a mutation leading to the replacement of Leu with Ala or Arg at position 235 according to the EU numbering, a mutation leading to the replacement of Ser with Lys at position 239 according to the EU numbering, and the Asn to Ala mutation at EU numbering position 297 are mutations designed to reduce affinity for the Fcγ receptor and complement (C1q). They are expected to inhibit Fab binding to CD3 and Fc-mediated cross-linking of the Fcγ receptor or complement and avoid the cytokine release syndrome that accompanies the increase in nonspecific effector functions.

An H chain introduced with deletion mutations at EU numbering positions 118 to 215 can be combined with the full length H chain sequence to produce an antibody that has only one Fab (monivalent antibody) and can be used for affinity assessment.

The mutation resulting in the replacement of Arg with Lys at position 409 according to the EU numbering, and the mutation resulting in the replacement of His with Arg at position 435 according to the EU numbering, are mutations that lead to modification of the properties of the antibody, bringing them closer to the properties of human IgG1 and human IgG3, respectively.

(3-1) Modification of the affinity of a humanized antibody to CD3 using point mutations

First, point mutations were introduced into the sequence FR1, FR2, FR3, CDR1, CDR2 and CDR3 of the humanized anti-human CD3ε chain antibody prepared according to the method described in Example 2, hCE115HA-ERY27_HE (SEQ ID NO: 55), to obtain modified antibodies. The affinity of these modified antibodies for the soluble human CD3ε chain was then determined. By combining sites that have the ability to increase the affinity of modified antibodies, modified antibodies were obtained, the affinities of which are presented in Table 8.

(3-2) Modification of the affinity of a humanized antibody to GPC3 using point mutations

First, point mutations were introduced into the CDR1, CDR2 and CDR3 sequence of the bispecific antibody prepared according to the method described in Example 2, H0000-ERY27_HK (SEQ ID NO: 54), to obtain modified antibodies. The affinity of these modified antibodies for soluble human GPC3 was then determined. By combining sites that have the ability to increase the affinity of modified antibodies, modified antibodies were obtained, the affinities of which are presented in Table 9.

(3-3) Modification of pI using point mutations

Commercial production of bispecific antibodies requires a high level of purity. When using ion exchange chromatography, modification of the isoelectric point (pI) of molecules is effective (PLoS One. 2013; 8(2):e57479). For this reason, mutations for pI modifications were introduced into the CDR1, CDR2 and CDR3 sequence of the humanized anti-human GPC3 antibody prepared according to the method described in Example 2, H0000-ERY27_HK (SEQ ID NO: 54), to obtain modified antibodies. The affinity of these modified antibodies for soluble human GPC3 was then determined.

As a result, it was determined that amino acid modifications that can lead to a decrease in pI while maintaining affinity for human GPC3 should be carried out at amino acid positions 19, 43, 53 and 61 according to Cabot numbering.

By combining sites that have the ability to maintain affinity for human GPC3 and reduce pI, antibodies were obtained whose affinities and pI are presented in Table 10.

(3-4) Modification of extracellular matrix binding ability by point mutation

It is known that nonspecific binding to the extracellular matrix (ECM), etc. may have an effect on pharmacokinetics (MAbs. 4(6), November-December 2012, pp. 753-760). Thus, the ECM-binding ability of the modified antibodies obtained according to the methods described in the examples was determined using the method presented in Example 4 given for reference. As a result, it was confirmed that the humanized bispecific antibody to the human CD3ε chain and to human GPC3, GPC3_ERY27_hCE115 (SEQ ID NO: 54, 55 and 56), had high ECM binding ability. In this way, any of the point mutations evaluated in Examples 3-1, 3-2 and 3-3 in the sequence of the humanized antibody to the human CD3ε chain hCE115HA-ERY27_HE (SEQ ID NO: 55) was studied as a combination to reduce ECM binding capacity. As a result, it was found that amino acids at positions 11, 16, 52a, 53, 98 and 100 according to Cabot numbering are involved in maintaining affinity for CD3ε and can influence the decrease in ECM-binding ability, and antibodies with reduced ECM-binding ability were obtained compared with the ability of a humanized bispecific antibody variant to the human CD3ε chain and to human GPC3, GPC3_ERY27_hCE115 (Table 11).

(3-5) Modifications of SuRe™ ligand binding ability via point mutations

There is a known example that the binding of an antibody to protein A depends on the sequence of the variable region (VH3) (J Biomol Tech. 22(2), July 2011, pp. 50-52). In the Protein A purification of a humanized anti-human CD3ε chain and anti-human GPC3 bispecific antibody, removal of the homomeric anti-CD3 antibody was important to suppress nonspecific responses by CD3. Thus, it appears desirable to inhibit the binding of homomeric anti-CD3 antibody to protein A. It is likely that SuRe™ ligand can be used in commercial production and therefore point mutations affecting binding to SuRe™ ligand have been introduced into the CDR2 H chain of humanized anti-CD3 antibody variants. TR01H082-E2702GsKsc and TR01H084-E2702GsKsc (SEQ ID NOs: 398 and 399), for the production of modified antibodies. The binding ability of these modified antibodies to the SuRe™ ligand was determined using the method presented in Reference Example 5. As a result, amino acids at positions 19, 57 and 59 according to Cabot numbering were found to be involved in maintaining affinity for CD3ε and may influence the ability bind to the Sure™ ligand, and produced antibodies with a reduced ability to bind to the Sure™ ligand compared to the ability of antibodies TR01H082-E2702GsKsc/L0011-k0 (SEQ ID NO: 398 and 410) or TR01H084-E2702GsKsc/L0011-k0 (SEQ ID NO : 399 and 410) (Table 12).

(3-6) Production of optimized bispecific antibodies by combining point mutations leading to improvement of various properties

Optimized modified antibodies can be obtained by combining point mutations that lead to improvements in various properties described in examples 3-1-3-5. As examples of these modified antibodies, the antibodies shown in Table 13 were prepared and assessed for T cell dependent cellular cytotoxicity (TDCC) using methods similar to those described in Example 1. The results are shown in FIG. 4-9. The result was optimized humanized bispecific antibodies to the human CD3ε chain and to human GPC3, having equivalent or higher T-cell-dependent cellular cytotoxicity than GPC3_ERY22_rCE115 before its humanization.

Examples 3-1-3-6 demonstrate that the following amino acid residues, for example, are important for maintaining the properties of optimized anti-human CD3ε chain and anti-human GPC3 bispecific antibodies that have equivalent or superior T-cell dependent cellular cytotoxicity than GPC3_ERY22_rCE115 before its humanization.

Antibodies to the human CD3ε chain contain, for example, Leu at position 11, Gly at position 16, Asp at position 52a, Gin at position 53, Ala at position 72, He at position 78, Ala at position 98, Gly at position 100 and Not at position 102. In antibodies to human GPC3 there are, for example, Thr at position 19, Glu at position 43, Gly at position 52a, Pro or Glu at position 53, Pro at position 55 and Glu at position 61. In addition, in common L-chains there are, for example, Pro at position 25, Pro at position 27a, Pro at position 27b, He at position 33, Gin at position 34, Arg or Trp at position 56 and Tyr at position 89 (all positions are designated according to Cabot numbering).

Example 4: In Vivo Efficacy Evaluation

The effectiveness of some of the antibodies described above was assessed in vivo using tumor-bearing animal models.

In vivo potency assessments were performed on representative antibodies shown in Table 13 that demonstrated cytotoxic activity when assessed using the in vitro assay described in Example 3-6. When evaluating in vivo efficacy, any influence caused by differences in the microenvironment due to the formation of tumor aggregates on the evaluation results should be taken into account. For the study, two types of human cancer cell lines with different sensitivity to the action of antibodies used as drugs were used, i.e. lines PC-10 and NCI-H446, despite the fact that the expression levels of GPC3 in these cells were almost the same. Cell lines were transplanted into severe combined immunodeficiency (NOD scid) mice and NOD scid mice confirmed to form tumors were transplanted with T cells grown by in vitro culture of human PBMCs. Mice (referred to as the T cell injected model) were treated with optimized bispecific antibodies to the human CD3ε chain and human GPC3.

More specifically, in experiments to evaluate the drug efficacy of optimized bispecific antibodies to the human CD3ε chain and human GPC3 using PC-10 cells in a T cell injected model, the experiments described below were performed. T cells were expanded by culture using PBMC isolated from the blood of healthy donors and a T cell activation/expansion kit/human (MACS Miltenyi biotec). The human cancer cell line PC-10 (1×10 ⁷ cells) was mixed with Matrigel™ basement membrane matrix (BD) and transplanted subcutaneously into the inguinal region of NOD scid mice (CLEA Japan, 6-week-old females). The day of transplantation was designated as day 0. One day before transplantation, mice were intraperitoneally injected with anti-asialo-ganglioside M1 (asialo-GM1) antibody (Wako Pure Chemical Industries) at a dose of 0.2 mg/mouse. 13-15 days after transplantation, mice were divided into groups depending on body weight and tumor size, and anti-Asialo-GM1 antibody was again intraperitoneally administered at a rate of 0.2 mg/mouse. The next day, T cells obtained by the above-described method of propagation by cultivation were transplanted intraperitoneally at a rate of 3×10 ⁷ cells/mouse. 4 hours after T-cell transplantation, optimized bispecific antibodies to the human CD3ε chain and human GPC3 were administered intravenously through the caudal vein at a dose of 1 mg/kg. Optimized bispecific antibodies to the human CD3ε chain and human GPC3 were administered only once.

As a result, a more pronounced antitumor activity was established in the group treated with optimized bispecific antibodies to the human CD3ε chain and human GPC3, compared to the solvent-treated group (Fig. 10a, b).

Similar methods were used in experiments to evaluate the effectiveness as drugs of optimized bispecific antibodies to the human CD3ε chain and human GPC3, in which NCI-H446 cells were used, in a model with injected T cells. To evaluate the effect on NCI-H446, optimized bispecific antibodies to the human CD3ε chain and human GPC3 were administered once via the caudal vein at a dose of 5 mg/kg.

As a result, a more pronounced antitumor activity was established in the group treated with optimized bispecific antibodies to the human CD3ε chain and human GPC3, compared with the group treated with the solvent (Fig. 11a, b).

Examples provided for reference

For reference, Example 1: Preparation of Antibody Expression Vectors and Antibody Expression and Purification

Amino acid substitutions were introduced by methods known to those skilled in the art, for example, using the QuikChange site-directed mutagenesis kit (Stratagene), PCR, or In-fusion Advantage PCR cloning kit (TAKARA), to create expression vectors. The nucleotide sequences of the resulting expression vectors were determined using a method known to those skilled in the art. The resulting plasmids were briefly introduced into cells of a cell line derived from human embryonic kidney HEK293H (Invitrogen) or FreeStyle293 (Invitrogen) for antibody expression. From the resulting culture supernatants, antibodies were purified using an rProtein A Sepharose™ Fast Flow column (GE Healthcare) using a method known to those skilled in the art. The absorbance at 280 nm of the purified antibody solutions was measured using a spectrophotometer, and the antibody concentration was calculated from the determined values using the absorbance coefficient calculated using the PACE method (Protein Science 4, 1995, pp. 2411-2423).

For reference, Example 2: Determination of the APCC activity of each antibody tested using human peripheral blood mononuclear cells as effector cells.

The ADCC activity of each antibody tested was determined using the method described below.

Human peripheral blood mononuclear cells (hereinafter referred to as human PBMC) were used as effector cells to measure the ADCC activity of each of the tested antibodies according to the method described below.

(1) Preparation of human PBMC solution

Samples of 50 ml of peripheral blood from healthy volunteers (adult men) were obtained using syringes pre-filled with 200 μl (1000 units/ml) of heparin solution (Novo-Heparin 5000 units per injection; Novo Nordisk). This peripheral blood was diluted twice with PBS(-), divided into four aliquots and added to Leucosep lymphocyte separation tubes (Greiner bio-one), into which 15 ml of Ficoll-Pak PLUS were previously added and centrifuged. After centrifugation of separation tubes containing peripheral blood aliquots at 2150 rpm for 10 min at room temperature, mononuclear cell fractions were collected. The cells in each fraction were washed once using Dulbecco's Modified Eagle's Medium (SIGMA) containing 10% FBS (hereinafter referred to as 10% FBS/D-MEM) and then the cell density was adjusted to 5×10 ⁶ cells/ml using 10% FBS/D-MEM. The cell suspension was used as the target cell in the experiments below. (2) Chromium release assay (APCC activity)

ADCC activity was assessed by the specific chromium release rate determined by the chromium release assay. First, antibody solutions at each of the prepared concentrations (0, 0.004, 0.04, 0.4, 4, and 40 μg/mL) were added to a 96-well U-bottom plate at a rate of 50 μL per well. Target cells were then seeded at a rate of 50 μl per well (1 x 10 ⁴ cells/well) and allowed to stand at room temperature for 15 min. The human PBMC solution prepared according to the method described in (1) was added at a rate of 100 μl per well (5 x 10 ⁵ cells/well) and the plate was incubated in an atmosphere containing 5% carbon dioxide at 37° C. for 4 hours, after which it was centrifuged. The radioactivity in 100 μl of culture supernatant in each well of the plate was measured using a gamma counter. The specific rate of chromium release was determined using the following formula:

specific chromium release rate (%)=(A-B)×100/(B-C),

where A represents the average radioactivity value (cpm) of 100 μl of culture supernatant in each well; B represents the average radioactivity value (cpm) of 100 μl of culture supernatant in wells in which 100 μl of a 2% aqueous solution of NP-40 (Nonidet P-40, Nacalai Tesque) and 50 μl of 10% aqueous solution were added to the target cells. medium FBS/D-MEM; and B represents the average radioactivity (cpm) of 100 μl of culture supernatant in wells in which 150 μl of 10% FBS/D-MEM medium was added to the target cells. The experiments were carried out in triplicate and, based on the results of the above experiment, the average value and standard deviation of the specific chromium release rate (%) reflecting ADCC activity were calculated for each of the tested antibodies.

Example 3 for reference: Determination of Tm modified antibodies using differential scanning fluorometry

In this assessment, the Tm value (thermal denaturation temperature) of the modified antibodies was determined using differential scanning fluorometry using a Rotor-Gene Q device (QIAGEN). This method is known to provide a favorable correlation with the Tm value determined using a differential scanning calorimeter, which is a well-known method for assessing the thermal stability of antibodies (Journal of Pharmaceutical Science 4, 2010, pp. 1707-1720).

5000x SYPRO™ orange concentrate (Molecular Probes) was diluted with PBS (Sigma) and then mixed with antibody solutions to obtain samples for measurement. Aliquots of 20 μl of each sample were added to measuring tubes and the temperature was increased from 30°C to 99°C at a temperature increase rate of 240°C/h. Fluorescence changes accompanying temperature increases were detected at 470 nm (excitation wavelength)/555 nm (fluorescence emission wavelength).

The data were analyzed using the Rotor-Gene Q Series program (QIAGEN) to calculate the temperature at which the fluorescence transition occurs, and this temperature was taken as Tm.

Example 4 for reference: Determination of ECM binding capacity

The determination was carried out according to the method described in WO 2012/093704. In general, the method was as follows: BD-Matrigel (BD Biosciences, no. 356237) was prepared at a concentration of 2 mg/ml using TBS (Takara, no. T903) and transferred to a 96-well measurement plate (Meso Scale Discovery , No. L15XB-3 (High Bind) at a rate of 5 µl per well and then allowed to stand overnight in a cold place. Then, 150 μl of ECL blocking buffer (PBS containing 0.05% Tween20, 0.5% BSA, and 0.01% sodium azide) was added to each well of the plate and allowed to stand at room temperature for 2 hours or more.

Goat anti-human IgG(γ) (Invitrogen, #628400) was labeled with ruthenium using NHS ester labeled MSD-SULFO (Meso Scale Discovery, #R91AN-2) according to the accompanying instructions. It was diluted with ECL dilution buffer (PBS containing 0.01% Tween20, 0.1% BSA, and 0.01% sodium azide) to a final concentration of 2 μg/ml. In addition, the standard antibody and test antibodies were diluted with PBS-T (PBS containing 0.05% Tween20 and 0.01% sodium azide) to a final concentration of 3 μg/ml.

In a 96-well reaction plate (Thermo scientific, Nunc No. 145399), 10 μl of ECL dilution buffer, 20 μl of standard antibody and test antibody (3 μg/ml) and 30 μl of ruthenium-labeled antibody (2 μg) were added sequentially. /ml), and allowed to react for 1 hour at room temperature with stirring in the dark.

The ECL blocking buffer was removed from the 96-well reaction plate by tipping, 50 μL of the sample solution from the 96-well reaction plate was added, and allowed to stand in the dark at room temperature for 1 hour. The sample solution was then removed from the 96-well reaction plate to measurement (measuring plate) by inverting and immediately adding 150 μl of 2×T buffer (4× MSD T-reading buffer (Meso Scale Discovery), diluted 2-fold with ECL dilution buffer), ECL measurements were carried out. A SECTOR Imager 2400 device (Meso Scale Discovery) was used for measurements.

Assays were performed by dividing the fluorescence intensity of the test antibody by the fluorescence intensity of the standard antibody to calculate and compare intensities, taking the value for the standard antibody as 1.

Reference Example 5 Determination of SuRe™ Ligand Binding Ability

SuRe™ ligand binding ability was assessed using Biacore™-T200 (GE Healthcare Japan). HBS-EP+ (GE Healthcare Japan) was used as a running buffer and an amine coupling kit (GE Healthcare Japan) was used to covalently couple the Mab Select SuRe™ ligand (GE Healthcare Japan) to the CM5 chip (carboxymethyldextran coated chip). . The antibody used as an analyte was prepared at a concentration of 5 μg/ml using HBS-EP+. To perform measurements, a solution with an antibody concentration of 5 μg/ml was first injected at a flow rate of 10 μl/min for 3 min, then the analyte was replaced with HBS-EP+ and the response (RU) was assessed after passing the flow for 0.5 min. After completion of the measurements, the sensor chip was regenerated by washing with 10 mM Gly-HCl at pH 1.5. In a control flow cell, the same experiment was performed without covalently binding the ligand to the chip, and affinity for the SuRe™ ligand was analyzed based on response differences (RU).

The sequences corresponding to the above-mentioned sequence numbers (SEQ ID NO:) are presented in the table below.

Industrial applicability

The present invention provides new multispecific antigen-binding molecules that retain the potent anticancer activity of BiTE and have very high safety, as evidenced by the absence of induction of a cancer antigen-independent cytokine storm, and also have a long half-life in the blood. Cytotoxicity-inducing agents that contain as an active substance the antigen-binding molecule of the present invention can target glypican 3-expressing cells and tumor tissues that contain such cells and induce cell damage. Administration of the multispecific antigen-binding molecules of the present invention to patients allows them to receive the desired treatment, which is not only highly safe, but also reduces the physical burden on the patient and is very convenient.

Claims (10)

1. An antibody that specifically binds to CD3 epsilon, wherein the heavy chain variable region and the light chain variable region of the antibody comprise a combination of the heavy chain CDR1, CDR2 and CDR3 and the light chain CDR1, CDR2 and CDR3, respectively, selected from the following (a1) - (a3): (a1) a combination of heavy chain CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 142; and light chain CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 223; (a2) a combination of heavy chain CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 164; and light chain CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 223; (a3) a combination of heavy chain CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 168; and light chain CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions contained in SEQ ID NO: 223. 2. The antibody according to claim 1, additionally containing an Fc region with reduced binding activity to the Fcγ receptor. 3. The antibody of claim 2, wherein the Fc region is a combination of (b1) the amino acid sequences set forth below: (b1) a combination of an amino acid sequence identical to the Fc region of the constant region containing the amino acid sequence of SEQ ID NO: 60 or 62, and the amino acid sequence identical to the Fc constant region region containing the amino acid sequence of SEQ ID NO: 61. 4. Nucleic acid encoding an antibody according to any one of paragraphs. 1-3. 5. An expression vector into which the nucleic acid according to claim 4 is introduced. 6. Cell for producing antibodies according to any one of paragraphs. 1-3, containing the vector according to claim 5. 7. A method for producing an antibody according to any one of claims. 1-3 by culturing the cell according to claim 6 and collecting the antibody from the culture supernatant.

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