EA041830B1 - BISPECIFIC ANTIBODY BINDING GLYPICAN 3 AND CD3ε (VERSIONS), PHARMACEUTICAL COMPOSITION INCLUDING IT, AND METHOD FOR PRODUCING THIS ANTIBODY - Google Patents

Description

The field of technology to which the invention belongs

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

Prerequisites for the creation of the invention

Antibodies are attracting attention as therapeutic agents due to their high plasma stability and few adverse reactions (Non-Patent Documents 1 and 2). Antibodies are known to induce not only antigen-binding activity, agonistic action, and antagonistic action, but also effector-mediated cytotoxic activities (also referred to as effector functions), such as antibody-mediated cell-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement dependent cytotoxicity (CDC), and have antitumor activities against cancer cells (Non-Patent Document 3). ADCC is effector cell-mediated cytotoxicity against antibody-loaded target cancer cells by binding the Fc region of an antibody to an Fc receptor present on effector cells such as NK cells and macrophages. The complement system complex binds to a complement binding site present in the antibody structure. CDC is detrimental to cells as a result of cellular degradation, in which the influx of water and ions into cells is enhanced by the formation of pores in the cell membrane of antibody-loaded cells by the complement components present in the complex. A number of therapeutic antibodies with very high antitumor activities have been developed as pharmaceuticals for the treatment of cancer (Non-Patent Document 4).

For an antibody to mediate ADCC, ADCP and CDC, there must be an Fc region in the antibody and the presence of a receptor for the antibody (FcyR) on effector cells such as NK cells and macrophages and various complement components suitable for binding. In humans, FcyRIa, FcyRIIa, FcyRIIb, FcyRIIIa, and FcyRIIIb isoforms are described as an FcyR protein family, and corresponding allotypes are also described (Non-Patent Document 5). Among these isoforms, FcyRIa, FcyRIIa and FcyRIIIa carry an immunoreceptor tyrosine-based activating motif (ITAM) in the intracellular domain and transmit activation signals. On the other hand, only FcyRIIb carries a domain known as the tyrosine receptor-based inhibitory motif (ITIM) in the intracellular domain and transmits inhibition signals. Each of the FcyRs is known to signal through cross-linking with immune complexes and the like. (non-patent document 6). When the antibodies actually exhibit effector function on cancer cells, the FcyRs on the effector cell membrane cluster on the Fc regions of several antibodies bound on the cancer cell membrane, and activation signals are transmitted by the effector cells. As a result, a cytocidal effect is exhibited, but since FcyRs are cross-linked only on effector cells that are present near cancer cells at this time, it is found that the activation of immunity occurs locally in cancer cells (Non-Patent Document 7).

Naturally occurring immunoglobulins bind to antigens in their variable regions and bind to receptors such as FcyR, FcRn, FcaR 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 has been found that since each of the heavy chains of an antibody binds to one FcRn molecule, two FcRn molecules bind to one IgG-type antibody molecule. However, unlike FcRn and others, FcyR interacts with the antibody hinge region and the CH2 domain, and only one FcyR molecule binds to one IgG-type antibody molecule (Non-Patent Document 8). In addition, a canonical naturally occurring IgG-type 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 cross-talk between proteins may occur. For example, several inflammatory cytokines (TNF, IL1 and IL6) are known to be involved in immunological diseases (Non-Patent Document 9). In addition, it is known that one of the mechanisms for acquiring drug resistance of cancer 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 (first antigen and 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 by cross-linking between cells that have cytotoxic activity and cancer cells. To date, a molecule containing an antigen-binding site added to the N- or C-terminus of the antibody has been described as molecular formats of a bispecific antibody (DVD-Ig and scFv-1 041830

IgG), a molecule having different sequences of two Fab regions of an antibody (containing a common L-chain bispecific antibody and hybridoma), a molecule in which one Fab region recognizes two antigens (IgG two-in-one), and a molecule having a loop CH3 region as a new antigen-binding site (Fcab) (Non-Patent Documents 12 and 13). Since all bispecific antibodies interact on their Fc regions with FcyR, the effector functions of the antibody are preserved. Thus, the bispecific antibody binds to any antigen it recognizes and simultaneously binds to FcyR and confers ADCC activity on cells expressing the antigen.

If all of the antigens recognized by the bispecific antibody are specifically expressed in cancer, then the bispecific antibody has a cytotoxic effect on cancer cells when it binds to any of the antigens. Thus, compared to a canonical pharmaceutical antibody that recognizes a single antigen, said 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 as a result of cross-linking with FcyR occurs. (non-patent document 14). As a result, strong undesirable reactions are induced.

A T cell redirecting antibody that exploits cytotoxicity by activating T cells as effector cells, which is its antitumor mechanism, has been known since the 1980s (Non-Patent Documents 15, 16 and 17). Unlike antibodies that use ADCC to activate 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 a target cancer cell. The cells come into contact with the cancer cells by the simultaneous binding of the CD3 epsilon chain and the cancer antigen by a T-cell redirecting antibody. This results in an antitumor effect on cancer cells due to cytotoxic activity mediated by T cells.

Catumaxomab, known as a T cell redirecting antibody, binds via two Fabs to the cancer antigen (EpCAM) and to the CD3ε- (CD3-epsilon) chain expressed on T cells. Catumaxomab induces T-cell mediated cytotoxic activity by concurrent binding to cancer antigen and CD3ε and induces cytotoxic activity mediated by antigen presenting cells such as NK cells and macrophages by concurrent binding to cancer antigen and FcyR. 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 allowed in Europe (non-patent document 18). In addition, there are cases where administration of catumaxomab is reported to result in cancer-reactive antibodies, which clearly demonstrate the induction of adaptive immunity (Non-Patent Document 19). In view of this result, antibodies that have both T-cell-mediated cytotoxic activity and FcyR-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 induction acquired immunity.

However, trifunctional antibodies bind simultaneously to CD3ε and FcyR even in the absence of a cancer antigen and therefore cross-link CD3ε expressing T cells with FcyR expressing cells even in the absence of cancer cells in the environment, leading to the production of large amounts 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, in a phase I clinical trial, it was found that the maximum tolerated dose for systemic administration of catumaxomab to patients with non-small cell lung cancer is a very low dose of 5 μg/body, and that administration of a higher dose causes serious adverse reactions (Non-Patent Document 21).

Thus, conventionally generated bispecific antibodies can bind to both antigens, with the first antigen being a cancer antigen (EpCAM) and the second antigen being CD3ε, while simultaneously binding to FcyR; and therefore, due to their molecular structure, it is not possible to avoid unwanted reactions caused by simultaneous binding to FcyR and the second CD3ε antigen.

At the same time, unlike catumaxomab, BiTE (a bispecific activator (conductor) of T cells) does not

- 2 041830 contains an Fcγ receptor binding site and therefore lacks cross-linking 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 cancer-antigen-independent cytokine induction, which is found with the introduction of catumaxomab. However, since BiTE is a modified low molecular weight antibody molecule without an Fco region, the problem is that its half-life in blood after administration to a patient is significantly shorter than for IgG-type antibodies that are commonly used as therapeutic antibodies. In fact, according to the published data, the blood half-life of BiTE when used in vivo is about several hours (Non-Patent Documents 22 and 23). In clinical trials, blinatumomab was administered by continuous intravenous infusion using a minipump. Such an administration method is not only extremely inconvenient for patients, but also has the potential risk of medical complications due to device malfunction or the like. Thus, this method of administration cannot be considered desirable.

In recent years, the use of an Fc region with a reduced ability to bind to FcyR has made it possible to maintain the strong antitumor activity inherent in BiTE and very good safety characteristics without inducing a cytokine storm in a cancer antigen-dependent manner, and has provided the creation of new polypeptide structures with a long half-life 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, such a route is very inefficient and complicated.

As a solution to this problem, a method has been described that preferentially secretes 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 IgG H chain (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. protrusions and depressions, and a method based on the repulsion of electric charges.

In order to obtain a molecule of interest with increased potency, methods have been described 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 (Patent Documents 8 and 9). However, affinity for an antigen can be significantly reduced when using common L chains and it is difficult to find common L chains that retain affinity for the antigen.

List of listed documents

Patent Documents.

Patent Document 1. WO2012/073985.

Patent document 2. WO96/27011.

Patent document 3. WO2006/106905.

Patent document 4. WO2007/147901

Patent document 5. WO2009/089004.

Patent document 6. WO2010/129304.

Patent Document 7. WO2013/065708.

Patent document 8. WO98/050431.

Patent document 9. WO2006/109592.

non-patent documents.

Non-Patent Document 1. Nat. Biotechnol. 23, 2005, p. 1073-1078.

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

Non-Patent Document 3. Drug Des Devel Ther 3, 2009, p. 7-16.

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

Non-Patent Document 5. Immunol. Lett. 82, 2002, p. 57-65.

Non-Patent Document 6. Nat. Rev. Immunol. 8, 2008, p. 34-47.

Non-Patent Document 7. Ann. Rev. Immunol. 6, 1988, p. 251-281.

Non-Patent Document 8. J. Bio. Chem., 276, 2001, p. 16469-16477.

Non-Patent Document 9. Nat. Biotech., 28, 2011, p. 502-510.

Non-Patent Document 10. Endocr Relat Cancer 13, 2006, p. 45-51.

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

Non-Patent Document 12. Nat. Rev. 10, 2010, p. 301-316.

Non-Patent Document 13. Peds 23(4), 2010, p. 289-297.

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

- 3 041830

Non-Patent Document 15. Nature 314 (6012), 1985, p. 628-631.

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

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

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

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

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

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

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

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

Non-Patent Document 24. Protein Engineering, vol. 9, 1996, p. 617-621.

Non-Patent Document 25. Nature Biotechnology, Vol. 16, 1998, c. 677-681.

Brief summary of the invention Objectives underlying the invention

The present invention has been made in view of the above circumstances. The object of the present invention was to provide multispecific antigen-binding molecules that have the ability to bring T cells into contact with cancer target cells and that can be used to treat cancer using the cytotoxic activity of T cells against cancer target tissues that contain expressing glypican 3 cells, and are molecular formats that can be produced with high efficiency; develop methods for obtaining antigen-binding molecules; and pharmaceutical compositions that contain antigen-binding molecules as an active ingredient.

Means for solving these problems

When creating the present invention, an L chain is described that is shared with a domain that contains the variable region of an antibody that binds to glypican 3, and a domain that contains the variable region of an antibody that binds to the T-cell receptor complex, while the common L chain has the ability to increase affinity to both antigens. This allows for high potency molecular forms, and further described are novel multi-specific antigen-binding molecules that retain the potent anti-tumor activity of T-cell redirecting antibodies such as BiTE and have very good safety characteristics without inducing a cytokine storm depending on the cancer. antigen, and also have a long half-life in the blood. In addition, the present invention has found 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 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 the following.

1. Multispecific antigen-binding molecule, which 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 Fc- a region with reduced Fcy receptor binding activity, in which the variable regions of the L chain contained in the variable region indicated in subparagraph (1) and the variable region indicated in subparagraph (2) have a common amino acid sequence; where the multispecific antigen-binding molecule has a cytotoxic activity equivalent to or greater than the activity 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 according to claim 1 or 2, wherein the T-cell receptor complex binding activity is T-cell receptor binding activity.

4. The multispecific antigen-binding molecule according to one of claims 1 to 3, wherein the T-cell receptor complex binding activity is CD3ε chain binding activity.

5. The multispecific antigen-binding molecule according to one of claims 1 to 4, wherein the antibody variable region specified in subparagraph (1) of claim 1 is an antibody variable region that contains any one of combinations of CDR1, CDR2, and CDR3 H- a chain selected from those listed in subparagraphs (a1)-(a5) below, or a functionally equivalent variable region of an antibody:

- 4 041830 (al) CDR1, CDR2 and CDR3 identical to the amino acid sequences of CDR1-, CDR2- and

CDR3-y4acTKoe contained in SEQ ID NO: 40;

(a2) CDR1, CDR2 and CDR3 identical to the amino acid sequences of CDR1-, CDR2- and

CDR3 regions as contained in SEQ ID NO: 197;

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

(a4) CDR1, CDR2, and CDR3 identical to the amino acid sequences of the CDR1, CDR2, and CDR3 y 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 y regions contained in SEQ ID NO: 215.

6. The multispecific antigen-binding molecule according to one of claims 1 to 4, wherein the antibody variable region specified in subparagraph (2) of claim 1 is an antibody variable region that contains any one of combinations of the amino acid sequences CDR1, CDR2, and CDR3 An H chain selected from (b1) to (b15) below, or a functionally equivalent antibody variable region:

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

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

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

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

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

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

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

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

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

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

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

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

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

(b14) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR3 regions contained in SEQ ID NO: 429; and (b15) CDR1, CDR2 and CDR3 identical to the amino acid sequences of the CDR3 y regions contained in SEQ ID NO: 430.

CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2i and

and and

and and

and and

and and

and and

and and and

7. The multispecific antigen-binding molecule according to one of claims 1 to 4, wherein the antibody variable regions specified in subparagraphs (1) and (2) in paragraph 1 are antibody variable regions that contain any one of combinations of CDR1, CDR2 and an H chain CDR3 selected from (c1) to (c19) below, or a functionally equivalent antibody variable region:

(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 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,

- 5 041830 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

CDR1, CDR2 and CDR3 regions 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;

(in 11) 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 amino acid sequences

- 6 041830 to the values of CDR1-, CDR2- and CDR3-y4acTKoe 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 identical to the amino acid sequences of the CDR1-, CDR2- and CDR3-regions, which are contained in SEQ ID NO: 424.

8. A multispecific antigen-binding molecule according to one of claims 5 to 7, wherein CDR1, CDR2 and CDR3 are CDR1, CDR2 and CDR3 regions determined based on Cabot numbering.

9. The multispecific antigen-binding molecule according to one of claims 1 to 4, wherein the antibody variable region specified in subparagraph (1) of claim 1 is an antibody variable region that contains any one of the H chain variable regions selected from specified in subparagraphs (a1)-(a5) below, or a 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. The multispecific antigen-binding molecule according to one of claims 1 to 4, wherein the antibody variable region specified in subparagraph (2) of claim 1 is an antibody variable region that contains any one of the H chain variable regions selected from specified in subparagraphs (b1)-(b15) below, or a functionally equivalent variable region of the antibody:

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

(b2) an H chain variable region that 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 that 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;

- 7 041830 (b11) variable NO: 421;

(612) variable NO: 424;

(613) variable NO: 426;

(b14) variable NO: 429; and (b15) variable NO: 430.

H chain region which is an H chain region which is an H chain region which is an H chain region which is an H chain region which has an amino acid has an amino acid has an amino acid has an amino acid has an amino acid sequence SEQ ID sequence SEQ ID sequence SEQ ID sequence SEQ ID sequence SEQ ID

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

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

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

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

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

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

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

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

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

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

(c10) the variable region of the H chain, 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: 206; and the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph 1, which has the amino acid sequence of SEQ ID NO: 168;

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

(c12) the variable region of the H chain, 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: 211; and the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph

- 8 041830 (2) in claim 1, which has the amino acid sequence of SEQ ID NO: 144;

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

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

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

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

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

(c18) the variable region of the H chain, 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 the variable region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in paragraph 1, which has the amino acid sequence of SEQ ID NO: 132; and (c19) the variable region of the H chain, 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: 424.

12. The multispecific antigen-binding molecule according to one of claims 1 to 11, wherein the total L chain of claim 1 is a general L chain that contains any one of the combinations of CDR1, CDR2, and CDR3 selected from those listed in sub-clauses below. (r1)-(r11), or the functional equivalent of the general L-chain:

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

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

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

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

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

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

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

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

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

(d10) CDR1, CDR2 and CDR3 identical to the amino acid CDR3 regions contained in SEQ ID NO: 310; and (d11) CDR1, CDR2 and CDR3 identical to the amino acid CDR3 regions contained in SEQ ID NO: 319.

sequences sequences sequences sequences sequences sequences sequences sequences sequences sequences

CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2CDR1-, CDR2 and

and and

and and

and and

and and and

13. The multispecific antigen-binding molecule according to one of claims 1 to 11, wherein the variable region of the L chain specified in claim 1 is the variable region of any one of the amino acid sequences of the L chain selected from those indicated in subparagraphs (d1) below. )-(r11):

(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;

- 9 041830 (g5) L-chain containing the amino acid sequence of SEQ ID NO: 302;

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

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

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

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

(d10) L-chain containing the amino acid sequence of SEQ ID NO: 310; and (d11) an L chain containing the amino acid sequence of SEQ ID NO: 319.

14. The multispecific antigen-binding molecule according to one of claims 1 to 4, wherein the antibody variable regions of subparagraphs (1) and (2) of claim 1 and the common L chain variable region are antibody variable regions 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 the following 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 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 common 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 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 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 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 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 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 that are contained in the variable region of an antibody listed under

- 10 041830 paragraph (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 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 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 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 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 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 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 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 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 identical to the amino acid sequences of the CDR1, CDR2 and CDR3 regions, which are contained in SEQ ID NO: 301;

- 11 041830 (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 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 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 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 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 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 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 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 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 common L chain of the antibody, and identical to the amino acid sequences of CDR1-, CDR2-, and CDR3-y4acTKoe, which are contained in SEQ ID NO: 53.

15. The multispecific antigen-binding molecule according to one of claims 1 to 4, wherein the antibody variable regions of subparagraphs (1) and (2) of claim 1 and the common L chain variable region are antibody variable regions containing any one of the combinations of variable regions selected from those listed in subparagraphs (e1)-(e26) below, or functionally equivalent antibody variable regions:

(e1) 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 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 identical to the amino acid sequence of SEQ ID NO: 128; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 53;

(e2) 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 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 identical to the amino acid sequence of SEQ ID NO: 128; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 299;

(e3) 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 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 identical to the amino acid sequence of SEQ ID NO: 128; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 310;

(e4) 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 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 identical to the amino acid sequence of SEQ ID NO: 128; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 319;

(e5) 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 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 identical to the amino acid sequence of SEQ ID NO: 142; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 223;

(e6) 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 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 identical to the amino acid sequence of SEQ ID NO: 144; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 223;

(e7) 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 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 identical to the amino acid sequence of SEQ ID NO: 164; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 223;

(e8) 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 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 identical to the amino acid sequence of SEQ ID NO: 168; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 223;

(e9) 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 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 identical to the amino acid sequence of SEQ ID NO: 142; and the variable region of the general L-chain of the antibody, identical to the amino acid sequence of the variable region, which is 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 identical to the amino acid sequence of SEQ ID NO: 211; variable-

- 13 041830 nay region of the H chain, which is contained in the variable region of the antibody specified in subparagraph (2) in

1, and identical to the amino acid sequence of SEQ ID NO: 142; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 299;

(e11 ) 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 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 identical to the amino acid sequence of SEQ ID NO: 144; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 223;

(e12 ) 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 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 identical to the amino acid sequence of SEQ ID NO: 164; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is 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 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 identical to the amino acid sequence of SEQ ID NO: 168; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 223;

(e14) 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 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 identical to the amino acid sequence of SEQ ID NO: 103; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 53;

(e15 ) 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 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 identical to the amino acid sequence of SEQ ID NO: 103; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 299;

(e16 ) 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 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 identical to the amino acid sequence of SEQ ID NO: 103; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 301;

(e17 ) 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 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 identical to the amino acid sequence of SEQ ID NO: 103; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 302;

(e18 ) 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 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 identical to the amino acid sequence of SEQ ID NO: 103; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is contained in SEQ ID NO: 304;

(e19 ) 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 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 identical to the amino acid sequence of SEQ ID NO: 103; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is 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 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 identical to the amino acid sequence of SEQ ID NO: 103; and variable region of common L-

- 14 041830 antibody chain identical to the amino acid sequence of the variable region, which is 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 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 identical to the amino acid sequence of SEQ ID NO: 103; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is 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 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 identical to the amino acid sequence of SEQ ID NO: 122; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is 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 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 the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is 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 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 identical to the amino acid sequence of SEQ ID NO: 132; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is 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 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 identical to the amino acid sequence of SEQ ID NO: 424; and the variable region of the general L chain of the antibody, identical to the amino acid sequence of the variable region, which is 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 a T-cell receptor complex that binds to the multispecific antigen-binding molecule specified in one of subparagraphs (e1)(e25) and which has a common L-chain.

16. The multispecific antigen-binding molecule according to one of claims 1 to 15, wherein the Fc region specified in subparagraph (3) of claim 1 is an Fc region with an amino acid mutation of any of the amino acid sequences forming the Fc region of SEQ ID NO: 23 -26 (IgG1-IgG4).

17. The multispecific antigen-binding molecule according to claim 16, wherein the Fc region specified in subparagraph (3) of claim 1 is an Fc region with a mutation of at least one amino acid selected from the amino acids at the following amino acid positions specified according to EU numbers: 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 claim 16, wherein the Fc region specified in subparagraph (3) of claim 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. The multispecific antigen-binding molecule according to one of claims 16 to 18, wherein the Fc region specified in subparagraph (3) of claim 1 further comprises 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 Fco 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. The multispecific antigen binding molecule according to one of claims 1 to 20, wherein the multispecific antigen binding molecule is a bispecific antibody.

22. A 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 53;

(h2) a bispecific antibody having an antibody H chain that has binding activity against 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 53;

(h3) a bispecific antibody having an antibody H chain that has binding activity against 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 the general L-chain of the antibody, which has 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 : 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 the total L-chain of the antibody, which has 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 the general L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 302;

(h6) a bispecific antibody having an antibody H chain that has binding activity against 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 304;

(h7) a bispecific antibody having an antibody H chain that has binding activity against 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

- 16 041830 Paradise has the amino acid sequence of SEQ ID NO: 60 or 62; and the general L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 306;

(h8) 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 the general L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 307;

(h9) a bispecific antibody having an antibody H chain that has binding activity against 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 L-chain of the antibody, which has 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 : 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 the total L-chain of the antibody, which has 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 : 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 53;

(h12) 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: 132 and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 53;

(h13) a bispecific antibody having an antibody H chain that has binding activity against 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 299;

(h14) a bispecific antibody having an antibody H chain that has binding activity against 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 310;

(h15) a bispecific antibody having an antibody H chain that has binding activity against 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; H chain of an antibody that has a binding active

- 17 041830 with respect to the T-cell receptor complex, containing the variable region of the H chain of the antibody, which has the amino acid sequence of SEQ ID NO: 128, and the constant region, which has the amino acid sequence of SEQ ID NO: 60 or 62; and the general L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 319;

(h16) 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 53;

(h17) a bispecific antibody having an antibody H chain that has binding activity against 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 the general L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 299;

(h18) 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 223;

(h19) 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: 144 and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 223;

(h20) a bispecific antibody having an antibody H chain that has binding activity against 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 223;

(h21) a bispecific antibody having an antibody H chain that has binding activity against 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 223;

(h22) a bispecific antibody having an antibody H chain that has binding activity against 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 223;

(h23) a bispecific antibody having an antibody H chain that has binding activity for glypican 3, containing an antibody H chain variable region that has

- 18 041830 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: 168 and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 223;

(h24) 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 the total L-chain of the antibody, which has the amino acid sequence of SEQ ID NO: 223; and (h25) 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: 168 and a constant region that has the amino acid sequence of SEQ ID NO: 60 or 62 ; and a common antibody L chain which has the amino acid sequence of SEQ ID NO: 223.

23. A nucleic acid that encodes a multispecific antigen-binding molecule according to one of claims 1 to 20 or a bispecific antibody according to claim 21 or 22.

24. A vector into which the nucleic acid of claim 23 has been introduced.

25. A cell containing a nucleic acid according to claim 23 or a vector according to claim 24.

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

27. Multispecific antigen-binding molecule or bispecific antibody obtained/obtained by the method of claim 26.

28. A pharmaceutical composition containing a multispecific antigen-binding molecule according to one of claims 1 to 20 or a bispecific antibody according to claim 21 or 22 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition according to claim 28 which induces cytotoxicity.

30. The pharmaceutical composition of claim 29, wherein the cytotoxicity is T-cell dependent cytotoxicity.

31. A 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 claim 21 or 22.

The present invention also relates to 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 obtained using the method of the present invention. The present invention also relates to the use of the multispecific antigen-binding molecule of the present invention or the multispecific antigen-binding molecule obtained by the method of the present invention for preparing a pharmaceutical composition for activating cytotoxic activity. The present invention also relates to a multispecific antigen-binding molecule of the present invention or a multispecific antigen-binding molecule obtained 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 a T-cell receptor complex;

in which the variable regions of the L chain 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 specified in subparagraph (1), which is more specifically a domain containing variable regions of the heavy chain and/or light chain of the antibody, which have binding

- 19 041830 activity against glypican 3, and which is part of a multispecific antigen-binding molecule. The present invention also relates to the domain specified in subparagraph (2), which is more specifically a domain containing the variable region of an antibody that has binding activity against the T-cell receptor complex, and which is part of a multispecific antigen-binding molecule. Components of the domains specified in subparagraphs (1) and (2) may include those specified in paragraphs 1-22 above. 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 mentioned in paragraphs 1-22 above. 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 a vector, a method for producing a multispecific antigen-binding molecule, which consists in culturing the cells, and the multispecific antigen-binding molecule or domains that contain an antibody variable region that has glypican 3 binding activity or T-cell receptor complex binding activity 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 may induce cell injury, the cell injury may be due to T-cell dependent cellular cytotoxicity, and the composition may be administered to a patient in need of a multispecific antigen-binding molecule.

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

With regard to the two Fc regions described in subparagraphs (g1) and (g2) of paragraph 20 above, the first Fc region may be included in the H chain of an antibody that has binding activity for glypican 3, and the second Fc 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 may 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 no induction of a cancer antigen-independent cytokine storm, etc. and have a long half-life in the blood. Pharmaceutical compositions that activate cytotoxic activity, containing the multispecific antigen-binding molecule of the present invention as an active ingredient, target cancer tissues that contain glypican 3 expressing cancer cells to cause cell damage, and can be used to treat or prevent various types of cancer. cancer. The invention provides the desired treatment, which not only has a high level of safety, but also reduces the physical burden on patients and is very convenient for patients.

Brief description of the drawings

The drawings show:

Fig 1 is a schematic diagram of 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. Solid diamonds (♦) and solid triangles (▲) indicate 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. Solid diamonds (♦) and solid triangles (▲) indicate the cytotoxic activity of GPC3_ERY22_rCE115 and GPC3_ERY27_hCE115, respectively;

in fig. 4 is a graph illustrating the cytotoxic activity of optimized antibodies in

- 20 041830 use as an NCI-H446 target cell;

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

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

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

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

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

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

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

in fig. 12 shows the relationship between the amino acid residues forming the Fc regions of IgG1, IgG2, IgG3 and IgG4 and the EU numbering system according to Cabot (referred to 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 given to facilitate understanding of the present invention. Antibody.

In the context of the present description, an antibody refers to a naturally occurring immunoglobulin or an immunoglobulin obtained 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 completely synthesized using techniques such as genetic recombination. Preferred antibodies are, for example, antibodies belonging to any 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 obtaining an antibody 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, it is also possible to obtain antibodies that bind to the T-cell receptor complex.

Anti-GPC3 antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Anti-GPC3 antibodies are preferably obtained as monoclonal antibodies derived from a mammalian organism. Said mammalian-derived antibodies include antibodies produced by 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, such as those described below. In particular, mammals are immunized using conventional immunization methods using the GPC3 protein as a sensitizing antigen. The resulting immune cells are fused with known parental 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 obtained according to the method described below. First, the GPC3 gene, the nucleotide sequence of which is presented in RefSeq under accession number NM_001164617.1 (SEQ ID NO: 1), can be expressed to obtain a GPC3 protein, the sequence of which is presented in RefSeq under accession number NP001158089.1 (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 appropriate 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, it is removed by deleting the amino acid at positions 564580, which form the hydrophobic region corresponding to the GPI anchoring sequence used to anchor GPC3 to the cell membrane, from the GPC3 polypeptide sequence of SEQ ID NO: 2, and then the resulting protein expressed instead of the GPC3 protein having SEQ ID NO: 2. Alternatively, a purified naturally occurring GPC3 protein can be used as the sensitizing antigen.

The purified GPC3 protein can be used as a sensitizing antigen for mammalian immunization. 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 a portion of the GPC3 gene into an expression vector and expressing therein. In addition, they can be obtained by cleavage of the GPC3 protein with a protease, however, a particular embodiment of the invention does not impose restrictions on the length and area of the GPC3 peptide used as a partial peptide. Any sequence from the amino acid sequence that corresponds to the amino acids at positions 524563, or more preferably any sequence from the amino acid sequence that corresponds to the amino acids at positions 537-563 in the amino acid sequence of SEQ ID NO: 2, can be selected as the preferred region. Preferably, any sequence can be selected from the amino acid sequence of a region that does not contain the amino acid sequence that corresponds to the amino acids at positions 550-663 in the amino acid sequence of SEQ ID NO: 2. Preferably, any sequence corresponding to positions 544-553 can be selected, and more preferably, any sequence can be selected from the amino acid sequence, corresponding to positions 546-551, in the amino acid sequence of SEQ ID NO: 2. The number of amino acids forming a peptide that can be used as a sensitizing th 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. Vectors for the expression of these fusion proteins can be constructed by fusing in frame the genes encoding two or more of the desired polypeptide fragments and inserting the fusion gene into the expression vector described above. Methods for making 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 producing GPC3 for use as a sensitizing antigen and immunization methods using GPC3 are specifically described in WO 2003/000883, WO 2004/022754 and WO 2006/006693.

There is no particular limitation regarding mammals to be immunized with the sensitizing antigen. However, it is preferable to select mammals based on their compatibility with the parental 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. Conventional methods for immunizing mammals are, for example, intraperitoneal or subcutaneous injection of a sensitizing antigen. In particular, the sensitizing antigen can be appropriately diluted in PBS (phosphate buffered saline), physiological 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 immunized with a sensitizing antigen, appropriate carriers may be used. In particular, when a low molecular weight partial peptide is used as the sensitizing antigen, it is sometimes required for immunization to combine the peptide representing the sensitizing antigen with a carrier protein such as albumin or keyhole limpet hemocyanin.

Alternatively, hybridomas producing the desired antibody can be obtained using the DNA immunization described below. DNA immunization is an immunization method that provides immunostimulation through the expression of a sensitizing antigen in the body of an immunized animal by introducing a DNA vector designed in such a way that it provides the expression of a gene encoding an antigenic protein in the body of an animal. Compared to conventional immunization methods, in which a protein antigen is administered to the animals to be immunized, DNA immunization appears to have the following advantages:

immunostimulation can be performed while maintaining the structure of a membrane protein such as GPC3; and there is no need to purify the antigen for immunization.

To obtain a 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 used expression vectors 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 an animal to be immunized. Antibodies recognizing GPC3 can also be obtained by the methods described in WO 2003/104453. After immunizing a mammal as described above, an elevated serum titer of GPC3-binding antibody is confirmed in the mammal. Thereafter, immune cells are obtained from the body of a mammal 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. Myeloma cells preferably contain an acceptable selection marker for screening. The selection marker gives the cells characteristics 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 a selection marker. Cells deficient in HGPRT or TK are sensitive to hypoxanthine-aminopterin-thymidine (abbreviated hereinafter as HAT-sensitivity). Cells with GAT-sensitivity cannot synthesize DNA in the GAT selection medium and die as a result. However, when cells are fused with healthy cells, they can continue DNA synthesis using the healthy cell recycling pathway, and as a result, they can grow even in HAT selection media.

HGPRT-deficient and TK-deficient cells can be selected in a medium containing 6-thioguanine, 8azaguanine (abbreviated hereinafter as 8AG) or 5'bromodeoxyuridine, respectively. Healthy cells are destroyed as they incorporate these pyrimidine analogs into their DNA. However, cells deficient in these enzymes can survive in the selection medium, since they cannot include these pyrimidine analogs. In addition, the selection marker includes resistance to G418, which is provided by the neomycin resistance gene, conferring 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 preferably be used as myeloma cells:

P3(P3x63 Ag8.653) (J. Immunol. 123 (4), 1979, pp. 1548-1550);

P3x63Ag8U.l (Current Topics in Microbiology and Immunology 81, 1978, pp.

1-Ό;

NS-l (C. Eur. J. Immunol. 6 (7), 1976, pp. 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, pp. 1-21);

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

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

Cellular fusion of immunocytes and myeloma cells is generally carried out using known methods, for example the method described by Kohler and Milstein et al. (Methods Enzymol. 73, 1981, p. 346).

More specifically, cell fusion can be performed, 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 (mouse haemagglutinating virus of Japan) (HVJ). If necessary, an auxiliary substance, such as dimethyl sulfoxide, is also added to improve the efficiency of the 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 media and MEM media, as well as other conventional culture media used for this type of cell culture. Additionally, 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 amounts, are well mixed in the above culture medium. Then, a PEG solution (eg, average molecular weight of about 1000 to 6000) preheated to about 37° C. is added thereto at a concentration typically between 30 and 60 w/v%. The mixture is gently stirred until the desired confluent cells (hybridomas) are obtained. Then, the above culture medium is gradually added to the cells and centrifuged again to remove the supernatant. In this way, fusion agents that are undesirable for hybridoma growth can be removed.

The hybridomas thus obtained can be selected by culturing using a conventional selective medium such as HAT medium (culture medium containing hypoxanthine, aminopterin and thymidine). Cells other than the desired hybridomas (non-fused cells) can be killed by subsequent culturing in the above HAT medium for a sufficient period of time. As a rule, the period is from several days to several weeks. Hybridomas producing the desired antibody are then screened for and individually cloned using conventional serial dilution techniques.

The hybridomas thus obtained can be selected using a selection medium based on a selection marker carried by the myeloma cells used for cell fusion. For example, HGPRT- or TK-deficient cells can be selected by culturing using HAT medium (culture medium containing hypoxanthine, aminopterin and thymidine). In particular, when HAT-responsive myeloma cells are used for cell fusion, cells that successfully fuse with healthy cells can selectively proliferate in the HAT medium. Cells other than the desired hybridomas (non-fused 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 for a period of time ranging from several days to several weeks. Hybridomas producing the desired antibody are then screened for and individually cloned using conventional serial dilution techniques.

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 a cell surface. You can screen these monoclonal antibodies using the method of cell separation based on fluorescence excitation (FACS). FACS is a system that evaluates antibody cell surface binding by analyzing cells in contact with a fluorescent antibody using a laser beam and by assessing the fluorescence emitted by individual cells.

For FACS screening for hybridomas that produce the monoclonal antibody of the present invention, GPC3 expressing cells are first obtained. Cells that are preferably used for screening are mammalian cells in which GPC3 is forced to be expressed. As a control, the ability of an antibody to bind to cell surface GPC3 can be selectively determined using non-transformed mammalian cells as host cells. In particular, anti-GPC3 monoclonal antibody-producing hybridomas can be isolated by selecting hybridomas that produce an antibody that binds to cells forcibly expressing 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 on the wells of an ELISA plate. Hybridoma culture supernatants are brought into contact with the immobilized cells in the wells and antibodies are detected that bind to the immobilized cells. When the monoclonal antibodies are of murine origin, the cell-bound antibodies can be detected with 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 the like.

Monoclonal antibody-producing hybridomas thus obtained can be subcultured into a conventional culture medium and stored in liquid nitrogen for an extended period of time.

The above hybridomas are cultured using a conventional method and the desired monoclonal antibodies can be obtained from culture supernatants. Alternatively, hybridomas are introduced and grown in 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 aforementioned hybridomas, can also be used. cloned gene

- 24 041830 antibodies are inserted into the appropriate vector and introduced into the host for expression of the antibody encoded by the gene. Techniques for isolating antibody genes, inserting genes into vectors, and transforming host cells have been developed (see, for example, Vandamme et al., Eur. J. Biochem. 192(3), 1990, p.

767-775). Methods for obtaining recombinant antibodies are also known and are described below.

For example, cDNA encoding the variable region (V region) of an anti-GPC3 antibody is obtained from hybridoma cells expressing an anti-GPC3 antibody. For this purpose, total RNA is first extracted from hybridomas. Methods that are used to extract mRNA from cells include, for example: the ultracentrifugation method in the presence of guanidine (Biochemistry 18(24), 1979, pp. 5294-5299) and the AGPC method (Anal. Biochem. 162(1), 1987, pp. 156-159). The extracted mRNAs can be purified using an mRNA purification kit (GE Healthcare Bioscience) or a 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. V-region-encoding cDNA antibodies can be synthesized from the resulting mRNAs using reverse transcriptase. cDNA can be synthesized using a Reverse Transcriptase First-strand cDNA Synthesis Kit (AMV) (Seikagaku Co.) or the like. In addition, the SMART RACE cDNA amplification kit (Clontech) and the PCR-based 5'δACE (rapid amplification of cDNA ends) method (Proc. Natl. Acad. Sci. USA 85(23), 1988, pp. 8998-9002; Nucleic Acids Res. 17(8), 1989, pp. 2919-2932). With this cDNA synthesis process, the appropriate restriction enzyme recognition 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 the 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 obtained from colony-forming E. coli. Then, using a known method such as the dideoxy method (nucleotide chain termination method), it is determined whether the recombinant vector has a cDNA nucleotide sequence of interest.

The 5'-RACE method, which uses primers to amplify a variable region gene, is generally used to isolate a gene encoding a variable region. First, a cDNA library used for 5'-RACE (5'-RACE cDNA library) 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-subclass with an available kit, such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

In particular, for example, to isolate genes encoding mouse IgG, primers are used that allow amplification of genes encoding γ1, γ2a, y2b and γ3 heavy chains and k and λ light chains. In general, a primer whose hybridization (annealing) site with the constant region is located near the variable region is used as a 3'-terminal primer for amplifying the IgG variable region gene. In this case, the primer attached to the set of 5'-RACE constructs of the cDNA library is used as the 5'-terminal primer.

The PCR products thus amplified 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 reshaped immunoglobulin as an indicator. For example, when the goal is to isolate an antibody to GPC3, it is more preferred that the binding of the antibody to GPC3 is specific. You can screen for GPC3-binding antibodies, for example, using the following steps, in which:

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

(2) determining antibody binding to a GPC3 expressing cell; and (3) selecting an antibody that binds to a 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 expressing GPC3 cells can be used to assess the binding activity of the antibody.

Preferred antibody screening methods that use binding activity in

- 25 041830 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. Genes encoding heavy chain and light chain variable regions can be fused with a suitable linker sequence to form a single chain Fv (scFv). Phages presenting scFv on their surface can be obtained by inserting the gene encoding scFv into a phage vector. The phages are brought into contact with the antigen of interest. DNA encoding an scFv that has binding activity of interest can then be isolated by harvesting antigen-bound phages. This process can be repeated as needed to enrich scFvs that have binding activity of interest.

After isolation of the cDNA encoding the V region of the anti-GPC3 antibody of interest, the cDNA is digested with restriction enzymes that recognize the restriction sites introduced at 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 one-copy cleavage fragment in the correct orientation, a restriction site for the enzyme that forms the sticky end is preferably introduced into the vector of interest. The cDNA coding for the anti-GPC3 antibody V region is digested as 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. In the context of the present description, a chimeric antibody means that the constant region and the variable region differ in their origin. Thus, in addition to murine/human heterochimeric antibodies to chimeric antibodies according to the present invention, human/human allochimeric antibodies are also included. An expression vector for a chimeric antibody can be created by inserting the above V-region gene into an expression vector that already contains the constant region. In particular, for example, a sequence recognized by a restriction enzyme that cuts out 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. An expression vector for a chimeric antibody is created by in-frame fusion of two genes cleaved with the same restriction enzyme combination.

To obtain a monoclonal antibody to GPC3, the antibody genes are inserted into an expression vector in such a way that the expression of the genes occurs under the control of the expression regulating region. An expression control region for expressing antibodies 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, you can attach other acceptable signal sequences. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence and the resulting polypeptide is secreted from the cells to the outside as the mature polypeptide. Acceptable host cells are then transformed with an expression vector to produce recombinant cells expressing DNA that encodes an anti-GPC3 antibody.

DNAs that encode an antibody heavy chain (H chain) and an antibody light chain (L chain) are inserted separately into different expression vectors to express the antibody gene. An antibody molecule having H and L chains can be expressed by co-transfecting the same host cell for this purpose with vectors into which the H chain and L chain genes are inserted, respectively. 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 the variable regions of an antibody of the present invention. Suitable eukaryotic cells that are used as host cells are 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.

Further, as a plant cell, an antibody gene expression system is known in which cells derived from members of the genus Nicotiana, such as Nicotiana tabacum, are used. Cultured callus cells can be used to transform plant cells.

- 26 041830

In addition, the following cells can be used as fungal cells: yeasts of the genus Saccharomyces, such as Saccharomyces cerevisiae, and the genus Pichia, such as Pichia pastoris, and filamentous fungi: the genus Aspergillus, such as Aspergillus niger. In addition, antibody gene expression systems are known that use prokaryotic cells. For example, according to the present invention, bacterial cells can be used, i. E. coli cells, Bacillus subtilis cells, and the like. 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 the culture of the 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 created as a fusion gene by inserting in frame into a gene that encodes a protein that is specifically produced in milk. As a protein secreted into milk, for example, goat β-casein can be used. DNA fragments containing the fusion gene containing the antibody gene are injected into a goat embryo and then the embryo is 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 a goat that is the recipient of the embryo (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, p. 699-702) .

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

The variable region of an antibody used to derive the domain of an antigen-binding molecule, which includes the variable region of an 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. The amino acid sequences of the CDRs are characterized by a high degree of variability. On the other hand, FR-forming amino acid sequences often have high identity even among antibodies with different binding specificities. Thus, in general, by CDR transplantation, the binding specificity of a particular antibody can be introduced into another antibody.

A humanized antibody is also referred to as a reshaped human antibody. In particular, humanized antibodies are known that are obtained by transplanting the CDR of an antibody of a non-human animal, such as a mouse antibody, into a human antibody, and the like. Conventional genetic engineering techniques for producing humanized antibodies are also known. In particular, for example, PCR with overlapping primers is a known method for transplanting mouse antibody CDRs into human FRs. 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. Get primers for each of the four FR. It is generally accepted that when a mouse CDR is transplanted into a human FR, it is advantageous to select a human FR having a high level of identity with the mouse FR to maintain CDR function. Thus, it is generally preferred to use a human FR that contains an amino acid sequence that has a high level of amino acid sequence identity with the amino acid sequence of the FR that is adjacent to the mouse CDR to be transplanted.

The nucleotide sequences to be ligated are designed to be joined to each other in reading frame. Human FRs are individually synthesized using appropriate primers. As a result, products are obtained in which the DNA encoding the murine CDR is fused to the DNA encoding individual FRs. The nucleotide sequences encoding the mouse CDR of each product are designed to overlap with each other. Then, a complementary chain synthesis reaction is carried out to anneal the overlapping CDRs of the products synthesized using the human antibody gene as a template. With 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 finally ligated, is amplified using primers hybridizing to the 5' or 3' end, which are added with sequences recognized by acceptable restriction enzymes. An expression vector for a humanized antibody can be obtained by inserting the

- 27 041830 method of DNA and DNA, which encodes the C-region of a human antibody, into an expression vector in such a way as to ligate them in reading frame. After the host is transfected with a recombinant vector to create recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed to produce the humanized antibody in cell culture (see European patent publication EP 239400 and international patent application publication WO 1996/002576). By qualitatively or quantitatively evaluating and measuring the antigen-binding activity of a humanized antibody prepared according to the method described above, human antibody FRs can be selected that allow the CDR to form a preferred antigen-binding site when ligated using the CDR. Amino acid residues in the FR can be changed as needed so that the CDRs of the reshaped human antibody form an acceptable antigen binding site. For example, amino acid sequence mutations can be introduced into FR using the PCR method used to transplant a mouse 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 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 obtained 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. You can select phages expressing scFv that binds to the antigen. The DNA sequence encoding the V region of a human antibody that binds to an antigen can be determined by gene analysis 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).

A domain containing the variable region of an antibody that has binding activity for glypican 3 (GPC3).

In the context of the present description, the phrase domain containing the variable region of an antibody that has binding activity against glypican 3 (GPC3) refers to the region of an antibody that contains a region that specifically binds to the GPC3 protein described above or to all or a portion of the partial peptide of the GPC3 protein. , and is also complementary to it. Domains containing the variable region of an antibody can be derived from the variable domains of one or more antibodies. Preferably, the antibody variable domains comprise the light chain and heavy chain variable regions (VL and VH) of the antibody. Suitable examples of such antibody variable domains are single chain Fv (scFv), single chain antibody, Fv, single chain Fv ₂ (scFv ₂ ), Fab, F(ab') ₂ , etc.

A domain containing the variable region of an antibody that has binding activity for the T-cell receptor complex.

As used herein, the phrase domain containing an antibody variable region that has binding activity for the T-cell receptor complex refers to the 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 him. 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.

A domain containing the variable region of an antibody that has T-cell receptor binding activity.

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

- 28 041830

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 the constant region sequence include the T cell receptor α chain, which has the accession number RefSeq. CAA26636.1 (SEQ ID NO: 4), T cell receptor P 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 AAB63312.1 (SEQ ID NO: 7), and T cell receptor 5 chain which has RefSeq AAA61033.1 (SEQ ID NO: : 8).

A domain containing the variable region of an antibody that has CD3-binding activity.

As used herein, the phrase domain containing 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 portion of CD3, and that is also complementary to it.

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

An inventive domain containing an antibody variable region that has CD3-binding activity can be any epitope-binding domain so long as the epitope is present in the γ-chain, δ-chain or ε-chain sequence that forms human CD3. Preferably, 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 can be preferably used in the present invention. In addition to the anti-CD3 antibody light chain (VL) variable region and the anti-CD3 antibody heavy chain variable region (VH) as described in the Examples section, known CD3 binding domains that comprise the anti-CD3 antibody light chain (VL) variable region and the variable region heavy chain (VH) anti-CD3 antibodies and derived from the OKT3 antibody (Proc. Natl. Acad. Sci. USA 77, 1980, pp. 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 a 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 a domain containing an antibody variable region that has CD3-binding activity. With regard to the structure of the γ chain, δ chain or ε chain forming human CD3, their polynucleotide sequences are shown in SEQ ID NOs: 9 (NM_000073.2), 10 (NM_000732.4) and 11 (NM_000733.3), and their polypeptide sequences are shown in SEQ ID NOs: 12 (NP_000064.1), 13 (NP_000723.1) and 14 (NP_000724.1) (registration numbers in the RefSeq database are in brackets).

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

Specificity.

The concept of 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 domain containing the variable region of an antibody binds is included in several different antigens, then the antigen-binding molecules containing the domain containing the variable region can bind to different antigens that contain the epitope.

Epitope.

An epitope means an antigenic determinant in an antigen and refers to an antigenic site to which the domain of an antigen-binding molecule, as provided herein, comprising the variable region of an antibody, binds. Thus, 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 from 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 in which the primary amino acid sequence is recognized. Said linear epitope typically contains at least three, and most often at least 5, eg about 8 to 10 or 6 to 20, amino acid residues in 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 recognized epitope (e.g., it is not necessary that the primary amino acid sequence of the conformational epitope be recognized by the epitope-specific antibody). Conformational epitopes may contain more amino acid residues than linear epitopes. An antibody that recognizes a conformational epitope 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 align and the epitope becomes recognizable by the antibody. Methods for determining epitope conformations include, but are not limited to, for example, X-ray crystallography, 2D nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, ed. Morris, v. 66, 1996).

An example of a method for evaluating epitope binding 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 epitope binding 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 in a 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 genetically engineered using a region in the GPC3 cDNA that encodes an amino acid sequence corresponding to the extracellular domain. The binding activity 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 is then assessed. 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 for the peptide. Alternatively, the binding activity for a linear peptide can be measured by the level of inhibition by the linear peptide of the binding of the 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 obtained. A test antigen-binding molecule containing a domain that contains an antibody variable region having GPC3-binding activity is said to recognize a conformational epitope if, upon contact, it binds strongly to GPC3-expressing cells, but binds little to an immobilized linear peptide that contains the amino acid sequence , which forms the extracellular domain of GPC3. In the context of the present description, binds slightly means that the binding activity is 80% or less, typically 50% or less, preferably 30% or less, and most preferably 15% or less, as compared to the binding activity to human GPC3 expressing cells. .

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

In an ELISA format, the binding activity of a tested antigen binding molecule containing a domain that binds to the GPC3 antigen on 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 cell-bound antigen-binding molecule to be tested is then detected with an enzyme-labeled antibody that recognizes the antigen-binding molecule to be tested. Alternatively, when FACS is used, serial dilutions of the antigen binding molecule to be tested 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 an antigen-binding molecule to be tested to an antigen that is expressed on the surface of cells suspended in buffer or a similar medium can be detected using a flow cytometer. Known flow cytometers are, for example, the following devices:

FACS Canto II,

FACSAria™,

FACS Array™,

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 on an antigen include, for example, the following method. GPC3-expressing cells are first reacted with the antigen-binding molecule to be tested and then stained with a FITC-labeled secondary antibody that recognizes the polypeptide complex. The antigen-binding molecule to be tested is appropriately diluted with a suitable buffer to obtain the complex at the desired concentration. For example, the complex can be used at a concentration of 10 μg/ml to 10 ng/ml. Then determine the intensity of fluorescence and the number of cells using FACSCalibur (firm BD). The fluorescence intensity, determined using analyzes based on the CELL QUEST program (company BD), namely, expressed as geometric mean values, reflects the level of antibody binding to cells. This means that the binding activity of a test antigen-binding molecule, which is characterized by the amount of bound test antigen-binding molecule, can be evaluated by determining the geometric mean.

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

In particular, when performing a cross-blockade assay, the GPC3 protein immobilized in the wells of a microtiter plate is pre-incubated with or without the antigen-binding molecule considered as a candidate competitor, and then the test antigen-binding molecule is added. The amount of GPC3 protein-bound test antigen-binding molecule in the wells is inversely correlated with the binding activity of the antigen-binding molecule considered as a competitor candidate that competes for binding to the same epitope. This means that the higher the affinity of an antigen-binding molecule considered as a candidate competitor for the same epitope, the lower the binding activity of the tested antigen-binding molecule to GPC3 protein-sensitized wells.

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

When an 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% by compared with the binding activity established in

- 31 041830 control experiment, which is carried out in the absence of an antigen-binding molecule considered as a competitor, then the tested antigen-binding molecule is considered to have a pronounced ability to bind to the same epitope to which the antigen-binding molecule considered as a competitor binds or competes for binding to the same epitope.

If the structure of an epitope bound to 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 to the peptide can be compared to decide 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, the binding activities of the test and control antigen-binding molecules are compared with the linear peptide in which the mutation is 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 a test and control antigen-binding molecule through the column and then assessing the amount of antigen-binding molecule eluted into the elution solution. Techniques are known for adsorbing a mutant peptide onto a column, for example in the form of a GST-fused 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, cells expressing GPC3 and cells expressing GPC3 with a mutation introduced into the epitope are obtained. The test and control molecules are added to a cell suspension obtained by suspending the cells in an acceptable buffer such as PBS. The cell suspensions are then washed appropriately with buffer and a FITC-labeled antibody that recognizes the test and control antigen-binding molecules is added. Determine the intensity of fluorescence and the number of cells stained with labeled antibody using FACSCalibur (firm BD). The test and control polypeptide complexes are appropriately diluted with an acceptable buffer and used at the desired concentration. For example, they can be used at a concentration of 10 μg/ml to 10 ng/ml. The fluorescence intensity is determined by an assay whose results are evaluated using the CELL QUEST program (BD), namely, the geometric mean is determined, 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 evaluating the geometric mean.

In carrying out the method described above, the following method can be used, for example, to decide whether an antigen-binding molecule exhibits negligible binding to cells that express the mutant GPC3. First, test and control antigen-binding molecules associated with cells that express the mutant GPC3 are stained with labeled antibody. Then determine the intensity of fluorescence in the cells. 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 in the presence of an antigen-binding molecule and without it, the comparative value (AGeo-Mean) can be calculated according to the formula below, which characterizes the degree of increase in fluorescence intensity as a result of binding to an antigen-binding molecule:

AGeo-Mean=Geo-Mean (in the presence of an antigen-binding molecule)/Geo-Mean (in the absence of an antigen-binding molecule).

The comparative geometric mean value (AGeo-Mean value for the mutant GPC3 molecule) determined by the assay described above, which reflects the amount of test antigen-binding molecule bound to cells that express the mutated GPC3, is compared with the relative AGeo-Mean value, which reflects the amount of test antigen-binding molecule associated with expressing GPC3 cells. In this case, the concentrations of the test antigen-binding molecule used to determine the relative AGeo-Mean values for GPC3 expressing cells and cells expressing mutant GPC3 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 comparative AGeo-Mean of the test antigen-binding molecule against cells expressing the mutant GPC3 is lower than the comparative AGeo-Mean of the test antigen-binding molecule against cells expressing GPC3 by at least 80%, preferably by 50%, more preferably 30% and most preferably 15%, then the antigen binding molecule tested is considered to bind insignificantly to cells that express the mutant GPC3. The formula for determining the value of Geo- 32 041830

Mean (geometric mean) is described in the CELL application manual

QUEST (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).

In the context of the present description, the term variable fragment (Fv) refers to the minimum unit derived from an antibody antigen-binding domain, which 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 can be obtained from the periplasmic fraction of E. coli by inserting the antibody gene downstream of the bacterial signal sequence and inducing gene expression in E. coli (Science 240(4855), 1988, pp. 1038-1041). In Fv derived from the periplasmic fraction, VH associates with VL in such a way as to bind to antigen.

According to the present description, Fv preferably includes, for example, a pair of Fv, which is an antigen-binding molecule or the like, containing:

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

(2) a domain containing an Fc domain which has no Fcγ receptor binding activity and which is derived from the amino acids forming the Fc domain of IgG1, IgG2a, IgG3, or IgG4, and (3) at least one univalent CD3 binding a domain where the light chain and heavy chain of the Fv fragments associate to form a CD3 binding domain that can bind to the CD3 antigen.

scFv, single chain antibody and sc(Fv) ₂ .

In the context of the present description, 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 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, allowing the formation of the desired structure, which is likely to provide 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, p. 269-315 (see also International Patent Publication WO 1988/001649; US Nos. 4946778 and 5260203). In a particular embodiment, the single chain antibody may be bispecific and/or humanized.

scFv is an antigen-binding domain in which VH and VL forming Fv are linked to each other by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. 85(16), 1988, p. 58795883). VH and VL can be held in close proximity using 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 one 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 include, for example, a bispecific sc(Fv)2 recognizing two epitopes that are present on the same antigen as described in Journal of Immunology 152(11), 1994, p. 5368-5374. sc(Fv)2 can be obtained by methods known to those skilled in the art. For example, sc(Fv) ₂ can be obtained by linking the scFv with a linker, such as a peptide linker.

According to the present description, 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 at the N-terminus of the single chain polypeptide. The arrangement order of the two VH units and the two VL units is not limited to the above shape, and they can be arranged in any order. An example of the arrangement order 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 detailed in WO 2006/132352. Thus, according to these descriptions, specialists in this field can receive the required sc(Fv)2, designed to create antigennegative molecules presented in the present description.

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

- 33 041830

Linkers for linking the variable regions of an antibody are arbitrary peptide linkers that can be introduced by genetic engineering, synthetic linkers, and the linkers described, for example, in Protein Engineering, 9(3), 1996, p. 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 those skilled in the art can choose it depending on the task at hand. The preferred length is five or more amino acids (with the upper limit being (but not limited to) generally 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, said 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 is an integer of 1 or more. Those skilled in the art may choose the length or sequence of the 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), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) ( sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis-[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES) and and bis-[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These cross-linking agents are commercially available.

Typically, three linkers are required to link 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'.

The Fab consists of a single light chain and a CH1 domain and a variable region from a single 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 refers to an antibody fragment obtained by cleaving an 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 to form two homologous antibody fragments in which the L chain containing VL (the variable region of the L chain) and CL (the constant region of the L chain ) are linked to an H chain fragment containing VH (variable region of the H chain) and CHyI (γ1 region in the constant region of the H chain) via a disulfide bridge at their C-terminal regions. Each of these two homologous antibody fragments is referred to as Fab'.

F(ab') ₂ consists of two light chains and two heavy chains containing a CH1 domain and a portion of CH2 domains in the constant region, resulting in the formation of disulfide bridges between the two heavy chains. F(ab') ₂ forming antigennegative molecule presented in the present description, preferably can be obtained as follows. Complete monoclonal antibody or

- 34 041830 a 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 to a protein A column. There is no limitation on a particular protease as long as it has the ability to selectively cleave a complete antibody to form F(ab')2 under appropriate reaction conditions for the enzyme, such as pH. Said proteases are, for example, pepsin and ficin.

Fc domain.

The Fc domain that forms the antigen-binding molecule described herein can preferably be obtained 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 appropriate reaction conditions for that enzyme, such as pH. Said proteases are, for example, pepsin and ficin.

The antigen-binding molecules provided herein comprise an Fc domain with reduced ability to bind the Fey receptor, 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 as Cy1, Cy2, Cy3, and Cy4, respectively. The amino acid sequences of the Fc domain of the polypeptides that form human Cy1, Cy2, Cy3 and Cy4 are exemplified in SEQ ID NOs: 23, 24, 25 and 26, respectively. The relationship between the amino acid residues constituting each amino acid sequence and Cabot's EU numbering (referred to herein as the EU index) is shown in FIG. 12.

The term Fc domain refers to the region outside of F(ab')2 that contains two light chains and two heavy chains, containing part of the constant region that 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 constituting the antigen-binding molecule described herein can preferably be obtained as follows. An IgG1, IgG2, IgG3, or IgG4 monoclonal antibody or similar antibody is partially cleaved with a protease such as pepsin, followed by elution of the fraction adsorbed on the protein A column. formation of F(ab') ₂ under appropriate reaction conditions for the enzyme, such as pH. Said proteases are, for example, pepsin and ficin.

Fcy receptor.

The term Fcy 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 Fcy receptor gene. In humans, the family includes FcyRI (CD64), including the FcyRIa, FcyRIb, and FcyRIc isoforms; FcyRII (CD32), including isoforms FcyRIIa (including allotype H131 and R131), FcyRIIb (including FcyRIIb-1 and FcyRIIb-2) and FcyRIIc; and FcyRIII (CD16), including the FcyRIIIa isoform (including the V158 and F158 allotypes), and FcyRIIIb (including the FcyRIIIb-NA1 and FcyRIIIb-NA2 allotypes); as well as all unidentified human FcyRs, FcyR isoforms and their allotypes. However, the Fcy receptor is not limited to these examples. FcyRs include, but are not limited to, FcyRs derived from human, mouse, rat, rabbit, and monkey antibodies. FcyR can be obtained from any organism. Mouse FcyR includes, but is not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII-2 (CD16-2), as well as all unidentified mouse FcyRs, FcyR isoforms, and allotypes thereof. Said preferred Fcy receptors include, for example, human FcyI (CD64), FcyIIA (CD32), FcyIIB (CD32), FcyIIIA (CD16) and/or FcyIIIB (CD16). The polynucleotide sequence and amino acid sequence of FcyI are shown in SEQ ID NOs: 27 (NM_000566.3) and 28 (NP_000557.1), respectively; the polynucleotide sequence and amino acid sequence of FcyIIA are shown in SEQ ID NOs: 29 (BC020823.1) and 30 (AAH20823.1), respectively; the polynucleotide sequence and amino acid sequence of FcyIIB are shown in SEQ ID NOs: 31 (BC 146678.1) and 32 (AAI46679.1), respectively; the polynucleotide sequence and amino acid sequence of FcyIIIA are shown in SEQ ID NOs: 33 (BC033678.1) and 34 (AAH33678.1), respectively, and the polynucleotide sequence and amino acid sequence of FcyIIIB are shown in SEQ ID NOs: 35 (BC 128562.1) and 36 (AAI28563.1) ) respectively (in parentheses is the registration number of each sequence in the RefSeq database). Whether the Fcy receptor has the ability to bind to the Fc domain of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be assessed using the ALPHA Screen® assay (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method based on surface plasmon resonance

- 35 041830 (SPR) and others (Proc. Nail. Acad. Sci. USA 103(11), 2006, pp. 4005-4010), in addition to the formats described above

FACS and ELISA.

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 an Fc ligand to an Fc preferably induces one or more effector functions. These ligands include, but are not limited to, Fc receptors, FcyR, FcaR, FcεR, FcRn, C1q and C3, mannan-binding lectin, mannose receptor, Staphylococcus A protein, Staphylococcus G protein, and viral FcyRs. Fc ligands also include the Fc receptor (FcRH) homologues (Davis et al., Immunological Reviews 190, 2002, pp. 123-136), which are a family of Fc receptors homologous to FcyR. Fc ligands also include unidentified molecules that bind to Fc.

Fcy receptor binding activity.

Disruption of Fc domain binding activity to any of the Fcy receptors FcyI, FcyIIA, FcyIIB, FcyIIIA and/or FcyIIIB can be assessed using the FACS and ELISA formats described above, as well as ALPHA Screen analysis (homogeneous analysis of proximity-enhanced luminescence, BIACORE method based on surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA 103(11), 2006, pp. 4005-4010).

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

For example, a biotin-labeled antigen-binding molecule is immobilized on donor beads, and a glutathione-S-transferase (GST) labeled Fcy receptor is immobilized on acceptor beads. In the absence of an antigen-binding molecule containing a competing mutant Fc domain, the Fcy 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 unlabeled mutant Fc domain competes with an antigen-binding molecule containing a wild-type Fc domain for interaction with the Fcy receptor. Relative binding affinity can be assessed by quantifying the reduction in fluorescence due to competition. Methods for biotinylating antigen-binding molecules such as antibodies with sulfo-NHSbiotin or the like are known. Acceptable methods for introducing a GST tag into the Fcy receptor include methods that involve in-frame fusion of Fcy and GST-encoding polypeptides, expression of the fusion gene using cells into which a vector carrying the gene has been introduced, and subsequent purification with a glutathione containing column. The induced signal can preferably be analyzed, for example, by fitting to a single site competition model based on non-linear 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 gold layer of the sensor chip. When light enters the rear surface of the sensor chip so that there is total reflection at the interface between the thin layer of gold and glass, then the intensity of the reflected light at a certain 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 (conversely, dissociation shifts the signal back to its original position). In the Biacore system, the level of the shift described above (i.e., mass change on the surface of the sensor chip) is plotted on the vertical axis, and thus a graph of mass change versus time (sensogram) is obtained as quantitative data. The kinetic parameters (association rate constant (k _a ) and dissociation rate constant (kd)) are determined from the sensorgram curves, and affinity (KD) is determined as the ratio of the two constants. BIACORE methods are preferably used for inhibition assays. Examples of such an inhibition assay are described in Proc. Natl. Acad. sci. USA 103(11), 2006, p. 4005-4010.

In the context of the present description, reduced binding activity to the Fcy receptor means

- 36 041830 for example, that when using the assay method described above, the competitive activity of the tested antigen-binding molecule 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 in the form of IgG1, IgG2, IgG3 or IgG4, respectively, can be used as control antigen-binding molecules. The Fc domain structures are shown in SEQ ID NO: 37 (A added to the N-terminus of the sequence listed in RefSeq accession number AAC82527.1), SEQ ID NO: 38 (A added to the N-terminus of the sequence listed in RefSeq accession number AAB59393.1), SEQ ID NO: 25 (A added to the N-terminus of the sequence listed in RefSeq accession CAA27268.1) and SEQ ID NO: 39 (A added to the N-terminus of the sequence listed in RefSeq accession number AAB59394.1). 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 Fcy receptor binding activity is evaluated using an antigen-binding molecule containing an Fc domain of the same isotype as a control. As described above, antigen-binding molecules containing a mutant Fc domain with truly reduced Fcy receptor binding activity are suitably prepared.

Such known mutants include, for example, amino acid deletion mutants 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).

Particularly preferred antigen-binding molecules are molecules 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 that form 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 the corresponding Fc domain derived from an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be used. Preferably, an Fc domain derived from IgG1 antibodies 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 the EU numbering (each number indicates the position of the amino acid residue according to the EU numbering; and the one-letter amino acid symbol that precedes the number indicates amino acid residue before its substitution, and the one-letter amino acid symbol following the number denotes the amino acid residue after substitution), among the amino acids that form the Fc domain of an IgG1 antibody:

(a) L234F, L235E, P331S;

(b) C226S, C229S, P238S;

(c) C226S, C229S;

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

(e) L234A, L235A or L235R, N297A;

(e) 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 an IgG2 antibody:

(g) H268Q, V309L, A330S and P331S;

(h) V234A;

(i) G237A;

(j) V234A and G237A;

(k) A235E and G237A;

(m) V234A, A235E and G237A.

Each number indicates the position of the amino acid residue according to the EU numbering; and the one-letter amino acid symbol that precedes the number indicates the amino acid residue before it was changed, and the one-letter amino acid symbol that follows the number indicates the amino acid residue after the change.

In addition, preferred antigen-binding molecules are 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 an IgG3 antibody:

(n) F241A;

- 37 041830 (o) D265A;

(p) V264A.

Each number indicates the position of the amino acid residue according to the EU numbering; and the one-letter amino acid symbol that precedes the number indicates the amino acid residue before it was changed, and the one-letter amino acid symbol that follows the number indicates the amino acid residue after the change.

In addition, preferred antigen-binding molecules are 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 an IgG4 antibody:

(p) L235A, G237A and E318A;

(c) L235E;

(r) F234A and L235A.

Each number indicates the position of the amino acid residue according to the EU numbering; and the one-letter amino acid symbol that precedes the number indicates the amino acid residue before it was changed, and the one-letter amino acid symbol that follows the number indicates the amino acid residue after the change.

Other preferred antigen binding molecules are molecules containing an Fc domain in which any amino acid at position 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) is among the amino acids that form 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 that form the Fc domain of an IgG1 antibody have been replaced by other amino acids . The invention is not limited to the type of amino acid after the substitution; however, most preferred are antigen-binding molecules containing an Fc domain in which one or more amino acids at positions 234, 235 and 297 have been replaced by alanine.

Preferred antigen-binding molecules are also molecules containing an Fc domain in which the amino acid at position 265 (EU numbering) among the amino acids that form the Fc domain of an IgG1 antibody has been replaced by another amino acid. The invention is not limited to the type of amino acid after the substitution; however, 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 variants of the multispecific antigen-binding molecule of the present invention include multispecific antibodies. When an Fc region with reduced Fcy receptor binding activity is used as the Fc region of a multispecific antibody, an Fc region derived from the multispecific antibody can be used accordingly. Bispecific antibodies are most preferred as 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 hybridoma (quadroma) obtained by fusing two types of hybridomas producing an IgG antibody (Milstein C. et al., Nature 305, 1983, pp. 537-540).

In addition, IgG-type bispecific antibodies are secreted by introducing four genes into cells in total, ie. L-chain and H-chain genes forming two types of IgGs of interest, and their co-expression. However, theoretically, there are ten combinations of H chains and L chains of IgG that can be obtained by such methods. However, it is difficult to purify IgG consisting of the required combination of H chains and L chains from ten IgG types. In addition, the theoretical amount of secreted IgG with the desired 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 applied to enhance association between H chains and between L and H chains having the desired combinations.

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

In a method for suppressing undesirable association of H chains by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues contacting at the interface with another H chain constant site include regions corresponding to residues located at positions 356 according to EU numbering. , 439, 357, 370, 399, and 409 in the CH3 region.

- 38 041830

More specifically, examples include an antibody containing two types of H chain CH3 regions, in which 1-3 amino acid residue pairs in the CH3 region of the first H chain are selected from the amino acid residue pairs 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 according to EU numbering at positions 356 and 439;

(2) amino acid residues in the CH3 region of the H chain, which are according to EU numbering at positions 357 and 370; and (3) amino acid residues in the CH3 region of the H chain, which are EU numbered at positions 399 and 409.

In addition, the antibody may be an antibody in which the pairs of amino acid residues in the CH3 region of the second H chain, which is different from the above 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 above pairs of amino acid residues in subparagraphs (1) to (3), bearing the same type of charge as indicated above for the CH3 region of the first H chain, carry opposite charges with respect to the corresponding amino acid residues in the above CH3 region of the first H chain.

The corresponding amino acid residues mentioned in subparagraphs (1) to (3) above are located close to each other when associated. For a desired H chain CH3 region or an H chain constant region, sites that correspond to the above residues specified in (1) to (3) can be determined by those skilled in the art through homology modeling and the like using sale program, and the amino acid residues of these sites can be subjected 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 regard to the above antibodies, that they have 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 having 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, 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 with 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 determine the amino acid residues that form the interface by 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 association. .

Other known techniques can also be applied to the association of multispecific antibodies proposed in 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 H chain of an antibody with a larger side chain (overhang) and replacing the amino acid side chain in the corresponding variable region with another H -chain onto a smaller side chain (depression) so that the protrusion fits into the depression (WO 1996/027011; Ridgway J.B. et al., Protein Engineering 9, 1996, p. 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 applied to the association of multispecific antibodies proposed in the present invention. Association of polypeptides having different sequences can be efficiently induced by complementary association of CH3 regions using a constructed CH3 chain exchange domain obtained by changing the CH3 portion of one of the antibody H chains to the corresponding IgA-derived sequence and introducing the CH3 complementary portion into the other. H chain of the corresponding IgA derived sequence (Protein Engineering Design & Selection, 23, 2010, pp. 195-202). This known technique can also be used to efficiently generate multispecific antibodies of interest.

In addition, the following techniques can be used to produce multispecific antibodies: techniques for producing antibodies based on the association of CH1 and CL of the antibody and the association of VH and VL, which

- 39 041830 are described in WO 2011/028952, WO2014/018572 and Nat Biotechnol. 32(2), February 2014, p. 191-198; techniques for producing bispecific antibodies using a combination of single-produced monoclonal antibodies (Fab arm exchange, FAE) described in WO 2008/119353 and WO 2011/131746; techniques for regulating association between the CH3 domain of antibody heavy chains as 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 from 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 efficiently produced, the multispecific antibody of the present invention can be obtained by separating the multispecific antibody of interest from the obtained antibodies and purifying it. For example, a method is described that allows the purification of two types of homomeric forms and a heteromeric antibody of interest using ion exchange chromatography, by establishing a difference in isoelectric points as a result of the introduction of amino acid substitutions in the variable regions of two types of H chains (WO 2007/114325). To date, a method based on the use of 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 has been described as a method for purifying heteromeric forms ( WO 98/050431 and WO 95/033844). In addition, a heterodimerized antibody can itself be efficiently purified using a protein A column by changing the interaction between each of the H chains and protein A, by using H chains in which the amino acid residues in the binding site of IgG protein A 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 indicated in the comparative example 5 to change the interaction of each of the H chains with protein A, and then apply the protein A column.

Alternatively, a common L chain, which can provide the ability to bind to many 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 this common L chain and many different H chains can be introduced into cells and the IgG expressed (Nature Biotechnology, 16, 1998, p. 677-681). The general L chain selection method, which has a pronounced ability to bind to any of the various H chains, can also be used to select the general H chain (WO 2004/065611).

Moreover, an Fc region having improved C-terminal heterogeneity can be used as an Fc region according to the present invention. More specifically, Fc regions are provided 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. In addition, these techniques can be appropriately and separately applied to the two H-strands to be associated. In addition, these techniques can be used in combination with the Fc region described above, which has reduced binding activity to the Fcy receptor. In addition, the antigen-binding molecule of the present invention may be a molecule obtained separately from an antigen-binding molecule subjected to the modifications 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 reduced ability to bind to the Fcy receptor, its structure is not limited to the specified.

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

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

Table 1

Sequence notation SEQID NO: Sequence notation SEQID NO: NOOOOO 40 GCH042 193 GCH003 170 GCH043 194 GCH005 171 GCH045 195 GCH006 172 GCH053 196 GCH007 173 GCH054 197 GCH008 174 GCH055 198 GCH010 175 GCH056 199 GCH012 176 GCH057 200 GCH013 177 GCH059 201 GCH014 178 GCH060 202 GCH015 179 GCH061 203 GCH016 180 GCH062 204 GCH019 181 GCH064 205 GCH022 182 GCH065 206 GCH023 183 GCH066 207 GCH025 184 GCH067 208 GCH026 185 GCH068 209 GCH027 186 GCH073 210 GCH029 187 GCH094 211 GCH032 188 GCH098 212 GCH034 189 GCH099 213 GCH035 190 GCH100 214 GCH039 191 H0610 215 GCH040 192

Examples of the preferred variable region of an antibody that has binding activity for the T cell receptor complex of the present invention include antibody variable regions that have binding activity for the T cell receptor. Of the T cell receptors, POPs are preferred and CD3e is most preferred. Examples of the variable region of the H chain of the antibody contained in these variable regions of the antibody include the variable regions of the H chain of the antibody, which are presented in table. 2, or antibody H chain variable regions that have CDR sequences whose CDR1, CDR2, and CDR3 amino acid sequences are the same as the CDR1, CDR2, and CDR3 amino acid sequences contained in the H chain variable regions shown in Table 2. 2 or antibody H chain variable regions that are functionally equivalent to the above variable regions.

-41 041830

table 2

Sequence designation SEQID NO: Sequence designation SEQID NO: Sequence designation SEQID NO: ICE115NA 52 TR01H036 99 TR01H074 135 SE115HA177 64 TR01H037 100 TR01H075 136 SE115HA178 65 TR01H038 101 TR01H076 137 SE115HA179 66 TR01H039 102 TR01H077 138 SE115HA180 67 TR01H040 103 TR01H079 139 hCE115HAa 68 TR01H041 104 TR01H080 140 TR01H006 69 TR01H042 105 TR01H081 141 TR01H007 70 TR01H043 106 TR01H082 142 TR01H008 71 TR01H044 107 TR01H083 143 TR01H009 72 TR01H045 108 TR01H084 144 TR01H010 73 TR01H046 109 TR01H090 145 TR01H011 74 TRO1H047 110 TR01H091 146 TR01H012 75 TR01H048 111 TR01H092 147 TR01H013 76 TR01H049 112 TR01H093 148 TR01H014 77 TR01H050 113 TR01H094 149 TR01H015 78 TR01H051 114 TR01H095 150 TR01H016 79 TR01H052 115 TR01H096 151 TR01H017 80 TR01H053 116 TR01H097 152 TR01H018 81 TR01H054 117 TR01H098 153 TR01H019 82 TR01H055 118 TR01H099 154 TR01H020 83 TR01H056 119 TR01H100 155 TR01H021 84 TR01H057 120 TR01H101 156 TR01H022 85 TR01H058 121 TR01H102 157 TR01H023 86 TR01H061 122 TR01H103 158 TR01H024 87 TR01H062 123 TR01H104 159 TR01H025 88 TR01H063 124 TR01H105 160 TR01H026 89 TR01H064 125 TR01H106 161 TR01H027 90 TR01H065 126 TR01H107 162 TR01H028 91 TR01H066 127 TR01H108 163 TR01H029 92 TR01H067 128 TR01H109 164 TR01H030 93 TR01H068 129 TR01H110 165 TR01H031 94 TR01H069 130 TR01H111 166 TR01H032 95 TR01H070 131 TR01H112 167 TR01H033 96 TR01H071 132 TR01H113 168 TR01H034 97 TR01H072 133 TR01H114 169 TR01H035 98 TR01H073 134 TR01H001 420 TR01H002 421 TR01H003 422 TR01H004 423 GSE115N 424 CE115HA121 425 CE115HA122 426 CE115HA124 427 CE115HA192 428 CE115HA236 429 CE115HA251 430 CE115HA252 431

The relationship between the amino acid residues of the CDRs forming the amino acid sequence of the antibody H chain and Cabot's EU numbering is shown in FIG. 13.

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

Examples of the common L chain variable region that can be used according to the present invention include the L chain variable regions shown in Table 1. 3, antibody L chain variable regions that have CDR sequences whose CDR1, CDR2, and CDR3 amino acid sequences are the same as the amino acid sequences of CDR1, CDR2, and CDR3 contained in the antibody L chain variable regions shown in Table 3. 3 and antibody L chain variable regions that are functionally equivalent to the above variable regions.

- 42 041830

Table 3

Sequence designation SEQID NO: L0000 53 L0002 217 L0003 218 L0006 219 L0007 220 L0008 221 L0009 222 L0011 223 L0012 224 L0013 225 L0014 226 L0015 227 L0016 228 L0032 229 L0038 230 L0039 231 L0041 232 L0042 233 L0043 234 L0044 235 L0045 236 L0046 237 L0047 238 L0062 239 L0063 240 L0064 241 L0065 242 L0066 243 L0069 244 L0075 245 L0079 246 L0082 247 L0085 248 L0089 249 L0090 250 L0091 251 L0093 252 L0104 253 L0106 254 L0107 255 L0109 256 L0113 257 L0115 258 L0117 259 L0120 260 L0122 261 L0123 262 L0124 263

Sequence designation SEQID NO: Sequence designation SEQID NO: L0125 264 L0214 312 L0126 265 L0215 313 L0127 266 L0216 314 L0129 267 L0217 315 L0132 268 L0218 316 L0134 269 L0219 317 L0136 270 L0220 318 L0137 271 L0222 319 L0138 272 L0223 320 L0139 273 L0224 321 L0140 274 L0226 322 L0141 275 L0227 323 L0143 276 L0228 324 L0144 277 L0229 325 L0145 278 L0230 326 L0147 279 L0231 327 L0148 280 L0232 328 L0149 281 L0233 329 L0151 282 L0234 330 L0152 283 L0235 331 L0154 284 L0236 332 L0155 285 L0237 333 L0157 286 L0238 334 L0160 287 L0239 335 L0161 288 L0240 336 L0163 289 L0241 337 L0167 290 L0242 338 L0168 291 L0243 339 L0173 292 L0246 340 L0175 293 L0247 341 L0180 294 L0248 342 L0181 295 L0249 343 L0186 296 L0250 344 L0187 297 L0258 345 L0200 298 L0259 346 L0201 299 L0260 347 L0202 300 L0261 348 L0203 301 L0262 349 L0204 302 L0263 350 L0205 303 L0264 351 L0206 304 L0265 352 L0207 305 L0266 353 L0208 306 L0267 354 L0209 307 L0268 355 L0210 308 L0269 356 L0211 309 L0270 357 L0212 310 L0271 358 L0213 311 L0272 359

The relationship between the amino acid residues of the CDRs forming the amino acid sequence of the antibody L chain and Cabot's EU numbering is shown in FIG. 14.

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

For example, the phrase antibody H chain variable region functionally equivalent to the antibody H chain variable region specified herein (i.e., the parent H chain variable region) means that the region has the same binding affinity when combined with an antibody L chain variable region as defined herein that 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 it used to obtain a multispecific antigen-binding molecule. In addition, the phrase 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 when its combined with variable

- 43 041830 region of the H chain of the antibody specified in the present description, which forms a pair with the original L chain, or alternatively means that the region has the same cytotoxic activity against expressing glypican 3 cells or tissues containing these cells, when used to obtain a multispecific antigen-binding molecule.

The concept of equivalent does not necessarily imply the same level of activity, and activity may be increased. In particular, antigen-binding affinity includes, for example, the case in which the ratio (KD value/K _D value of the parent antibody) obtained by comparison with the binding affinity of the variable region of the antibody that serves as a control (K _D value of the parent antibody) is 1 .5 or less. The K _D value/K _D value ratio 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 here, examples include coefficients of 10 ^_1 ,10' ² ,10' ³ ,10' ⁴ ,10' ⁵ , or 10 ^-6 . More specifically, according to the present invention, the ratio KD value/K _D value of the parent antibody is preferably from 10 ^-6 to 1.5x100, more preferably from 10 ^-6 to 10 ^-1 , even more preferably from 10 ^-6 to 10 ^-2 and more more preferably from 10 ^-6 to 10 ^-3 . Regarding the cytotoxic activity, examples include the case in which the ratio (rate of cell growth inhibition/rate of cell growth inhibition by parent antibody) obtained by comparison with the cell growth inhibition rate of the multispecific antigen-binding molecule that serves as a control (rate of cell growth inhibition by parent antibody) , is 0.7 or more. The concentration of the multispecific antigen-binding molecule added can be determined as appropriate, but is preferably, for example, 0.01, 0.05, 0.1, 0.5, or 1nM; and preferably measurements are made at a concentration of 0.05 or 0.1 nM. The cell growth inhibition rate/cell growth inhibition rate ratio 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. In this case, there is no upper limit, the coefficient can be 10, 10 ² , 10 ³ , 10 ⁴ , 105 or 106.

In addition, with regard to cytotoxic activity, examples include the case in which the ratio (concentration causing 50% inhibition of cell growth/concentration of parental antibody causing 50% inhibition of cell growth) obtained by comparison with the concentration of the original multispecific antigen-binding molecule causing inhibition of cell growth by 50% (the concentration of the parent antibody that causes inhibition of cell growth by 50%) is 1.5 or less. The concentration causing inhibition of cell growth by 50% means the concentration of the multispecific antigen-binding molecule necessary to reduce the rate of cell proliferation by half compared to the case in which the multispecific antigen-binding molecule is not added. The ratio of concentration causing 50% inhibition of cell growth/concentration of parent antibody causing 50% inhibition of cell growth 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. Here, ^{there is} no lower limit, examples include coefficients of ^{10_1,10'2,10'3,10'4,10'5} , or ^10-6 . In particular ^, the coefficient is preferably ^10-6 to 1.5x100, more preferably 10 ^-6 to 10 ^-1 , even more preferably 10 ^-6 to 10 ^-2 and even more preferably 10 ^-6 to 10 ^-3 .

Regarding the domain containing the variable region of an antibody that has GPC3 binding activity, the K _D value characterizing binding to GPC3 (for example, human GPC3) can be, for example, 5x10-9 M or less, preferably 4x10-9 M or less, for example 3x10- 9 M or less, 2x10-9 M or less, 1x10-9 M or less, 8x10 ^-10 M or less, 5x10 ^-10 M or less, 4x10 ^-10 M or less, 3x10' ¹⁰ M or less, 2x10 ^-10 M or less, 1x10 ^-10 M or less, 8x10 ^-11 M or less, 5x10 ^-11 M or less, 4x10 ^{-11 M or less, 3x10 -11} M or less, ^{2x10 -11 M} or less, 1x10 ^-11 M or less, 8x10 ^-12M or less, 5x10 ^-12M or less, 4x10 ^-12M or less, 3x10 ^-12M or less, 2x10 ^-12M or less, 1x10 ^-12M or less, 8x10 ^-13M or less less, 5x10 ^-13 M or less, 4x10 ^-13 M or less, 3x10 ^-13 M or less, 2x10 ^-13 M or less, or 1x10 ^-13 M or less.

With respect to a domain containing a variable region that has binding activity for a T cell receptor complex, a K _D value indicative of binding to a human T cell receptor complex, such as the human T cell receptor or more specifically, for example, human CD3ε, may be , for example, 2x10 ^-7 M or less, preferably 1.5x10 -7 M or less, for example, 1.4x10 -7 M or less, 1.3x10 -7 M or less, 1.2x10 -7 M or less, 1x10 ^{- 7M} or less, 3x10-8M or less, 2x10-8M or less, 1x10-8M or less, 8x10-9M or less, 5x10-9M or less, 4x10-9M or less, 3x10-9 M or less, 2x10-9 M or less, 1x10-9 M or less, 8x10 ^-10 M or less, 5x10 ^-10 M or less, 4x10' ¹⁰ M or less, 3x10' ¹⁰ M or less, 2x10 ^-10 M or less, 1x10' ^10M or less, 8x10 ^-11M or less, 5x10 ^-11M or less, 4x10 ^-11M or less, 3x10 ^-11M

- 44 041830 or less, 2x10 ^-11 M or less, 1x10 ^-11 M or less, 8x10 ^-12 M or less, 5x10 ^-12 M or less, 4x10 ^-12

M or less, 3x10 ^-12 M or less, 2x10 ^-12 M or less, or 1x10 ^-12 M or less.

The multispecific antigen-binding molecules of the present invention preferably have KD values indicating binding to human GPC3 and human T-cell receptor complex (e.g., human CD3ε chain) of 5 x 10 ^-9 M or less and 2 x 10 ^-7 M or less, respectively, and more preferably 1x10 ^-9 M or less and 5x10 ^-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 meet the above requirements. Examples of such antibody variable regions include regions obtained by introducing a substitution, deletion, addition, and/or insertion of one or more amino acids (e.g., 1, 2, 3, 4, 5, or 10 amino acids) into the amino acid sequences of the variable regions as set out above in tab. 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 generate variable regions that are functional equivalents of antibody variable regions having the above functions by introducing appropriate mutations into amino acid sequences using techniques 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 B., 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 a nd efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad. sci. USA. 82, 1985, p. 488-492).

When an amino acid residue is changed, the amino acid is preferably mutated to another amino acid(s) that retains 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 side chains containing a hydroxyl group (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 referred to as conservative substitutions. It is well known that a polypeptide containing a modified amino acid sequence in which one or more amino acid residues in said amino acid sequence is deleted, added and/or replaced with other amino acids can retain its 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, p. 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl. Acad. Sci. USA) 79, 1982, p. 6409-6413). The variable regions of the present invention containing said 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 CDR sequences, FR sequences of the variable region or complete variable regions prior to 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. Amino acid sequence identity 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 to nucleic acids that contain a nucleotide sequence encoding the amino acid sequence of the variable region indicated in Table 1 above. 1-3. The 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,

- 45 041830

0.5xSSC and 37° C. or hybridization conditions substantially equivalent to those indicated. The selection of nucleic acids with the highest possible homology can be expected when applying more stringent conditions, for example the following conditions: 6 M urea, 0.4% SDS, 0.1xSSC and 42°C. The washing conditions after hybridization are, for example, washing with 0.5xSSC (1xSSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at 60°C, more preferably washing with using 0.2xSSC and 0.1% SDS at 60°C, even more preferably washing using 0.2xSSC and 0.1% SDS at 62°C, even more preferably washing using 0.2xSSC and 0.1% SDS at 65°C and even more preferably washing using 0.1xSSC and 0.1% SDS at 65°C. The sequence of the selected 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 assessing the degree of sequence identity.

Nucleic acids that hybridize under stringent conditions to a nucleic acid containing a nucleotide sequence that encodes an amino acid sequence for a variable region can also be isolated by using gene amplification methods such as polymerase chain reaction (PCR) instead of the hybridization-based methods described above, in which use primers synthesized based on information about the nucleotide sequence 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 by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90, 1993, pp. 5873-5877). Based on this algorithm, the BLASTN and BLASTX programs have been developed (Altschul et al., J. Mol. Biol. 215, 1990, pp. 403-410). For analysis of nucleotide sequences according to BLASTN based on BLAST, parameters are set, such as, for example, score=100 and wordlength=12. On the other hand, the parameters used for the analysis of amino acid sequences using BLASTX based on BLAST include, for example, score=50 and word length=3. The default parameters apply per program 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 binding activity for glypican 3 and an antibody variable region having binding activity for a T-cell receptor complex 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. In the context of the present description, equivalent does not necessarily mean the same degree of the above activity, and the activity may be higher. The molecule is equivalent to GPC3_ERY22_rCE115, for example, when the ratio (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 greater than, 0.9 or greater, 1.0 or greater, 1.2 or greater, 1.5 or greater, 2 or greater, 3 or greater, 5 or greater, 10 or greater, or 20 or greater. While there is no upper limit, the index may be, for example, 10, 10 ² , 103, 10 ⁴ , 105, or 106. The concentration of the multispecific antigen-binding molecule added may be determined as appropriate, but is preferably, for example, 0.01, 0 .05, 0.1, 0.5 or 1nM; and preferably measurements are made at a concentration of 0.05 or 0.1 nM.

In addition, examples include the case where the ratio of concentration causing 50% inhibition of cell growth/concentration (GPC3_ERY22_rCE115) causing inhibition of cell growth by 50%, obtained by comparison with the concentration (GPC3_ERY22_rCE115) that causes inhibition of cell growth by 50% ( the concentration (GPC3_ERY22_rCE115) causing 50% inhibition of cell growth is 1.5 or less. The concentration ratio causing 50% inhibition of cell growth/concentration (GPC3_ERY22_rCE115) causing 50% inhibition of cell growth 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 here, examples include factors of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 or 10 ^-6 . ^{In particular} , ^the ratio is preferably ^{10 -6 to 1.5x10 3} .

Preferred specific K _D values characterizing binding to human GPC3 and

- 46 041830 complex of the human T-cell receptor (for example, with the human CD3ε chain) also correspond to the above. Appropriate GPC3 expressing cells or appropriate tissue containing said cells can be used, and for example human cancer cell lines PC-10 or NCI-H446 that express GPC3 can be used.

Examples of said combination of an antibody variable region having glypican 3 binding activity and an antibody variable region having T-cell receptor complex binding activity include combinations of the antibody H chain variable regions shown in Table 1. 4, combinations of antibody H chain variable regions that have CDR sequences whose CDR1, CDR2, and CDR3 amino acid sequences are the same as the amino acid sequences of CDR1, CDR2, and CDR3 that are included in the antibody H chain variable regions shown in Table 4. . 4 and combinations of antibody H chain variable regions functionally equivalent to the indicated variable regions. In the context of the present description, the concept of functionally equivalent has the meaning indicated above.

Table 4

SR3 arm/T-cell receptor complex arm SEQID NO: H0000/hCE115HA 40/52 H0000/CE115HA251 40/500 H0000/CE115HA236 40/429 H0000/TR01H002 40/421 H0000/CE115HA122 40/426 H0610/rCE115H 215/424 H0610/TR01H040 215/103 H0610/TR01H061 215/122 H0610/TR01H068 215/129 H0610/TR01H071 215/132 GCH054/TR01H067 197/128 GCH094/TR01H082 211/142 GCH094/TR01H084 211/144 GCH065/TR01H084 206/144 GCH065/TR01H082 206/142 GCH094/TR01H109 211/164 GCH065/TR01H109 206/164 GCH094/TR01H113 211/168 GCH065/TR01H113 206/168

A 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 include, for example, L0000, L0011, L0201, L0203, L0204, L0206, L0208, L0209, L0211, L0212, L0222 and a common L chain which has CDR sequences (amino acid sequences of CDR1, CDR2 and CDR3) identical to the amino acid sequences of CDR1, CDR2 and CDR3 mentioned above for the common L chain. Specific combinations include, for example, combinations of the variable regions of the H chain of the antibody and the total L chain, which are presented in table. 5, combinations of antibody variable regions that have CDR sequences (amino acid sequences of CDR1, CDR2, and CDR3) identical to the amino acid sequences of CDR1, CDR2, and CDR3 that are included in the common L chain variable regions shown in Table 5. 5 and combinations of antibody H chain and common L chain variable regions that are functionally equivalent to said variable regions. In the context of the present description, the concept of functionally equivalent has the meaning indicated above.

- 47 041830

Table 5

SR3 arm/arm of the T-cell receptor complex/common L chain SEQIDNO: H0610/rCE115H/L0000 215/424/53 H0610/TR01H040/L0000 215/103/53 H0610/TR01H040/L0201 215/103/299 H0610/TR01H040/L0203 215/103/301 H0610/TR01H040/L0204 215/103/302 H0610/TR01H040/L0206 215/103/304 H0610/TR01H040/L0208 215/103/306 H0610/TR01H040/L0209 215/103/307 H0610/TR01H040/L0211 215/103/309 H0610/TR01H061/L0000 215/122/53 H0610/TR01H068/L0000 215/129/53 H0610/TR01H071/L0000 215/132/53 GCH054/TR01H067/L0201 197/128/299 GCH054/TR01H067/L0212 197/128/310 GCH054/TR01H067/L0222 197/128/319 GCH054/TR01H067/L0000 197/128/53 GCH094/TR01H082/L0201 211/142/299 GCH094/TR01H082/L0011 211/142/223 GCH094/TR01H084/L0011 211/144/223 GCH065/TR01H084/L0011 206/144/223 GCH065/TR01H082/L0011 206/142/223 GCH094/TR01H109/L0011 211/164/223 GCH065/TR01H109/L0011 206/164/223 GCH094/TR01H113/L0011 211/168/223 GCH065/TR01H113/L0011 206/168/223

There are no particular restrictions on the Fc region included in the multispecific antigen-binding molecule of the invention as long as it is an Fc region with reduced Fcy receptor binding activity, but examples of a preferred Fc region of the present invention include a combination of the Fc region E22Hh region and E22Hk Fc region region, combination of E2702GsKsc Fc region region and E2704sEpsc Fc region region, and combination of E2702sKsc Fc region region and E2704sEpsc Fc region region.

Examples of the preferred multispecific antigen-binding molecule of the present invention include bispecific antibodies that comprise an antibody variable region with glypican 3 binding activity and an antibody variable region with CD3e binding activity. More preferably, the cytotoxic activity is the same as or greater than that of the GPC3_ERY22_rCEl 15 bispecific antibody. 13, and bispecific antibodies that bind to an epitope overlapping with an epitope that binds to the above antibodies and that contain an Fc region with reduced Fcy receptor binding activity.

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. Antibody competition can be assessed using competitive binding assays such as ELISA, Fluorescent Resonance Energy Transfer (FRET), and Fluorometric Microvolume Assay (FMAT (registered trademark)) technology. The amount of antibody bound to an antigen indirectly correlates with the binding capacity of a competing candidate antibody (test antibody) that competitively binds to the overlapping epitope. In other words, if the amount or affinity of the 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 an antibody to be studied are added simultaneously to an antigen, and the antibody that binds as a result is detected by a label. The amount of antibody bound to an antigen can easily be determined using a prelabeled antibody. Said label is not limited to a specific label, and the labeling method is selected depending on the analysis technique used. In particular, the labeling method includes the introduction of 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 immobilized with GPC3 or CD3s, and the labeled antibody is detected using fluorometric microvolume analysis technology.

In the context of the present description, "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, the concentration at which the unlabeled antibody reduces by 50% the amount of bound labeled antibody (1C ₅₀ ).

-48041830

Multispecific antigen-binding molecules that have antibody antigen-binding sites that bind epitopes that overlap with epitopes that bind the above antibodies can have very high cytotoxic activity.

The multispecific antigen-binding molecules of the present invention are prepared using the same technology as described above as a method for producing recombinant antibodies.

The present invention also relates to polynucleotides encoding the antigen-binding molecules of the present invention and 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 obtained by culturing cells that express antigen-binding molecules and collecting expression products from culture supernatants. Thus, the present invention relates to vectors that contain a polynucleotide encoding an antigen-binding molecule of the present invention, to cells that carry said vector, and to methods for producing antigen-binding molecules, which consist in culturing cells and collecting antigen-binding molecules from supernatants. culture. They can be obtained using the same technologies as described above for the production of recombinant antibodies.

pharmaceutical compositions.

Another object of the present invention are 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 a binding activity against the T cell receptor complex, and (3) a domain containing an Fc region with reduced Fcγ receptor binding activity. In addition, the present invention relates to pharmaceutical compositions that induce cell damage, containing an antigen-binding molecule as an active ingredient. Pharmaceutical compositions of the present invention which induce the desired cell damage, especially T-cell dependent cellular cytotoxicity, are preferably administered to an individual who suffers from a disease in which said activities are needed to prevent or treat, or to an individual who is likely to relapse. diseases.

In addition, according to the present invention, cytotoxicity-inducing agents and cell growth-inhibiting agents, and anti-cancer 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 a T-cell receptor complex, and (3) a domain containing an Fc region with reduced Fcy receptor binding activity can be used in a method for inducing cell damage, which comprises administering an antigen-binding molecule to an individual, or the antigen-binding molecule can be used to prepare a cytotoxicity-inducing agent and a cell growth-inhibiting agent.

In the present invention, the phrase contains 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 a T-cell receptor complex, and (3) a domain containing an Fc region with reduced Fcy 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 the 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) and the like) and can be incorporated into components of colloidal drug administration systems (liposomes). , albumin microspheres, microemulsions, nanoparticles and nanocapsules) (for example, see Remington's Pharmaceutical Science 16th ed., ed. Oslo, 1980)). In addition, methods for preparing agents in the form of sustained release agents are well known and can be used for multispecific antigen-binding molecules of the present invention (J. Biomed. Mater. Res. 15, 1981, p. 267-277; Chemtech. 12 , 1982, pp. 98-105, US No. 3773719, published European patents (EP) EP 58481 and EP

- 49 041830

133988; Biopolymers 22, 1983, p. 547-556).

Pharmaceutical compositions of the present invention or cytotoxicity-inducing agents and cell growth-inhibiting agents may be administered orally or parenterally to a patient, and parenteral administration is preferred. These 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 applied topically or systemically by injection. The method of administration can be selected depending on the patient's age and symptoms. The dose can be selected, for example, from the following range: from 0.0001 to 1000 mg per 1 kg of body weight for each administration. Alternatively, the dose may be selected, for example, from the following range: 0.001 to 100,000 mg/patient. However, the doses of the pharmaceutical composition of the present invention or the cytotoxicity-inducing agent and the cell growth-inhibiting agent are not limited to these doses.

Pharmaceutical compositions of the present invention, or cytotoxicity-inducing agents and cell growth-inhibiting 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 of these include, but are not limited to, surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, suspending agents, isotonicizing agents, binders, disintegrants, lubricants, flow agents and flavoring agents, and others may be used. common carriers. Specific examples of carriers are light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carmellose, sodium carmellose, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salt, and the like.

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 said 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 the 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 the antigen, are not limited to specific cells, as long as those cells express the antigen.

According to the present invention, the contacting can be carried out, for example, by adding the multispecific antigen-binding molecule of the present invention, which binds to the antigen, to the culture medium of GPC3 antigen-expressing cells that are cultured in vitro. In this case, the added antigen-binding molecule can be used in a form suitable for this purpose, 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, colorants, flavors, preservatives , stabilizers, buffers, suspending agents, isotonicizing agents, binders, disintegrants, lubricants, flow agents and flavoring agents. The concentration of the added molecule is not subject to special restrictions; 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.

In addition, according to another embodiment of the present invention, contacting is also carried out by treating non-human animals into which GPC3 antigen-expressing cells are transplanted and animals in which the antigen is expressed endogenously. The method of administration may be oral or parenteral, and parenteral administration is preferred. These 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 applied topically or systemically, for example, by injection. The method of administration can be chosen depending on the age under

- 50 041830 experimental animal and symptoms. When administered as an aqueous solution, an aqueous solution containing only the multispecific antigen-binding molecule of the present invention may be used, or a solution may be used that also contains the above surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, suspending agents, imparting isotonicity agents, binders, leavening agents, lubricants, flow agents and flavoring agents, and the like. The dose can be selected from the following range: from 0.0001 to 1000 mg per 1 kg of body weight per administration.

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

The method described below is preferably used as a method for evaluating 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 against glypican 3, which is included in an antigen-binding molecule, which is the result of contacting cells with a multispecific antigen-binding molecule of the present invention. Examples of a method for evaluating or measuring cytotoxic activity in vitro include methods for measuring cytotoxic T cell activity or the like. Whether a 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. Immunologic studies in humans, ed. John E, Coligan et al. ., 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 in the cells used for evaluation, can be used as a control, which is analyzed in the same way as the multispecific antigen-binding molecule proposed in of the present invention, and activity can be determined by evaluating whether the multispecific antigen-binding molecule of the present invention has more cytotoxic activity than the antigen-binding molecule used as a control.

To assess or measure cytotoxic activity in vivo, for example, cells expressing glypican 3 antigen are intradermally or subcutaneously transplanted into a non-human experimental animal, and then the antigen-binding molecule to be tested is administered intravenously or intraperitoneally every day or at intervals of several days, starting from the day transplantation or the next day after it. Cytotoxic activity can be determined by daily measurement of the size of the tumors, determining the difference in the change in the size of the tumors. Similar to the method used for in vitro evaluation, an antigen-binding molecule of the present invention can be considered to have cytotoxic activity if, upon administration of the control antigen-binding molecule, it is found that the size of the tumors in the group administered with the antigen-binding molecule of the present invention is significantly smaller than the size of the tumors in the group that received the control antigen-binding molecule.

As a method for evaluating or measuring an inhibitory effect on the proliferation of cells expressing glypican 3 as an antigen, the thymidine isotopically labeled uptake cell method or the MTT method can be used. As a method for evaluating or measuring an inhibitory effect on cell proliferation in vivo, a method similar to that described above for evaluating 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, which 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, kits may be packaged with an 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 obtained 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, which 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 relates to 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 H and L chains (six CDRs in total) that are included in the molecule. The present invention also relates to molecules having binding activity for the T-cell receptor complex, which contain a domain containing an antibody variable region that has binding activity for the T-cell receptor complex,

- 51 041830 multispecific antigen-binding molecule proposed in the present invention. In addition, the present invention relates to a molecule that has binding activity against the T-cell receptor complex, which contains the variable regions of the H - and L-chains of the antibody, respectively, containing three CDRs of H - and L-chains (six CDRs in total), which enter the molecule. These molecules may be antibodies or polypeptides containing antigen-binding antibody fragments. The present invention also relates to antibodies that bind to epitopes that overlap or compete with said 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 have to be multispecific (bispecific), but can only bind to either GPC3 or T-cell receptor complex (eg, CD3ε chain).

Said molecules include a molecule that contains a domain containing an antibody variable region that has GPC3 binding activity, a multispecific antigen binding molecule detailed in the Examples section of this specification (which contains H chain variable regions that have GPC3 binding activity and variable region of the common L chain), a molecule that contains a domain containing the variable region of an antibody that has binding activity against the T-cell receptor complex, a multispecific antigen-binding molecule, a detailed description of which is presented in the Examples section of the present description (which contains variable regions H chains that have binding activity on the T-cell receptor complex and the variable region of the common L-chain), as well as a molecule that has the ability to bind to the same antigenic protein (GPC3 or T-cell receptor complex), which contains lives 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 (shared) with the CDRs of the multispecific antigen-binding molecule of the present invention; and therefore they are expected to bind to an epitope overlapping with that of the multispecific antigen-binding molecule of the present invention. Thus, these molecules can compete with multispecific antigennegative molecules of the present invention, when they are present together with multispecific antigennegative molecules of the present invention. Thus, these molecules can be used, for example, as regulatory agents to suppress various activities (such as antigen-binding activity, cytotoxic activity and antitumor activity) of the multispecific antigen-binding molecules of the present invention. In addition, the molecule can be pre-bound to a target protein (GPC3 or T-cell receptor complex), and after the addition of the multispecific antigen-binding molecule of the present invention, competition-dissociated molecules can be detected. In this application, the molecule is useful as an agent for assessing the binding of a multispecific antigen-binding molecule of the present invention to a target protein. According to the present invention, 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 that overlap with epitopes that bind to the multispecific antigen-binding molecules of the present invention. As mentioned above, the specified molecule can be previously associated with a target protein (GPC3 or T-cell receptor complex), and then the test antibody is added, while if there is a dissociation of the bound molecules, then the test antibody is promising (candidate) as an antibody , which binds to an epitope that overlaps 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 exemplary 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 (K _D values) is preferably exemplified by the KD values of the multispecific antigen-binding molecule of the present invention, but is not limited to these values.

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

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

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Examples

Below, the present invention is described 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) Preparation of GPC3 ERY22 rCE115.

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

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

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

A series of expression vectors with an inserted polynucleotide encoding each of the constructs GL4-ERY22_Hk (SEQ ID NO: 47), H0000-ERY22L (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: GL4ERY22_Hk, H0000-ERY22L, rCE115H-ERY22_Hh, rCE115L-k0.

(1-2) Purification of GPC3 ERY22 rCE115.

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

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

The cytotoxic activity of GPC3_ERY22_rCE115 was evaluated in vitro.

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

Received samples of 50 ml of peripheral blood of healthy volunteers (adults) 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 twice with PBS(-), divided into four aliquots, and added to Leucosep Lymphocyte Separation Tubes (catalog no. 227290; Greiner bio-one), which were preloaded with 15 ml of Ficoll-Pak PLUS and centrifuged. After centrifuging 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 (referred to hereinafter as 10% FBS/D-53 041830

MEM) and then the cell density was adjusted to 4x10 ⁶ cells/ml with 10% FBS/D-MEM. The cell suspension thus obtained was used as the human PBMC solution in subsequent experiments.

(1-3-2) Analysis of cytotoxic activity.

Cytotoxic activity was evaluated based on the degree of inhibition of cell growth using a real-time cell analyzer xCELLigence (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 detached from the plates and seeded in an E-Plate 96 (Roche Diagnostics) as aliquots of 100 μl/well, adjusted to 1 x 10 ⁴ , and analysis of viable cells was started using a cell analyzer 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 at different concentrations (0.004, 0.04, 0.4, and 4 nM) was added to the plate. After reacting for 15 min at room temperature, 50 µl of human PBMC solution (2x10 ⁵ cells/well) prepared according to the method described in section (1-2) was added and the cells were again placed in the xCELLigence real-time cell analyzer and analysis was started. viable cells. 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 following formula using the cell index value determined 72 hours after the addition of human PBMC. The cell index value used for the 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) x 100/(A-1)

A is the average cell index in wells without antibody addition (target cells and human PBMCs only), and B is the average cell index 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 GPC3_ERY22_rCE115 cytotoxicity assay, very strong activity was found (FIG. 2).

Example 2 Humanization of the H chain of the anti-CD3 antibody, rCE115, and sharing of the common L chain.

(2-1) Creation of hCE115HA, a humanized H chain variable region of the rCE115 antibody.

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 rCE115 variable region sequence. The IMGT database (http://www.imgt.org/) and NCBI GenBank (http://www.ncbi.nlm.nih.gov/genbank/) were used as the database. A humanized H chain variable region sequence was generated by ligating the rCE115 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 the database of rat and human germline sequences (IMGT database (http://www.imgt.org/)) found only a few sequences that contain Arg in this site. It is known that the amino acid residue at position 94 according to Cabot numbering is involved in the stabilization of the structure of the antibody by the formation of the upper core (Ewert and others, Methods 34(2) October 2004, pp. 184-199). Based on this information, a new humanized H chain variable region sequence was generated in which amino acid residues at Cabot positions 93 and 94 in the FR3 H chain were replaced with residues present in the rCE115 variable region sequence. In this way, a humanized hCE115HA H chain variable region sequence was obtained (SEQ ID NO: 52).

(2-2) Construction of the common L chain L0000 for the anti-CD3 antibody rCE115 and the anti-GPC3 antibody.

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

The GL4 FR sequence was chosen as the L chain FR sequence. The CDR2 of the L chain was the same in rCE115L and GL4. L chain CDR1s were selected from GL4 CDR sequences and L chain CDR3s were selected from 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 generated by ligation of the selected FRs and CDRs described above. This resulted in a humanized L chain variable region sequence, L0000 (SEQ ID NO: 53).

(2-3) Affinity assessment for human GPC3.

The binding activity for human GPC3 was evaluated 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 an antibody carrying a single Fab heterodimerized in the Fc region of human IgG1 using knobs-into-hole technology was used. H0000 (SEQ ID NO: 40) was used as the variable region of the H chain of an anti-GPC3 antibody.

The affinity and binding constants of the anti-GPC3 antibody for the antigen were measured by surface plasmon resonance multicyclic kinetics using a Biacore™-T200 device (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 bind the A/G protein to the CM5 chip (chip coated with carboxymethyl dextran). Each anti-GPC3 antibody was prepared so that approximately 100 RU was captured by the A/G protein. Human GPC3 used as analyte was prepared at 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 minutes, allowing the reaction to proceed. The solution was then replaced with HBS-EP+ buffer and the dissociation phase was evaluated for 15 minutes. After completion of the dissociation phase evaluation, the sensor chip was regenerated by washing with 10 mM Gly-HCl at pH 1.5. Similarly, a measurement was made at 0 concentration, allowing the A/G protein to capture in the antibody solution, injecting HBS-EP+ for 3 min, allowing reactions to proceed, and then replacing the solution with HBS-EP+ to evaluate the dissociation phase for 15 min. After completion of the dissociation phase evaluation, 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 binding reaction rate constant (k _a ), dissociation reaction rate constant (kd) and rate constant ratio based on the obtained sensorgrams. The results are presented in table. 6.

Table 6

Variable region Affinity for human CD3 Variable region of Nchain Variable region of the L chain KD (M) ka (1/ms) kd (1/s) NOOOOO GL4 ^4.2x10'9 4.3* ^{105 1.8x10'3} NOOOOO L0000 ^3.6x10'8 3.0x10 ⁵ Ι,ΙχΙΟ' ²

(2-4) Assessing affinity for human CD3.

Human CD3 binding activity was evaluated 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 an antibody carrying a single Fab heterodimerized in the Fc region of human IgG1 using knobs-into-hole technology was used.

The affinity and binding constants of the anti-CD3 antibody for the antigen were measured by the single-cycle kinetics method based on surface plasmon resonance using a Biacore™-F200 device (GE Healthcare Japan). HBSEP+ (GE Healthcare Japan) was used as a running buffer, and an amine coupling kit (GE Healthcare Japan) was used to covalently bind human CD3 to a CM5 chip (carboxymethyl dextran coated chip). Anti-CD3 antibody used as analyte was prepared at concentrations of 5 and 20 μg/ml using HBS-EP+. Measurements were made by first injecting each of the anti-CD3 antibody solutions at concentrations of 5 and 20 μg/ml for 3 minutes 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 evaluated for 3 minutes. After completion of the dissociation phase evaluation, 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 binding reaction rate constant (k _a ), dissociation reaction rate constant (k _d ) and ratio of rate constants based on obtained sensorgrams . The results are presented in table. 7.

Table 7

Variable region Affinity for human CD3 Variable region of Nchain Variable region of the L chain KD(M) ka (1/ms) kd (1/s) GSE115N rCE115L Ι,ΟχΙΟ ⁷ 5.9x10 ⁴ 6.0x10' ³ hCE115HA L0000 1.2x10 ⁷ 1.9x10 ⁵ 2,3xu' ²

(2-5) Preparation of GPC3 ERY27 hCE115.

Anti-cancer antigen IgG4 (GPC3) was used as a base structure to generate the ERY27 molecule (FIG. 16), in which the H chain variable region of one of the Fab was replaced with a CD3 epsilon binding domain and the L chain was shared. for both Fab. In this case, the IgG4 Fc that was used as the main structure was a silent Fc with reduced affinity for FcgR (Fey-(Fc-gamma) receptor). H0000 (SEQ ID NO: 40) was used as the H chain variable region of the GPC3 binding domain, and hCE115HA (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 variable region of the L chain. Mutations D356K and K439E 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 has been introduced to efficiently separate heteromer and homomer (WO 2011/078332).

A series of expression vectors with an inserted polynucleotide encoding each of constructs f H0000-ERY27HK (SEQ ID NO: 54), hCE115HA-ERY27_HE (SEQ ID NO: 55) and L0000-k0 (SEQ ID NO: 56) 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: H0000ERY27HK, hCE115HA-ERY27HE and L0000-k0 (2-6) Purification of GPC3 ERY27 hCE115.

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

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

(2-7-1) Obtaining 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) Assessment of cytotoxic activity.

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

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

Example 3 Generation 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 be the result of weakened affinity for GPC3 and the CD3ε chain as a result of humanization and sharing of the common L chain. Regarding antigens such as GPC3 and CD3ε chains, which have sequences independent of each other, there is currently no evidence of humanized bispecific antibodies with increased T-cell dependent cytotoxic activity and improved affinity for both antigens using a common antibody L chain. . Thus, it is difficult to obtain dual specificity humanized antibodies that have drug efficacy equivalent to or greater than that of the GPC3_ERY22_rCE115 antibody.

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

Point mutation introduction, antibody expression and purification, antigen affinity assessment, and determination of T-cell dependent cellular cytotoxicity while optimizing humanized bispecific antibodies were performed 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 purpose, 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/N97A/D356C/T366S/L368A/Y407V/G446 -deletion/K447-deletion in human IgG1; E22Hk (SEQ ID NO: 58), which was obtained by introducing mutations

- 56 041830

L234A/L235A/N97A/Y349C/T366W/G446-deletion/K447-deletion, and Ser-Ser insertion mutation just before position 118 in human IgG1; G1dh was obtained by introducing mutations

D356C/T366S/L368A/Y407V/G446-deletion/K447-deletion in 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 obtained by introducing mutations

L235R/S239K/N297A/E356K/R409K/H435R/L445P/G446-deletion/K447-deletion in human IgG4; E2704sEpsc (SEQ ID NO: 61) was obtained by introducing mutations

K196Q/L235R/S239K/N297A/R409K/K439E/l445P/G446-deletion/K447-deletion in human IgG4; and E2702sKsc (SEQ ID NO: 62) were obtained by introducing mutations

L235R/S239K/N297A/E356K/R409K/L445P/G446-deletion/K447-deletion in human IgG4. In addition, human k chain (kappa chain) k0 (SEQ ID NO: 63) and E22L (SEQ ID NO: 432) were obtained by introducing R108A/T109S mutations into the human k chain used as L chain constant regions antibodies.

Mutation resulting in the replacement of Asp by Cys at position 356 according to the EU numbering, mutation resulting in the replacement of The by Ser at position 366 according to the EU numbering, mutation resulting in the replacement of Leu by Ala at position 368 according to the EU numbering, mutation, a Tyr to Val substitution at EU position 407, a Tyr to Cys substitution at EU position 349, a Thr to a Tip substitution at EU position 366, and a mutation resulting in to the Ser-Ser insert just before position 118 according to the EU numbering, are mutations designed to efficiently form heterodimeric molecules of each H chain when creating heteromeric antibodies. Similarly, a mutation resulting in the replacement of Glu with Lys at position 356 according to the EU numbering, and a 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. As expected, they should increase the efficiency of production of bispecific antibodies.

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

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

A mutation resulting in the change of Arg to Lys at position 409 according to the EU numbering, and a mutation resulting in the change of His to Arg at position 435 according to the EU numbering, are mutations that lead to a modification of the property 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 anti-CD3 antibody by point mutations.

First, point mutations were introduced into the sequence of FR1, FR2, FR3, CDR1, CDR2, and CDR3 of a humanized anti-human CD3ε chain antibody prepared according to the method described in Example 2, hCE115HA-ERY27HE (SEQ ID NO: 55), to obtain modified antibodies. The affinity of these modified antibodies to the soluble human CD3ε chain was then determined. By combining sites that have the ability to increase the affinity of the modified antibodies, modified antibodies were obtained, the affinities of which are presented in table. 8.

- 57 041830

Table 8

Name of the antibody KD (human CD3) hC Е115HA-E22Hh//-Hi-KoO10G3/L0000-k0 1.43E-07 TR01H083-E22Hh/none-Hi-Kn010G3/L0212-k0 5.86E-11 TR01H040-E22Hh/none-Hi-Kn010G3/^ 2.17E-O9 TROlH002-E22Hh/GLS3l08-kO/GL4-E22Hk/H0610-E22L 2.04E-09 TR01H040-E22Hh^one-Hi-Kn010G3/L0212-k0 2.17E-Z9 TR0lH040-E22Hh/none-Hi-Kn0l0G3/L0235-k0 2.81 E-09 TR01H040-E22Hh/none-Hi-Kn010G3/L0238-k0 2.91 E-09 TR01H040-E22Hh>none-Hi-Kn010G3/TR01L015-kD 2.52E-O9 TR01H083-E22Hh/none-Hi-Kn010G3/La262-k0 2.45E-09 TR01H04O-E22Hh/none-Hi-Kri010G3/L0207-k0 2.6OE-O9 TR0lH040-E22Hh/none-Hi-Kn0l0G3/L0241-k0 3.48E-09 TR01H040-E22Hh/none-Hi-Kn010G3/L0242-k0 E.58E-09 TRQ1HO4O-E22Hh/none-Hi-KnO1OG3/LQ2O6-kO 2.9QE-Q9 TR01H040-E22Hh/none-Hi-Kn010G3/TR01L019-k0 3.20ΕΌ9 TR01H080-E22Hh/none-Hi-Kn010G3/L0000-k0 3.25E-09 TR01H040-E22Hh/none-Hi-Kn010G3/L0211-k0 3.22ΕΌ9 TRO1HOO2-E22Hh//-Hi-KnD10G3/GLC31O8-kO 4.61 E-09 TR01H040-E22Hh/none-Hi-Kn010G3/L0209-k0 4.25E-09 TR01HC40-E22Hh/none-Hi-Kn010G3/L020S-W 4.16E-09 TR01H040-E22Hh/none-Hi-Kn010G3/L0224-k0 5.06E-09 TR01H040-E22Hh/nane-Hi-Kn010G3/LQ236-k0 5.64E-O9 TR01H083-E22Hh/nane-Hi-Kn010G3/L0201-k0 4.42E-09 TR01H084-E2702GsKsc/none-Hi-E2704sE/L0011-kO 4.14E-09 TR01H040-E22Hh/none-Hi-Kn010G3/L02l0-k0 5.06E-09 TR01H114.E2702GsKsc/nQne.H!-E2704sE/L0011-kO 4.22E-09 CE115HA236-E22Hh/GLS3108-KO/CE4-E22Hk/H06U-E22E 6.08E-09 TR01H077-E22Hh/none-Hi-Kn010G3/L0200-k0 6.12E-09 TR01H071-E22Hh/none-Hi-Kn010G3/L0200-k0 6.13E-09 TR01H111-E2702GsKsc/none-Hi-E2704sE/L0011-k0 4.91 E-09 TR01H081-E22Hh/none-Hj-Kn010G3/L0262-k0 5.76E-09 TR01H001-E22Hh/AHi-Kn010G3/GLC3108-kO 8.22E-O9 CE115HA179-G1dh/M4l-Kn010G3/L0Q00-k0 8.35E-09 TR01H112-E2702GsKsc/nore-Hf-E2704sE/L0011-k0 5.12E-O9 TR01H113-E2702GsKsc/none-Ht-E2704sE/L0011-k0 5.14E-09 TR01H082-E22Hh/none-Hi-Kn010G3/L0212-k0 4.75E-09 CE115HA236-E22Hh//-Hi-Kn010G3/GLC3108-k0 9.10E-09 TR01H040-E22Hh/none-Hi-Kn010G3/L0231-k0 7.75E-09 TR0lH037-E22Hh/none-Hi-Kn0l0G3/L0000-k0 6.93E-09 CE115H A252-E22Hh//-Hi-KnO WG3/LQOOO-kO 9.48E-09 TR01H083-E22Hh/none-Hi-Kn010G3/L0011-kO 6.70E-09 TR01H040-E22Hh/none-Hi-Kn010G3/L0223-kO 8.15E-09 Τ^01Ηΰθ3-£22ΗΜ/ηοηθ-ΗρΚη0ΐ0ΰ3/10ΰ00-ω 6.83E-O9 TR01H071-E22Hh/none-Hi-Kn010G3/L0000-k0 E.85E-09 TR01H067-E22Hh/none-Hi-Kn010G3/L0212-k0 5.88E-O9 CE115HA178-G1dh//-Hi-Kn010G3/L0000-k0 1.09E-08 TR01H040-E22Hh/none-Hi-Kn010G3/L0237-k0 1.02E-0B TR01H083-E22Hh/none-Hi-Kn010G3/L0222-k0 9.42E-09 TR01H084-E22Hh/none-Hi-Kn010G3/L0262-k0 8.51E-O9 TR0lH071-E22Hh/none-Hi-Kn0l0G3/L0215-k0 9.51 E-09 TR01H040-E22Hh/none-Hi-Kn010G30218-kO 8 20E-09 TRQ1HO81-E22Hh/none-Hi-KnO1OG3/L02O1-kO 9.46E-09 TR01H071-E22Hh/none-Hi-Kn010G3/L0222-k0 1.04E-08 TR01H040-E22Kh/none-Hi“Kri010G3/L0220“k0 9.15E-O9 TR01H067-E22Hh/none-Hi-Kn010G3/TR01L018-k0 1.09E-08 TRO1HOO2-E22Hh/GLS31384<O/GL4-E22Hk/HD0OO-E22L 4.78E-09 TR01H067-E22Hh/none-Hi-Kn010G3/TR0U019-k0 1.21E-08 TRO1HO38-E22Hh/none-Hi-KnO1OG3/L0OOO-kO 1.24E-08 TR01H06l-E22Hh/none-Hi-Kn010G3/L0200-k0 1.27E-08 TR01H082-E2702GsKsc/nQne-Ht-E27Q4sE/L(X!11 -k0 1.01 E-08 CE115HA18O-G1dh/AHi-KnO1OG3/LD0OO-kO 1.68E-08 CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L 1.37E-08 TROlHl0O-E2702GsKsc/nore-Hi-E2704sE/L00l1-k0 1.11 E-08 TR01H040-E22Hh/none-Hi-Kn010G3/L0228-k0 1.60E-08 TR01H081-E22Hh/none-Hi-Kn010G3/L0011-k0 1.35E-08 TR01H061-E22Hh/none-Hi-Kn010G3/L0215-k0 1.54E-08 TR01H110-E2702GsKsG/none-Ht-E2704sE/L0011-kO 1.26E-O8 TR0lH043-E22Hh/non©-Hi-Kn0l0G3/L0000-k0 1.52E-08 TR01H081-E22Hh/none-Hi-Kn010G3/L0000^ 1.56E-08 CE115HA251-E22Hh//-Hi-Kn010G3/L000D-k0 2.23E-08 TR01H091-E27Q2GsKsc/none-Ht-E2704sE/LQ011-k0 1.E9E-08 CE115HA236-E22Hh/GLS31 D8-kO/GL4-E22Hk/HOOOO-E22L S.95E-09 TR01H084-E22Hh/none-Hi-Kn0l0G3/L0201-No. 1.65E-08 TRO1HO72-E22Hh/none-Hi-KnO1OG3/L0OOO-kO 2.03E-0S TR01H099-E2702GsKsc/nons>HHE2704sE/L0011-kO 14SE-08 TR01H061-E22Hh/non©-Hi-Kn010G3/L0222-kO 1.88E-08 TR01HW0-E22Hh/none-Hi-Kn010G3/L0239-k0 2.31 E-08 TR01H040-E22Hh/none-Hi-Kn010G3/L0262-kO 1.81 E-08 TR01H040-E22Hh/none-Hi-Kn010G3/L0234-k0 2.40E-08 TRO1HO12-E22Hh/nane-Hi-KnO1OG3/L0OOO-kO 794E-D9 TR01H061-E22Hh/none-Hi-Kn010G3/L0000-k0 1.71 E-08 TRO1HO4O-E22Hh/none-Hi-KnO1OG3/L0243-kO 2.4SE-08 TR0lH109-E2702GsK8c/none-Hi-E2704sE/L0011-k0 1.84E-08 TR01H047-E22Hh/none-Hi-Kn010G3/L0000-k0 2.04E-08 TR01H082-E22Hh/none-Hi-Kn010G3/L0267-k0 2.29E-08 TR01H082-E22Hh/none-Hi-Kn010G3/L0266-k0 2.29E-08 TR01H064-E22Hh/none-Hi“Kri010G3/L0011-kO 1.98E-08 TR01H040-E22Hh/none-Hi-Kn010G3/L0250-k0 2.15E-08 TR01H040-E22Hh/none-Hi-Kn010G3/L0204-k0 2.21E-08 TR01H084-E22Hh/none-Hi-Kn010G3/LM00-k0 2.13E-08 TR01H040-E22Hh/none-Hi-Kn010G3/LD213-k0 2.01E-08

- 58 041830 ^iCEDSHA^gHh^^^________

TR01H040-E22Hh/n0ne-Hi-Kn010G3/L0214-k0

TRD1H040-E22Hh/none-Hi-Kn010G3/L0217-k0

TR01H071-E22Hh/none-Hi-Kn010G3/L0226-k0

TKO1N040-E22NP/pope-NcKp0UOZ/k0200-k0

TRO1H074-E22Hh/none'Hi'Kn010G3/LO000'k0

TR01 H039-E22Hh/none-HI-Kn010G3/LO00G-kQ

CE115HA177-G1dh/AHi-KnO1OG3/LOOOO-kO

TR01H040-E22Hh/none-Hi-Kri010G3/L0201-k0

TR01H082-E22Hh/none-Hi-Kn010G3/L02S3-k0

TKO1N040-E22N/pope-NcKp010SZL_0000-k0

TK01N040E22NP/POPE-NcKp010OZ/k0216-k0

TR01H051-E22Hh/none-Hi-Kn010G3/L0000-k0

TR01 H003-E22Hh/AHi-Kn01 OG3/LOOOO-kO

TR01 Η082Έ22ΗΚ/ποπδΉί Κη010Ο3/10264-Κ0

TR01 H04Q-E22Hh>none-Hi-Kn010G3/L0232 k0

TRO1H041 -E22Hh/rK»ne-Hi-KnO1OG3/LO0OO'kO

CE115HA122-E22Hh//-Hi-KnO1 DG3/L000D-k0

TR01H040-E22Hh/none-Hi-Kn010G3/L0233-k0

TRD1H04G-E22Hh/none-Hi-Kn010G3/L0215-k0

TRDlH040-E22Hh/none-Hi-Kn0l0G3/L0203-k0

TRO1HO15-E27O2GsKsc/GCHO19-E27iMsEpsG/L0OOO-kO TR01H04C-E22Hh/rt0n6-Hi-Kn010G3/TR01 L008-k0

TR01H040-E22Hh/none.Hi-Kn010G3?L0205-kO

TR01H015-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L

TR01H064-E22Hh/none-Hi-Kn010G3/L0000-k0

TR01HO44-E22HIVnone-Hi-Kn010G3/LQ000-k0

TR01H082-E22Hh/none-Hi-Kn010G3/L0262-k0

TR01H062-E22Hh/none-HhKn010G3/LO000-k0

CE115HA251-E22HhiL0000-k0/GL4-E22Hk/H0000-E22L

TR01H040-E22Hh/rK>ne-Hi-Kn010G3/L0011-kO

TRO1 HQ40-E22Hh/rtone-Hi-Kn010G3/L0222-k0

CE115HA192-E22Hh/FHi-KnO10G3/L0000-k0

TR01H04G-E22Hh/none-Hi-Kn010G3/TR01 LOIO-kO

TR01H025-E22Hh/none-Hi-Kn010G3/L0000-k0

TR01 H082-E22Hh/none-Hi-Kn010G3rTR01 L023-kD

TR01H040-E22Hh/nane-Hi-Kn010G3/TR01L015-k3

TRO1H055-E22Hh/none-Hi-Kn010G3/LtXl00-k0

TR0lH082*E22Hh/none-Hi-Kn010G3/L0260-k0

TR01 H040-E22Hh/none-Hi-Kn010G3/TR01 L009-k0

TR01H040-E22Hh/none-Hi-Kn010G3/TRCHLC11-kO

TR01 HOI 7.E22Hhinone-Hi-Kn010G3/L0000-k0

CE115HA122*E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L

TR01H076-E22Hh/none-Hi-Kn010G3/LO000-k0

TR01H082-E22Hh/none-Hi-Kn010G3/L0258-k0

TR01H046-E22Hh/none-Hi-Kn010G3/L0000-k0 rCE115H-G1dW-Hi-Kn010G3/L0000-k0

TR01 H082-E22Hh/none-Hi-Kn0l0G3/TR0l L024-k0

TR01H016-E22HtVnone-Hi-Kn010G3/L000O-k0

TR01H040-E22Hh/none-Hi-KnOlOG3?TR01LGl8kO

TR01 H084-E22Hh/none-Hi-Kn010G3/L0271-k0

TRO1HO84-E22Hh/none-Hi-KnO1OG3/LO27O-kO

TRCl1H040-E22Hh/none-Hi-Kn010G3/L0000vk1-k0

TRO1HO4G-E22Hh/none-Hi-KnO1OG3/LO219-kO

TR01 HOI 4-E22Hh/none-Hi-Kn010G3/LO000-k0

TRO1H061-E22Hh(none-Hi-Kn010G3/L0226-k0

TR01H048-E22Hh/none-Hi-Kn010G3/LM00-k0

TRD1H082-E22Hh/none-Hi-Kn010G3/L0259-k0

TN01N028^22NK/gupe-NnKp010YE/1SKU0-c0

TR01H082-E22Hh/none-Hi-Kn010G3/L0201-k0

TRD1H040-E22Hh/none-Hi-Kn010G3/TR01 L013-k0

TR01H033-E22Hh/none-Hi-Kn010G3/L0a00-k0 hCEl 15HA-G1 dh//-HbKn010G3rLC00CM<0

TR01H040-E22Hh?none-Hi-Kn010G3/TR0U012-k0

TR01HQ65-E22H^non^Hi-Kn010G30000-kO

TRD1H079-E22Hh/none-Hi-Kn010G3/LM00-k0

TR01 H042-E22Hh<none’Hi’Kn010G3/L0a00'-k0

TRO1 HO63^22Hb/POPE-NnKpO1OSZ/1.SIO0O-k0

TR01H084-E22Hh/none-Hi-Kn010G3/L0272-k0

CE115HA121 -E22Hh//-Hi-KnD1 DG3/LOOOD-kO

TR01H026-E22Hh/none-Hi-Kn010G3/LOO00-k0

TR01H067-E22Hh/nane-Hi-Kn010G3/LQ262-k0

TR01H073*E22Hh/none-Hi-Kn010G3/Lt)000-kO

7R01H045-E22Hh/none-Hi-Kn010G3/LM00-k0

TR01H007-E22Hh/none-Hi-Kn010G3/LtXK)0-k0

TR01H082Έ22Hh/πone-Hl Kn010GЗ/L0203-k0

TR01H032-E22Hh/none-HI-Kn010G3/LCK}00-k0

TR01H006-E22Hh/none-Hi-Kn010G3/L0000-k0

TRO1 HOI 3-E22Hh/none-Hi-Kn010G3/LM00-k0

TR01H05G-E22Hh/none-Hi-Kn010G3/LOa00-k0

TR01H067-E22Hh/nane-Hi-Kn010G3/L0200-k0

TR01 HOI 5-E22Hh/none-Hi-Kn010G3/L0000-k0 hCEl 15HA-E22Hh/LOOOO-kO/GL4-E22Hk/HQOOO-E22L hCE115ΗΑ-Ε22δΐΗη/ηοη&-Ηΐ·&ίΚηη0003/04:0

TR01H069-E22Hh/none'HhKn010G3/L0000-k0

TRO1 HOI 5*E22Hb/male-NkKpO1OSZ/TNO1 L003-k0

TR01H040-E22Hh/none-Hi-Kn010G3/L0202-k0

TRO1H067-E22Hh/none-Hi-Kn010G3/L0201-k0

TRO1 H02G-E22Hh/non^Hi-Kn010G3/L0000-k0

TR01 H082-E22Hh/none-Hi-Kri01QG3/L0011-k0

43E-07 2 02E-O8 207E-08 2.51E-0B 287E-08 2.91E-08 2.61EΌ8 3 55E-08 2.81E-08 309E-08 3 60E-0B 253EΌ8 2.91EΕΌΕ 403E-08 3.44

16E-08 428E-08

4.01E-08

3.37E-08 3 24E-0& 2.96E-08 2.93E-08

42E-O8 357E-08

3.07E-0B 352E-08 39SE-O8

13ΕΌ8 1 48ΕΌ8

48E-08 465E-08 50&E-0B E28E-08 386E-08

4.25E-08

3.95E-O&

3.Θ8Ε-08 453E-O8 356E-O8 357E-08

3.50E-0B

69ΕΌ8 478E-08 4.70E-08 423E-O8

5.76E-OB 476E-08 369E-0B 451E-0&

2.76E-08

2.76E-08 4.69ΕΌ8 3.94ΕΌ8 387E-08 471E-08 452E-08 5.07E-08 480E-08 449E-08 4.15E-O8

4.48E-0&

4.35ΕΌ8 3 10E-08 678E-08

5.12E-0B

92ΕΌ8 597ΕΌ8 5.22E-O8 2.17E-08

4.07E-0B 573E-08 230E-0& 4.94E-08

5.76E-08 603E-08

13E-08 6 16E-08

5.17E-O8

7.11ΕΌ8

6.34ΕΌ8

6.19E-08

93E-08 6.48E-0B 595E-0B

- 59 041830

hCE115HA-E22Hh//-Hi-KnO10G3/LM00-k0 1.43E-07 TRC1H0B2-E22Hh/none-Hi-Kn010G37TR0lL018-k0 4.72E-08 TR01H015-E22Hh/none-Hi-Kn010G34R01L005-k0 6.53E-08 TRG1 H052-E22NI/poge-NnKp01 OG3/LOOOO-kO 6.27Ε-0Θ TR01 H036-E22Hh/none-Hi-Kn0l GG3A0000-k0 6.50E-08 TR01 H067-E22Hh/none-Hi-Kn0l GG3/L0203-kO 4.79E-C6 TR01 H03G-E22Hh/none-HiKn01 GG3/L0000-kG 6.54E-08 TR01H0l5-E22Hh/none-Hi-Kn0l0G3/TR0lL001-k0 6.56E-08 TRG1 Hl00-E22Hh/none-Hi-Kn010G3/LOO11-kO 6.25E-08 TR01 H029-E22Hh/none-Hi-Kn0l OG3/LOOOO-kO 6.70E-08 TR01 H019-E22Hh/none-Hi-Kn01 QG3/L0000-kG 6.&5E-08 TRG1 H082-E22Hh/none-Hi-Kn0l GG3/L0000-kG 7.37E-08 TRG1 H0l8-E22Hh/none-Hi-Kn0l OG3/LOOOO-kO 6.93E-08 TR01 H027-E22Hh/none-Hi-Kn01 OG3/LOOOO-kO 6.95E-08 TR01 H049-E22Hh/rwe-Hi-Kn010G3/L000frk0 6.79E-08 TR01 H066-E22Hh/none-Hi-Kn0l GG3/L0000-kG 6.02E-08 TR01 H091-E22Hh/none-Hi-Kn0l OG3/L0011-kO 6.67E-08 rCE115H-E22Hh/ncne-Hi-Kn010G3/L0000-k0 8.00E-08 TR01H015-E22Hh/none-Hi-Kn010G3/TR01L002-k0 7.14E-08 TR01 H040-E22Hh/nore-Hi-Kn01 OG3/L0226-kO 8.01E-08 TR01H067-E22Hh/none-Hi-Kn010G3HR0lL018-k0 5.26E-08 TRG1 H093-E22Hh/nore-Hi-Kn01 OG3/L0011-kO 6.80E-08 TR01 H067-E22Hh/none-Hi-Kn010G3/L0215-kO 7.41E-08 TR01H015-E22Hh/none-Hi-Kn010G3/TRC1L004-k0 7.34Ε-0Θ TR01 H107-E22Hh/none-Hi-Kn01 OG3/L0011-kO 6.91E-08 TR01 H105-E22Hh/none-Hi-Kn01 OG3/L0011-kO 6.95E-08 TR01 HQ90-E22Hh/none-Hi-Kn01 OG3/L0011-kO 6.95E-08 TR01 H108-E22Hh/none-Hi-Kn01 OG3/L0011-kO 6.98Ε-0Θ TR01 H094-E22Hh/none-Hi-Kn01 OG3/L0011-kO 7.00E-08 TR01 HI 09-E22Hh/nore-Hi-Kn01 OG3/L0011-kO 7.06E-08 TR01 H056-E22Hh/none-Hi-Kn01 OG3/LOOOO-kO 7.32E-08 TR01 H031-E22Hh/none-Hi-Kn01 OG3/LOOOO-kO 7.55E-08 TR01 H022-E22Hh/none-Hi-Kn01 OG3/LOOOO-kO 7.58E-08 TR01 H092-E22Hh/none-Hi-Kn01 OG3/L001 FkO 7.21E-08 TR01 H067-E22Hh/none-Hi-Kn01 OG3/LOOOO-kO 7.15E-08 TR01 H067-E22Hh/nore-Hi-Kn010G3A0Q1 FkO 7.1 BE-08 TR01 H040-E22Hh/nore-Hi-Kn010G3A.O248-k0 7.89E-08 TR01 Η009Έ22ΗΗ/πογθ-Ηϊ-Κπ01 OG3/LOOOO-kO 3.15E-08 TR01 H023-E22Hh/rone-Hi-Kn01 0G3/L00OT-k0 7.94E-08 TR01 7.47E-08 TR01 H040E22Hh/none-Hi-Kn010G34RD1LDO7-kO 6.B2E-08 TR01 H054-E22Hb/pope-NbKp01 OG3/LOOOO-kO 7.79E-08 TR01 Η021Έ22ΗΜποπβ-Ήϊ-Κπ01 OG3/LOOOO-kO 8.05E-08 TR01H W3-E22Hh/rore-Hi-Kn010G3/L0011-kO 7.72E-08 TRG1 H09>E22Hb/Gope-HnKp01 0G3/L0Q1 FkO 7.74E-08 gSE115N-E22NMpop<^NyKp010SZL_0000¥k1-k0 8.52E-08 TR01H101-E22Hh/none-Hi-Kn01OG3/L0011-kO 7.B7E-08 TR01 N0BZ-E22N/gope-NkKp01 OG3A.OOOO-kO Β.23Ε-Ό8 TR01 H03S-E22Hh/none-Hi-Kn01 OG3AOOOO-kO B.49E-Q8 TRG1H067-E22Hh/none-Hi-Kn010G3/TRQ1L015-k0 B.64E-08 TR01 Η104-Ε22Ηή/ΓθΓθΉί-Κη01 OG3/L0011-kO B.26E-08 TR01 H075-E22NI/pope-NyKp01 OG3/LOOOO-kO 9.BBE-08 TR01 HD40-E22Hh/none-Hi-Kn010G3/1.0227-k0 1.01E-07 TR01H1D2-E22Hh/none-Hi-Kn010G3/L0011-k0 B.54E-Q8 TR01 Η034-Ε22ΗΜ/ΓΟΓ&Ήί-Κη01 OG3/LOOOD-kO 9.11E-08 TR01 H082-E22Hh/none-Hi-Kn010G3/L0222-k0 1.01E-07 rCE115H-E22HhirCE115L-kO/GL4-E22Hk/HOOOO-£22L 9.37E-08 TRQ1 H015-E22Hb/pope-N|-Kl01063/TK01E006-k0 9.30E-0B TRG1 H040-E22Hh/none-Hi-Kn010G3A_0246-k0 9 2ВЁ-С0 TRG1 H097-E22Hh/none-Hi-Kn01 OG3/L0011-kO B.76E-08 TRG1 HOI 1-E22Hh/rore-Hi-Kn01 OGS/tOOOO-kO 3.71E-0B TR01 H010-E22Hh/none-Hi-KnD1 OG3/L0000-RO 3.73E-08 TRG1 H095-E22Hh/none-Hi-KnGl OG3/L0011-kG 9.09ё-08 TR01 H082-E22Hh/none-Hi-KnD1 OG37TROt LO2O-kO 1.08E-07 TR01 H098-E22Hh/none-Hi-Kn010G3/LOO11-ktJ 9.14E-08 TR01 H082-E22Hh/none-Hi-Kn01QG3/TR01L017-k0 1.09E-07 TR01 H04CbE22Hh/mna-Hi-Kn010G3/L0247-kD 1.00E-07 GSE115H-E22Hh/ncne-Hi-KnO1OG3/fCE115L-k0 1.24E-07 TRQ1 H004-E22Hh//-Hi-Kn01 OG3/LOOOO-kO 1.35E-07 TR01 H067-E22Hb/pope-N|-Kl01 OG3fl_0222-kO 7.63E-08 rCE115H-E22HM-HE-KriO1OG3/rCE115L-kO 1.38E-07 TRD1 H008-E22Hh/none-Hi-Kn01 OG3A_OQOO-kO 4.22E-08 TR01 HO70-E22HMnora-Hi-Kn01 OG3/LOOOO-kO 1.20E-07 TR01 H106-E22Hh/none-Hi-Kn01 OG3A0011-kO 1.00E-07 TR01 H024-E22Hh/mns-Hi-Kn01 OG3/LOOOO-kO 1.OSE-07 CE115HA124-E22HW/-Hi-Kn010G3/L0000-k0 1.43E-07 TR01 H040-E22Hh/none-Hi-Kn010G3/LO249-k0 1.11E-07 TR01 H082-E22Hh/none-Hi-KnD10G3/LO271-k0 8.82E-08 TR01 H057-E22Hh/non&-Hi-Kn01 OG37LOOOO-kO 1.12E-07 TRD1 HO58-E22Hh/mne-Hi-KnO1 OG3/LOO<JO-kO 1.15E-07 TR01 H068-E22Hh/none-Hi-Kn01 OG3/LOOOO-kO 1.01E-07 TR01 H082-E22Hh/none-Hi-Kn01 OG3/L0270-kO 7.42E-08 TRC1 H082-E22Hh/none-Hi-Kn01 OG3/L0272-kO 7.44E-08 hCE115HA-E22Hh/none-Hi-Kn010G3/L0000-k0 1.24E-07 TRC1 H082-E22Hh/none-Hi-Kn01 OG3/L026&-kO 1.36E-07 hCE115HAa-E22Hh/none-Hi-KnC1CG3/L0000-k0 1.08E-07 TR01 H067-E22Hh/norre-Hi-Kn01 OG3A0226-kO 1.32E-07 TR01 H067-E22Hh/none-Hi-Kn010G3A024B-k0 1.39E-07

(3-2) Modification of the affinity of a humanized anti-GPC3 antibody by point mutations.

First, point mutations were introduced into the sequence of CDR1, CDR2 and CDR3 of the bispecific antibody prepared according to the method described in Example 2, H0000-ERY27HK (SEQ ID NO: 54), to obtain modified antibodies. The affinity of these modified antibodies to soluble human GPC3 was then determined. By combining sites that have the ability to increase the affinity of the modified antibodies, modified antibodies were obtained, the affinities of which are presented in table. 9.

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Table 9

Name of the antibody KD (human GPC3) H0610-G1dh/none-Hi-Kn010G3/L0000-k0 3&7E-09 H0610-G1dh/none-Hi-Kn010G3/L0222k0 1.40E-13 H0610-G1dh/none-Hi-Kn010G3/L0259-k0 3.52E-13 GCH054-G1dh/none-Hi-Kn010G3/L0262-k0 5.25E-13 GCH060-G1dh/none-Hi-Kn010G3/L0222-k0 6.42E-13 H06W-Gldh/none-Hi-Kn010G3/L0246-k0 1.21E-12 GCH0&7-Gldh/none-Hi^n0l0G3/L0222-k0 1.B5E-12 GCH054-G1dh/nons-Hi-Kn010G3/L0249-k0 3.31 EC 2 GCH055-G1dh/none-Hi-Kn010G3/L0222-k0 3.90E-12 GCH094-G1dh/none-Hi-Kn010G3/L0248-k0 4.12E-12 H0610-G1dh/none-Hi-Kn010G3/L0249-k0 6.86E-12 H0610-G1dh/none-Hi-Kn010G3HR01L017-k0 8.27E-12 H0610-G1 dh/none-Hi-Kn010GM0265-k0 8.70E-12 H06l0-G1d№one-Hi-Kn010G3A026l-k0 1.07E-11 GCH065-G1dh/none-Hi-Kn010G3/L0262-k0 1.18E-11 GCH056-G1dh/none-Hi-Kn010G3iL0262-kD 1.19E-11 H0610-G1dh/none-Hi-Kn0WG3/L026a-k0 1.69E-11 H0610-G1dh/none-Hi-Kn010G3nR01L020-k0 2.24E-11 GCH0M-G1dh/none-Hi-Kn010G3/L024S-k0 3.15ΕΊ1 SSN054-S1yb/pope-NcKp010yZ/Yu222-k0 3.15ΕΊ1 GCH073-G1dh/none-Hi-Kn0l0G3/L020l-k0 3.50E-11 H0610-G1dh/none-HhKn010G3/L0248k0 5.55E-11 GCH065-G1dh/none-Hi-Kn010G3/L020l-k0 7.74E-11 H0610-G1dh/none-Hi-Kn010G3/L022S-k0 9.ZOE-11 H0610-G1dh/none-Hi-Kn010G3/L0093-k0 1.06E-10 GCH098-G1dh/none-Hi-Kn010G3)L0201-k0 1.11E-10 H0610-G1dh/non&-Hi-Kn010G3/LC267-k0 179E-10 H0610-G1dh/none-Hi-Kn010G3/L0228-k0 2.O2E-10 H0610-G1dh/none-Hi-Kn010G3/LD262-k0 2.11E-Yu H0610-G1dh/none-Hi-Kn010G3/L0266-k0 2.13E-10 H0619-G1dh/none-Hi-Kn010G3/L0264-k0 2.19E-10 HO6 W-G1 dh/none-Hi-Kn010G3/L0224-k0 2.43E-10 H0610'Gldh/noneHi-Kn010G3/L0167-k0 2.11E-10 CE115HA251-E22Hh/LOOOO-kO/GL4’E22Hk/HD6lO-E22L 2.36EL0 TROlHOl5-E22Hh/LOOOOAO/GL4-E22Hk/H06lO-E22L 2.S3E-10 CE116HA236-E22Hh/GLS3l 0&-kO/GL4-E22Hk/H0610-E22L 2.67E-10 H0610-G1dh/none-Hi-Kn010G3/L0259-k0 3.34EL0 H0610-G1dh/none-Hi-Kn010G3/L0227-k0 4.08E-1O GCH065-G1dh/none-Hi-Kn010G3/L0272-k0 3.93E-10 H0610-G1dh/none-Hi-Kn010G3/L0269-k0 4.59E-10 HQ610-Gldh/none-Hi-Kn010G3/L0223-k0 4.75E-10 TR01 HC02-E22Hh/GLS3108-kC/GL4-E22Hk/H0610-E22L 475E-S GCH054-G1 dh/none-Hi-Kn010G3/LC212-k0 5.17E-S H0610-G1dh/none-Hi-Kn010G3/L0208-k0 5.30E-10 H0610-G1db/none-Hi-Kn010G3/L0263-k0 5.84E-10 H0610-G1 dh/none-Hi-Kn010G3/L0231-k0 5.89E-10 H0610-G1dh/none-Hi-Kn010G3/L0143-k0 5.73E-10 GCH055-G1dh/Wie-Hi-KnO1OG3iLO212-k0 6.14E-10 H0610-Gidh/none-Hi-Kn0i0G3/L0211-k0 6.47E-10 H0610-G1dh/none-Hi-Kn010G3/LQ238-kO 6.37E-10 H0610-G1dh/none-Hi-Kn010G3/L0214-k0 6.57E-10 H0610-G1dh/none-Hi-Kn010G3/L0243-kD 6.49E-10 GCH025-G1dh/none-Hi-Kn010G3/L0204-k0 6.70E-10 GCH054-G1 dh/none-Hi-Kn010G3HR01 L016-kO 7.63E-10 H0610-G1dh/none-Hi-Kn010G3/L016S-k0 6.99E-10 GCH094-G1db/none-Hi-Kn0l0G3/L0271-k0 6.92E-10 GCH054-G1 dh/none-Hi-KnOIOGO/TROI L019-k0 8.71 ELO H0610-G1db/nons-Hi-Kn010G3/L0234-k0 7.78E-10 GCH098-G1 dh/none-Hi-Kn010G3/L0011-k0 8.O2E-10 H0610-G1dh/none-Hi-Kn010G3/L0204-k0 7.27E-10 H0610-G1dh/none-Hi-Kn010G3/L0240-k0 8.48E-10 H3610-G1dh/none-Hi-Kn010G3/L0239-k0 8.74E-10 H0610-G1dh/none-Hi-Kn010G3/L0212-k0 9.94E-10 GCH065-G1 dh/none-Hi-Kn010G3/L0011-kO 8.S4E-10 H0610-G1dh/none-Hi-Kn010G3/L0200-k0 1.O4E-O9 H0610-G1dh/none-Hi-Kn010G3/L0124-k0 9.72E-10 GCH073-G1 dh/none-Hi-Kn010G3/L0011-kO 9.10E-10 H0610-G1dh/none-Hi-Kn010G3HR01L016-k0 1.08E-O9 GCH054-Gldh/none-Hi-Kn010G3/L0201-k0 1.08ΕΌ9 H06l0-Gldh/none-Hi-Kn0l0G3/L0090-k0 1.12E-09 N9610-S^Mpope-NnKp010SZA0209-c0 1.12E-09 H0610-G1dh/none-Hi-Kn010G3/L0201-k0 1.1 SE-09 H0610-G1dh/none-Hi-Kn010G3/L0161-k0 9.73Е-10 H0610-G1dh/none-Hi-Kn010G3/L0206-k0 8.65Е-10 H06l0-Gldh/none-Hi-Kn010G3/L0186-k0 1Ό8Ε-09 H06l0-G1db/none-Hi-KnO10G3/TR01L019-kQ 1.15E-09 H0610-G1dh/none-Hi-Kn010G3/L0085-k0 1.17E-09 GCH055-G1dh/none-Hi-Kn010G3/L0200-kD 1.13E-09 H0ei0-G1dh/none-Hi-Kn010G3/L0154-k0 1.01Е-09 H0610-G1dh/none'Hi-Kn010G3/L0229-k0 1.2OE-09 GCH054-G1dh/none-Hi-Kn010G3/L0200-k0 1.18E-09 GCH094-Gldh/none-Hi-Kn010G3/L0201-k0 1.17E-09

- 61 041830

H0610-G1dh/none-Hi-Kn010G3/L000C^0 3.97ΕΌ9 H0610-G1dh/none-Hi-Kn010G3/L0205-k0 1.01E-09 GCHC99-G1dh/ncne-Hi-KnCHOG3/LO2O1-kQ 1.29E-0E H0610-G1dh/none-Hi-Kn010G3/L0242-k0 1.19E-09 GCH056-G1dh/none-Hi-KnGlOG3/L0201-kO 1 16E-09 H0610-G1dh/none-Hi-Kr010G3/L0213-k0 1.25E-09 GCH060-G1dh/none-Hi-Kn010G3/L0200-kO 1.34E-09 GOH065-G1dh/none-Hi-Kn010G3/L0000-k0 1 41E-O9 GCHWO-G1dh/ncne-Hi-KnD1OG3/LO201-kO 1.37E-09 H0310-G1dhinone-Hi‘Kn010G3/L00154<0 1.31 E-09 HQ610-G1dh/none-Hi-Kn010G3/L0151-k0 1.25E-09 H0S10-G1dh/none-Hi-Kn010G3/L0237-k0 1.31 E-09 H0Sl0-Gldhinone-Hi-Kn0lOG3/LO220-k0 1.36E-09 H0610-G1dhinone-Hi-Kn010G3/L0155-k0 1.28E-09 GCH055-G1dhinone-Hi-Kn010G3/L0215-kO 1.52E-09 H0B10-Gldh/none-Hi-Kn01OG3/LO202-k0 1.22E-09 GCH056-G1dh/none-Hi-Kn010G3/L0215-kO 1.59E-09 H0510-G1dh/none-Hi-Kn010G3/L0012-k0 1.55E-O9 GCH054-G1dhincne-HkKnD10G3/L0215-kO 1.62E-09 H0610-G1dh/none-Hi-Kn010G3/L0215-k0 1.84E-09 GCHC98-G1dhrnone-HkKnOlOG3/LOOOO-kO 1.77E-09 H0610-G1dh/none-Hi-Kn010G3/L0125-k0 1.71 E-09 GCH057-G1dh/non©-Hi-Kn010G3/L0215-k0 1.83E-09 HQ610-G1dh/none-Hi-Kii010G3/L0217-k0 1.79E-09 H0610-G1dh/none-Hi-Kn010G3/L0014-k0 1.82E-09 HO610-Gldh/none-Hi-Kn0l0G3/L0216-k0 1.86E-O9 TR01HQ15-E2702GsKsc/GCHO19-E2704sEpSC/LO0OQ-k0 1.64E-09 H0610-G1dhinone-Hi-Kn010G3/TR01L015-k0 2.16E-09 H06l0-Gldh/none-Hi-Kn0l0G3/TRO1L0l8-k(> 2.17E-09 H0610-G1dh/none-Hi-Kn010G3/L0218-k0 1.99E-09 HO610-G1dh/none-Hi-Kn010G3/L0000vk1-k0 2.16E-09 H0B10'G1dh/none-Hi-Kn010G3/L0160'k0 2.12E-09 H0610-G1dh/none-Hi-Kn010G3/L0047-k0 2.23E-09 GCH073-G1dh/ncne-HkKnO1OG3/L0OD0-kO 2.00E-09 GCH054-G1dh/ncne-Hi-Kn010G3/TRC1L015-kO 2.45E-09 H0610-G1dh/none-Hi-Kr010G3/L0219-k0 2.28E-09 GCHQ94-G1dh/none-Hi-KnO1OG3/LO272-kO 2.YUE-09 H0610-G1dh/none-Hi-Kn010G3/L0149-K0 2.16E-09 GCHO54-Gldh/none-HkKn010G3/TR0lL018*k0 2.59E-09 GCHC54-G1dh/none-Hi-Kn010G3/L0203-kC 2.48E-09 H0B10-G1dh/none-Hi-Kn010G3/L0122-k0 2.42E-09 HOBlO-Gldh/none-Hi-KnOlOG3/LOl34-kO 2.53E-09 H0610-G1dh/none-Hi-Kn010G3/L0152-k0 2.36E-09 HO610G1dh/none-Hi-Kn010G3/L0203-k0 2.11E-09 HO610-G1dh/none-Hi-Kr010G3/L0075-K0 2.85E-09 H0B10-G1dh/none-Hi-Kn010G3/L0038-k0 2.75E-09 H0Sl0-Gldh/none-Hi-Kn0lOG3/L0011-k0 2.76E-09 H0610-G1dh/none-Hi-Kn010G3/L0157-K0 2.60E-09 H0610-G1dhinone-Hi-Kn010G3/L0145-k0 2.66E-09 HO610-Gldh/norie-Hi-Kr010G3/TR01LOl0-kC 2.92E-09 H0B10-G1dh/none-Hi-Kn010G3/L0009-k0 2.99E-09 GCH099-G1dh/none-Hi-Kn010G3/L0011-kD 2.78E-09 H0B10-G1dh/none-Hi-Kn010G3/L0006-K0 3.04E-09 H0610-G1dhinone-Hi-Kn010G3/L0173-k0 2.83E-09 H0810-G1dh/none-Hi-KnQ10G3/L0127-kO 3.12E-09 HO610-G1dhinone-Hi-Kr01QG3/L0082A0 3.43E-09 H0S10-Gldhinone-Hi-Kn010G3/L0064-k0 E.37E-09 H0B10-G1dhinone-Hi4<n010G3/L0008-k0 3.30E-09 H0610-G1dh/none-Hi-Kn010G3/L0013-kD 3.35E-09 H0610-Gldh/none-Hi-Kn010G3/L0140-kO 3.38E-09 H0610-G1dh/none-Hi-Kn010G3/L0039-k0 3.41 E-09 GCHO43-G1dhinone-Hi-Kn010G3/L0O00-kO 3.74E-09 H0610'G1dh/none'Hi'Kr010G3/TR01LOQ8'k(} 3.48ΕΌ9 H0B10-G1dh/none-Hi-Kn010G3/L01484;0 3.28E-09 GOH062-G1dh/none-HkKn010G3/L0000-k0 3.73E-09 H0610-G1dh/none-Hi-Kr010G3/L0163-kD 3.38ΕΌ9 H0610-G1dh/none-Hi-Kn010G3/L0233-k0 3.55E-09 HQ610-G1dh/none-Hi-Kn010G3/L.O230-k0 400E-09 GCH006-G1dh/ncne-Hf-KnD10G3/L0030-kQ 4.06ΕΌ9 H0610-G1dh/none-Hi*Kn010G3/L0032-k0 3.72E-09 H0B10-G1dh/none-Hi-Kn010G3/L0181-k0 3.51 E-09 HO610-G1dh/none-Hi-Kn010G3/TR01L009-k0 3.81 E-09 H06l0-Gldhinone-Hi*Kn010G3/L0l4l-k0 3.86E-09 H0610-G1dhinone-Hi-Kn010G3/L0079-k0 4.23E-09 GCH094-G1dh/nono-Hi-Kn010G3/L0270-k0 3.60E-09 GCH066-Gldh/ncne-Hi-KnO1OG3/L0O00-kC 4.2SE-09 GCHO64-G1dh/ncne-Hi-KnO1OG3/LOO00-kO 4.14E-09 H0B10-G1dh/none-Hi-Kn010G3/L00S6-k0 4.20E-09 GCH027'G1dh/noneHkKnD10G3/LOOQO'kO 3.83E-09 H0610-G1dh/none-Hi-Kn010G3/L0003-k0 4.01 E-09 H05l0-G1dMnone-Hi-Kfi010G3/LQ042-k0 427E-09 H0610-G1dh/none-Hi-Kn010G3/TRD1L011-kO 4.02E-09

- 62 041830

H0610-G1dh/none-Hf-Kn010G3/L0000-k0 3.97E-09 GCH015-G1dh/none-Hi-Kn010G3/L0000-k0 4 14E-09 H0610-G1dh/none-H(-KnC10G3/L0175-kC 3.84Е-09 GCH100-G1 dh/none-Hi-Kn010G3/LO011-kC 3.81 E-09 GCH014-G1dh/none-Hi-Kn010G3/L00D0-k0 4.20E-09 GCH053-G1dh/none-Hi-Kn010G3/L0000-k0 4.05Е-09 hCE115HA-E22Hh/LOOQO-kO/GL4-E22Hk/HOOOO-E22L 428E-09 GCH094-G1dh/none-H-Kn010G3/L0G11-kC 3.8ΘΕ-09 GCH045-Gldh/none-Hi-Kn0l0G3/L0000-k0 4.63E-09 H0Sl0-GldWnone4TKnCl0G3^R01L012di0 4.25ED9 H0610-G1dh/nona-Hi-Kn010G3/L0115-k0 4.34Е-09 H0610-G1dh/none-H>Kn010G3/L0044-k0 4.57E-09 H0610-G1dh/none-Hi-Kn010G3/L0107-k0 438E-09 H0510-G1dh/none-Hf-KnC10G3/LC007-k0 4.39E-09 GCH0l3-Gldh/none-HkKn0l0G3/L0000-kC 4 44E-09 H0610-G1dh/none4d^Kn010G3/L0045-k0 4 66E-09 GCH010-G1 dh/none-Hi-Kn010G3/L0000-k0 4.12E-09 GCH04O-G1 dh/none-Hi-KnOI 0G3/L0G00-kG 4Θ0Ε-09 H0S10-G1dh/none-HFKn010G3/L0G02-k0 443ΕΌ9 H06W-Gldh/none^H^KnG10G3/LGG16-kO 4.44Е-09 GCH3O7Gldh/noneHKnClOG3/LOCOO0O 4.93E-09 GCH042-G1dh/none4d^n010G3/L0000-k0 4 89E-09 rCE115H-E22Hh/rCE115L-kO/GL4-E22Hk/HOOOO-E22L 4.57E-09 H0610-G1dh/none-Hi-Kn010G3/L0120-k0 4.54Е-09 H0610-G1dh/none-Hf-Kn010G3/L0065-k0 4.79E-09 GCH016-G1 dh/none-Hi-KnO 10G3/L0CQ0-kC 4.59Е-09 GCH035 G1dh/noneHKn0l0G3/L0G00kC 4.94Е-09 GCH039-G1dh/none-Hi-Kn010G3/L0000-k0 4.95E-09 GCH099-G1dh/none-Hi-Kn010G3/L0000-k0 424E-09 H0610-G1dh/non^H^Kn010G3/L0041-k0 4.85E-09 GCH019-G1dh/none-HI-Kn010G3/L0000-k0 4.36Е-09 GCH029-G1dh/none-Hi-Kn010G3/L0O00-k0 501E-09 GCH056-G1dh/none-Hi-Kn010G3/L0011-k0 4.31 E-09 H061D-G1 dh/nona-Ht-Kn010G3/L0147-k0 4.38E-09 GCH034-G1dh/none-Hi-Kn010G3/L0000-k0 5.O9E-09 GCH0O3-G1 dh/none-H-KnC 10G3/L0G00-kG 520E-09 HOSLO-Gldh/none-Hs-KnOlOGa/LOiag-kC 4 78E-09 H0610-Gldh/none-HF-Kn010G3/L0C89-kC 524E'O9 H0610-G1dh/nona-Hi-Kn010G3/L0113-kO 4.82E-09 H0610-G1dh/none-Hi-Kn010G3/L0180-k0 44ΘΕ-09 GCH005-G1dh/none^HKn010G3/L0000-k0 532E-09 GCH067-G1dh/none-HI-KnC10G3/L0000-K0 524ΕΌ9 H06i0-G1dh/none-Hr-Kn0l0G3/LCl87-k0 4 92E-09 H0610-G1dh/none-H^Kn010G3/L0043-k0 5.14Е-09 H0610-G1dh/nona-Ht-Kn010G3/L0117-kG 4.92E-09 GCH061-G1dh/none-Hf-Kn010G3/L0000-k0 5.1 SE-09 GCH022-G1d h/none-H-KnC 1OG3/LOGOO0O 4.92ΕΌ9 N06Yu-b1yG/pope-NnKp010yZ/1.S091-k0 5.43E-09 GCH023-G1dh/none-Ht-Kn0l0G3/L0000-k0 4.94Е-09 H0610-G1dh/none-HFKn010G3/L0G62~k0 5.28E-09 H0810-G1dh/nona-Hf-Kn010G3/L0136-k0 5.04Е-09 H0610-G1 dh/none-Ηί-ΚηΟ 10G3/TR01 L003-k0 5O8E-09 H0610-G1dh/none-Hi-Kn010G3/L0069-k0 532E-09 N0610-SSH/POPE-NnKp010SZ/10123-k0 5 08E-09 GCH025-G1 dh/none-HrKnO 10G3/L0000‘k0 5.O5E-09 GCH100-G1dh/nona-Hj-Kn010G3/L0000*k0 539E-09 H0810-G1dh/none-Hi-Kn010G3/LG046-k0 545E-09 H0610-G1dh/none-HF-KnC10G3/L0144-k0 4.84Е-09 GCH026-Gldh/none-Hi-Kn010G3/L0000-k0 5.17E-09 HO6W-Gidh/none-Ht-KnCiOG3/LOi30-kO 5 24E-09 GCHO56-G1dh/npne-Hi-KnC1OG3/L0GO0-kO 5Q3E-09 H061D-G1dh/none-Hi-Kn010G3/L0129-k0 5.2SE-09 GCH032-G1dh/none'Hi-Kn010G3/L0000-k0 5.74Е-09 HOBTO-Gldh/none-HLKnCIOGa/TROILOOS-kG 537ΕΌ9 GCH012-G1 dh/none-Hi-KnO l0G3/L0G00-kG &.40E-09 GCH055-G1dh/none-Hi-Kn010G3/L0000-kG 5 63E09 H06i0-G1dh/nona4-h-Kn010G3/L0104-k0 590E-09 GCH059-G1dh/nona-Hi-Kn010G3/L0000-k0 5.70E-09 GCH054-G1 dh/none-Hi-ΚπΟ 1CG3/LOOOO-kO 530E-09 GCH0G8-G1dh/none-Hi-Kn010G3/L0000-kC 555E-09 H06l0-G1dh/none-Hi-KnCl0G3/LC232-k0 5.33E-09 H0610-G1dh/none-Hi-KnC10G3/L0126-k0 5.62E-09 GCH0S4-G1dh/none-Hi-Kn010G3/L0000-k0 5.89E-09 H0610-G1dh/none-H4Kn010G3/L0132-k0 5.65E-09 H0S1D-G1dWnone^Ht-KnC10G3/LC106-kG 56SE-09 GCH054-G1dh/none-Hi-Kn0l0G3/L0011-k0 5.25E-09 N0610-SSh/pope-NnKp010SZ/1X109-cS 5.70E-09 H0810-G1 dh/nona-Hr-Kn010G3/L0083-k0 6OSE-09 GCH068-G1dh/nona-Hi-Kn010G3/L0000-k0 623E-09 GCH057-G1dh/none-Hi-Kn010G3/L0000-k0 5.61E-09 H0610-G1 dh/none-HnKp010G3/L0137-kO____________________________________ (3-3) Modification of pI with point mutations. 587E-09_________ In the commercial production of bispecific antibodies a high level of purity is required. When using ion-exchange chromatography, the effective is a modification of the isoelectric point (pI) of molecules (PLoS One. 2013;8(2):e57479). For this reason, mutations have been introduced for pI modifications to the sequence of CDR1, CDR2 and CDR3 of a humanized anti-human antibody GPC3 obtained 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 to soluble human GPC3 was then determined. As a result, it was found that amino acid modifications, which can lead to a decrease in

pI reduction while maintaining affinity for human GPC3 should be subjected to amino acids at positions 19, 43, 53 and 61 according to Cabot numbering.

Combining sites that have the ability to maintain affinity for human GPC3 and reduce pI, antibodies were obtained, affinities and pI of which are presented in table. 10.

- 63 041830

Table 10

Antibody name Calc. PI value Antibody name KD human. GPC3 (homomeric antibody) (homomeric antibody (single chain antibody) (single chain antibody)

Mutation sites based on H0610-E2704sEpsc

H0610-E27D4sEp5C/L00OD-k0 7.8 HO61(Wdh/non^Hi4<naiOG3/LO# 4.16E-09

GCH054-E2704sEpsc/LCD11-k0 6.2 GCH054-G1dh/none-Hi-Kn010G3/L0011-k0 5.25E-09 K19T/O43E/P52aG/K53E/G55P/Q61E

GCH065-E2704sEpsc/LOQ11 -kO 6.4 GCH06&G1dh/n _Q ne-Hi-KnO1 QG3/L0011-kO 8.84E-10 K19T/Q43E/P52aG/K53P/G55P/Q61 E

GCH094432704sEpsc^0114cO 6 _; 2 GCH094 _; G1d!Vn^^ (3-4) Modification of the ability to bind the extracellular matrix using a point mutation.

It is known that non-specific binding to the extracellular matrix (ECM) and the like. may influence pharmacokinetics (MAbs. 4(6), Nov-Dec 2012, pp. 753-760). Thus, the ECM-binding capacity of the modified antibodies obtained according to the methods described in the Examples was determined using the method shown in Reference Example 4. As a result, it was confirmed that the humanized anti-human CD3ε chain and anti-human GPC3 bispecific antibody, GPC3_ERY27_hCE115 (SEQ ID NO : 54, 55 and 56) had a high ECM binding capacity. In this way, any of the point mutations assessed in Examples 3-1, 3-2 and 3-3 in the sequence of the humanized anti-human CD3ε chain antibody hCE115HAERY27HE (SEQ ID NO: 55) were studied as a combination to reduce ECM binding capacity. As a result, it was found that the amino acids at positions 11, 16, 52a, 53, 98 and 100 according to Cabot numbering are involved in maintaining affinity for CD3ε and can affect the decrease in ECM-binding ability, and antibodies with reduced ECM-binding ability compared to the ability to humanized bispecific antibody variant to human CD3ε chain and to human GPC3, GPC3_ERY27_hCE115 (Table 11).

Table 11

Name of the antibody ECM binding ratio (standard = 1) GPC3 ERY22 CE115 (rCE115H-E22Hh/rCE115L-k0/GL4-E22Hk/H0000-E22L) 4.0 GPC3 ERY27 (hCE115HA-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L) 50.9 CE 115HA236-E22 Hh/G LS3108-k0/G L4-E22Hk/H0610-E 22L 429.9 CE 115HA236-E22 Hh/G LS3108-k0/G L4-E22Hk/H0000-E 22L 414.8 CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 346.9 CE 115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0610-E22L 334.4 TR01 H002-E22Hh/GLS3108-k0/GL4-E22Hk/H0610-E22L 301.1 TR01H002-E22H h/GLS 3108-k0/GL4-E22 Hk/H0000-E22 L 216.9 TR01H015-E22H h/L0000-k0/GL4-E22Hk/ H0610-E22L 185.7 TR01H040-E2702GS Ksc/H0610-E2704S E ps c/L0208-k0 50.4 CE115HA122-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L 47.0 TR01H040-E2702GS Ksc/H0610-E2704S E ps C/L0211-kO 15.5 TR01H040-E2702Gs Ksc/H0610-E2704s E ps c/L0206-k0 15.4 TR01H040-E2702Gs Ksc/H0610-E2704S E ps c/L0209-k0 7.4 rCE 115H-E22H h/rCE 115L-k0/GL4-E22Hk/ H0610-E 22L 4.6 TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0204-k0 4.4 TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0212-k0 3.3 TR01H113-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 2.5 TR01 H082-E2702GsKsc/GCH065-E2704sEpsc/L0011-kO 1.7 TR01H113-E2702GsKsc/GCH094-E2704sEpsc/L0011-kO 1.6 rCE 115H-E22Hh/rCE 115L-k0/L0000-E22Hk/H0610-E22L 1.4 TR01 H084-E2702GsKsc/GCH065-E2704sEpsc/L0011-kO 1.3 TR01 H084-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 1.2 TR01H082-E2702GsKsc/GCH094-E2704sEpsc/L0201-k0 1.1 TR01H040-E2702GS Ksc/H0610-E2704S E ps c/LOOOO-kO 0.8 TR01H040-E2702GS Ksc/H0610-E2704S E ps C/L0201-kO 0.8 TR01H040-E2702GS Ksc/H0610-E2704S E ps c/L0203-k0 0.8 TR01 H082-E2702GsKsc/GCH094-E2704sEpsc/L0011-kO 0.7 TR01H109-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 0.7 TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0222-k0 0.6 TR01H067-E2702GsKsc/GCH054-E2704sEpsc/L0201-k0 0.5 TR01 H109-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0 0.4 TR01H113-E2702S Ksc/GCH065-E2704s E psc/L0011-kO 0.3 MRAN-S1s1/MRAE-k01(standard) 1

(3-5) Modifications in the ability to bind to the SuRe ligand by point mutations.

An example is known 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). When purified on protein A humanized bispecific antibodies to human CD3ε chain and to human GPC3, the removal of homomeric anti-CD3 antibody was important to suppress non-specific reactions by CD3. Thus, it seems desirable to suppress the binding of a homomeric anti-CD3 antibody to protein A.

It is likely that the SuRe™ ligand can be used commercially and therefore point mutations affecting binding to the SuRe™ ligand have been introduced into the CDR2 H chain of the humanized anti-CD3 antibody variants, TR01H082-E2702GsKsc and TR01H084-E2702GsKsc (SEQ ID NOS: 398 and 399) , to obtain modified antibodies. The binding capacity of these modified antibodies to the SuRe™ ligand was determined using the method presented in reference Example 5. As a result, it was found that the amino acids at positions 19, 57 and 59 according to Cabot numbering are involved in maintaining affinity for CD3ε and may affect the ability bind to the Sure™ ligand, and received antibodies with a reduced ability to bind to the Sure ligand by

- 64 041830 compared with the ability of antibodies TR01H082-E2702GsKsc/L0011-k0 (SEQ ID NOS: 398 and 410) or

TR01H084-E2702GsKsc/L0011-k0 (SEQ ID NOS: 399 and 410) (Table 12).

Table 12

Name of the antibody Bonding SuRe™ (RU) Binding sites based on CE115HA000 TR01H084-E2702GsKsc/L0011-k0 5065.8 R16G/A52aD/N53Q/D72A/L78l/G98A/Y100G/A102l TR01H082-E2702GsKsc/L0011-k0 4469.2 V11 UA52aD/N53Q/G98A/Y 100G TR01H090-E2702GsKsc/L0011-k0 3606.3 V11L/ R16G/A52aD/N53Q/G98A/Y 100G TR01H093-E2702GsKsc/L0011-k0 2459.7 V11 L/A52aD/N53Q/K64Q/G98A/Y 100G TR01H094-E2702GsKsc/L0011-k0 2351.9 V11 L/A52aD/N53Q/K64S/G98A/Y 100G TR01H114-E2702GS Ks C/L0011-k0 1485.5 R16G/A52aD/N53Q/T57S/D72A/L78l/G98A/Y100G/A102l TR01H092-E2702GsKsc/L0011-k0 1159.5 V11L/A52aD/N53Q/K64A/G98A/Y100G TR01H100-E2702Gs Ks c/L0011-kO 383.0 V11 L/A52aD/N53Q/T57S/G98A/Y 100G TR01H111-E2702Gs Ks c/L0011-kO 50.7 R16G/R19K/A52aD/N53Q/D72A/L78l/G98A/Y100G/A102l TR01H110-E2702GS Ks C/L0011-kO 29.5 R19K/A52aD/N53Q/G98A/Y100G TR01H091-E2702GsKsc/L0011-k0 27.5 V11L/R19K/A52aD/N53Q/G98A/Y100G TR01H091-E2702GS Ks C/L0011-kO 15.0 V11L/R19K/A52aD/N53Q/G98A/Y100G TR01H112-E2702GS Ks c/LOO11 -kO 8.8 R16G/A52aD/N53Q/T57Q/D72A/L78l/G98A/Y100G/A102l TR01H113-E2702GS Ks C/L0011-kO 7.0 R16G/A52a D/N53Q/Y59V/D72A/L78I/G98A/Y100G/A1021 TR01H096-E2702GsKsc/L0011-k0 2.7 V11L/A52aD/N53Q/T57G/G98A/Y100G TR01H109-E2702GS Ks C/L0011-kO 2.2 V11L/A52aD/N53Q/Y59V/G98A/Y100G TR01H098-E2702GsKsc/L0011-k0 1.6 V11 L/A52aD/N53Q/T57P/G98A/Y 100G TR01H107-E2702GSKSC/L0011-k0 1.4 V11 L/A52aD/N53Q/Y59Q/G98A/Y 100G TR01H103-E2702Gs Ks c/L0011-kO 1.4 V11 L/A52aD/N53Q/Y59G/G98A/Y 100G TR01H104-E2702GS Ks C/L0011-kO 1.0 V11 UA52aD/N53Q/Y59l/G98A/Y100G TR01H105-E2702GS Ks C/L0011-kO 0.8 V11 L/A52aD/N53Q/Y59L/G98A/Y 100G TR01H099-E2702GsKsc/L0011-k0 0.6 V11L/A52aD/N53Q/T57Q/G98A/Y100G TR01H102-E2702Gs Ks c/L0011-kO 0.5 V11L/A52aD/N53Q/Y59F/G98A/Y100G TR01H101-E2702GS Ks C/L0011-kO 0.5 V11 L/A52aD/N53Q/T57 V/G98A/Y1 DOG TR01H108-E2702GS Ks C/L0011-kO 0.4 V11 L/A52aD/N53Q/Y59T/G98A/Y 100G TR01H097-E2702GsKsc/L0011-k0 0.1 V11L/A52aD/N53Q/T57UG98A/Y100G TR01H106-E2702Gs Ks c/L0011-kO 0.0 V11L/A52aD/N53Q/Y59P/G98A/Y100G TR01H095-E2702GsKsc/L0011-k0 -0.2 V11 UA52aD/N53Q/T57F/G98A/ Y100G

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

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

Table 13

Sample number in TDCC assay Antibody name

GPC3_ERY22_CE115 (rCE115H-E22Hh/rCE115L-k0/GL4-E22Hk/H0000-E22L)

GPC3_ERY27 (hCE115HA-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L)

CE115HA251-E22Hh/L0000-k0/GL4-E22Hk/H0000-E22L

CE115HA236-E22Hh/GLS3108-k0/GL4-E22Hk/H0000-E22L

TR01H002-E22Hh/GLS3108-k0/GL4-E22Hk/H0000-E22L

CE115HA122-E22Hh/LOOOO-kO/GL4-E22Hk/HOOOO-E22L rCE115H-E22Hh/rCE115L-kO/LO0OO-E22Hk/HO61O-E22L rCE115H-E22Hh/rCE115L-k0/GL4-E22Hk/H0610-E22L

TR01 HMO-E2702GsKsc/H0610-E2704sEpsc/L(X}00-k0

TR01 H04O-E2702GsKsc/H0610-E2704sEpsc/L0201-k0

TR01H040-E2702GsKsc/H0610-E2704sEpsc/L0203-k0

TR01 H04O-E2702GsKsc/H0610-E2704sEpsc/L0204-k0

TR01 H040-E2702GsKsc/H0610-E2704sEpsc/L0206-k0

TR01 H04O-E2702GsKsc/H0610-E2704sEpsc/L0208-k0

TR01 H040-E2702GsKsc/H0610-E2704sEpsc/L0209-k0

TR01 H040-E2702GsKsc/H0610-E2704sEpsc/L0211-kO rCEH5H-E2702GsKsc/H0610-E2704sEpsc/L0000-k0

TR01 H061-E2702GsKso/H0610-E2704sEpsc/L0000-k0

TR01 H068-E2702GsKsc/H0610-E2704sEpsc/L0O00-k0

TR01 H071-E2702GsKsc/H0610-E2704sEpsc/L0O00-k0

TR01 H067-E2702GsKsc/GCH054-E2704sEpsc/L0201-k0

TR01 H067-E2702GsKsc/GCH054-E2704sEpsc/L0212-k0

TR01 H067-E2702GsKsc/GCH054-E2704sEpsc/L0222-k0

TR01H067-E2702GsKsc/ GCH054-E2704sEpsc/L0000-k0

TR01 H082-E2702GsKsc/GCH094-E2704sEpsc/L0201-k0

TR01 H082-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0

TR01 H084-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0

TR01 H084-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0

TR01 H082-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0

TR01 H109-E2702GsKsc/GCH094-E2704sEpsc/L0011-k0

TR01 H109-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0

TR01H113-E2702GsKsc/GCH094-E2704sEpsc/L0011-kO

TR01H113-E2702GsKsc/GCH065-E2704sEpsc/L0011-k0 ______________38______________TR01H113-E2702sKsc/GCH065-E2704sEpsc/L0011-kQ____________________

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 higher T-cell dependent cellular cytotoxicity than GPC3_ERY22_rCE115 before it was humanized.

In antibodies to the human CD3ε chain, for example, Leu at position 11, Gly at position 16, Asp at position 52a, Gln at position 53, Ala at position 72, He at position 78, Ala at position 98, Gly at position 100 and Ile at position 102. Anti-human GPC3 antibodies have, 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, common L strands present, for example, Pro at position 25, Pro at position 27a, Pro at position 27b, Not at position 33, Gln at position 34, Arg or Trp at position 56, and Tyr at position 89 ( all positions are designated according to Cabot numbering).

Example 4 Evaluation of In Vivo Efficacy.

The efficacy of some of the antibodies described above was evaluated in vivo using tumor-bearing model animals.

Evaluation of in vivo efficiency was carried out for representative antibodies from those presented in table. 13, which demonstrated the presence of cytotoxic activity when assessed using the in vitro assay described in examples 3-6. When evaluating in vivo efficacy, any influence caused by differences in the microenvironment due to the formation of tumor aggregates on the results of the assessment 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 levels of GPC3 expression in these cells were almost the same. The cell lines were transplanted into severely combined immunodeficient (NOD scid) mice and NOD scid mice that were confirmed to have tumors were transplanted with T cells grown by in vitro culture of human PBMCs. Mice (designated as the T cell injected model) were treated with optimized bispecific antibodies to human CD3ε chain and human GPC3.

More specifically, in experiments evaluating the efficacy as drugs of optimized bispecific antibodies to human CD3ε chain and human GPC3, in which PC-10 cells were used in a model with injected T cells, the experiments described below were carried out. T cells were expanded by culturing using PBMCs isolated from the blood of healthy donors and a T cell activation/expansion kit/human (MACS Miltenyi biotec). The PC-10 human cancer cell line (1 x 10 ⁷ cells) was mixed with basement membrane matrix Matrigel (BD) and transplanted subcutaneously into the groin of NOD scid mice (CLEA Japan, 6 weeks old females). The day of transplantation was designated as day 0. Anti-asialoganglioside M1 (asialo-GM1) antibody (Wako Pure Chemical Industries) was intraperitoneally injected 1 day before transplantation at a rate of 0.2 mg/mouse. 1315 days after transplantation, mice were divided into groups depending on body weight and tumor size, and anti-asialo-GM1 antibody was again injected intraperitoneally at a rate of 0.2 mg/mouse. The next day, T cells obtained by the above-described culture propagation method were transplanted intraperitoneally at a rate of 3×10 ⁷ cells/mouse. 4 hours after transplantation of T cells, optimized bispecific antibodies to the human CD3ε chain and human GPC3 were injected intravenously through the caudal vein at a rate of 1 mg/kg. Optimized bispecific antibodies to human CD3ε chain and human GPC3 were administered only once.

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

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

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

Examples provided for reference.

Reference Example 1 Preparation of antibody expression vectors and expression and purification of antibodies.

Amino acid substitutions were introduced by methods known to those skilled in the art, eg using the QuikChange site-directed mutagenesis kit (Stratagene), PCR, or the In-fusion Advantage PCR cloning kit (TAKARA) to generate 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 with a spectrophotometer and the antibody concentration was calculated

- 66 041830 on the basis of determined values, applying the absorption coefficient calculated using the method

PACE (Protein Science 4, 1995, pp. 2411-2423).

Reference Example 2 Determination of the ADCC activity of each of the test antibodies using human peripheral blood mononuclear cells as effector cells The ADCC activity of each of the test antibodies was determined by the method described below.

Human peripheral blood mononuclear cells (hereinafter referred to as human PBMCs) 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.

Received samples of 50 ml of peripheral blood of healthy volunteers (adult men) using syringes pre-filled with 200 μl (1000 units/ml) of heparin solution (NovoHeparin 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), which were previously filled with 15 ml of Ficoll-Pak PLUS and centrifuged. After centrifugation of separation tubes containing peripheral blood aliquots at 2150 rpm for 10 minutes at room temperature, mononuclear cell fractions were collected. Cells in each fraction were washed once with 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 5x10 ⁶ cells/ ml with 10% FBS/D-MEM. The cell suspension was used as the target cell in the experiments below.

(2) Chromium release assay (ADCC activity).

ADCC activity was evaluated from the specific chromium release rate determined by the chromium release method. 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-bottomed plate at a rate of 50 μl per well. Target cells were then plated at 50 μl per well (1 x 10 ⁴ cells/well) and allowed to stand at room temperature for 15 minutes. A solution of human PBMC prepared according to the method described in subparagraph (1) was added at the rate of 100 μl per well (5x105 cells/well) and the plate was incubated in an atmosphere containing 5% carbon dioxide at 37°C for 4 hours and then centrifuged. The radioactivity in 100 μl of the culture supernatant in each well of the plate was measured using a gamma counter. The specific release rate of chromium was determined by the following formula:

specific chromium release rate (%)=(A-B)x100/(B-C), where A is the average radioactivity (cpm) of 100 μl culture supernatant in each well; B is the mean radioactivity (cpm) of 100 µl of culture supernatant in wells in which 100 µl of 2% NP-40 aqueous solution (Nonidet P-40, Nacalai Tesque) and 50 µl of 10%- FBS/D-MEM environment; and B is the mean radioactivity (cpm) of 100 μl of culture supernatant in wells in which 150 μl of 10% FBS/D-MEM medium was added to target cells. The experiments were carried out in triplicate, and from the results of the above experiment, the mean value and standard deviation of the specific chromium release rate (%), reflecting ADCC activity, were calculated for each of the tested antibodies.

Reference Example 3 Determination of T _m of modified antibodies by differential scanning fluorometry.

In this assessment, the T _m (heat denaturation temperature) of the modified antibodies was determined by differential scanning fluorometry using a Rotor-Gene Q device (QIAGEN). This method is known to provide a favorable correlation with the T _m 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).

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

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

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Reference Example 4. 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, high binding capacity)) 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(y) (Invitrogen, No. 628400) was labeled with ruthenium using an NHS ester labeled with MSD-SULFO (Meso Scale Discovery, No. R91AN-2) according to the instructions provided. 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.

To 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 /ml), and allowed to react for 1 h at room temperature with stirring in the dark.

The blocking ECL buffer was removed from the 96-well measurement 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. measurement (measuring plate) by inversion and immediately add 150 μl of 2xT buffer (4xMSD T-reading buffer (Meso Scale Discovery) 2-fold diluted with ECL dilution buffer), ECL measurements were performed. For measurements, a SECTOR Imager 2400 (Meso Scale Discovery) was used.

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

Reference Example 5 Determination of SuRe™ Ligand Binding Ability.

The ability to bind to the SuRe ligand was evaluated 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 bind the Mab Select SuRe™ ligand (GE Healthcare Japan) to the CM5 chip (carboxymethyl dextran coated chip) . The antibody used as analyte was prepared at a concentration of 5 μg/ml using HBS-EP+. To carry out measurements, a solution with an antibody concentration of 5 μg/ml was first injected with a flow rate of 10 μl/min for 3 min, then the analyte was replaced with HBS-EP+ and the response (RU) was evaluated after passing the flow for 0.5 min. After the measurements were completed, 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 carried out without covalent binding of the ligand to the chip, and the affinity for the SuRe™ ligand was analyzed based on the difference between responses (RU).

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

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Table 14

SEQ ID NO: title SEQ _ID MO: _Title SEQID NO ^; Name 1 nucleotide sequence of GPC3 (NP_991164617.1) 81 TROT H018 154 TR01H099 2 amino acid sequence of GPC3 (NP_991158089.1) 82 TROT H019 155 TR01H100 3 signal sequence 83 TR01H020 156 TR01H101 4 α-chain peptide of the T-cell receptor (САА 26636.1) 84 TR01H021 157 TR01H102 5 β-chain peptide of the T-cell receptor (С25777) 85 TR01H022 158 TR01H103 6 peptide of the γΐ chain of the T-cell receptor (А26659) 86 TR01H023 159 TR01H104 7 y2-chain peptide of the T-cell receptor (AAB633 12.1) 87 TR01H024 160 TR01H105 8 δ-chain peptide of the T-cell receptor (AAA61033.1) 88 TR01H025 161 TR01H106 9 CD3 γ-chain nucleotide (NM_000073.2) 89 TR01H026 162 TR01H107 10 nucleotide of the CD3 δ-chain (NM_000732.4) 90 TR01H027 163 TR01H108 eleven nucleotide of the CD3 ε-chain (NM_000733.3) 91 TR01H028 164 TR01H109 12 CD3 γ-chain peptide (NP_000064.1) 92 TR01H029 165 TR01H110 13 CD3 δ-chain peptide (NP_000723.1) 93 TR01H030 166 TR01H111 14 CD3 ε-chain peptide (NP_000724.1) 94 TR01H031 167 TR01H112 15-22 peptide linker 95 TR01H032 168 TR01H113 23 human cyl 96 TR01H033 169 TR01H114 24 human su2 97 TR01H034 170 GCH003 25 human SuZ 98 TR01H035 171 GCH005 26 human su4 99 TR01H036 172 GCH006 27 nucleotide FcyR! (NM_000566.3) 100 TR01H037 173 GCH007 28 FcyRI peptide (NM_000557.1) 101 TR01H038 174 GCH008 29 FcyRIIA nucleotide (BC020823.1) 102 TR01H039 175 GCH010 thirty FcyRIIA peptide (AAH20823.1) 103 TR01H040 176 GCH012 31 FcyRIIB nucleotide (BC146678.1) 104 TR01H041 177 GCH013 32 peptide FcyRIIB (AAI46679.1) 105 TR01H042 178 GCH014 33 nucleotide FcyRIIIA (BC033678.1) 106 TR01H043 179 GCH015 34 peptide FcyRIIIA (AAN033678.1) 107 TR01H044 180 GCH016 35 nucleotide FcyRIIIB (BC128562.1) 108 TR01H045 181 GCH019 36 peptide FcyRIIIB (AAI28563.1) 109 TR01H046 182 GCH022 37 Fc region (add. A to the N-terminus of RefSeq per. number AAC82527.1) 110 TR01H047 183 GCH023 38 Fc region (add. A to the N-terminus of RefSeq per. number AAB59393.1) 111 TR01H048 184 GCH025 39 Fc region (add. A to the N-terminus of RefSeq per. number AAB59394.1) 112 TR01H049 185 GCH026 40 NOOOOO, GPC3 H chain variable region 113 TR01H050 186 GCH027 41 GL4, GPC3 L chain variable region 114 TR01H051 187 GCH029 42 rSE115H, variable region of the CE115 H chain 115 TR01H052 188 GCH032 43 rCE115L, CE115 L chain variable region 116 TR01H053 189 GCH034 44 G1dh 117 TR01H054 190 GCH035 45 ERY22_Hk 118 TR01H055 191 GCH039 46 ERY22_Hh 119 TR01H056 192 GCH040 47 GL4-ERY22_Hk 120 TR01H057 193 GCHO42 48 H0000-ERY22L 121 TR01H058 194 GCHO43 49 rCE115H-ERY22_Hh 122 TR01H061 195 GCH045 50 rCE115L-ko 123 TR01H062 196 GCH053 51 hCE115HL (humanized CE115 heavy chain) 124 TR01H063 197 GCH054 52 hCE115HA (humanized CEllS heavy chain) 125 TR01H064 198 GCH055 53 L0000 (humanized CE115 light chain) 126 TR01H065 199 GCH056 54 H0000-ERY27_HK 127 TR01H066 200 GCH057 55 hCEl 15HA-ERY27_HE 128 TR01H067 201 GCH059 56 LOOOC-ko 129 TR01H068 202 GCH060 57 E22Hh 130 TRO1H069 203 GCH061 58 E22Hk 131 TR01H070 204 GCH062 59 Hi-Kn010G3 132 TR01H071 205 GCH064 60 E2702GsKsc 133 TR01H072 206 GCH065 61 E2704sEpsc 134 TR01H073 207 GCH066 62 E2702SKSC 135 TR01H074 208 GCH067 63 kO 136 TR01H075 209 GCH068 64 CE115HA177 137 TR01H076 210 GCH073 65 CE115HA178 138 TR01H077 211 GCH094 66 CE115HA179 139 TR01H079 212 GCH098 67 CE115HA180 140 TR01H080 213 GCH099 68 hCE115HAa 141 TR01H081 214 GCH100 69 TR01H006 142 TR01H082 215 H0610 70 TR01H007 143 TR01H083 216 LOOOOvkl 71 TR01H008 144 TR01H084 217 L0002 72 TR01H009 145 TR01H090 218 L0Q03 73 TR01H010 146 TR01H091 219 L0006 74 TR01H011 147 TR01H092 220 L0007 75 TR01H012 148 TR01H093 221 L0008 76 TR01H013 149 TR01H094 222 L0009 77 TR01H014 150 TR01H095 223 LO011 78 TR01H015 151 TR01H096 224 L0012 79 TR01H016 152 TR01H097 225 LO013 80 TR01H017 153 TR01H098 226 L0014

- 69 041830

SEQID NO: Name SEQID NO: Name SEQ ID 1® _{: name} 227 L0015 300 L0202 373 TR01L015 228 L0016 301 L0203 374 TR01L016 229 L0032 302 L0204 375 TR01L017 230 L0038 303 L0205 376 TR01L018 231 L0039 304 L0206 377 TR01L019 232 L0041 305 L0207 378 TR01L02O 233 L0042 306 L020B 379 TR01L023 234 L0043 307 L0209 380 TR01L024 235 L0O44 308 LO210 381 CE115HA122-E22Hh 236 L0045 309 L0211 382 CE115HA236-E22Hh 237 L0046 310 L0212 383 CE115HA251-E22Hh 238 L0047 311 L0213 384 GCH054-E2704sEpsc 239 L0062 312 L0214 385 GCH065-E2704sEpsc 240 L0O63 313 L0215 386 GCH094-E2704sEpsc 241 L0064 314 L0216 387 HQ610-E2704sEpsc 242 L0065 315 L0217 388 hCE115HA-E22Hh 243 L0066 316 L0218 389 rCE115H-E22Hh 244 L0069 317 L0219 390 r0E115H-E2702GsKsc 245 L0O75 318 LO220 391 TR01Η002Έ22ΗΊ 246 L0079 319 L0222 392 TR01H015-E22Hh 247 L0082 320 L0223 393 TR01H040-E2702GSKSC 248 L0085 321 L0224 394 TR01H061-E2702GsKsC 249 L0089 322 L0226 395 TR01 H067-E2702GsKsc 250 L0090 323 L0227 396 TR01 H06B-E2702GsKsc 251 L0091 324 L0228 397 TR01 H071-E2702GsKsc 252 L0093 325 L0229 398 TR01H082-E2702GSKSC 253 L0104 326 L023D 399 TR01 H034-E2702GsKsc 254 L0106 327 L0231 400 TR01 H109-E2702GsKsc 255 L0107 328 L0232 401 TR01H113-E2702GsKsc 256 L0109 329 L0233 402 TR01H113-E2702sKsc 257 L0113 330 L0234 403 GL4-E22Hk 258 L0115 331 L0235 404 LOOOO-E22Hk 259 L0117 332 L0236 405 H0000-E22L 260 L0120 333 L0237 406 H0610-E22L 261 L0122 334 L0238 407 rCE115L-ko 262 L0123 335 L0239 408 GLS3108-k0 263 L0124 336 L0240 409 LOOOO-ko 264 L0125 337 L0241 410 L0011-k0 265 L0126 338 L0242 411 L0201-k0 266 L0127 339 L0243 412 L0203-k0 267 L0129 340 L0246 413 L0204-k0 268 L0132 341 L0247 414 L0206-k0 269 L0134 342 L024B 415 L0208-k0 270 L0136 343 L0249 416 L0209-k0 271 L0137 344 L0250 417 L0211-k0 272 L0138 345 L0258 418 L0212-k0 273 L0139 346 L0259 419 L0222-k0 274 L0140 347 L0260 420 TR01H001 275 L0141 348 L0261 421 TR01H002 276 L0143 349 L0262 422 TR01H003 277 L0144 350 L0263 423 TR01H004 278 L0145 351 L0264 424 GSE115N 279 L0147 352 L0265 425 CE115HA121 280 L0148 353 L0266 426 CE115HA122 281 L0149 354 L0267 427 CE115HA124 282 L0151 355 L0268 428 CE115HA192 283 L0152 356 L0269 429 CE115HA236 284 L0154 357 L0270 430 CE115HA251 285 L0155 358 L0271 431 CE115HA252 286 L0157 359 L0272 432 E22L 287 L0160 360 TR01L001 288 L0161 361 TR01L002 289 L0163 362 TR01L003 290 L0167 363 TR01L004 291 L0168 364 TR01L005 292 L0173 365 TR01L006 293 L0175 366 TR01L007 294 L0180 367 TR01L008 295 L0181 368 TR01L009 296 L0186 369 TR01L010 297 L0187 370 TR01L011 298 L0200 371 TR01L012 299 L0201 372 TR01L013

Industrial Applicability

The present invention provides novel multi-specific antigen-binding molecules that retain the potent anti-cancer activity of BiTE and have very high safety, as evidenced by no induction of a cancer-antigen-independent cytokine storm, and have a long blood half-life. Cytotoxicity-inducing agents that contain the antigen-binding molecule of the present invention as an active ingredient 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 allows patients to receive the desired treatment, which not only has a high level of safety, but also reduces the patient's physical burden and is very convenient.

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Claims (3)

CLAIM (1) a bispecific antibody that has an antibody heavy chain comprising a heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 206 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising a heavy chain variable region of an antibody with CD3ε binding activity having the amino acid sequence of SEQ ID NO: 142 and a constant region having the amino acid sequence of SEQ ID NO: 60; and two identical light chains having the amino acid sequence of SEQ ID NO: 410; (1) a bispecific antibody that has an antibody heavy chain comprising a heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 206 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising a heavy chain variable region of an antibody with CD3ε binding activity having the amino acid sequence of SEQ ID NO: 142 and a constant region having the amino acid sequence of SEQ ID NO: 60; and two identical antibody light chain variable regions having the amino acid sequence of SEQ ID NO: 223; And - 74 041830 (2) a bispecific antibody which has an antibody heavy chain comprising the heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 206 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising a heavy chain variable region of an antibody with CD3ε binding activity having the amino acid sequence of SEQ ID NO: 164 and a constant region having the amino acid sequence of SEQ ID NO: 60; and two identical antibody light chain variable regions having the amino acid sequence of SEQ ID NO: 223; and (3) a bispecific antibody that has an antibody heavy chain comprising the heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 206 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising a heavy chain variable region of an antibody with CD3ε binding activity having the amino acid sequence of SEQ ID NO: 168 and a constant region having the amino acid sequence of SEQ ID NO: 60; and two identical antibody light chain variable regions having the amino acid sequence of SEQ ID NO: 223; and (4) a bispecific antibody that has an antibody heavy chain comprising the heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 206 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising a heavy chain variable region of an antibody with CD3ε binding activity having the amino acid sequence of SEQ ID NO: 168 and a constant region having the amino acid sequence of SEQ ID NO: 62; and two antibody light chain variable regions identical, having the amino acid sequence of SEQ ID NO: 223; (5) a bispecific antibody which has an antibody heavy chain comprising a heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 211 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising a heavy chain variable region of an antibody with CD3ε binding activity having the amino acid sequence of SEQ ID NO: 142 and a constant region having the amino acid sequence of SEQ ID NO: 60; and two identical light chain variable regions having the amino acid sequence of SEQ ID NO: 223; and (6) a bispecific antibody that has an antibody heavy chain comprising the heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 211 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising a heavy chain variable region of an antibody with CD3ε binding activity having the amino acid sequence of SEQ ID NO: 164 and a constant region having the amino acid sequence of SEQ ID NO: 60; and two identical antibody light chain variable regions having the amino acid sequence of SEQ ID NO: 223. 8. A bispecific antibody that binds glypican 3 and CD3ε selected from the group consisting of: (1) a bispecific antibody, wherein the variable region having glypican 3 binding activity has a variable region - 73 041830 heavy chain, comprising the amino acid sequence of SEQ ID NO: 206; variable region having CD3ε binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142, and both variable regions having glypican 3 binding activity and having CD3ε binding activity have a common light chain variable region comprising the amino acid sequence of SEQ ID NO: 223; (1) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-57 (CDR2) and 97-104 (CDR3) SEQ ID NO: 206 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-59 (CDR2) and 99111 (CDR3) of SEQ ID NO: 142, respectively; and both variable regions, both having glypican 3 binding activity, so. and having CD3ε binding activity, include common light chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94-102 (CDR3) of SEQ ID NO: 223, respectively; (1) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 50-66 (CDR2) and 99-104 (CDR3) SEQ ID NO: 206 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 50-68 (CDR2) and 101111 (CDR3) of SEQ ID NO: 142, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94-102 (CDR3) SEQ ID NO: 223 respectively; 1. A bispecific antibody that binds glypican 3 and CD3ε, which contains an antibody variable region having glypican 3 binding activity, an antibody variable region having CD3ε binding activity selected from (1)-(5): (2) a bispecific antibody that has an antibody heavy chain comprising the heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 206 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising a heavy chain variable region of an antibody with CD3ε binding activity having the amino acid sequence of SEQ ID NO: 164 and a constant region having the amino acid sequence of SEQ ID NO: 60; and two identical light chains having the amino acid sequence of SEQ ID NO: 410; (2) a bispecific antibody, wherein the variable region having glypican 3 binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 206; the variable region having CD3ε binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164, respectively; and both variable regions, both having glypican 3 binding activity and having CD3ε binding activity, share a common light chain variable region comprising the amino acid sequence of SEQ ID NO: 223; (3) a bispecific antibody, wherein the variable region having glypican 3 binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 206; a variable region having CD3ε binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 168; and both variable regions, both having glypican 3 binding activity and having CD3ε binding activity, share a common light chain variable region comprising the amino acid sequence of SEQ ID NO: 223; (4) a bispecific antibody, wherein the variable region having glypican 3 binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 211; a variable region having CD3ε binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 142; and both variable regions, both having glypican 3 binding activity and having CD3ε binding activity, share a common light chain variable region comprising the amino acid sequence of SEQ ID NO: 223; (5) a bispecific antibody, wherein the variable region having glypican 3 binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 211; a variable region having CD3ε binding activity has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 164; and both variable regions, both having glypican 3 binding activity and having CD3ε binding activity, share a common light chain variable region comprising the amino acid sequence of SEQ ID NO: 223. 5. A bispecific antibody according to any one of claims 1 to 4, wherein the bispecific antibody further has an Fc region with at least one amino acid mutation of any of the amino acid sequences forming the Fc region of SEQ ID NOs: 23-26 (IgG1-IgG4), where at least one mutation is selected from amino acids in the following amino acid positions according to the 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, regulation 329, regulation 330, regulation 331 and regulation 332. 6. The bispecific antibody of claim 5, wherein the Fc region of the bispecific antibody 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. 7. A bispecific antibody that binds glypican 3 and CD3ε selected from the group consisting of: (2) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 47-59 (CDR2) and 97-103 (CDR3) SEQ ID NO: 206 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 4761 (CDR2) and 99-110 (CDR3) of SEQ ID NO: 164, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 35-41 (CDR1), 51-56 (CDR2) and 94- 101 (CDR3) SEQ ID NO: 223 respectively; (3) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 47-59 (CDR2) and 97-103 (CDR3) SEQ ID NO: 206 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 4761 (CDR2) and 99-110 (CDR3) of SEQ ID NO: 168, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 35-41 (CDR1), 51-56 (CDR2) and 94- 101 (CDR3) SEQ ID NO: 223 respectively; (4) a bispecific antibody, wherein the variable region having glypican 3 binding activity that includes CDR1, CDR2, and CDR3 heavy chain regions identical to the amino acid sequences of positions 3035 (CDR1), 47-59 (CDR2) and 97-103 (CDR3) of SEQ ID NO: 211 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 4761 (CDR2) and 99-110 (CDR3) of SEQ ID NO: 142, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 35-41 (CDR1), 51-56 (CDR2) and 94- 101 (CDR3) SEQ ID NO: 223 respectively; (5) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 47-59 (CDR2) and 97-103 (CDR3) SEQ ID NO: 211 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 4761 (CDR2) and 99-110 (CDR3) of SEQ ID NO: 164, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 35-41 (CDR1), 51-56 (CDR2) and 94- 101 (CDR3) SEQ ID NO: 223, respectively, wherein the heavy chain numbering of CDR1, CDR2 and CDR3 and the light chain of CDR1, CDR2 and CDR3 are based on pin numbering. 4. A bispecific antibody that binds glypican 3 and CD3ε, which contains an antibody variable region having glypican 3 binding activity and an antibody variable region having CD3ε binding activity selected from (1)-(5): (2) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-57 (CDR2) and 97-104 (CDR3) SEQ ID NO: 206 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-59 (CDR2) and 99111 (CDR3) of SEQ ID NO: 164, respectively; and both variable regions, both having glypican 3 binding activity, so. and having CD3ε binding activity, include common light chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94-102 (CDR3) of SEQ ID NO: 223, respectively; (3) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-57 (CDR2) and 97-104 (CDR3) SEQ ID NO: 206 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-59 (CDR2) and 99111 (CDR3) of SEQ ID NO: 168, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common light chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94- 102 (CDR3) SEQ ID NO: 223 respectively; (4) a bispecific antibody, wherein the variable region having glypican 3 binding activity comprises heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-57 (CDR2) and 97-104 (CDR3) SEQ ID NO: 211 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-59 (CDR2) and 99111 (CDR3) of SEQ ID NO: 142, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94-102 (CDR3) SEQ ID NO: 223 respectively; (5) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-57 (CDR2) and 97-104 (CDR3) SEQ ID NO: 211 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 26-32 (CDR1), 52-59 (CDR2) and 99111 (CDR3) of SEQ ID NO: 164, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94-102 (CDR3) SEQ ID NO: 223, respectively, wherein the heavy chain numbering of CDR1, CDR2 and CDR3 and the light chain of CDR1, CDR2 and CDR3 are based on Chothia numbering. 3. A bispecific antibody that binds glypican 3 and CD3ε, which contains an antibody variable region having glypican 3 binding activity and an antibody variable region having CD3ε binding activity selected from (1)-(5): - 72 041830 (1) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes CDR1, CDR2 and Heavy chain CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 47-59 (CDR2) and 97-103 (CDR3) of SEQ ID NO: 206, respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 30-35 (CDR1), 4761 (CDR2) and 99-110 (CDR3) of SEQ ID NO: 142, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 35-41 (CDR1), 51-56 (CDR2) and 94- 101 (CDR3) SEQ ID NO: 223 respectively; 2. A bispecific antibody that binds glypican 3 and CD3ε, which contains an antibody variable region having glypican 3 binding activity and an antibody variable region having CD3ε binding activity selected from (1)-(5): (2) a bispecific antibody, wherein the variable region having glypican 3 binding activity comprises heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 50-66 (CDR2) and 99-104 (CDR3) SEQ ID NO: 206 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 50-68 (CDR2) and 101111 (CDR3) of SEQ ID NO: 164, respectively; and both variable regions, like. having glypican 3 binding activity, so. and having CD3ε binding activity, include common light chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94-102 (CDR3) of SEQ ID NO: 223, respectively; (3) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 50-66 (CDR2) and 99-104 (CDR3) SEQ ID NO: 206 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 50-68 (CDR2) and 101-111 (CDR3) of SEQ ID NO: 168, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94 -102 (CDR3) SEQ ID NO: 223 respectively; (4) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 50-66 (CDR2) and 99-104 (CDR3) SEQ ID NO: 211 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 5068 (CDR2) and 101-111 (CDR3) of SEQ ID NO: 142, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common CDR1, CDR2 and CDR3 light chain regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94 -102 (CDR3) SEQ ID NO: 223 respectively; (5) a bispecific antibody, wherein the variable region having glypican 3 binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 50-66 (CDR2) and 99-104 (CDR3) SEQ ID NO: 211 respectively; the variable region having CD3ε binding activity includes heavy chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 31-35 (CDR1), 5068 (CDR2) and 101-111 (CDR3) of SEQ ID NO: 164, respectively; and both variable regions having glypican 3 binding activity and having CD3ε binding activity include common light chain CDR1, CDR2 and CDR3 regions identical to the amino acid sequences of positions 24-39 (CDR1), 55-61 (CDR2) and 94- 102 (CDR3) SEQ ID NO: 223, respectively, wherein the heavy chain numbering of CDR1, CDR2 and CDR3 and the light chain of CDR1, CDR2 and CDR3 are based on Cabot numbering. - 71 041830 (3) a bispecific antibody that has an antibody heavy chain comprising a heavy chain variable region of an antibody with glypican 3 binding activity having the amino acid sequence of SEQ ID NO: 206 and a constant region having the amino acid sequence of SEQ ID NO: 61; an antibody heavy chain comprising an antibody heavy chain variable region with CD3ε binding activity having the amino acid sequence of SEQ ID -