WO2025169945A1 - Cd3-targeting antigen-binding molecule with improved stability - Google Patents

Abstract

The present disclosure provides a novel CD3-binding domain with superior stability compared to CD3-binding domains used in conventional T cell redirecting antibodies, and an antigen-binding molecule comprising the CD3-binding domain. A bispecific antigen-binding molecule prepared by combining the CD3-binding domain of the present disclosure with an antigen-binding domain binding to an antigen expressed on the surface of a target cell such as a cancer cell can induce T-cell-dependent cytotoxicity to the target cell and can be used for the treatment or prevention of various cancers.

Description

CD3-targeted antigen-binding molecules with improved stability

The present disclosure relates to an antigen-binding molecule comprising a CD3-binding domain with improved stability, a pharmaceutical composition comprising the antigen-binding molecule, and uses thereof.

Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and minimal side effects (Non-Patent Documents 1 and 2). Antibodies not only bind to antigens and have agonistic and antagonistic effects, but are also known to exert antitumor effects against cancer cells by inducing cytotoxic activity (also known as effector function) by effector cells, such as ADCC (Antibody Dependent Cytotoxicity), ADCP (Antibody Dependent Cell phagocytosis), and CDC (Complement-Dependent Cytotoxicity) (Non-Patent Document 3). ADCC is the cytotoxicity exerted by effector cells against antibody-bound target cancer cells when the Fc region of an antibody binds to Fc receptors present on effector cells such as NK cells and macrophages.

Natural immunoglobulins bind to antigens through their variable regions, and to receptors such as FcγR, FcRn, FcαR, and FcεR, or to complements, through their constant regions. Normal natural IgG antibodies recognize and bind to a single epitope through their variable region (Fab), and can therefore only bind to a single antigen. However, it is known that many different proteins are involved in cancer and inflammation, and crosstalk between these proteins can occur.

Bispecific antibodies, which bind to two or more antigens with a single molecule, are being researched as molecules that inhibit multiple targets. By improving natural IgG antibodies, it is possible to confer binding activity to two different antigens (a first antigen and a second antigen) (Non-Patent Document 4). Therefore, it is possible to neutralize two or more antigens with a single molecule, or to enhance anti-tumor activity by cross-linking cytotoxic cells with cancer cells.

T cell-redirecting antibodies, a type of bispecific antibody known since the 1980s, are antibodies whose antitumor effect is mediated by cytotoxicity, which recruits T cells as effector cells (Non-Patent Documents 5, 6, 7). Unlike antibodies whose antitumor effect is mediated by ADCC, which recruits NK cells and macrophages as effector cells, T cell-redirecting antibodies are bispecific antibodies that contain an antibody against one of the constituent subunits of the T cell receptor (TCR) complex on T cells, specifically an antibody that binds to the CD3ε chain, and an antibody that binds to an antigen on the target cancer cell. The simultaneous binding of T cell-redirecting antibodies to the CD3ε chain and the cancer antigen allows T cells to approach the cancer cells. As a result, it is believed that the cytotoxic activity of T cells exerts an antitumor effect on cancer cells.

Catumaxomab, a T cell-redirecting antibody, uses two Fab fragments to bind to a cancer antigen (EpCAM) and the CD3ε chain expressed on T cells. Catumaxomab is a trifunctional antibody that simultaneously binds to both a cancer antigen and CD3ε, thereby inducing cytotoxic activity by T cells, and to both a cancer antigen and FcγR, thereby inducing cytotoxic activity by antigen-presenting cells such as NK cells and macrophages. However, because trifunctional antibodies simultaneously bind to CD3ε and FcγR even in the absence of cancer antigens, CD3ε-expressing T cells and FcγR-expressing cells are cross-linked, resulting in the mass production of various cytokines, even in an environment where cancer cells are not present. Due to this cancer antigen-independent induction of cytokine production, trifunctional antibody administration is currently limited to intraperitoneal administration (Non-Patent Document 8), and systemic administration is extremely difficult due to the severe cytokine storm-like side effects.

On the other hand, unlike catumaxomab, BiTE (bispecific T-cell engager) does not have a binding site for Fcγ receptors, and therefore does not cross-link T cells with receptors expressed on NK cells, macrophages, etc. in a cancer antigen-independent manner. As a result, it has been shown that the cancer antigen-independent cytokine induction observed when catumaxomab is administered does not occur. However, because BiTE is a low-molecular-weight modified antibody molecule lacking the Fc region, there is a problem in that the half-life of BiTE administered to patients in the blood is significantly shorter than that of IgG antibodies, which are commonly used as therapeutic antibodies.

Recently, new polypeptide complexes have been provided that use an Fc region with reduced FcγR-binding activity to bring T cells into close proximity with target cancer cells, exhibiting excellent safety characteristics by not inducing cytokine storms or other conditions in a cancer antigen-independent manner, and have a long blood half-life (Patent Document 1). It has been reported that new T cell redirecting antibodies with such advantageous effects are useful not only for targeting cancer cells, but also for targeting regulatory T cells and exhausted T cells, which are cells present in the cancer microenvironment that function to suppress immune responses (Patent Documents 2 and 3).

WO2012/073985 WO2016/047722 WO2015/174439

Nat. Biotechnol. (2005) 23, 1073-1078 Eur J Pharm Biopharm. (2005) 59 (3), 389-396 Drug Des Devel Ther (2009) 3, 7-16 MAbs. (2012) Mar 1, 4(2) Nature (1985) 314 (6012), 628-31 Int J Cancer (1988) 41 (4), 609-15. Proc Natl Acad Sci USA (1986) 83 (5), 1453-7 Cancer Immunol Immunother. (2007) 56(9), 1397-406

The present invention was made in light of the above circumstances, and focuses particularly on the CD3-binding domain used in T cell redirecting antibodies. It aims to provide a novel CD3-binding domain that has greater stability than the CD3-binding domains used in conventional T cell redirecting antibodies, an antigen-binding molecule containing said CD3-binding domain, a method for producing said antigen-binding molecule, a pharmaceutical composition containing said antigen-binding molecule as an active ingredient, and uses thereof.

The present inventors introduced various modifications into the CDRs of the CD3-binding Fab contained in the bispecific antibody described in WO2015174439 and succeeded in obtaining modified antibodies that showed a smaller degree of decline in CD3-binding activity when stored at room temperature or above than the parent Fab. The present inventors further combined these modified CD3-binding domains with other antigen-binding domains, such as a cancer-specific antigen-binding domain, to prepare bispecific antigen-binding molecules and found that the degree of decline in T cell-dependent cytotoxicity when stored at room temperature or above was also small. Based on these findings, the present inventors have demonstrated that the multispecific antigen-binding molecules of the present invention can damage tissues containing target cells expressing a target antigen, such as a cancer-specific antigen.

The present disclosure is based on these findings and specifically includes the embodiments exemplified below.
[1] An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises an antibody H chain variable region and L chain variable region that have binding activity to CD3;
the heavy chain variable region
H chain CDR1 comprising the amino acid sequence NAWMH (SEQ ID NO: 1);
an H chain CDR2 comprising the amino acid sequence _{QIX1DKSQNYATX2VAESVKG} (SEQ ID NO: ₂ ), wherein _X1 is K or R, and _X2 is Y or F; and an H chain _CDR3 comprising the amino acid sequence _{VHYX3AGYGVDX4} (SEQ ID NO: 3), wherein _X3 is A or P, and _X4 is I, M, or L.
Including,
the light chain variable region
an L chain CDR1 comprising the amino acid sequence _{RSX5X6X7VVHENRX8TYLH} ( _SEQ ID NO: ₄ ), wherein _X5 is _S or T, _X6 is Q or M, _X7 is S or T, and _X8 is Q or N;
L chain CDR2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 5); and L chain CDR3 comprising the amino acid sequence GQGTQVPYT (SEQ ID NO: 6).
An antigen-binding molecule comprising:
[2] The antigen-binding molecule of [1], wherein the antibody H chain variable region and L chain variable region having CD3-binding activity comprise any combination of H chain CDR1, CDR2, and CDR3, and L chain CDR1, CDR2, and CDR3 selected from the following (a1) to (a8):
(a1) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a2) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a3) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a4) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a5) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a6) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a7) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a8) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.
[3] The antigen-binding molecule of [1] or [2], wherein the antibody H chain variable region and L chain variable region having CD3-binding activity comprise any combination of H chain variable region and L chain variable region selected from the following (a1) to (a8):
(a1) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a2) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a3) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26;
(a4) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a5) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a6) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27;
(a7) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28;
(a8) A combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29.
[4-1] The antigen-binding molecule of any of [1] to [3], further comprising an Fc region.
[4-2] the antigen-binding molecule of any of [1] to [3], further comprising an Fc region with reduced binding activity to an Fcγ receptor;
[4-3] The antigen-binding molecule of [4-1] or [4-2], which comprises any one of the combinations of heavy chains and light chains selected from the following (a1) to (a8):
(a1) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a2) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a3) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 36;
(a4) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a5) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 33 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a6) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 37;
(a7) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 38;
(a8) A combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 39.
[5-1] The antigen-binding molecule of any of [1] to [4], which is a monospecific antigen-binding molecule.
[5-2] the antigen-binding molecule of any one of [1] to [4], wherein the first antigen-binding domain and the second antigen-binding domain comprise the same amino acid sequence;
[5-3] the antigen-binding molecule of any one of [1] to [4], wherein the first antigen-binding domain and the second antigen-binding domain comprise amino acid sequences that are different from each other;
[6-1] The antigen-binding molecule of any of [1] to [4], which is a multispecific antigen-binding molecule.
[6-2] The antigen-binding molecule of any of [1] to [4], which is a bispecific antigen-binding molecule.
[6-3] The antigen-binding molecule of any of [1] to [4], which is an antigen-binding molecule with triple or higher specificity.
[7-1] the antigen-binding molecule of [6], wherein the second antigen-binding domain comprises an antibody variable region having binding activity against a cancer antigen;
[7-2] the antigen-binding molecule of [6], wherein the second antigen-binding domain comprises an antibody variable region that has binding activity toward a molecule expressed on the surface of a cell that has the function of suppressing an immune response;
[8] The antigen-binding molecule of any one of [1] to [7], which is an antibody.
[9] A nucleic acid encoding the antigen-binding molecule of any one of [1] to [8].
[10] A vector into which the nucleic acid according to [9] has been introduced.
[11] A cell comprising the nucleic acid of [9] or the vector of [10].
[12] A method for producing the antigen-binding molecule of any one of [1] to [8], comprising the step of culturing the cell of [11].
[13] An antigen-binding molecule produced by the method according to [12].
[14] A pharmaceutical composition comprising the antigen-binding molecule of any of [1] to [8] and a pharmaceutically acceptable carrier.
[15-1] The pharmaceutical composition according to [14], for inducing cell damage.
[15-2] The pharmaceutical composition according to [14], for inducing T cell-dependent cytotoxicity.
[15-3] The pharmaceutical composition according to [14], for use in the treatment or prevention of cancer.
[16-1] A method for inducing cytotoxicity, comprising the step of administering the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of [14].
[16-2] A method for inducing T cell-dependent cytotoxicity, comprising the step of administering the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of [14].
[16-3] A method for treating or preventing cancer, comprising the step of administering the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of [14].
[17-1] A kit for inducing cytotoxicity, comprising the antigen-binding molecule of any one of [1] to [8] or the pharmaceutical composition of [14], and instructions for use.
[17-2] A kit for inducing T cell-dependent cytotoxicity, comprising the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of [14], and instructions for use.
[17-3] A kit for treating or preventing cancer, comprising the antigen-binding molecule of any of [1] to [8] or the pharmaceutical composition of [14], and instructions for use.
[18-1] The antigen-binding molecule of any one of [1] to [8], for use in inducing cytotoxicity.
[18-2] The antigen-binding molecule of any one of [1] to [8], for use in inducing T cell-dependent cytotoxicity.
[18-3] the antigen-binding molecule of any one of [1] to [8], for use in the treatment or prevention of cancer.
[19-1] Use of the antigen-binding molecule of any of [1] to [8] in the production of a cytotoxicity-inducing agent.
[19-2] Use of the antigen-binding molecule of any of [1] to [8] in the production of an inducer of T cell-dependent cytotoxicity.
[19-3] Use of the antigen-binding molecule of any of [1] to [8] in the manufacture of a cancer treatment or prevention agent.
[A1] A monovalent antigen-binding molecule comprising a single antigen-binding domain, wherein the single antigen-binding domain comprises an antibody H chain variable region and an L chain variable region that have binding activity to CD3;
the heavy chain variable region
H chain CDR1 comprising the amino acid sequence NAWMH (SEQ ID NO: 1);
an H chain CDR2 comprising the amino acid sequence _{QIX1DKSQNYATX2VAESVKG} (SEQ ID NO: ₂ ), wherein _X1 is K or R, and _X2 is Y or F; and an H chain _CDR3 comprising the amino acid sequence _{VHYX3AGYGVDX4} (SEQ ID NO: 3), wherein _X3 is A or P, and _X4 is I, M, or L.
Including,
the light chain variable region
an L chain CDR1 comprising the amino acid sequence _{RSX5X6X7VVHENRX8TYLH} ( _SEQ ID NO: ₄ ), wherein _X5 is _S or T, _X6 is Q or M, _X7 is S or T, and _X8 is Q or N;
L chain CDR2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 5); and L chain CDR3 comprising the amino acid sequence GQGTQVPYT (SEQ ID NO: 6).
An antigen-binding molecule comprising:
[A2] The antigen-binding molecule of [A1], wherein the antibody H chain variable region and L chain variable region having CD3-binding activity comprise any combination of H chain CDR1, CDR2, and CDR3, and L chain CDR1, CDR2, and CDR3 selected from the following (a1) to (a8):
(a1) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a2) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a3) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a4) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a5) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a6) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a7) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a8) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.
[A3] The antigen-binding molecule of [A1] or [A2], wherein the antibody H chain variable region and L chain variable region having CD3-binding activity comprise any combination of H chain variable region and L chain variable region selected from the following (a1) to (a8):
(a1) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a2) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a3) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26;
(a4) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a5) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a6) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27;
(a7) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28;
(a8) A combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29.
[A4-1] The antigen-binding molecule of any of [A1] to [A3], which further comprises an Fc region.
[A4-2] The antigen-binding molecule of any of [A1] to [A3], further comprising an Fc region with reduced binding activity to an Fcγ receptor.
[A4-3] The antigen-binding molecule of [A4-1] or [A4-2], which comprises any one of the combinations of heavy chains and light chains selected from the following (a1) to (a8):
(a1) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a2) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a3) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 36;
(a4) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a5) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 33 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a6) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 37;
(a7) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 38;
(a8) A combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 39.
[A5] A nucleic acid encoding the antigen-binding molecule of any of [A1] to [A4].
[A6] A vector into which the nucleic acid described in [A5] has been introduced.
[A7] A cell comprising the nucleic acid according to [A5] or the vector according to [A6].
[A8] A method for producing the antigen-binding molecule of any of [A1] to [A4], comprising the step of culturing the cell of [A7].
[A9] An antigen-binding molecule produced by the method described in [A8].

I. Definitions The following definitions are provided to facilitate understanding of the invention described herein.

The term "antigen-binding molecule" refers to a molecule capable of binding to its antigen with sufficient affinity. In one embodiment, the extent of binding of an antigen-binding molecule to proteins unrelated to the antigen is less than about 10% of the binding of the antigen-binding molecule to the antigen, as measured (e.g., by radioimmunoassay (RIA)). In certain embodiments, the antigen-binding molecule has a dissociation constant (Kd) for the antigen of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ M). In certain embodiments, the antigen-binding molecule binds to an epitope of the antigen that is conserved among the antigens from different species. In some embodiments, the antigen-binding molecule is an antibody.

As used herein, the term "antibody" refers to an immunoglobulin that is natural or partially or fully synthetically produced. Antibodies can be isolated from natural sources such as plasma or serum where they occur, or from the culture supernatant of antibody-producing hybridoma cells, or can be partially or fully synthesized using techniques such as genetic recombination. Suitable examples of antibodies include immunoglobulin isotypes and their isotype subclasses. Nine known classes (isotypes) of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the present invention may include IgG1, IgG2, IgG3, and IgG4.

An antibody's "class" refers to the type of constant domain or constant region present in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

"Native antibodies" refer to immunoglobulin molecules with a variety of naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

The terms "variable region" or "variable domain" refer to the domains of an antibody heavy or light chain that are involved in binding the antibody to antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a complementary library of VL or VH domains, respectively, with a VH or VL domain from an antibody that binds to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and/or forms structurally defined loops (the "hypervariable loops") and/or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following:
(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
(d) A combination of (a), (b), and/or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).
Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residue at the Fc region may or may not be present. In one embodiment, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residue at the Fc region may be removed by degradation. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% sequence identity with the native-sequence Fc region and/or the Fc region of the parent polypeptide, more preferably at least about 90% sequence identity thereto, and most preferably at least about 95% sequence identity thereto.

"Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997).) FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126:330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO2004/92219 (Hinton et al.)).

In vivo binding to human FcRn and plasma half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with mutant Fc regions are administered. Instead of plasma half-life, blood half-life or serum half-life can be measured. WO2000/42072 (Presta) describes antibody variants with increased or decreased binding to FcRs. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant manipulation of the nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region according to the present invention can include an antibody with G446-K447, an antibody with G446 but no K447, an antibody from which G446-K447 have been completely removed (in the case of IgG, an antibody whose C-terminus is P445 or an antibody with an amide group (NH2) added to P445, resulting in proline amide (Pro-NH2)), or a mixture of the above three types of antibodies.

"Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, in vitro ADCC assays, such as those described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), may be performed. Effector cells useful for such assays include PBMCs and NK cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and/or bind the same epitope (determinant), except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are typically present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma technology, recombinant DNA technology, phage display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from the human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain that includes an Fc region as defined herein.

An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antigen-binding molecules formed from antibody fragments.

As used herein, the term "Fv (variable fragment)" refers to the smallest unit of an antibody-derived antigen-binding domain consisting of a pair of an antibody light chain variable region (VL (light chain variable region)) and an antibody heavy chain variable region (VH (heavy chain variable region)). In 1988, Skerra and Pluckthun discovered that homogeneous and active Fv could be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). In the Fv prepared from the periplasmic fraction, VH and VL were associated in a manner that allowed them to bind to antigens.

As used herein, the term "scFv,""single-chainantibody," or "sc(Fv) ₂ " refers to an antibody fragment that contains, in a single polypeptide chain, the variable regions from both the heavy and light chains, but lacks a constant region. Generally, single-chain antibodies further comprise a polypeptide linker between the VH and VL domains, which enables them to form the desired structure that may enable antigen binding. Single-chain antibodies are discussed in detail by Plückthun in *The Pharmacology of Monoclonal Antibodies*, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994). See also International Patent Application Publication No. WO 1988/001649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may also be bispecific and/or humanized.

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

sc(Fv) ₂ is a single-chain antibody in which four variable regions, two VL and two VH, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). The two VH and VL may be derived from different monoclonal antibodies. Suitable examples include bispecific sc(Fv) _2s that recognize two different epitopes present in the same antigen, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv) _2s can be produced by methods known to those skilled in the art. For example, they can be produced by linking scFvs with a linker such as a peptide linker.

As used herein, the configuration of the antigen-binding domain constituting sc(Fv) ₂ includes antigen-binding molecules in which two VHs and two VLs are arranged in the following order, starting from the N-terminus of the single-chain polypeptide: VH, VL, VH, VL ([VH] linker-[VL] linker-[VH] linker-[VL]). However, the order of the two VHs and two VLs is not limited to the above configuration and may be arranged in any order. For example, the following order configurations are also possible:
[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 structure of sc(Fv) ₂ is also described in detail in WO2006/132352, and those skilled in the art can prepare the desired sc(Fv) ₂ appropriately for producing the antigen-binding molecules disclosed herein based on these descriptions.

The antigen-binding molecules of the present invention may also be conjugated to carrier polymers such as PEG or organic compounds such as anticancer drugs. Furthermore, by inserting a glycosylation sequence into the amino acid sequence of the antigen-binding molecules of the present invention, a glycosylation sequence can be suitably added to the antigen-binding molecules in order to obtain the desired effects of the glycosylation.

The linker linking the antibody variable regions can be any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (e.g., the linkers disclosed in Protein Engineering, 9 (3), 299-305, 1996). In the present invention, however, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be selected appropriately by those skilled in the art depending on the purpose. A preferred length is 5 amino acids or more (the upper limit is not particularly limited, but typically 30 amino acids or less, preferably 20 amino acids or less), with 15 amino acids being particularly preferred. When sc(Fv) ₂ contains three peptide linkers, all of the peptide linkers may be the same length, or peptide linkers of different lengths may be used. A widely known example of a peptide linker is a flexible polypeptide linker composed of glycine and serine. The length and sequence of the peptide linker can be selected appropriately by those skilled in the art depending on the purpose.

Synthetic chemical linkers (chemical crosslinkers) are crosslinkers commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available.

When linking four antibody variable regions, three linkers are usually required, but the same linker may be used for all three, or different linkers may be used.

"Fab" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

The terms "F(ab') ₂ " and "Fab'" refer to antibody fragments produced by treating an immunoglobulin (monoclonal antibody) with a proteolytic enzyme such as pepsin or papain, resulting in digestion across the disulfide bond between the two heavy chains in the hinge region. For example, treating IgG with papain cleaves it upstream of the disulfide bond between the two heavy chains in the hinge region, producing two homologous antibody fragments in which an light chain consisting of a VL (light chain variable region) and a CL (light chain constant region) and an heavy chain fragment consisting of a VH (heavy chain variable region) and a CHγ1 (the γ1 region of the heavy chain constant region) are linked by a disulfide bond at their C-terminal regions. These two homologous antibody fragments are each referred to as Fab'.

"F(ab') ₂ " comprises two light chains and two heavy chains comprising constant regions, i.e., portions of the CH1 and CH2 domains, such that interchain disulfide bonds are formed between the two heavy chains. The F(ab') ₂ constituting the antigen-binding molecule disclosed herein can be suitably obtained by partially digesting a full-length monoclonal antibody or the like having the desired antigen-binding domain with a protease such as pepsin, followed by removal of the Fc fragment by adsorption onto a protein A column. Such a protease is not particularly limited, as long as it can digest a full-length antibody so as to produce F(ab') ₂ in a limited manner by appropriately setting the enzyme reaction conditions, such as pH, and examples thereof include pepsin and ficin.

"Affinity" refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

"Specific binding" refers to the state in which one of the specifically binding molecules does not exhibit any significant binding to any molecules other than the one or more molecules to which it binds. The term is also used when a domain containing an antibody variable region is specific to a particular epitope among multiple epitopes contained in an antigen. Furthermore, when the epitopes to which the domain containing an antibody variable region binds are contained in multiple different antigens, an antigen-binding molecule having the domain containing the antibody variable region can bind to various antigens containing that epitope.

The term "epitope" refers to a site on an antigen to which an antigen-binding molecule (e.g., an antibody), whether proteinaceous or non-proteinaceous, binds. For example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of an antigen-binding molecule (e.g., an antibody) that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that make up the epitope. When the epitope is a glycan, the epitope can also be identified by a specific glycan structure.

Linear epitopes are epitopes that contain a recognized primary amino acid structure. Linear epitopes typically contain at least three, and most usually at least five, e.g., about 8 to about 10, or 6 to 20 amino acids in a unique sequence.

In contrast to linear epitopes, conformational epitopes are epitopes in which the amino acids constituting the epitope are typically not contiguous as a primary structure but are composed of discontinuous amino acid residues (sometimes referred to as discontinuous epitopes). Conformational epitopes may encompass an increased number of amino acids compared to linear epitopes. In recognizing conformational epitopes, antigen-binding molecules (e.g., antibodies) recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and/or polypeptide backbones that form a conformational epitope are juxtaposed, allowing antigen-binding molecules (e.g., antibodies) to recognize the epitope. Methods for determining the conformational structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-directed spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

The structure of an antigen-binding molecule (e.g., an antibody) that binds to an epitope is called the "paratope," "antigen-binding site," "antigen-binding region," or "antigen-binding domain." The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, and other forces that act between them. The binding strength between these epitopes and paratopes is called affinity. When multiple antigen-binding molecules (e.g., antibodies) bind to multiple antigens, the sum of their binding strengths is called avidity. For example, when an antigen-binding molecule containing multiple antigen-binding domains (i.e., a multivalent antigen-binding molecule) binds to multiple epitopes, the affinities act synergistically, resulting in avidity being higher than affinity.

An "antigen-binding molecule that competes with a reference antigen-binding molecule" refers to an antigen-binding molecule that inhibits the binding of the reference antigen-binding molecule to its own antigen by 50% or more in a competition assay; conversely, a reference antigen-binding molecule inhibits the binding of the aforementioned antigen-binding molecule to its own antigen by 50% or more in a competition assay. Competitive antigen-binding molecules include antigen-binding molecules that bind to the same or overlapping epitope as the reference antigen-binding molecule, and antigen-binding molecules that bind to an adjacent epitope that is sufficiently proximal to the epitope bound by the reference antigen-binding molecule and cause steric hindrance. An exemplary competition assay is provided herein.

An "isolated" antibody is one that has been separated from components of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as measured, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

"Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains that nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an antigen-binding molecule" refers to one or more nucleic acid molecules that encode an antigen-binding molecule, preferably comprising an antibody heavy chain variable region and a light chain variable region, and includes nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Some vectors are capable of conferring expression of a nucleic acid to which they are operatively linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells using methods such as viruses or electroporation, but vector introduction is not limited to ex vivo introduction; vectors can also be introduced directly into a living body.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to whom the formulation is administered.

"Pharmaceutically acceptable carrier" refers to any ingredient, other than an active ingredient, in a pharmaceutical formulation or composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to the amount, at the dosage and for the period of time necessary, that is effective to achieve the desired therapeutic or prophylactic result.

The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and/or warnings regarding the use of such therapeutic product.

As used herein, "treatment" (and its grammatical derivatives, such as "treat" and "treating") refers to a clinical intervention intended to alter the natural course of the individual being treated and may be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

"Inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, including inducing cell death.

"Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and after not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

The term "and/or" is used herein to refer to each of the objects listed before and after "and/or" or any combination thereof. For example, "A, B and/or C" includes each of the objects "A," "B," and "C," as well as the combinations "A and B," "A and C," "B and C," and "A and B and C."

II. Detailed Description of the Invention The invention of this disclosure generally relates to antigen-binding molecules comprising a CD3-binding domain with excellent stability, pharmaceutical compositions comprising them, and uses thereof. Specifically, the antigen-binding molecules of the present invention comprise a CD3-binding domain with improved stability compared to the CD3-binding domain contained in conventional T cell-redirecting antibodies (e.g., the bispecific antibodies described in WO2015174439). The structure of the antigen-binding molecules of the present invention is not particularly limited, as long as they comprise the CD3-binding domain of the present invention with improved stability. The antigen-binding molecules may be molecules having a natural antibody structure or polypeptides having other artificially designed structures.

In some embodiments, the antigen-binding molecule of the present invention is an antibody. In some embodiments, the antigen-binding molecule of the present invention is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the antigen-binding molecule of the present invention is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, single-domain antibody, or F(ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody, a complete IgG2 antibody, a complete IgG3 antibody, or a complete IgG4 antibody.

Antigen-binding molecules comprising a CD3-binding domain with excellent stability . In one aspect, the present invention provides antigen-binding molecules comprising a first antigen-binding domain (CD3-binding domain) having binding activity to CD3 and a second antigen-binding domain. In another aspect, the present invention provides monovalent antigen-binding molecules comprising a single antigen-binding domain (CD3-binding domain) having binding activity to CD3. The CD3-binding domain contained in the antigen-binding molecules of the present invention is characterized by excellent stability and a small decrease in CD3-binding activity after storage at room temperature or higher. In one embodiment, the CD3-binding activity of the CD3-binding domain contained in the antigen-binding molecules of the present invention after storage at room temperature or higher (e.g., 50°C) for a predetermined period of time (e.g., 3 days) maintains 65% or more, 70% or more, or 75% or more of the CD3-binding activity after storage at a low temperature (e.g., 4°C). In some embodiments, the CD3-binding domain contained in the antigen-binding molecules of the present invention has binding activity to the CD3 epsilon chain. In some embodiments, the CD3 is mammalian CD3. In certain embodiments, the CD3 is human CD3. In some embodiments, the antigen-binding domain comprised in the antigen-binding molecule of the present invention is Fv, Fab, Fab', or scFv.

CD3-binding domain In one embodiment, the CD3-binding domain contained in the antigen-binding molecule of the present invention comprises an antibody H chain variable region and L chain variable region that have binding activity to CD3,
The H chain variable region
H chain CDR1 comprising the amino acid sequence NAWMH (SEQ ID NO: 1);
an H chain CDR2 comprising the amino acid sequence _{QIX1DKSQNYATX2VAESVKG} (SEQ ID NO: ₂ ), wherein X1 is K or R, and _X2 is Y or F; and an H chain CDR3 comprising the amino acid sequence _{VHYX3AGYGVDX4} (SEQ ID NO: ₃ ), wherein _X3 is A or P, and _X4 is I, M, or L.
Including,
The light chain variable region
an L chain CDR1 comprising the amino acid sequence _{RSX5X6X7VVHENRX8TYLH} ( _SEQ ID NO: ₄ ), wherein _X5 is _S or T, _X6 is Q or M, _X7 is S or T, and _X8 is Q or N;
L chain CDR2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 5); and L chain CDR3 comprising the amino acid sequence GQGTQVPYT (SEQ ID NO: 6).
In this specification, the terms H chain and heavy chain are used interchangeably, and the terms L chain and light chain are used interchangeably.

In certain embodiments, the antibody heavy chain variable region and light chain variable region having binding activity to CD3 comprise any combination of heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 selected from the following (a1) to (a8):
(a1) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a2) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a3) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a4) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a5) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a6) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a7) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a8) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

In one embodiment, the antibody heavy chain variable region and light chain variable region having binding activity to CD3 comprise any combination of heavy chain variable region and light chain variable region selected from the following (a1) to (a8):
(a1) a combination of an H chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 20 and an L chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25;
(a2) a combination of a heavy chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 and a light chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25;
(a3) a combination of a heavy chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 and a light chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26;
(a4) a combination of a heavy chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 and a light chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25;
(a5) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23 and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25;
(a6) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27;
(a7) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28;
(a8) A combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 29.

In one embodiment, the antibody H chain variable region and L chain variable region having binding activity to CD3 comprise any combination of an H chain variable region and an L chain variable region selected from the following (a1) to (a8):
(a1) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 20, and comprising an H-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and an H-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 25, and comprising an L-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a2) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 21 and comprising an H-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and an H-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 25 and comprising an L-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a3) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 21 and comprising an H-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and an H-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 26 and comprising an L-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a4) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and comprising an H-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and an H-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 25 and comprising an L-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a5) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 23 and comprising an H-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and an H-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 25 and comprising an L-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a6) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and comprising an H-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and an H-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 27 and comprising an L-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a7) a combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and comprising an H-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and an H-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 28 and comprising an L-chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L-chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a8) A combination of an H-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 22 and comprising an H-chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H-chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H-chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and an L-chain variable region having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 29 and comprising an L-chain CDR1 having the amino acid sequence shown in SEQ ID NO: 19, an L-chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L-chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

In certain embodiments, the antibody heavy chain variable region and light chain variable region having binding activity to CD3 comprises any one of the combinations of heavy chain variable region and light chain variable region selected from the following (a1) to (a8):
(a1) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a2) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a3) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26;
(a4) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a5) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a6) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27;
(a7) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28;
(a8) A combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29.

In one embodiment, the antigen-binding molecule of the present invention comprises, as heavy and light chains having binding activity to CD3, any of the combinations of heavy and light chains selected from the following (a1) to (a8):
(a1) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35;
(a2) a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35;
(a3) a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36;
(a4) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35;
(a5) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 33 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35;
(a6) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37;
(a7) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38;
(a8) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32 and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39.

In one embodiment, the antigen-binding molecule of the present invention comprises, as a heavy chain and a light chain having binding activity to CD3, any combination of a heavy chain and a light chain selected from the following (a1) to (a8):
(a1) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 30 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 7, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 10, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6;
(a2) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 31 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6;
(a3) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 31 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 11, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 36 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6;
(a4) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6,
(a5) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 33 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 13, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 35 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6,
(a6) a combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 37 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6;
(a7) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 38 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6,
(a8) A combination of a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 32 and comprising an H chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 having the amino acid sequence shown in SEQ ID NO: 9, and an H chain CDR3 having the amino acid sequence shown in SEQ ID NO: 12, and a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 39 and comprising an L chain CDR1 having the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

In a specific embodiment, the antigen-binding molecule of the present invention comprises, as a heavy chain and a light chain having binding activity to CD3, any of the following combinations of heavy chains and light chains selected from (a1) to (a8):
(a1) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a2) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a3) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 36;
(a4) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a5) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 33 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 35;
(a6) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 37;
(a7) a combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 38;
(a8) A combination of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 39.

Second antigen-binding domain In the antigen-binding molecules of the present invention, the structure of the second antigen-binding domain is not particularly limited, as long as it has binding activity to a target antigen. In some embodiments, the second antigen-binding domain comprises an antibody heavy chain variable region and light chain variable region.

Monospecific antigen-binding molecules In some embodiments, the antigen-binding molecules of the present invention are monospecific antigen-binding molecules, and both the first and second antigen-binding domains have binding activity to CD3 (e.g., human CD3). In one embodiment, the first and second antigen-binding domains have binding activity to the same epitope on CD3. In a specific embodiment, both the first and second antigen-binding domains have binding activity to the CD3ε chain (e.g., human CD3ε chain).
In a particular embodiment, the first antigen-binding domain and the second antigen-binding domain comprise the same amino acid sequence. In another particular embodiment, the first antigen-binding domain and the second antigen-binding domain comprise different amino acid sequences from each other.

Multispecific antigen-binding molecules In some embodiments, the antigen-binding molecules of the present invention are multispecific antigen-binding molecules having binding specificities at at least two different sites. In certain embodiments, the multispecific antigen-binding molecules of the present invention are bispecific antigen-binding molecules. In another specific embodiment, the multispecific antigen-binding molecules of the present invention are antigen-binding molecules with three or more specificities, such as trispecific, tetraspecific, pentaspecific, or hexaspecific antigen-binding molecules.

In one embodiment of the multispecific antigen-binding molecule of the present invention, the first antigen-binding domain has binding activity to CD3, and the second antigen-binding domain has binding activity to an antigen other than CD3. In this embodiment, the "antigen other than CD3" recognized and bound by the second antigen-binding domain is preferably a molecule expressed on the surface of cells that are targeted by the cytotoxic activity of T cells (T cell-dependent cytotoxicity), and examples include molecules specifically expressed on the surface of cancer cells (also referred to herein as cancer antigens or cancer-specific antigens) and molecules expressed on the surface of cells that have the function of suppressing immune responses (also referred to herein as immunosuppressive cell surface antigens).

In one embodiment, the second antigen-binding domain has binding activity to a cancer antigen. In such an embodiment, the multispecific antigen-binding molecule of the present invention binds to CD3 via the first antigen-binding domain and binds to a cancer antigen via the second antigen-binding domain, thereby bringing T cells into proximity with target cancer cells and enabling the treatment or prevention of cancer through the cytotoxic activity of T cells against tissues containing the target cancer cells.
In another embodiment, the second antigen-binding domain has binding activity to an immunosuppressive cell surface antigen. In such an embodiment, the multispecific antigen-binding molecule of the present invention binds to CD3 via the first antigen-binding domain and binds to the immunosuppressive cell surface antigen via the second antigen-binding domain, thereby bringing T cells into proximity with cells that have the function of suppressing immune responses (also referred to herein as immunosuppressive cells), and enabling the treatment or prevention of cancer through the cytotoxic activity of T cells against the immune response-suppressing cells.
In the multispecific antigen-binding molecules of the present invention, known antigen-binding domains and other antigen-binding domains, including those to be identified in the future, that have binding activity for cancer antigens or immunosuppressive cell surface antigens can be used as the second antigen-binding domain.

In a preferred embodiment, the multispecific antigen-binding molecule of the present invention has a cytotoxic activity that is equivalent to or greater than that of a control multispecific antigen-binding molecule that comprises, as a first antigen-binding domain having binding activity to CD3, an antibody heavy chain variable region (e.g., an heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20) comprising an heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 1, an heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 7, and an heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 10, and an antibody light chain variable region (e.g., an light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24) comprising an light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 14, an light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6. Examples of such multispecific antigen-binding molecules include multispecific antigen-binding molecules (e.g., bispecific antigen-binding molecules, e.g., bispecific antibodies) that comprise, as a first antigen-binding domain, the heavy and light chain CDRs, heavy and light chain variable regions, or heavy and light chains of antibody Nos. 2, 14, 25, 29, 30 to 32, and 34 listed in Table 5 below.

Domain containing an antibody variable region with binding activity to CD3. Herein, the terms "antibody variable region with binding activity to CD3" and "domain containing an antibody variable region with binding activity to CD3" are used interchangeably and refer to a portion of an anti-CD3 antibody comprising a region that specifically binds to and is complementary to part or all of CD3, an adaptor molecule that forms the T cell receptor complex together with the T cell receptor. Preferably, the domain contains the light chain variable region (VL) and heavy chain variable region (VH) of an anti-CD3 antibody. Suitable examples of such domains include "scFv (single chain Fv),""single chain antibody,""Fv,"" _scFv2 (single chain Fv2),""Fab," and "F(ab') ₂ ."

The CD3-binding domain of the present invention (a domain containing an antibody variable region having binding activity to CD3) may bind to any epitope present in the gamma, delta, or epsilon chain sequence that constitutes CD3. In the present invention, a domain containing the light chain variable region (VL) and heavy chain variable region (VH) of an anti-CD3 antibody that binds to an epitope present in the extracellular domain of the epsilon chain of the human CD3 complex is preferably used. The light chain variable region (VL) and heavy chain variable region (VH) of an anti-CD3 antibody described in the Examples are preferably used as such a domain. As described above, an appropriate humanized antibody or human antibody is used as the anti-CD3 antibody that is the source of the domain containing an antibody variable region having CD3-binding activity. The polynucleotide sequences of the gamma, delta, and epsilon chains that make up CD3 are registered under RefSeq accession numbers NM_000073.2, NM_000732.4, and NM_000733.3, respectively, and their polypeptide sequences are registered under RefSeq accession numbers NP_000064.1, NP_000723.1, and NP_000724.1, respectively.

Domains Comprising Antibody Variable Regions with Binding Activity to Antigens Other Than CD3: Herein, the terms "antibody variable region with binding activity to antigens other than CD3" and "domains comprising antibody variable regions with binding activity to antigens other than CD3" are used interchangeably and refer to portions of antibodies comprising a region that specifically binds to and is complementary to part or all of the antigen (e.g., a cancer antigen; also referred to herein as a cancer-specific antigen). A domain comprising an antibody variable region can be provided by one or more antibody variable domains. Preferably, a domain comprising an antibody variable region comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Suitable examples of such domains comprising antibody variable regions include "scFv (single chain Fv),""single chain antibody,""Fv,"" _scFv2 (single chain Fv2),""Fab," and "F(ab') ₂ ."

Antigen : As used herein, the term "antigen" is limited to including only the epitope to which the antigen-binding domain binds. Suitable examples of antigens include, but are not limited to, peptides, polypeptides, and proteins derived from animals or humans. Suitable examples of antigens used to treat diseases caused by target tissues include, but are not limited to, molecules expressed on the surface of target cells (e.g., cancer cells and inflammatory cells), molecules expressed on the surface of other cells in tissues containing target cells, molecules expressed on the surface of cells that play an immunological role in target cells and tissues containing target cells, and large molecules present in the interstitium of tissues containing target cells.

In one embodiment, the antigen may be derived from any animal species (e.g., human; or non-human animal, e.g., mouse, rat, hamster, guinea pig, rabbit, monkey, cynomolgus monkey, rhesus monkey, hamadryas baboon, chimpanzee, goat, sheep, dog, horse, pig, cow, or camel), or any bird; preferably, the antigen is derived from human, rabbit, monkey, rat, or mouse.

Unless otherwise specified, the "antigen" (e.g., CD3) recognized and bound by the antigen-binding molecules of the present invention refers to any naturally occurring "antigen" (e.g., CD3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length" unprocessed "antigen" (e.g., CD3) as well as any form of "antigen" (e.g., CD3) that results from processing within a cell. These terms also encompass naturally occurring variants of the "antigen" (e.g., CD3), such as splice variants and allelic variants.

In some embodiments, antigens include 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3/osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6, Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, C CL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD 4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, claudin 6, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CX CL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXC R2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatory factor (Decay accelerating factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DLL3, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, Ef Phospholipase B2/EphB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractal chi FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2 , IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor receptor 1 (IGFR), insulin-like growth factor receptor 2 (IGFR), insulin-like growth factor receptor 3 (IGFR), insulin-like growth factor receptor 4 (IGFR), insulin-like growth factor receptor 5 (IGFR), insulin-like growth factor receptor 6 (IGFR), insulin-like growth factor receptor 7 (IGFR), insulin-like growth factor receptor 8 (IGFR), insulin-like growth factor receptor 9 (IGFR), insulin-like growth factor receptor 1 ... factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4/beta 1, integrin alpha 4/beta 7, integrin alpha 5 (alpha V), integrin alpha 5/beta 1, integrin alpha 5/beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MG DF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NCadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid-stimulating hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-α connectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-like receptor) 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von・Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT 11, WNT16, XCL1, XCL2, K9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, Examples of antigens include fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, and S1P. In some embodiments, the antigen includes receptors for hormones and growth factors. In certain embodiments, the antigen is an antigen molecule expressed or secreted by cells contained in tumor tissue (e.g., tumor cells, immune cells, stromal cells, etc.), particularly an antigen specifically expressed by cancer cells (cancer-specific antigens). In certain embodiments, the antigen is an antigenic molecule expressed on the surface of a cell that functions to suppress an immune response.

" Cancer-specific antigens " as non-limiting examples of antigens on target cells refer to antigens expressed by cancer cells that enable the distinction between cancer cells and healthy cells. Examples include antigens expressed as cells become malignant, and abnormal sugar chains that appear on the cell surface or protein molecules when cells become cancerous. Specific examples include ALK, pleiotrophin (PTN), EpCAM, CA125, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), TYRP1, HMW-MAA, prostate-specific membrane antigen (PSMA), CEA, MUC1, HMFG1, TAG-72, GICA (CA19-9), NY-ESO-1, LEA, CD15, CD17, CD19, CD20, CD22, CD30, CD33, CD38, CD77, CD79b, CD147, CD228, GD2, GD3, GM2, GM3, TSTA, and virus-induced tumors. Antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), alpha-fetoprotein (AFP), 5T4, differentiation antigens (e.g., L6 and L20 antigens), CD165, EGFR, ANKRD17, ErbB2, APO-1, SSEA-1, SCP-1, LeY, oligosaccharide antigens, SSEA-3, SSEA-4, CTAGE1, MART-1, sialyl Tn (STn), NY-CO-45, NY-LU-12, ART1, MA2, NOVA2, TSPAN8, MAGE-C1, MAGE-B1, MAGE-B2, MAGE-4A, Examples of polypeptides include MAGE-X2, YKL-40, EREG, CA15-3, CLEC12A, Nectin4, Trop2, BCMA, Tissue factor, FRα (FOLR1), ErbB3, Claudin18 (Claudin18.2), B7-H3 (CD276), MET, PSCA, PTK7, MSLN (Mesothelin), CCR4, CDH6, IL13RA2, GPC3, XPR1, NOX1, MARVELD3 isoform 1, MARVELD3 isoform 2, SPINT2, MANSC1, SLC12A2, CDCP1, SEZ6L2, FLVCR1, SLC7A5, STEAP1, MMP14, TNFRSF21, TMPRSS4, claudin 6, and DLL3, as well as any fragments of these polypeptides and modified structures thereof.

Antigens expressed on the surface of cells that have the function of suppressing an immune response "cells that have the function of suppressing an immune response" are not particularly limited as long as they have the function of suppressing an immune response, and examples include regulatory T cells (Treg), exhausted T cells, myeloma-derived stromal cells (MDSC), tumor-associated macrophages (TAM), induced regulatory T cells (Tr1), tumor-associated dendritic cells (TADC), tumor-associated neutrophils (TAN), cancer-associated fibroblasts (CAF), and regulatory B cells (Breg). Regulatory T cells and exhausted T cells are particularly preferred as target cells of the present invention. Specific examples of molecules expressed on the surface of cells that have the function of suppressing these immune responses include CTLA4, PD1, TIM3, LAG-3, CD244 (2B4), CD160, GARP, OX40, CD137 (4-1BB), CD25, VISTA, VISATA, BTLA, TNFR25, CD57, KLRG1, CCR2, CCR5, CCR6, CD39, CD73, CD4, CD18, CD49b, CD1d, CD5, CD21, TIM1, CD19, CD20, CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, PD-L1, CD11b, Ly6G, ICAM-1, FAP, PDGFR, podoplanin, and TIGIT. Among these molecules, preferred target molecules for the binding domains of the present invention include, for example, CTLA4, TIM3, LAG3, CD137 (4-1BB), CD25, CCR5, CCR6, CD38, and TIGIT, which are cell surface molecules specifically expressed on cell fractions (CD4 ⁺ , CD25 ^high , CD45RA ^- ) that have been reported to have high immune response suppression function. Preferred target molecules for the binding domains of the present invention include, in particular, CTLA4, LAG3, and OX40.

Antigen-binding activity: Methods for confirming epitope binding by test antigen-binding molecules having domains containing antibody variable regions with binding activity to CD3 are exemplified below. Methods for confirming epitope binding by test antigen-binding molecules having domains containing antibody variable regions with binding activity to other antigens can also be appropriately performed in accordance with the examples below.

For example, whether a test antigen-binding molecule containing a domain comprising an antibody variable region with binding activity to CD3 recognizes a linear epitope present in the CD3 molecule can be confirmed, for example, as follows. For this purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of CD3 is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region in CD3 cDNA that encodes the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide consisting of the amino acid sequence constituting the extracellular domain to a test antigen-binding molecule containing a domain comprising an antibody variable region with binding activity to CD3 is assessed. For example, the binding activity of the antigen-binding molecule to the peptide can be assessed by ELISA using immobilized linear peptide as the antigen. Alternatively, the binding activity of the antigen-binding molecule to the linear peptide can be determined based on the level of inhibition by the linear peptide of binding of the antigen-binding molecule to CD3-expressing cells. These tests can determine the binding activity of the antigen-binding molecule to the linear peptide.

Furthermore, whether a test antigen-binding molecule having a domain containing an antibody variable region that has binding activity to CD3 recognizes a conformational epitope can be confirmed as follows. For this purpose, CD3-expressing cells are prepared. Examples of such cases include when a test antigen-binding molecule having a domain containing an antibody variable region that has binding activity to CD3 binds strongly to CD3-expressing cells upon contact with the cells, but does not substantially bind to a linear peptide consisting of the amino acid sequence that constitutes the extracellular domain of immobilized CD3. Here, "not substantially binding" refers to a binding activity that is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity toward human CD3-expressing cells.

Methods for measuring the binding activity of a test antigen-binding molecule containing a CD3 antigen-binding domain to CD3-expressing cells include, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, evaluation can be performed using ELISA or FACS (fluorescence activated cell sorting) principles, using CD3-expressing cells as antigens.

In the ELISA format, the binding activity of a test antigen-binding molecule containing a CD3 antigen-binding domain for CD3-expressing cells is quantitatively assessed by comparing the signal levels generated by the enzymatic reaction. That is, the test antigen-binding molecule is added to an ELISA plate on which CD3-expressing cells have been immobilized, and the test antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding molecule. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared, and the antibody binding titer for CD3-expressing cells is determined, allowing the binding activity of the test antigen-binding molecule for CD3-expressing cells to be compared.

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

For example, the following method is an example of a suitable method for measuring the antigen-binding activity of a test antigen-binding molecule containing a CD3 antigen-binding domain. First, the test antigen-binding molecule is reacted with CD3-expressing cells and stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding molecule. The test antigen-binding molecule is diluted with an appropriate buffer solution to prepare the complex at the desired concentration. For example, it can be used at a concentration between 10 μg/ml and 10 ng/ml. Next, the fluorescence intensity and cell count are measured using a FACSCalibur (BD). The amount of antibody binding to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the Geometric Mean value, the binding activity of the test antigen-binding molecule, represented by the amount of binding of the test antigen-binding molecule, can be measured.

Whether a test antigen-binding molecule containing a CD3 antigen-binding domain shares an epitope with another antigen-binding molecule can be confirmed by competition between the two for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. A competitive ELISA assay is a preferred cross-blocking assay.

Specifically, in a cross-blocking assay, CD3 protein coated on the wells of a microtiter plate is pre-incubated in the presence or absence of a candidate competing antigen-binding molecule, after which the test antigen-binding molecule is added. The amount of test antigen-binding molecule bound to CD3 protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding molecule that competes for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to CD3 protein-coated wells.

The amount of test antigen-binding molecules bound to the wells via CD3 protein can be easily measured by labeling the antigen-binding molecules in advance. For example, biotin-labeled antigen-binding molecules can be measured using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are specifically referred to as competitive ELISA assays. Antigen-binding molecules can also be labeled with other detectable or measurable labeling substances. Specific examples include radiolabels and fluorescent labels.

If a competitor antigen-binding molecule can block the binding of a test antigen-binding molecule comprising an antigen-binding domain to CD3 by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity obtained in a control test performed in the absence of the candidate competitor antigen-binding molecule, the test antigen-binding molecule binds to substantially the same epitope as the competitor antigen-binding molecule, or is an antigen-binding molecule that competes for binding to the same epitope.

If the structure of the epitope to which a test antigen-binding molecule containing a CD3 antigen-binding domain binds has been identified, whether the test antigen-binding molecule and a control antigen-binding molecule share the same epitope can be assessed by comparing the binding activity of both antigen-binding molecules to a peptide in which amino acid mutations have been introduced into the peptide constituting the epitope.

Such binding activity can be measured, for example, by comparing the binding activity of test and control antigen-binding molecules to a mutated linear peptide in the ELISA format described above. As a method other than ELISA, the binding activity to the mutant peptide bound to a column can also be measured by passing the test and control antigen-binding molecules down the column and then quantifying the antigen-binding molecules eluted in the eluate. Methods for adsorbing mutant peptides to a column, for example as fusion peptides with GST, are known.

Furthermore, if the identified epitope is a conformational epitope, whether the test and control antigen-binding molecules share the same epitope can be assessed using the following method. First, CD3-expressing cells and cells expressing CD3 with an epitope mutation introduced are prepared. These cells are suspended in an appropriate buffer solution such as PBS, and the test and control antigen-binding molecules are added to the cell suspension. Next, an FITC-labeled antibody that can recognize the test and control antigen-binding molecules is added to the cell suspension after washing with an appropriate buffer solution. The fluorescence intensity and cell count of cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test and control antigen-binding molecules are diluted with an appropriate buffer solution to the desired concentration before use. For example, they are used at a concentration between 10 μg/ml and 10 ng/ml. The amount of labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the geometric mean value, obtained by analysis using CELLQUEST Software (BD). In other words, by obtaining the Geometric Mean value, it is possible to measure the binding activity of the test antigen-binding molecule and the control antigen-binding molecule, which is represented by the amount of bound labeled antibody.

In this method, for example, "substantially no binding to mutant CD3-expressing cells" can be determined by the following method. First, test and control antigen-binding molecules bound to cells expressing mutant CD3 are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used for flow cytometry to detect fluorescence, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The percentage increase in fluorescence intensity due to antigen-binding molecule binding can be determined by calculating the comparative value (ΔGeo-Mean) from the Geometric Mean values in the presence and absence of the antigen-binding molecule using the formula below.

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

The Geometric Mean comparison value (mutant CD3 molecule ΔGeo-Mean value) obtained by analysis, which reflects the binding amount of the test antigen-binding molecule to mutant CD3-expressing cells, is compared with the ΔGeo-Mean comparison value, which reflects the binding amount of the test antigen-binding molecule to CD3-expressing cells. In this case, it is particularly preferable that the concentrations of the test antigen-binding molecule used to determine the ΔGeo-Mean comparison values for mutant CD3-expressing cells and CD3-expressing cells are adjusted to be identical or substantially identical to each other. An antigen-binding molecule that has been previously confirmed to recognize an epitope in CD3 is used as a control antigen-binding molecule.

If the ΔGeo-Mean comparison value of a test antigen-binding molecule for mutant CD3-expressing cells is at least 80%, preferably 50%, more preferably 30%, and especially preferably 15% of the ΔGeo-Mean comparison value of the test antigen-binding molecule for CD3-expressing cells, the antigen-binding molecule is deemed to "not substantially bind to mutant CD3-expressing cells." The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). If the comparison values are substantially equivalent, the epitopes of the test antigen-binding molecule and the control antigen-binding molecule can be determined to be identical.

Antigen-binding molecules containing an FcRn-binding domain contained in the Fc region Preferred examples of the antigen-binding molecules of the present invention include antigen-binding molecules containing an FcRn-binding domain contained in the Fc region of an antibody. A well-known method for extending the blood half-life of a protein administered to the body involves adding an antibody FcRn-binding domain to the protein of interest and utilizing the FcRn-mediated recycling function.

In the present invention, the term "FcRn-binding domain" is not particularly limited as long as it has binding activity to FcRn, and may be a domain that directly binds to FcRn or a domain that indirectly binds to FcRn. Examples of domains that directly bind to FcRn include the variable region of an antibody whose antigen is FcRn, Fab, the Fc region of an antibody, fragments thereof, albumin, albumin domain 3, human serum albumin (HSA), and transferrin. Examples of domains that indirectly bind to FcRn include domains that have binding activity to the above-mentioned domains that directly bind to FcRn. In the context of the present invention, one embodiment of an FcRn-binding domain is the Fc region of an antibody, or a fragment of the Fc region that contains the FcRn-binding region. Examples of "Fc region" include IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM Fc regions, and an Fc region derived from a native IgG can be used. Native IgG refers to a polypeptide that includes the same amino acid sequence as IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. For example, native human IgG refers to native human IgG1, native human IgG2, native human IgG3, native human IgG4, etc. Native IgG also includes naturally occurring variants thereof. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotype sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, and any of these may be used in the present invention. In particular, for the sequence of human IgG1, the amino acid sequence at EU numbering positions 356 to 358 may be DEL or EEM.

In some embodiments, the antigen-binding molecules of the present invention comprise an antibody Fc region. When the antigen-binding molecules of the present invention comprise a single antigen-binding domain, the antigen-binding domain may be linked to one of the two polypeptides constituting the dimeric Fc region. When the antigen-binding molecules of the present invention comprise a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain may be linked to one of the two polypeptides constituting the dimeric Fc region, and the second antigen-binding domain may be linked to the other, but this is not a limitation. In certain embodiments, the antigen-binding molecules of the present invention comprise an antibody Fc region with reduced Fcγ receptor-binding activity. When the multispecific antigen-binding molecules of the present invention comprise an antibody Fc region, it is preferable to use an antibody Fc region with reduced Fcγ receptor-binding activity to avoid the target antigen-independent induction of various cytokine production, as seen in trifunctional antibodies such as catumaxomab.
Various amino acid mutations (modifications) in the Fc region of an antibody that result in reduced binding activity to Fcγ receptors are known, and Fc regions bearing such known amino acid mutations (modifications) or other mutations (modifications) that will be identified in the future can be used in the antigen-binding molecules of the present invention. Furthermore, the strength of the effector function acting through the binding of the antibody Fc region to the Fcγ receptor varies depending on the IgG subclass, and in humans, it is known to be high in IgG1 and IgG3 and low in IgG2 and IgG4. Therefore, native Fc regions of human IgG2 or IgG4 can also be used as the Fc region of an antibody with reduced binding activity to Fcγ receptors that is included in the antigen-binding molecules of the present invention.

Fcγ Receptor An Fcγ receptor refers to a receptor that can bind to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody, and refers to any member of a family of proteins substantially encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγRs or FcγR isoforms or allotypes. FcγRs may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγRs or FcγR isoforms or allotypes. Preferred examples of such Fcγ receptors include human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and/or FcγRIIIB (CD16). The polynucleotide and amino acid sequences of FcγRI are registered under RefSeq accession numbers NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIA are registered under RefSeq accession numbers BC020823.1 and AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIB are registered under RefSeq accession numbers BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIA are registered under RefSeq accession numbers BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIB are registered under RefSeq accession numbers BC128562.1 and AAI28563.1, respectively. Whether an Fcγ receptor has binding activity to the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be confirmed by the FACS or ELISA formats described above, as well as by ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method using surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

Fcγ Receptor-Binding Activity: Reduced binding activity of an Fc region to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and/or FcγIIIB can be confirmed by the FACS and ELISA formats described above, as well as ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method using surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

The ALPHA screen is performed using ALPHA technology, which uses two beads, donor and acceptor, based on the following principle: A luminescent signal is detected only when a molecule bound to the donor bead biologically interacts with a molecule bound to the acceptor bead and the two beads are in close proximity. A photosensitizer inside the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches a nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately resulting in the emission of light. If the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescent reaction occurs.

For example, biotin-labeled antigen-binding molecules are bound to donor beads, and glutathione S-transferase (GST)-tagged Fcγ receptors are bound to acceptor beads. In the absence of competing antigen-binding molecules with mutant Fc regions, antigen-binding molecules with wild-type Fc regions interact with Fcγ receptors, generating a signal at 520-620 nm. Antigen-binding molecules with untagged mutant Fc regions compete with the interaction between antigen-binding molecules with wild-type Fc regions and Fcγ receptors. Relative binding affinity can be determined by quantifying the decrease in fluorescence that occurs as a result of competition. It is known to biotinylate antigen-binding molecules such as antibodies using sulfo-NHS-biotin or similar. Methods for tagging Fcγ receptors with GST include expressing a fusion gene in which a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST are fused in-frame in cells harboring an expression vector, followed by purification using a glutathione column. The resulting signals are suitably analyzed by fitting them to a one-site competition model using nonlinear regression analysis, for example, using software such as GRAPHPAD PRISM (GraphPad, San Diego).

One of the substances (ligand) whose interaction is to be observed is immobilized on the gold film of a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light shows a decrease in reflection intensity (SPR signal). When the other substance (analyte) whose interaction is to be observed is poured over the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, when the bond dissociates, the signal position returns). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The kinetics (binding rate constant (ka) and dissociation rate constant (kd)) can be calculated from the sensorgram curve, and affinity (KD) can be calculated from the ratio of these constants. Inhibition measurement methods are also suitable for use with the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

As used herein, "decreased binding activity to an Fcγ receptor" means, for example, that the competitive activity of a test 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 particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the binding activity of a control antigen-binding molecule having an Fc region, based on the above-mentioned analytical method.

Antigen-binding molecules having the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be used as a control, as appropriate. These Fc regions may have structures in which an A is added to the N-terminus of the amino acid sequences registered under RefSeq accession numbers AAC82527.1, AAB59393.1, CAA27268.1, and AAB59394.1, respectively. In one embodiment, when an antigen-binding molecule having a mutant Fc region of an antibody of a certain isotype is used as a test substance, the effect of the mutation in the mutant on the binding activity to Fcγ receptors can be verified by using an antigen-binding molecule having the Fc region of the antibody of that specific isotype as a control. As described above, antigen-binding molecules having Fc region mutants verified to have reduced binding activity to Fcγ receptors can be appropriately prepared. In another embodiment, the Fc region of an antibody of a certain isotype is compared with the Fc region of an antibody of another certain isotype in terms of Fcγ receptor-binding activity, and the Fc region with lower Fcγ receptor-binding activity can be used in the antigen-binding molecules of the present invention. Furthermore, amino acid mutations can be introduced into such an Fc region with low Fcγ receptor-binding activity to obtain an Fc region with even lower Fcγ receptor-binding activity.

Known examples of such mutations in the Fc region include deletion of amino acids 231A-238S, identified according to EU numbering (WO 2009/011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54), and C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192).

Preferred examples of such antigen-binding molecules include those having an Fc region in which any of the following amino acids, as specified by EU numbering, have been substituted among the amino acids constituting the Fc region of an antibody of a specific isotype: 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. The antibody isotype from which the Fc region originates is not particularly limited, and Fc regions derived from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used as appropriate.

For example, any of the following substitutions, specified according to EU numbering, among the amino acids constituting the Fc region of an IgG1 antibody (the numbers indicate the positions of the amino acid residues specified according to EU numbering, the single-letter amino acid code preceding the number indicates the amino acid residue before substitution, and the single-letter amino acid code following the number indicates the amino acid residue before substitution):
(a) L234F, L235E, P331S,
(b) C226S, C229S, P238S,
(c) C226S, C229S,
(d) C226S, C229S, E233P, L234V, L235A
(e) L234A, L235A or L235R, N297A
(f) L235A or L235R, S239K, N297A
Alternatively, antigen-binding molecules having an Fc region in which the amino acid sequence at positions 231 to 238 has been deleted may also be used as appropriate.

Furthermore, any of the following substitutions, specified according to EU numbering, among the amino acids constituting the Fc region of an IgG2 antibody (the numbers indicate the amino acid residue positions specified according to EU numbering, the single-letter amino acid code preceding the number indicates the amino acid residue before substitution, and the single-letter amino acid code following the number indicates the amino acid residue before substitution):
(g) H268Q, V309L, A330S, P331S
(h) V234A
(i) G237A
(j) V234A, G237A
(k) A235E, G237A
(l) V234A, A235E, G237A
Antigen-binding molecules having an Fc region modified with α-glucan may also be used appropriately.

Furthermore, any of the following substitutions, specified according to EU numbering, among the amino acids constituting the Fc region of an IgG3 antibody (the numbers indicate the amino acid residue positions specified according to EU numbering, the single-letter amino acid code preceding the number indicates the amino acid residue before substitution, and the single-letter amino acid code following the number indicates the amino acid residue before substitution):
(m) F241A
(n) D265A
(o) V264A
Antigen-binding molecules having an Fc region modified with α-glucan may also be used appropriately.

Furthermore, any of the following substitutions, specified according to EU numbering, among the amino acids constituting the Fc region of an IgG4 antibody (the numbers indicate the amino acid residue positions specified according to EU numbering, the single-letter amino acid code preceding the number indicates the amino acid residue before substitution, and the single-letter amino acid code following the number indicates the amino acid residue before substitution):
(p) L235A, G237A, E318A
(q) L235E
(r) F234A, L235A
Antigen-binding molecules having an Fc region modified with α-glucan may also be used appropriately.

Other preferred examples include antigen-binding molecules having an Fc region in which any of the following amino acids, identified according to EU numbering, that constitute the Fc region of an IgG1 antibody are substituted with amino acids corresponding to the corresponding EU numbering in the corresponding IgG2 or IgG4: 233, 234, 235, 236, 237, 327, 330, and 331.

Other preferred examples include antigen-binding molecules having an Fc region in which one or more of the amino acids at positions 234, 235, and 297, identified according to EU numbering, among the amino acids constituting the Fc region of an IgG1 antibody, have been substituted with other amino acids. The type of amino acid present after substitution is not particularly limited, but antigen-binding molecules having an Fc region in which one or more of the amino acids at positions 234, 235, and 297 have been substituted with alanine are particularly preferred.

Another preferred example is an antigen-binding molecule having an Fc region in which the amino acid at position 265, as specified by EU numbering, among the amino acids constituting the Fc region of an IgG1 antibody, has been substituted with another amino acid. The type of amino acid present after substitution is not particularly limited, but antigen-binding molecules having an Fc region in which the amino acid at position 265 has been substituted with alanine are particularly preferred.

In certain embodiments, the antigen-binding molecules of the present invention comprise an Fc region with reduced binding activity to Fcγ receptors. Examples of such embodiments include antibodies with reduced effector functions attributable to the Fc region, which are desirable when certain effector functions (such as complement and ADCC) are unnecessary or deleterious. Examples of antibodies with reduced effector functions include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

For other examples of Fc region variants, see also U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94/29351.

Method for Producing Antibodies One preferred embodiment of the antigen-binding molecule of the present invention is an antibody comprising the variable region of the antibody of the present invention.

Methods for producing antibodies with the desired binding activity are known to those skilled in the art, and they can be obtained as polyclonal or monoclonal antibodies. Monoclonal antibodies derived from mammals are preferably produced as antibodies of the present invention. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed by genetic engineering techniques with an expression vector containing an antibody gene.

The mammal to be immunized to obtain antibodies is not limited to a specific animal, but it is preferable to select it taking into consideration its compatibility with the parent cells used in cell fusion to produce hybridomas. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

The above-mentioned animals are immunized with the sensitizing antigen according to known methods. For example, a common method is to administer the sensitizing antigen to the mammal by intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen is diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, and if desired, mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. An appropriate carrier may also be used when immunizing with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered to the immunized animal, and a sensitizing antigen is expressed in the immunized animal's body, thereby conferring immune stimulation. Compared to general immunization methods in which a protein antigen is administered to the immunized animal, DNA immunization is expected to have the following advantages:
- Immunostimulation can be achieved by maintaining the structure of membrane proteins - No need to purify the immunizing antigen

To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA that expresses the antigen protein is administered to the animal to be immunized. DNA encoding the antigen protein can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. As the expression vector, commercially available expression vectors such as pcDNA3.1 can be suitably used. Commonly used methods can be used to administer the vector into the living body. For example, DNA immunization is performed by introducing gold particles to which the expression vector has been adsorbed into the cells of the animal to be immunized using a gene gun.

After a mammal is immunized in this manner and an increase in the antibody titer that binds to the antigen in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. Splenocytes, in particular, are preferred as immune cells.

Mammalian myeloma cells are used as the cells to be fused with the immune cells. It is preferable that the myeloma cells have an appropriate selection marker for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells that are HGPRT or TK deficient are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells are unable to synthesize DNA in HAT selective medium and die, but when fused with normal cells, they can continue to synthesize DNA using the salvage pathway of normal cells, allowing them to grow even in HAT selective medium.

HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in selective media. Another selection marker, called G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cell lines suitable for cell fusion are known.

Such myeloma cells include, for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), and MPC-11 (Cell ( 1976) 8 (3), 405-415), SP2/0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194/5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. can be suitably used.

The immune cells are fused with myeloma cells essentially according to known methods, such as the method of Köhler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).

More specifically, the cell fusion can be carried out in a standard nutrient culture medium in the presence of a cell fusion promoter. Fusion promoters such as polyethylene glycol (PEG) and Sendai virus (HVJ) can be used, and if desired, an adjuvant such as dimethyl sulfoxide can be added to further enhance fusion efficiency.

The ratio of immune cells to myeloma cells can be set as desired. For example, a ratio of immune cells to myeloma cells of 1 to 10 is preferred. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or any other conventional culture medium used for this type of cell culture, which is suitable for growing the myeloma cell line. Furthermore, serum supplements such as fetal calf serum (FCS) may be suitably added.

Cell fusion is performed by thoroughly mixing a predetermined amount of the immune cells and myeloma cells in the culture medium, and then adding a PEG solution (for example, an average molecular weight of approximately 1000 to 6000) that has been preheated to approximately 37°C, usually at a concentration of 30 to 60% (w/v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, the appropriate culture medium listed above is added sequentially, and the mixture is centrifuged and the supernatant is removed repeatedly, allowing cell fusion agents and other substances that are undesirable for hybridoma growth to be removed.

The hybridomas obtained in this manner can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture can be continued using the above-mentioned HAT culture medium for a sufficient period of time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibody are then screened and single-cloned using the conventional limiting dilution method.

Hybridomas obtained in this manner can be selected using a selective culture medium that corresponds to the selection marker possessed by the myeloma used in cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing in HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT culture medium. Culture in the above HAT culture medium is continued for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Hybridomas producing the desired antibody can then be screened and single-cloned using the conventional limiting dilution method.

Screening and single cloning of the desired antibody can be suitably carried out by known screening methods based on antigen-antibody reactions. The desired antibody can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that analyzes cells that have been brought into contact with a fluorescent antibody using laser light, allowing the binding of the antibody to the cell surface to be measured by measuring the fluorescence emitted by individual cells.

To screen hybridomas that produce the monoclonal antibodies of the present invention using FACS, first prepare cells that express the antigen to which the produced antibody binds. Preferred cells for screening are mammalian cells that have been forced to express the antigen. By using untransformed mammalian cells that have been used as host cells as a control, the binding activity of the antibody to the antigen on the cell surface can be selectively detected. In other words, by selecting hybridomas that produce antibodies that do not bind to host cells but that bind to cells that have been forced to express the antigen, hybridomas that produce the desired monoclonal antibody can be obtained.

Alternatively, cells expressing a target antigen can be immobilized, and the binding activity of an antibody to the antigen-expressing cells can be evaluated based on the principles of ELISA. For example, antigen-expressing cells are immobilized in the wells of an ELISA plate. Hybridoma culture supernatant is contacted with the immobilized cells in the wells, and antibodies binding to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas producing the desired antibody capable of binding to the antigen, selected by these screening methods, can be cloned by limiting dilution or other methods.
The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a conventional culture medium and can be stored for a long period of time in liquid nitrogen.

The hybridoma can be cultured according to conventional methods, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to grow, and the monoclonal antibody can be obtained from the ascites fluid. The former method is suitable for obtaining highly pure antibodies.

Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody gene is incorporated into a suitable vector and introduced into a host, whereby the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

To obtain cDNA encoding the variable region (V region) of an antibody, total RNA is usually extracted from the hybridoma. mRNA can be extracted from cells using the following methods, for example.
- Guanidine ultracentrifugation method (Biochemistry (1979) 18 (24), 5294-5299)
-AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

The extracted mRNA can be purified using, for example, an mRNA Purification Kit (manufactured by GE Healthcare Biosciences). Alternatively, kits for extracting total mRNA directly from cells, such as the QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Biosciences), are also commercially available. Using such kits, mRNA can be obtained from hybridomas. cDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) or the like. Additionally, the SMART RACE cDNA Amplification Kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be used appropriately to synthesize and amplify cDNA. Furthermore, during the cDNA synthesis process, appropriate restriction enzyme sites can be introduced at both ends of the cDNA, as described below.

The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. A recombinant vector is thus created, introduced into E. coli or other bacteria, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Whether the recombinant vector contains the base sequence of the desired cDNA is then confirmed using known methods, such as the dideoxynucleotide chain termination method.

The easiest way to obtain genes encoding variable regions is to use the 5'-RACE method, which uses primers for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize the 5'-RACE cDNA library.

Using the resulting 5'-RACE cDNA library as a template, antibody genes are amplified by PCR. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

Specifically, for example, when the goal is to obtain a gene encoding mouse IgG, primers can be used that are capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains, and κ and λ light chains. To amplify the IgG variable region gene, the 3' primer generally anneals to a region corresponding to the constant region close to the variable region. Meanwhile, the 5' primer used is one that comes with the 5' RACE cDNA library construction kit.

The PCR products thus amplified can be used to reconstitute immunoglobulins consisting of a combination of heavy and light chains. The antigen-binding activity of the reconstituted immunoglobulins can be used as an index to screen for the desired antibody. For example, screening can be performed as follows:
(1) contacting an antibody containing a V region encoded by a cDNA obtained from a hybridoma with a cell expressing a desired antigen;
(2) detecting the binding of the antigen-expressing cells to the antibody; and (3) selecting an antibody that binds to the antigen-expressing cells.

Methods for detecting the binding between an antibody and the antigen-expressing cells are known. Specifically, the binding between an antibody and the antigen-expressing cells can be detected by techniques such as the FACS mentioned above. Fixed specimens of the antigen-expressing cells can be used as appropriate to evaluate the binding activity of the antibody.

Panning methods using phage vectors are also suitable as a screening method for antibodies using binding activity as an indicator. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, screening methods using phage vectors are advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with an appropriate linker sequence to form single-chain Fvs (scFvs). By inserting a gene encoding an scFv into a phage vector, a phage that expresses scFv on its surface can be obtained. After contacting this phage with the desired antigen, DNA encoding an scFv with the desired binding activity can be recovered by recovering the phage that has bound to the antigen. By repeating this procedure as necessary, scFvs with the desired binding activity can be concentrated.

After obtaining cDNA encoding the V region of the desired antibody, the cDNA is digested with a restriction enzyme that recognizes restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that appear infrequently in the nucleotide sequences constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into a vector in the correct orientation, it is preferable to insert a restriction enzyme that generates a cohesive end. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the antibody digested as described above into an appropriate expression vector. If the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in frame, a chimeric antibody can be obtained. Here, a chimeric antibody refers to an antibody in which the constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already contains the constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately placed on the 5' side of an expression vector carrying DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusing the two genes in frame after digestion with the same combination of restriction enzymes.

To produce a monoclonal antibody, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. In addition, an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly. The signal sequence is cleaved from the carboxyl terminal portion of the expressed polypeptide, allowing the antibody to be secreted extracellularly. Next, by transforming an appropriate host cell with this expression vector, recombinant cells expressing DNA encoding the antibody can be obtained.

To express antibody genes, DNA encoding the antibody heavy chain (H chain) and light chain (L chain) are each incorporated into separate expression vectors. By simultaneously transforming (co-transfecting) the same host cell with vectors incorporating the H chain and L chain, antibody molecules equipped with both H chains and L chains can be expressed. Alternatively, host cells can be transformed by incorporating DNA encoding the H chain and L chain into a single expression vector (see International Publication WO 94/11523).

Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into suitable hosts. All of these expression systems can be applied to isolating antigen-binding domains contained in the antigen-binding molecules of the present invention.

When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be used as appropriate. Specific examples of animal cells include the following:
(1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc. (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

Alternatively, an antibody gene expression system using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, is known. For transformation of plant cells, callus cultured cells can be used as appropriate.

Furthermore, the following fungal cells can be used:
- Yeasts: Saccharomyces genus such as Saccharomyces cerevisiae, Pichia genus such as Pichia pastoris - Filamentous fungi: Aspergillus genus such as Aspergillus niger

Antibody gene expression systems using prokaryotic cells are also known. For example, when using bacterial cells, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be used as appropriate. An expression vector containing the desired antibody gene is introduced into these cells by transformation. By culturing the transformed cells in vitro, the desired antibody can be obtained from the culture of the transformed cells.

In addition to the host cells described above, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein specifically produced in milk. Examples of proteins secreted into milk include goat beta-casein. A DNA fragment containing the fusion gene with the antibody gene inserted is injected into a goat embryo, and the injected embryo is then introduced into a female goat. The transgenic goat (or its offspring) born to the goat that received the embryo produces milk from which the desired antibody can be obtained as a fusion protein with a milk protein. Furthermore, hormones can be administered to transgenic goats to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio/Technology (1994), 12 (7), 699-702).

When the antigen-binding molecules described herein are administered to humans, for example, when domains containing antibody variable regions are used as various binding domains in the molecules, antigen-binding domains derived from recombinant antibodies that have been artificially modified to reduce heterologous antigenicity to humans can be appropriately used. Recombinant antibodies include, for example, humanized antibodies. These modified antibodies can be appropriately produced using known methods.

The antibody variable regions used to create the various binding domains in the antigen-binding molecules described herein are typically composed of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDRs are essentially the regions that determine the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. On the other hand, the amino acid sequences that make up FRs often show high identity even between antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of one antibody can be transferred to another antibody by CDR grafting.

Humanized antibodies are also called reshaped human antibodies. Specifically, known humanized antibodies include those in which the CDRs of non-human animals, such as mouse antibodies, are grafted onto human antibodies. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a well-known method for grafting the CDRs of mouse antibodies onto human FRs. In overlap extension PCR, the base sequence encoding the CDR of the mouse antibody to be grafted is added to a primer used to synthesize the FR of the human antibody. Primers are prepared for each of the four FRs. In general, when grafting mouse CDRs onto human FRs, selecting a human FR that is highly identical to the mouse FR is considered advantageous in terms of maintaining CDR function. In other words, it is generally preferable to use a human FR whose amino acid sequence is highly identical to the amino acid sequence of the FR adjacent to the mouse CDR to be grafted.

The base sequences to be linked are also designed to be connected in-frame to each other. Human FRs are synthesized individually using each primer. As a result, products are obtained in which DNA encoding mouse CDRs is added to each FR. The base sequences encoding the mouse CDRs of each product are designed to overlap with each other. Next, the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed to each other to carry out a complementary strand synthesis reaction. Through this reaction, the human FRs are linked via the mouse CDR sequences.

The V-region gene, which ultimately contains the three CDRs and four FRs, is amplified in its entirety using primers that anneal to its 5' and 3' ends and have appropriate restriction enzyme recognition sequences added. A human antibody expression vector can be created by inserting the DNA obtained as described above into an expression vector so that it is fused in-frame with DNA encoding the human antibody C-region. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed, resulting in the production of the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400 and International Publication WO 1996/002576).

By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, it is possible to select a suitable FR of a human antibody that will form a good antigen-binding site when linked via the CDR. If necessary, amino acid residues in the FR can be substituted so that the CDR of the reshaped human antibody forms a suitable antigen-binding site. For example, the PCR method used to graft mouse CDRs onto human FRs can be applied to introduce amino acid sequence mutations into the FR. Specifically, partial nucleotide sequence mutations can be introduced into primers that anneal to the FR. The nucleotide sequence mutations will be introduced into the FR synthesized using such primers. By measuring and evaluating the antigen-binding activity of mutant antibodies with amino acid substitutions using the above method, mutant FR sequences with desired properties can be selected (Sato, K. et al., Cancer Res, 1993, 53, 851-856).

In addition, transgenic animals carrying the entire repertoire of human antibody genes (see International Publications WO1993/012227, WO1992/003918, WO1994/002602, WO1994/025585, WO1996/034096, and WO1996/033735) can be used as immunized animals, and the desired human antibodies can be obtained by DNA immunization.

Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv) using phage display. Phages expressing scFvs that bind to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of a human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, an expression vector can be created by fusing the V region sequence in frame with the sequence of the C region of the desired human antibody and then inserting it into an appropriate expression vector. The expression vector is introduced into a suitable expression cell such as those listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see International Publications WO1992/001047, WO1992/020791, WO1993/006213, WO1993/011236, WO1993/019172, WO1995/001438, and WO1995/015388).

In addition to phage display, other known techniques for obtaining human antibodies by panning using a human antibody library include techniques using cell-free translation systems, techniques that display antigen-binding molecules on the surface of cells or viruses, and techniques that use emulsions. For example, techniques that use cell-free translation systems include ribosome display, which forms a complex between mRNA and the translated protein via ribosomes by removing stop codons, cDNA display, which covalently binds a gene sequence to the translated protein using compounds such as puromycin, and mRNA display, and CIS display, which forms a complex between a gene and the translated protein using a nucleic acid-binding protein. Furthermore, in addition to phage display, other techniques for displaying antigen-binding molecules on the surface of cells or viruses include E. coli display, Gram-positive bacteria display, yeast display, mammalian cell display, and virus display. Techniques that use emulsions include in vitro virus display, which involves encapsulating genes and translation-related molecules in an emulsion. These methods are already known (Nat Biotechnol. 2000 Dec;18(12):1287-92, Nucleic Acids Res. 2006;34(19):e127, Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2806-10, Proc Natl Acad Sci U S A. 2004 Jun 2 2;101(25):9193-8, Protein Eng Des Sel. 2008 Apr;21(4):247-55, Proc Natl Acad Sci U S A. 200 0 Sep 26;97(20):10701-5, MAbs. 2010 Sep-Oct;2(5):508-18, Methods Mol Biol. 2012;911:183-98).

Method for Producing Multispecific Antibodies One preferred embodiment of the antigen-binding molecules of the present invention is a multispecific antibody. When an Fc region with reduced Fcγ receptor-binding activity is used as the Fc region of a multispecific antibody of the present invention, an Fc region derived from a known multispecific antibody can also be used as appropriate. Bispecific antibodies are particularly preferred as multispecific antibodies of the present invention.

To form multispecific antibodies, a technique can be applied that suppresses undesired association between heavy chains by introducing charge repulsion at the interface of the second constant region (CH2) or third constant region (CH3) of the antibody heavy chain (WO2006/106905).

In the technology for suppressing unintended association between H chains by introducing charge repulsion at the CH2 or CH3 interface, examples of amino acid residues that come into contact at the interface of other H chain constant regions include the regions corresponding to residues 356 (EU numbering), 439 (EU numbering), 357 (EU numbering), 370 (EU numbering), 399 (EU numbering), and 409 (EU numbering) in the CH3 region.

More specifically, for example, an antibody containing two types of H chain CH3 regions can be an antibody in which one to three pairs of amino acid residues selected from the following pairs of amino acid residues (1) to (3) in the first H chain CH3 region have the same charge: (1) amino acid residues contained in the H chain CH3 region, which are amino acid residues at positions 356 and 439 (EU numbering), (2) amino acid residues contained in the H chain CH3 region, which are amino acid residues at positions 357 and 370 (EU numbering), and (3) amino acid residues contained in the H chain CH3 region, which are amino acid residues at positions 399 and 409 (EU numbering).

Furthermore, the antibody may have a second H chain CH3 region different from the first H chain CH3 region, and the second H chain CH3 region has a set of amino acid residues selected from the sets of amino acid residues shown in (1) to (3), wherein one to three sets of amino acid residues corresponding to the sets of amino acid residues shown in (1) to (3) that have the same charge in the first H chain CH3 region have an opposite charge to the corresponding amino acid residues in the first H chain CH3 region.

The amino acid residues described in (1) to (3) above are close to each other when associated. Those skilled in the art can find the positions corresponding to the amino acid residues described in (1) to (3) above for the desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can modify the amino acid residues at those positions as appropriate.

In the above-described antibody, the "charged amino acid residue" is preferably selected from amino acid residues included in either group (a) or (b) below:
(a) glutamic acid (E), aspartic acid (D),
(b) Lysine (K), arginine (R), histidine (H).

In the above-mentioned antibodies, "having the same charge" means, for example, that two or more amino acid residues all have amino acid residues included in either group (a) or (b) above. "Having opposite charges" means, for example, that when at least one amino acid residue among two or more amino acid residues has an amino acid residue included in either group (a) or (b) above, the remaining amino acid residues have amino acid residues included in a different group.

In a preferred embodiment, the antibody may have the first H chain CH3 region and the second H chain CH3 region cross-linked by a disulfide bond.

The amino acid residues that can be modified in the present invention are not limited to those in the antibody variable region or constant region described above. Those skilled in the art can identify amino acid residues that form an interface between polypeptide mutants or heteromultimers by homology modeling using commercially available software, and can modify the amino acid residues at those sites to control association.

Furthermore, other known techniques can also be used to aggregate the multispecific antibodies of the present invention. By substituting a larger side chain (knob) for an amino acid side chain in the variable region of one of the antibody's heavy chains and a smaller side chain (hole) for an amino acid side chain in the opposing variable region of the other heavy chain, the knob can be positioned in the hole, thereby efficiently facilitating aggregation between polypeptides with different amino acids that have Fc domains (WO1996/027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al., Nature Biotechnology (1998) 16, 677-681, US20130336973).

In addition, other known techniques can also be used to form the multispecific antibodies of the present invention. By using a strand-exchange engineered domain CH3, in which part of the CH3 of one antibody H chain is replaced with a corresponding IgA-derived sequence, and the complementary part of the CH3 of the other H chain is replaced with a corresponding IgA-derived sequence, it is possible to efficiently induce association of polypeptides with different sequences through complementary association of CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently form the desired multispecific antibodies.

Other methods for forming multispecific antibodies include antibody production techniques that utilize the association of antibody CH1 and CL, and VH and VL, as described in WO2011/028952, WO2014/018572, and Nat Biotechnol. 2014 Feb;32(2):191-8.; techniques for producing bispecific antibodies using separately prepared monoclonal antibodies (Fab Arm Exchange), as described in WO2008/119353 and WO2011/131746; and techniques described in WO2012/058768 and Other techniques that can be used include the technology described in WO2013/063702 for controlling the association between CH3s of antibody heavy chains, the technology described in WO2012/023053 for producing bispecific antibodies consisting of two types of light chains and one type of heavy chain, and the technology described in Christoph et al. (Nature Biotechnology Vol. 31, pp. 753-758 (2013)) for producing bispecific antibodies using two bacterial cell lines that each express one half of an antibody chain consisting of one H chain and one L chain.

As mentioned above, one embodiment of multispecific antibody formation involves mixing two types of monoclonal antibodies in the presence of a reducing agent, cleaving the disulfide bonds in the core hinge, and then reassociating them to obtain a heterodimerized bispecific antibody (FAE). However, by introducing electrostatic interactions (WO2006/106905) into the interaction interface of the CH3 region, heterodimerization can be induced more efficiently during reassociation (WO2015/046467). With FAE using native IgG, reassociation occurs randomly, so theoretically bispecific antibodies can only be obtained with a 50% efficiency, but this method allows for the production of bispecific antibodies with a high yield.

Even if the desired multispecific antibody cannot be efficiently formed, it is still possible to obtain the multispecific antibody of the present invention by isolating and purifying it from the produced antibodies. For example, a method has been reported in which amino acid substitutions are introduced into the variable regions of two types of H chains to impart a difference in isoelectric point, making it possible to purify two types of homoantibodies and the desired heteroantibody by ion exchange chromatography (WO2007114325). Furthermore, a method for purifying heteroantibodies has been reported in which a heterodimerized antibody consisting of a mouse IgG2a H chain that binds to Protein A and a rat IgG2b H chain that does not bind to Protein A is purified using Protein A (WO98050431, WO95033844). Furthermore, by using H chains in which the amino acid residues at EU numbering positions 435 and 436, which are the binding sites between IgG and Protein A, have been replaced with amino acids such as Tyr and His that have different binding strengths to Protein A, the interaction between each H chain and Protein A can be changed, and by using a Protein A column, it is possible to efficiently purify only the heterodimerized antibody.

Alternatively, a common L chain capable of conferring binding ability to multiple different H chains can be obtained and used as the common L chain for a multispecific antibody. By expressing IgG by introducing such a common L chain and multiple different H chain genes into cells, it becomes possible to efficiently express multispecific IgG (Nature Biotechnology (1998) 16, 677-681). When selecting a common H chain, a method can also be used to select a common L chain that corresponds to any different H chain and exhibits high binding ability (WO2004/065611).

Furthermore, as the Fc region of the present invention, an Fc region in which C-terminal heterogeneity has been reduced can be used as appropriate. More specifically, an Fc region in which glycine at position 446 and lysine at position 447, as specified according to EU numbering, in the amino acid sequences of two polypeptides that constitute an Fc region originating from IgG1, IgG2, IgG3, or IgG4, are deleted, is provided.

Several of these techniques, for example, two or more in combination, can also be used. These techniques can also be applied separately, as appropriate, to the two H chains to be associated. Furthermore, these techniques can also be used in combination with the above-mentioned Fc region with reduced binding activity to Fcγ receptors. The antigen-binding molecule of the present invention may also be an antigen-binding molecule having the same amino acid sequence as the antigen-binding molecule having the above-mentioned modifications added thereto, which has been separately prepared.

The structure of a multispecific antigen-binding molecule of the present invention is not limited, as long as it comprises: (1) a first antigen-binding domain comprising the antibody H-chain variable region and L-chain variable region and having CD3-binding activity; (2) a second antigen-binding domain comprising an amino acid sequence different from that of the first antigen-binding domain; and, optionally, (3) a domain comprising an Fc region with reduced Fcγ receptor-binding activity.
In the present invention, the above-mentioned domains can be directly linked via peptide bonds. For example, when F(ab') ₂ is used as the antigen-binding domain in (1) and (2), and these Fc regions are used as the domain containing an Fc region with reduced Fcγ receptor-binding activity in (3), when the antigen-binding domains described in (1) and (2) and the domain containing an Fc region in (3) are linked via peptide bonds, the linked polypeptide forms an antibody structure. To produce such an antibody, the antibody can be purified from the culture medium of the above-mentioned hybridoma, or from the culture medium of a desired host cell in which a polynucleotide encoding the polypeptide constituting the antibody is stably maintained.

Preferred antibody variable regions of the present invention that have CD3-binding activity include antibody variable regions that have binding activity to CD3ε. Examples of such antibody variable regions include the heavy chain variable regions of antibody Nos. 2, 14, 25, 29, 30-32, and 34 listed in Table 5 below, or antibody H chain variable regions having the amino acid sequences of heavy chain CDR1, CDR2, and CDR3; and the light chain variable regions of antibody Nos. 2, 14, 25, 29, 30-32, and 34 listed in Table 5, or antibody L chain variable regions having the amino acid sequences of light chain CDR1, CDR2, and CDR3; or antibody H chain variable regions and L chain variable regions functionally equivalent to these variable regions.

In the present invention, "functionally equivalent" means that the binding affinity to an antigen under specified conditions is equivalent, or that when used as a multispecific antigen-binding molecule, the cytotoxic activity against cells expressing the target antigen or tissues containing said cells is equivalent. Binding affinity and cytotoxic activity can be measured based on the descriptions herein. The cells used to measure cytotoxic activity may be desired cells expressing the target antigen or desired tissues containing said cells, but for example, human cancer cell lines expressing the target antigen can be used. Furthermore, with respect to antibody constant regions, the reduction in binding activity to Fcγ receptors may also be equivalent.

For example, an antibody H-chain variable region functionally equivalent to an antibody H-chain variable region described herein (the original H-chain variable region) means that when combined with the antibody L-chain variable region described herein as the pair of the original H-chain, it has equivalent binding affinity, or when used as a multispecific antigen-binding molecule, it has equivalent cytotoxic activity against cells expressing the target antigen or tissues containing said cells. Furthermore, an antibody L-chain variable region functionally equivalent to an antibody L-chain variable region described herein (the original L-chain variable region) means that when combined with the antibody H-chain variable region described herein as the pair of the original L-chain, it has equivalent binding affinity, or when used as a multispecific antigen-binding molecule, it has equivalent cytotoxic activity against cells expressing the target antigen or tissues containing said cells.

Furthermore, "equivalent" does not necessarily mean the same level of activity; it may also mean that the activity is enhanced, and therefore can also be expressed as "equivalent or greater." Specifically, in the case of binding affinity to an antigen, "equivalent" refers to a value (KD value/parent KD value) of 2.0 or less compared to the binding affinity (parent KD value) of a control antibody variable region. The KD value/parent KD value is preferably 1.5 or less, more preferably 1.3 or less, 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, but it may be, for example, ^10-1 , ^10-2 , ^10-3 , ^10-4 , ^10-5 , or ^10-6 . Specifically, in the present invention, the KD value/parent KD value is preferably 10 ^-6 to 2.0, more preferably 10 ^-3 to 1.5, more preferably 10 ^-1 to 1.3, more preferably 0.8 to 1.2, more preferably 0.9 to 1.1, and most preferably 1.0. In the case of cytotoxic activity, "equivalent" refers to a value (cell growth inhibition rate/parent cell growth inhibition rate) of 0.7 or higher compared to the cell growth inhibition rate of a control multispecific antigen-binding molecule (parent cell growth inhibition rate). The concentration of the added multispecific antigen-binding molecule is determined appropriately, but is preferably measured at, for example, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, or 1 nM, preferably 0.05 nM or 0.1 nM. The value of cell proliferation inhibition rate/parent cell proliferation inhibition rate is preferably 0.8 or more, more preferably 0.9 or more, 1.0 or more, 1.2 or more, 1.5 or more, 2 or more, 3 or more, 5 or more, 10 or more, or 20 or more. There is no upper limit, but it may be, for example, 10, ¹⁰² , ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ , or ¹⁰⁶ .

In the case of cytotoxic activity, examples of such activity include a value (concentration for 50% cell growth inhibition/concentration for 50% parent cell growth inhibition) of 1.5 or less, relative to the concentration of the original multispecific antigen-binding molecule that inhibits 50% cell growth (concentration for 50% parent cell growth inhibition). The concentration of multispecific antigen-binding molecule required to reduce the cell growth rate by half compared to when the multispecific antigen-binding molecule is not added. The value of "concentration for 50% cell growth inhibition/concentration for 50% parent cell growth inhibition" is preferably 1.3 or less, and 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, but it may be, for example, ^10-1 , ^10-2 , ^10-3 , ^10-4 , ^10-5 , or ^10-6 . Specifically, it is preferably 10 ⁻⁶ to 1.5×10 ⁻⁰ , more preferably 10 ⁻⁶ to 10 ⁻¹ , more preferably 10 ⁻⁶ to 10 ⁻² , and even more preferably 10 ⁻⁶ to 10 ⁻³ .

Cytotoxic activity can be assessed by in vitro TDCC activity as described in Example 4, and in this case, "equivalent" refers to a value (TDCC activity/parent TDCC activity) of 0.7 or greater compared to the TDCC activity of a control multispecific antigen-binding molecule. The TDCC activity/parent TDCC activity value is preferably 0.8 or greater, and more preferably 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.

Furthermore, the KD value of a second antigen-binding domain having binding activity to a target antigen may 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 ^-12 M or less, 5x10 ^-12 M or less, 4x10 ^-12 M or less, 3x10 ^-12 M or less, 2x10 ^-12 M or less, 1x10 ^-12 M or less, 8x10 ^-13 M or 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.

Furthermore, with regard to an antigen-binding domain comprising an antibody variable region that has binding activity to CD3, the KD value for CD3, for example, human CD3, more specifically, for example, human CD3ε chain, may be, for example, 5x10 ⁻⁷ M or less, preferably 2x10 ⁻⁷ M or less, for example, 1.5x10 ⁻⁷ M or less, 1.4x10 ⁻⁷ M or less, 1.3x10 ⁻⁷ M or less, 1.2x10 ⁻⁷ M or less, 1x10 ⁻⁷ M or less, 3x10 ⁻⁸ M or less, 2x10 ⁻⁸ M or less, 1x10 ⁻⁸ M or less, 8x10 ⁻⁹ M or less, 5x10 ⁻⁹ M or less, 4x10 ⁻⁹ M 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 ^-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 ^-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 for the target antigen and human CD3 (e.g., human CD3ε chain) of ^5x10-9 M or less and ^5x10-7 M or less, respectively, and more preferably ^1x10-9 M or less and ^5x10-8 M or less, respectively.

In the present invention, a "functionally equivalent" antibody variable region is not particularly limited as long as it is an antibody heavy chain variable region and/or antibody light chain variable region that meets the above-mentioned conditions. Such antibody variable regions may, for example, have one or more amino acids (e.g., 1, 2, 3, 4, 5, or 10 amino acids) substituted, deleted, added, and/or inserted into the amino acid sequence of the variable regions of antibodies Nos. 2, 14, 25, 29, 30-32, and 34 listed in Table 5. Methods for substituting, deleting, adding, and/or inserting one or more amino acids into an amino acid sequence that are well known to those skilled in the art include methods for introducing mutations into proteins. For example, those skilled in the art are familiar with site-directed mutagenesis methods (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275; Zoller, MJ, and S mith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymo l. 100, 468-500, Kramer,W, Drutsa,V, Jansen,HW, Kramer,B, Pflugfelder,M, and Fritz,HJ(1984) The gapped duple x DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W, and Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 82, 488-492), etc., can be used to appropriately introduce mutations into the amino acid sequence, thereby preparing variable regions functionally equivalent to the antibody variable regions having the above-mentioned functions.

When modifying an amino acid residue, it is desirable to mutate it to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), amino acids with sulfur-containing side chains (C, M), amino acids with carboxylic acid- and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (the numbers in parentheses represent the single-letter symbols of the amino acids). Substitution of amino acids within each of these groups is referred to as a conservative substitution. It is already known that polypeptides having modified amino acid sequences by deletion, addition, and/or substitution of one or more amino acid residues with other amino acids can retain their biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81:5662-6; Zoller, M. J. and Smith, M., Nucleic Acids Res. (1982) 10:6487-500; Wang, A. et al., Science (1984) 224:1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409-13). Variable regions of the present invention containing such amino acid modifications have at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity with the CDR sequence, FR sequence, or entire variable region amino acid sequence before modification. As used herein, sequence identity is defined as the percentage of residues identical to those in the original H-chain variable region or L-chain variable region amino acid sequence, after aligning the sequences as necessary to maximize sequence identity and introducing gaps as appropriate. Amino acid sequence identity can be determined by the method described below.

Furthermore, "functionally equivalent antibody variable regions" can also be obtained from nucleic acids that hybridize under stringent conditions to nucleic acids consisting of nucleotide sequences encoding the amino acid sequences of the variable regions of antibodies Nos. 2, 14, 25, 29, 30 to 32, and 34 listed in Table 5, for example. Examples of stringent hybridization conditions for isolating nucleic acids that hybridize under stringent conditions to nucleic acids consisting of nucleotide sequences encoding the amino acid sequences of the variable regions include 6 M urea, 0.4% SDS, 0.5 x SSC, and 37°C, or hybridization conditions of equivalent stringency. Higher stringency conditions, such as 6 M urea, 0.4% SDS, 0.1 x SSC, and 42°C, are expected to result in the isolation of nucleic acids with higher homology. Washing conditions after hybridization include, for example, 0.5xSSC (1xSSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) and 0.1% SDS at 60°C, more preferably 0.2xSSC and 0.1% SDS at 60°C, more preferably 0.2xSSC and 0.1% SDS at 62°C, more preferably 0.2xSSC and 0.1% SDS at 65°C, and more preferably 0.1xSSC and 0.1% SDS at 65°C. The sequence of the isolated nucleic acid can be determined by known methods described below. The homology of the isolated nucleic acid is at least 50% or more, more preferably 70% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, 99% or more) sequence identity across the entire base sequence.
Instead of the above-mentioned method utilizing hybridization techniques, it is also possible to isolate nucleic acids that hybridize under stringent conditions with nucleic acids consisting of a nucleotide sequence encoding the amino acid sequence of the variable region by using gene amplification methods, such as polymerase chain reaction (PCR), which use primers synthesized based on the nucleotide sequence information encoding the amino acid sequence of the variable region.

The identity of nucleotide sequences and amino acid sequences can be determined using the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-7). Programs called BLASTN and BLASTX have been developed based on this algorithm (Altschul et al., J. Mol. Biol. (1990) 215:403-10). When analyzing nucleotide sequences using BLASTN based on BLAST, the parameters are, for example, score = 100 and wordlength = 12. When analyzing amino acid sequences using BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific techniques for these analysis methods are publicly known (see the NCBI (National Center for Biotechnology Information) BLAST (Basic Local Alignment Search Tool) website; http://www.ncbi.nlm.nih.gov).

Nucleic Acids, Vectors, and Cells In one aspect, the present invention relates to nucleic acids (polynucleotides) encoding the antigen-binding molecules of the present invention. The antigen-binding molecules of the present invention can be incorporated into any expression vector. An appropriate host can be transformed with the expression vector to create cells expressing the antigen-binding molecules. The antigen-binding molecules encoded by the nucleic acids (polynucleotides) can be obtained by culturing the cells expressing the antigen-binding molecules and recovering the expression product from the culture supernatant. Specifically, the present invention relates to vectors containing nucleic acids (polynucleotides) encoding the antigen-binding molecules of the present invention, cells harboring the vectors, and methods for producing antigen-binding molecules, which include culturing the cells and recovering the antigen-binding molecules from the culture supernatant. These can be obtained, for example, by techniques similar to those used for the recombinant antibodies described above.

Pharmaceutical Compositions In one aspect, the present invention provides pharmaceutical compositions comprising, as an active ingredient, an antigen-binding molecule of the present invention (particularly a multispecific antigen-binding molecule). In one embodiment, the present invention provides pharmaceutical compositions that induce cytotoxicity, comprising, as an active ingredient, the antigen-binding molecule. The pharmaceutical compositions of the present invention induce cytotoxicity, particularly T-cell-dependent cytotoxicity, and are preferably administered to subjects suffering from or at risk of recurrence of a disease for which such cytotoxicity is necessary for prevention or treatment. In one embodiment, the pharmaceutical compositions of the present invention are for use in the treatment or prevention of cancer.

Pharmaceutical compositions containing an antigen-binding molecule of the present invention (particularly a multispecific antigen-binding molecule) as an active ingredient can also be described as cytotoxicity inducers and cytostatic agents containing the antigen-binding molecule as an active ingredient; methods for inducing cytotoxicity and methods for inhibiting cell proliferation comprising the step of administering the antigen-binding molecule to a subject; the antigen-binding molecule for use in inducing cytotoxicity and inhibiting cell proliferation; or use of the antigen-binding molecule in the production of cytotoxicity inducers and cytostatic agents.
Furthermore, pharmaceutical compositions containing an antigen-binding molecule of the present invention (particularly a multispecific antigen-binding molecule) as an active ingredient can also be described as a cancer therapeutic or preventive agent containing the antigen-binding molecule as an active ingredient, a method for treating or preventing cancer which comprises administering the antigen-binding molecule to a subject, the antigen-binding molecule for use in cancer therapeutic or preventive purposes, or use of the antigen-binding molecule in the manufacture of a cancer therapeutic or preventive agent.

In the present invention, "containing an antigen-binding molecule as an active ingredient" means containing the antigen-binding molecule as the main active ingredient, and does not limit the content of the antigen-binding molecule.

In one embodiment, the antigen-binding molecule of the present invention can be administered by administering or incorporating a nucleic acid encoding the antigen-binding molecule of the present invention into a living body using a vector or the like, thereby directly expressing the antigen-binding molecule of the present invention in the living body; however, the antigen-binding molecule may also be administered without using a vector. Examples of vectors include viral vectors and plasmid vectors, and further examples include adenovirus vectors and adeno-associated virus vectors. The nucleic acid encoding the antigen-binding molecule of the present invention may be administered directly to a living body, or cells into which a nucleic acid encoding the antigen-binding molecule of the present invention has been introduced may be administered to a living body. For example, the antigen-binding molecule of the present invention can be administered by chemically modifying mRNA encoding the antigen-binding molecule of the present invention to increase mRNA stability in the living body, and then directly administering the mRNA to a human to express the antigen-binding molecule of the present invention in the living body (see EP2101823B and WO2013/120629). Alternatively, B cells into which a nucleic acid encoding the antigen-binding molecule of the present invention has been introduced may be administered (Sci Immunol. (2019) 4(35), eaax0644). Alternatively, bacteria into which nucleic acids encoding the antigen-binding molecules of the present invention have been introduced may be administered (Nature Reviews Cancer (2018) 18, 727-743).

If necessary, the antigen-binding molecules of the present invention can be encapsulated in microcapsules (such as microcapsules made of hydroxymethylcellulose, gelatin, or poly(methyl methacrylate)) to form colloid drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remington's Pharmaceutical Science ^16th edition," Oslo Ed. (1980)). Furthermore, methods for producing sustained-release drugs are also known, and these methods can be applied to the antigen-binding molecules of the present invention (J. Biomed. Mater. Res. (1981) 15, 267-277; Chemtech. (1982) 12, 98-105; U.S. Pat. No. 3,773,719; European Patent Publications EP 58481 and EP 133988; Biopolymers (1983) 22, 547-556).

The pharmaceutical composition, cytotoxicity inducer, cytostatic agent, or cancer treatment or prevention agent (hereinafter collectively referred to as the pharmaceutical composition of the present invention) of the present invention can be administered to a patient either orally or parenterally. Parenteral administration is preferred. Specific examples of such administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection methods include, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition of the present invention can be administered systemically or locally by injection. Furthermore, an appropriate administration method can be selected depending on the patient's age and symptoms. The dosage can be selected, for example, from a range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from a range of 0.001 mg/body to 100,000 mg/body per patient. However, the pharmaceutical composition of the present invention is not limited to these dosages.

The pharmaceutical compositions of the present invention can be formulated in accordance with conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.) and may contain pharmaceutically acceptable carriers and additives. Examples include surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. Furthermore, without being limited to these, other commonly used carriers can be used as appropriate. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts.

A non-limiting example of a technique that can be combined with the antigen-binding molecules of the present invention is the creation of T cells that secrete T cell-redirecting antibodies using a CD3-binding domain (Trends Immunol. (2019) 40(3) 243-257). One non-limiting creation method involves introducing nucleic acid encoding a bispecific antibody comprising the CD3-binding domain described herein and a cancer antigen-binding domain into effector cells such as T cells using gene modification techniques.

In one aspect, such as a method for treatment , the present invention provides a method for inducing (inducing) damage to a target cell or a method for inhibiting the proliferation of the cell, the method comprising the step of contacting the target cell with a multispecific antigen-binding molecule of the present invention that binds to an antigen specifically expressed on the surface of the cell. In one aspect, the present invention provides a method for inducing (inducing) damage to a cell expressing a target cancer-specific antigen or a tumor tissue containing the expressing cell, or a method for inhibiting the proliferation of the cell or tumor tissue, the method comprising the step of contacting the cell expressing the target cancer-specific antigen with a multispecific antigen-binding molecule of the present invention that binds to the antigen. In another aspect, the present invention provides a method for inducing (inducing) damage to a cell or a method for inhibiting the proliferation of the cell, the method comprising the step of contacting a cell having the function of suppressing an immune response with a multispecific antigen-binding molecule of the present invention that binds to an antigen expressed on the surface of the cell. The multispecific antigen-binding molecule that binds to the antigen is as described above for the antigen-binding molecule of the present invention contained in the cytotoxicity-inducing agent and cytostatic agent of the present invention. The cells to which the multispecific antigen-binding molecule of the present invention that binds to the antigen are not particularly limited, as long as the antigen is expressed.

In the present invention, "contact" is performed, for example, by adding a multispecific antigen-binding molecule of the present invention that binds to the antigen to the culture medium of target antigen-expressing cells cultured in a test tube. In this case, the antigen-binding molecule to be added may be in the form of a solution or a solid obtained by lyophilization or the like, as appropriate. When added as an aqueous solution, it may be an aqueous solution containing only the multispecific antigen-binding molecule of the present invention, or it may be a solution containing, for example, the surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc. described above. The concentration to be added is not particularly limited, but a final concentration in the culture medium of 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, is suitable.

In another embodiment, "contact" in the present invention can also be achieved by administering to a non-human animal into which cells expressing the target antigen have been transplanted, or to an animal that has cells endogenously expressing the antigen. Administration can be performed either orally or parenterally. Parenteral administration is particularly preferred, and specific examples of such administration methods include injection, intranasal administration, pulmonary administration, and transdermal administration. Injection can be, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical composition of the present invention, or the cytotoxicity inducer and cell proliferation inhibitor can be administered systemically or locally by injection. An appropriate administration method can be selected depending on the age and symptoms of the subject animal. When administered as an aqueous solution, the solution may contain only the multispecific antigen-binding molecule of the present invention, or may contain, for example, the above-mentioned surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents. The dosage can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg/body per patient. However, the dosage of the multispecific antigen-binding molecules of the present invention is not limited to these dosages.

The following methods are preferably used to evaluate or measure the cytotoxicity induced in target cells (particularly cells expressing a target antigen other than CD3, to which the second antigen-binding domain of the multispecific antigen-binding molecule of the present invention binds) by contact with the multispecific antigen-binding molecule of the present invention. Methods for evaluating or measuring the cytotoxic activity in vitro include methods for measuring cytotoxic T cell activity. Whether or not the multispecific antigen-binding molecule of the present invention has T cell mediated cytotoxic activity can be measured by known methods (e.g., Current Protocols in Immunology, Chapter 7. Immunologic Studies in Humans, Editor, John E., Coligan et al., John Wiley & Sons, Inc., (1993)). When measuring activity, an antigen-binding molecule that binds to an antigen different from the target antigen and that is not expressed by the cells used in the test is used as a control in the same manner as the multispecific antigen-binding molecule of the present invention, and activity can be determined if the multispecific antigen-binding molecule of the present invention exhibits stronger cytotoxic activity than the antigen-binding molecule used as the control.

Furthermore, to evaluate or measure cytotoxic activity in vivo, cells expressing the target antigen are transplanted intradermally or subcutaneously into a non-human test animal, and the test antigen-binding molecule is then administered intravenously or intraperitoneally daily or at intervals of several days from the same day or the following day. By measuring the size of the tumor over time, the difference in the change in size of the tumor can be defined as cytotoxic activity. As with in vitro evaluation, a control antigen-binding molecule is administered, and cytotoxic activity can be determined if the tumor size in the group administered with the antigen-binding molecule of the present invention is significantly smaller than the tumor size in the group administered with the control antigen-binding molecule.

Methods for assessing or measuring the inhibitory effect on the proliferation of cells expressing a target antigen include measuring the uptake of isotope-labeled thymidine into cells and the MTT assay. Furthermore, methods for assessing or measuring cell proliferation inhibitory activity in vivo can be suitably the same as the methods for assessing or measuring cytotoxic activity in vivo described above.

The present invention also provides kits for use in the methods of the present invention, which comprise the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention. The kits may also contain other packaged components such as pharmaceutically acceptable carriers, vehicles, and instructions for use.
The present invention also relates to the antigen-binding molecules of the present invention or antigen-binding molecules produced by the production methods of the present invention, for use in the methods of the present invention.

The foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

The present invention will be described in more detail below using examples, but these examples are not intended to limit the scope of the present invention.

[Example 1] Preparation of CDR-modified antibodies with improved binding stability to CD3 antigen Bispecific antibodies that damage target cells by recognizing the T cell marker CD3 and an antigen on the target cell have been reported, but there was still room for improvement in the binding stability of the reported CD3-binding antibody variable regions. Improving this profile was expected to have benefits such as sustained in vivo efficacy and ease of in vitro evaluation. Specifically, the antibody variable region described in WO2015174439 lost its binding ability over time in buffer, and further modification to improve binding stability was considered necessary.
First, the antibody heavy chain gene TR01H113-F760mnP17 (SEQ ID NO: 30) was constructed, which contains the heavy chain variable region of an antibody against human CD3 and the constant region sequence of human IgG1, as described in WO2015174439. The antibody light chain gene L0011-KT0 (SEQ ID NO: 34) contained the light chain variable region of an antibody against human CD3 and the constant region sequence of human κ chain. Plasmids encoding CDR-modified antibodies were obtained by introducing modifications into the bases encoding the CDR amino acid sequences of this antibody gene using methods known to those skilled in the art. The heavy and light chain-encoding plasmids were mixed and transfected into human embryonic kidney cell-derived HEK293 cells. The supernatant was cultured for 4 days and purified using methods known to those skilled in the art to obtain antibodies. The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antibody was calculated from the obtained measurement values using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

[Example 2] Evaluation of binding of antibodies with altered CDRs to CD3 antigen A polynucleotide encoding an antigen consisting of human CD3ε and CD3γ and a FLAG tag was introduced into HEK293 cells, and the culture supernatant was purified by ion exchange chromatography, FLAG tag affinity chromatography, and gel filtration chromatography to obtain the antigen (hereinafter referred to as CD3eg-linker).
The interaction analysis between the prepared antibodies and CD3eg-linker was performed using Octet HTX as follows. Phosphate-buffered saline (pH 7.4) containing 0.05% TWEEN 20 was used as the buffer. Protein A chips (Sartorius) were used as the sensor chip. The antibodies listed in Table 1 were prepared in buffer at 10 μg/mL, and human CD3eg-linker was serially diluted to 900, 300, 100, and 33 nM. The antibodies were loaded onto the sensor chip for 60 seconds after a 30-second baseline. Antigen binding characteristics were measured by a 30-second baseline step followed by 180-second antigen binding and dissociation steps. The binding value was defined as the magnitude of the response at 295 seconds into the measurement. The equilibrium dissociation constant (KD) was determined by global fitting of the response curve at multiple concentrations (Octet BLI Analysis Version: 12.2.2.4). In this global fitting, the 1:1 binding model was used for all regions of the association and dissociation phases.

[Example 3] Evaluation of binding stability of antibodies with modified CDRs to CD3eg-linker The prepared antibodies were diluted to 0.1 mg/mL and stored at 4°C for 3 days or at 50°C for 3 days, and antigen binding was evaluated using the same method as in Example 2.

The "binding H/I ratio" in the table is the ratio of the binding measurement values for each antibody after storage at 4°C to those after storage at 50°C, and indicates the extent to which each antibody maintains its binding activity at 50°C. Antibodies Nos. 2, 14, 25, 29, 30-32, and 34 created in the present invention had binding values at 50°C that were equal to or greater than 0.078, the value of the previously reported control antibody 1. Furthermore, the binding H/I values for these antibodies were 75% or greater, exceeding the 61% value of the previously reported control antibody 1. Therefore, it was confirmed that these modified antibodies showed a significant improvement over the decrease in binding ability at 50°C in buffer.

Example 4: Evaluation of antibodies with modified CDRs by TDCC Reporter Bioassay. Bispecific antibodies were prepared using each antibody prepared in the present invention and an anti-Marvel D3 antibody using methods known to those skilled in the art. The prepared antibodies were diluted to 0.044 mg/mL and stored at 4°C or 50°C for 3 days, as in Example 3. The in vitro TDCC activity of these antibodies was measured to assess differences in TDCC activity depending on storage temperature. In vitro TDCC activity was measured using TCR/CD3 Effector Cells (NFAT) and the Bio-Glo luciferase assay system (Promega), and TDCC activity was quantified by luminescence from the reporter gene luciferase. The reaction was initiated by adding 10 μL of NFAT-RE-luc2 Jurkat cells (prepared in medium at a concentration of 3 × 10 ⁶ /mL) and LS 174T cells (ATCC) (prepared at a concentration of 5 × 10 ⁵ mL) to each well of a 384-well plate. LS 174T is a cell line expressing the MarvelD3 antigen. The reaction was carried out overnight at 37°C. After incubation, reporter gene expression was quantified by adding luciferase substrate to each well and measuring luminescence using a plate reader. The luciferase response (counts per second: CPS) represents the average of the values measured for the 10 nM and 100 nM antibody solutions.

NA indicates that the measurement was not performed.

The "activation H/I ratio" in the table is the ratio of the luciferase response values for each antibody after storage at 4°C to those after storage at 50°C, and indicates the extent to which each antibody lost its binding activity at 50°C.
Antibody Nos. 2, 14, 25, 29, 30-32, and 34 selected in Example 3 exhibited luciferase responses of 4.3.E+05 or higher when stored at 4°C, which corresponds to the response of the previously reported control antibody 1 when stored at 50°C. Furthermore, antibody Nos. 14, 25, 29, 30-32, and 34 exhibited activated H/I TDCC activity ratios of 67% or higher when stored at 50°C. This was superior to the previously reported value of control antibody 1, indicating that these antibodies were modified antibodies that maintained TDCC activity while improving the activated H/I ratio. The amino acid sequences of antibody Nos. 1, 2, 14, 25, 29, 30-32, and 34 are shown in Table 5.

The present invention provides a novel CD3-binding domain that has superior stability compared to CD3-binding domains used in conventional T cell-redirecting antibodies, and an antigen-binding molecule containing said CD3-binding domain. Bispecific antigen-binding molecules prepared by combining the CD3-binding domain of the present invention with an antigen-binding domain that binds to an antigen expressed on the surface of target cells such as cancer cells can induce T cell-dependent cytotoxicity against the target cells and can be used to treat or prevent various cancers. Antigen-binding molecules containing the CD3-binding domain of the present invention show only a small decrease in CD3-binding activity at room temperature or above, and are expected to maintain their T cell-dependent cytotoxic activity for a long period of time when administered to patients.

Claims (15)

An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises an antibody heavy chain variable region and an antibody light chain variable region having binding activity to CD3;
the heavy chain variable region
H chain CDR1 comprising the amino acid sequence NAWMH (SEQ ID NO: 1);
an H chain CDR2 comprising the amino acid sequence _{QIX1DKSQNYATX2VAESVKG} (SEQ ID NO: ₂ ), wherein X1 is K or R, and _X2 is Y or F; and an H chain CDR3 comprising the amino acid sequence _{VHYX3AGYGVDX4} (SEQ ID NO: ₃ ), wherein _X3 is A or P, and _X4 is I, M, or L.
Including,
the light chain variable region
an L chain CDR1 comprising the amino acid sequence _{RSX5X6X7VVHENRX8TYLH} ( _SEQ ID NO: ₄ ), wherein _X5 is _S or T, _X6 is Q or M, _X7 is S or T, and _X8 is Q or N;
L chain CDR2 comprising the amino acid sequence KVSNRFS (SEQ ID NO: 5); and L chain CDR3 comprising the amino acid sequence GQGTQVPYT (SEQ ID NO: 6).
An antigen-binding molecule comprising: The antigen-binding molecule of claim 1, wherein the antibody H chain variable region and L chain variable region having binding activity to CD3 comprise any combination of H chain CDR1, CDR2, and CDR3, and L chain CDR1, CDR2, and CDR3 selected from the following (a1) to (a8):
(a1) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 10, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a2) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a3) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a4) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a5) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 15, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a6) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a7) a combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;
(a8) A combination of an H chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, an H chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, an H chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, an L chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, an L chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and an L chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6. The antigen-binding molecule of claim 1 or 2, wherein the antibody H chain variable region and L chain variable region having CD3-binding activity comprise any combination of H chain variable region and L chain variable region selected from the following (a1) to (a8):
(a1) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a2) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a3) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 26;
(a4) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a5) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 25;
(a6) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27;
(a7) a combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28;
(a8) A combination of an H-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 and an L-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29. The antigen-binding molecule of any one of claims 1 to 3, further comprising an Fc region. The antigen-binding molecule of any one of claims 1 to 4, which is a monospecific antigen-binding molecule. The antigen-binding molecule of any one of claims 1 to 4, which is a bispecific antigen-binding molecule. The antigen-binding molecule of claim 6, wherein the second antigen-binding domain comprises an antibody variable region having binding activity against a cancer antigen. The antigen-binding molecule of any one of claims 1 to 7, which is an antibody. A nucleic acid encoding the antigen-binding molecule of any one of claims 1 to 8. A vector incorporating the nucleic acid described in claim 9. A cell containing the vector described in claim 10. A method for producing the antigen-binding molecule of any one of claims 1 to 8, comprising the step of culturing the cell of claim 11. An antigen-binding molecule produced by the method of claim 12. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 1 to 8 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 14 for use in the treatment or prevention of cancer.