Pharmaceutical compositions, antigen-binding molecules, treatment methods, and screening methods

Abstract

To provide antibodies whose effector functions are more highly specific for disease-associated cells than for normal cells, thereby resulting in greater reduction in side effects.SOLUTION: The present invention relates to a pharmaceutical composition comprising a combination of a first antigen-binding molecule and a second antigen-binding molecule. The first antigen-binding molecule and the second antigen-binding molecule are not covalently bound but are more likely to form a heterodimer than a homodimer when mixed in a liquid. Further the present invention relates to antigen-binding molecules, therapeutic methods, and screening methods, which are related to such a combination.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical compositions, antigen-binding molecules, therapeutic methods, and screening methods. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and minimal side effects. Many IgG-type antibody drugs are on the market, and many more are currently being developed (Non-Patent Documents 1 and 2). The functions required of therapeutic antibodies include blocking interactions between specific molecules by antibody binding to targets, and eliminating target cells using antibody-dependent cellular cytotoxicity (hereinafter also referred to as ADCC) and complement-dependent cytotoxicity (hereinafter also referred to as CDC) activities, which are the effector functions of antibodies.

[0003] Native antibodies and immunoglobulins (hereinafter referred to as IgG) typically consist of two identical light (L) chains and two identical heavy (H) chains. The L and H chains are linked by disulfide bonds, and the L and H chain complexes also form disulfide bonds between the H chains, forming a homodimer of the complex with a molecular weight of approximately 150,000 daltons. The L chains consist of an L chain variable region and an L chain constant region (hereinafter referred to as CL), while the H chains consist of an H chain variable region and an H chain constant region consisting of CH1, CH2, CH3, and a hinge region. CH1 and CH2 of the H chain are separated by a hinge region that is involved in disulfide bonding between the H chains. IgGs with this structure have two antigen-binding sites (bivalent antibodies).

[0004] Structurally, the Fc region contributes to dimer formation via the hinge region and CH3 interface. For this reason, examples have been reported in which heterodimer formation was promoted by introducing mutations into the CH3 interface (Non-Patent Documents 3, 4, and 5), and in which monovalent antibodies were produced by substituting cysteine ​​residues in the hinge region with other amino acids and then introducing modifications that inhibit CH3 dimer formation (Non-Patent Document 6).

[0005] In terms of function, it contributes to prolonging blood circulation through interaction with fetal Fc receptors (hereinafter also referred to as FcRn) (Non-Patent Document 7), and is involved in the expression of effector functions such as Fc receptor-mediated ADCC, CDC, and antibody-dependent cell-mediated phagocytosis (ADCP). In recent years, modification techniques to improve effector functions have also been reported (Non-Patent Document 8), and these have been utilized to enhance the efficacy of antibody drugs.

[0006] Antibody molecules bind to antigens expressed on cancer cells and exert cytotoxic activity against cancer cells through ADCC and other mechanisms. It is known that cytotoxic activity through ADCC and other mechanisms depends on the number of antigens expressed on the target cells of a therapeutic antibody (Non-Patent Document 9). Therefore, a high expression level of the target antigen is preferable from the perspective of the efficacy of a therapeutic antibody. However, even if the expression level of an antigen is high, if the antigen is expressed in normal tissues, ADCC and other cytotoxic activities will be exerted against normal cells, resulting in serious side effects. Therefore, it is preferable that the antigen targeted by a therapeutic antibody used as a cancer therapeutic drug is specifically expressed in cancer cells. For example, antibody molecules against the EpCAM antigen, a known cancer antigen, were thought to be promising as a cancer therapeutic drug. However, the EpCAM antigen is also known to be expressed in the pancreas. In fact, clinical trials have shown that administration of anti-EpCAM antibodies can cause pancreatitis as a side effect due to cytotoxic activity against the pancreas (Non-Patent Document 10).

[0007] Following the success of antibody pharmaceuticals that exert cytotoxic activity through ADCC activity, second-generation improved antibody molecules that exert potent cytotoxic activity have been reported, such as those that enhance ADCC activity by removing fucose from the N-glycosylation chains in the Fc region of native human IgG1 (Non-Patent Document 11) and those that enhance ADCC activity by enhancing binding to FcγRIIIa through amino acid substitution in the Fc region of native human IgG1 (Non-Patent Document 12). As antibody pharmaceuticals that exert cytotoxic activity against cancer cells by mechanisms other than the above-mentioned NK cell-mediated ADCC activity, improved antibody molecules that exert even more potent cytotoxic activity have also been reported, such as antibody drug conjugates (ADCs) in which a drug with potent cytotoxic activity is conjugated to an antibody (Non-Patent Document 13), and small molecule antibodies that exert cytotoxic activity against cancer cells by recruiting T cells to the cancer cells (Non-Patent Document 14).

[0008] While these antibody molecules exhibit more potent cytotoxic activity, they can also exert cytotoxic activity against cancer cells with low antigen expression, they also exert cytotoxic activity against normal tissues with low antigen expression. In fact, compared to cetuximab, a natural human IgG1 directed against the EGFR antigen, EGFR-BiTE, a bispecific antibody directed against CD3 and EGFR, exerts potent cytotoxic activity against cancer cells by recruiting T cells to cancer cells, thereby exerting antitumor effects. However, because EGFR is also expressed in normal tissues, serious side effects have been observed when EGFR-BiTE was administered to cynomolgus monkeys (Non-Patent Document 15). Furthermore, bivatuzumab mertansine, an ADC consisting of mertansine conjugated to an antibody directed against CD44v6, which is highly expressed in cancer cells, has been shown to cause severe skin and liver toxicity in clinical trials because CD44v6 is also expressed in normal tissues (Non-Patent Document 16).

[0009] When using an antibody that can exert strong cytotoxic activity even against cancer cells with low antigen expression, the target antigen must be expressed in an extremely cancer-specific manner, but the number of cancer antigens that are expressed in an extremely cancer-specific manner is thought to be limited, as HER2, the target antigen of Herceptin, and EGFR, the target antigen of cetuximab, are also expressed in normal tissues. Therefore, although cytotoxic activity against cancer can be strengthened, side effects due to cytotoxic effects on normal tissues can become a problem.

[0010] Recently, ipilimumab, which enhances tumor immunity by inhibiting CTLA4, which contributes to immunosuppression in cancer, has been shown to prolong overall survival in metastatic melanoma (Non-Patent Document 17). However, because ipilimumab systemically inhibits CTLA4, while tumor immunity is enhanced, it also has the problem of exhibiting severe autoimmune disease-like side effects due to systemic immune activation (Non-Patent Document 18).

[0011] Various technologies applicable to second-generation antibody drugs have been developed, and technologies for improving effector function, antigen-binding ability, pharmacokinetics, and stability, or for reducing the risk of immunogenicity have been reported (Non-Patent Document 19). However, there have been few reports of technologies that enable antibody drugs to act on target tissues with high specificity in order to resolve the side effects described above.

[0012] As a technique aimed at imparting high selectivity, a strategy of simultaneously targeting two types of targets has been reported (Non-Patent Documents 20, 21, 22, 23). Studies of bispecific antibodies using CD4 and CD70 double-positive cells or HER2 and EGFR double-positive cells have shown that the cytotoxic activity is approximately 10-fold improved compared to single-positive cells (Non-Patent Documents 24 and 25). In this context, various techniques for producing bispecific antibodies have been developed in recent years (Patent Documents 1 to 6). [Prior art documents] [Patent documents]

[0013]

Patent Document 1

Patent document 2

Patent document 3

Patent document 4

Patent document 5

Patent document 6

Non-licensed literature

[0014] [Non-licensed document 1] Monoclonal antibody successes in the clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073 - 1078 [Non-licensed document 2] The therapeutic antibodies market to 2008. Pavlou AK, Belsey MJ., Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396 [Non-licensed document 3] 'Knobs-into-hole' engineering of antibody CH3 domains for heavy chain heterodimerization. Ridgway JB, Presta LG, Carter P, Protein Eng. (1996) 9 (7), 617-621

Non-licensed Document 4

Direct Environment 5

[0015]

Outdoor Configuration6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

[0016]

Outdoor Content11

Outdoor Tools 12

Outdoor Content13

Outdoor Tools 14

[0017]

Non - Patent Document 15

Non - Patent Document 16

Non - Patent Document 17

[0018] [Non-Patent Document 21] Smarter drugs: a focus on pan-specific monoclonal antibodies. Fagete S, Fischer N, BioDrugs (2011) 25 (6), 357-364 [Non-Patent Document 22] "NextGen" Biologics: Bispecific Antibodies and Emerging Clinical Results. Thakur A, Lum LG, Expert Opin. Biol. Ther. 2016 16 (5), 675-688. [Non-Patent Document 23] Bispecific antibodies and their applications. Fan G, Wang Z, Hao M, Li J. J Hematol Oncol. (2015) 8:130.

Non-Patent Document 24

Non-Patent Document 25

Summary of the Invention

Problems to be Solved by the Invention

[0019] As described above, the usefulness of bispecific antibodies is expected to increase, and there is a demand for antibodies whose effector functions are more highly specific for disease-associated cells than for normal cells, thereby resulting in significantly reduced side effects. [Means for solving the problem]

[0020] The present inventors have created a combination of antigen-binding molecules that do not interact with each other when present in plasma in an antigen-unbound state, but exist separately as antibody half molecules, and form bispecific antibodies on the surface of target cells, thereby completing the present invention. The following embodiments of the present invention are exemplified: [1] to

[15] .

[0021] [1] A first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising a first CH2 and / or a first CH3, and a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising a second CH2 and / or a second CH3, A pharmaceutical composition, wherein the first antigen-binding molecule and the second antigen-binding molecule are not covalently bound to each other and are more likely to form heterodimers than homodimers when mixed in liquid. [2] Surface plasmon resonance at 1 mm 2 The composition according to [1], wherein when the affinity of both antigen-binding molecules is measured using a sensor chip on which 50 pg of the first antigen-binding molecule is immobilized per 1000 kJ / mol and a measurement solution containing 2.5 mg / mL of the second antigen-binding molecule, the binding amount of the second antigen-binding molecule to the first antigen-binding molecule is within a molar ratio of 1:0.1 to 1:0.9.

[0022] [3] The composition according to [1] or [2], wherein the amount of the heterodimer formed in the presence of cells expressing the first antigen and the second antigen is greater than in the absence of the cells. [4] The composition of any of [1] to [3], wherein the FcγR-binding activity of the heterodimer, when formed, is higher than the FcγR-binding activity of a monomer of the first antigen-binding molecule or a monomer of the second antigen-binding molecule, or the FcγR-binding activity of the homodimer, when formed.

[0023] [5] A composition described in any of [1] to [4], wherein the first polypeptide comprises the first CH3, the second polypeptide comprises the second CH3, and the first CH3 and the second CH3 have at least one of the following modifications (i) to (iii) that make the heterodimer more likely to form than the homodimer when mixed in a liquid. (i) a modification in which one of the first CH3 and the second CH3 has a positively charged region and the other has a negatively charged region, and the positively charged region interacts with the negatively charged region when the heterodimer is formed; (ii) a modification in which one of the first CH3 and the second CH3 has a convex portion and the other has a concave portion, and the convex portion fits into the concave portion and interacts with the heterodimer when the heterodimer is formed; (iii) The first CH3 and the second CH3 are CH3s of modified IgG, a portion of the modified IgG CH3 is replaced with a portion of IgA CH3, and when the heterodimer is formed, the portion of the IgA CH3 replaced with the first CH3 and the portion of the IgA CH3 replaced with the second CH3 interact with each other. [6] The composition described in any of [1] to [5], wherein either or both of the first CH3 and the second CH3 further have a substitution with at least one other amino acid residue among the amino acid residues at positions 357, 397, and 409 in the EU numbering system.

[0024] [7] The composition described in any of [1] to [6], wherein either or both of the first polypeptide and the second polypeptide further comprises a hinge region portion of an antibody half molecule. [8] The composition described in [7], wherein the hinge region portion in either or both of the first polypeptide and the second polypeptide has a modification of either or both of the cysteine ​​residues at positions 226 and 229 in the EU numbering system to another amino acid residue.

[0025] [9] The composition according to any one of [1] to [8], wherein the first polypeptide and the second polypeptide each comprise a constant region portion of an antibody half molecule.

[10] The composition according to any one of [1] to [9], wherein the effector function in the presence of cells expressing the first antigen and the second antigen is higher than that in the presence of cells expressing the first antigen but not the second antigen, or cells expressing the second antigen but not the first antigen.

[0026]

[11] A first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising a first CH3, and a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising a second CH3; the first antigen-binding molecule and the second antigen-binding molecule are not bound by a covalent bond; A pharmaceutical composition, wherein the first CH3 and the second CH3 have at least one of the following modifications (iv) to (vi): (iv) a modification in which one of the first CH3 and the second CH3 has a positively charged region and the other has a negatively charged region, and the positively charged region interacts with the negatively charged region when the heterodimer is formed. (v) a modification in which one of the first CH3 and the second CH3 has a convex portion and the other has a concave portion, and the convex portion fits into the concave portion and interacts with each other when the heterodimer is formed; (vi) The first CH3 and the second CH3 are CH3s of modified IgG, a portion of the modified IgG CH3 is replaced with a portion of IgA CH3, and when the heterodimer is formed, the portion of the IgA CH3 replaced with the first CH3 and the portion of the IgA CH3 replaced with the second CH3 interact with each other.

[0027]

[12] A first antigen-binding molecule comprising a first antigen-binding region that binds to a first antigen and a first polypeptide comprising either or both of a first CH2 and a first CH3, A first antigen-binding molecule that, when mixed in a liquid with a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising a second CH2 and / or a second CH3, is more likely to form a heterodimer with the second antigen-binding molecule than a homodimer with the first antigen-binding molecule, and in the heterodimer, the first antigen-binding molecule and the second antigen-binding molecule are not covalently bound.

[13] A second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising either or both of a second CH2 and a second CH3, A second antigen-binding molecule that, when mixed in a liquid with a first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising a first CH2 and / or a first CH3, is more likely to form a heterodimer with the first antigen-binding molecule than a homodimer with the second antigen-binding molecule, and in the heterodimer, the first antigen-binding molecule and the second antigen-binding molecule are not covalently bound.

[0028]

[14] A method for treating a disease caused by pathogenic cells in a subject, the method comprising simultaneously or sequentially administering to the subject a first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising a first CH2 and / or a first CH3, and a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising a second CH2 and / or a second CH3, the method comprising: the first antigen-binding molecule and the second antigen-binding molecule are not covalently bound to each other before and after administration, and form a heterodimer on the surface of the pathogenic cell to exert an effector function.

[0029]

[15] From a group of variants of a first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising either a first CH2 or a first CH3, and a group of variants of a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising either a second CH2 or a second CH3, (a) the first antigen-binding molecule and the second antigen-binding molecule are not bound by a covalent bond; (b) the first antigen-binding molecule and the second antigen-binding molecule more likely form heterodimers between the first antigen-binding molecule and the second antigen-binding molecule than homodimers between the first antigen-binding molecules or between the second antigen-binding molecules; (c) Surface plasmon resonance at 1 mm 2 When the affinity of both antigen-binding molecules is measured using a sensor chip on which 50 pg of a first antigen-binding molecule is immobilized per molecule and a measurement solution containing 2.5 mg / mL of a second antigen-binding molecule, the binding amount of the second antigen-binding molecule to the first antigen-binding molecule is within the molar ratio range of 1:0.1 to 1:0.9. A method for selecting a combination of a first antigen-binding molecule and a second antigen-binding molecule. [Effects of the Invention]

[0030] According to the present invention, a combination of antigen-binding molecules with reduced side effects is provided. [Brief explanation of the drawings]

[0031] [Figure 1-1] This figure shows the results of confirming whether antibody half molecules form dimers (whole antibodies) by size exclusion chromatography. W indicates the elution position of the whole antibody, H indicates the elution position of the antibody half molecules, and E indicates the elution position of a molecular state presumed to be in equilibrium between the whole antibody and the antibody half molecules. [Figure 1-2] This is a figure showing a continuation of Figure 1-1. [Figure 1-3] This is a diagram showing a continuation of Figure 1-2. [Figure 2] This figure shows the results of an ADCC reporter assay to examine the ADCC activity of combinations of antibody half molecules in the presence of cells expressing two types of antigens (EREG_SK-pca60_#2) or cells expressing one type of antigen (SK-pca60 or SKE-4B2). [Figure 3] FIG. 1 shows the time course of blood concentrations in normal mice of antibody half molecules or whole antibodies containing the variable region against mouse CD19. [Figure 4] This figure shows the quantification by FACS of the proportion of B cells in the blood of normal mice administered with antibody half molecules or whole antibodies having the variable region against mouse CD19. [Figure 5-1] This figure shows the results of confirming whether antibody half molecules form dimers (whole antibodies) by size exclusion chromatography. W indicates the elution position of the whole antibody, and H indicates the elution position of the antibody half molecules. [Figure 5-2] This is a figure showing a continuation of Figure 5-1. [Figure 6]This figure shows the results of an ADCC reporter assay to examine the ADCC activity of combinations of antibody half molecules in the presence of cells expressing two types of antigens (EREG_SK-pca60_#2) or cells expressing one type of antigen (SK-pca60 or SKE-4B2). DETAILED DESCRIPTION OF THE INVENTION

[0032] A.Definition As used herein, the term "polypeptide" encompasses all peptides in which multiple amino acids are linked by peptide bonds. Polypeptides may also be referred to as "peptides" or "proteins" herein. As used herein, the term "antigen-binding region" refers to a compound that has the activity of binding to an antigen. The antigen-binding region may be peptidic or non-peptidic.

[0033] As used herein, "CH1" refers to the polypeptide of one chain of antibody CH1. Specifically, CH1 is the region represented by amino acid residues 118 to 215 of the H chain according to the EU numbering system, and in this specification, it encompasses not only wild-type but also variants in which amino acid residues have been substituted, added, or deleted from the wild-type. As used herein, "CH2" refers to the polypeptide of one chain of antibody CH2. Specifically, CH2 is the region represented by amino acid residues 231 to 340 in the H chain according to the EU numbering system, and includes not only wild-type but also variants in which amino acid residues have been substituted, added, or deleted from the wild-type. As used herein, "CH3" refers to the polypeptide of one chain of antibody CH3. Specifically, CH3 is the region represented by amino acid residues from position 341 of the H chain to the C-terminus according to the EU numbering system, and as used herein, it encompasses not only wild-type but also variants in which amino acid residues have been substituted, added, or deleted from the wild-type. As used herein, "CL" refers to a polypeptide chain of an antibody's CL. Specifically, CL is the region of amino acid residues from position 108 of the L chain to the C-terminus according to the EU numbering system. In this specification, CL encompasses not only wild-type variants but also variants in which amino acid residues have been substituted, added, or deleted from the wild-type.

[0034] As used herein, "antibody half molecule" refers to a single molecule obtained by dissociating the bond between the H chains of an antibody, and is sometimes generally referred to as a monovalent antibody. When the antibody is an IgG, an example of an antibody half molecule is a complex consisting of one H chain and one L chain. Antibody half molecules also include molecules consisting of one H chain obtained by dissociating the bond between the H chains of antibodies consisting of two H chains found in antibodies of camelids, etc., so-called heavy-chain antibodies (also called VHH (VH originating from heavy-chain antibody) antibodies). In one embodiment, the antibody half molecules include those derived from chimeric or humanized antibodies. In one embodiment, the antibody half molecules include those derived from various isotypes such as IgG, IgM, IgA, IgD, and IgE. The antibody half molecules are preferably derived from IgG. IgG includes IgG1, IgG2, IgG3, and IgG4. The antibody half molecules may be derived from any of these subtypes. From the viewpoint of easily exerting effector function, the antibody half molecules are preferably derived from IgG1 or IgG2.

[0035] As used herein, the term "hinge region" refers to the region located between CH1 and CH2 in an antibody. Specifically, the hinge region is the region represented by amino acid residues 216 to 230 in the EU numbering system, and includes not only wild-type regions but also variants in which amino acid residues have been substituted, added, or deleted from the wild-type. As used herein, the term "hinge region in an antibody half molecule" refers to the hinge region in one H chain, and refers to a region consisting of a single polypeptide chain.

[0036] As used herein, the term "constant region" refers to a region of an antibody comprising CH1, CH2, CH3, CL, and hinge regions. As used herein, the term "constant region portion of an antibody half molecule" refers to the constant region portion of an antibody half molecule.

[0037] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, the 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) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to 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.

[0038] "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.

[0039] "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 binds to 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 (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), 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.

[0040] The term "covalent bond" as used herein includes all commonly known covalent bonds, such as disulfide bonds and carbon-carbon bonds.

[0041] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g.,211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioactive isotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents, as disclosed below.

[0042] B. Pharmaceutical Compositions In one aspect, the present invention provides a pharmaceutical composition containing either or both of a first antigen-binding molecule and a second antigen-binding molecule.

[0043] 1. First antigen-binding molecule The first antigen-binding molecule has a first antigen-binding region and a first polypeptide.

[0044] a. First antigen-binding region The first antigen-binding region is a region that binds to a first antigen. Preferably, the first antigen-binding region comprises the variable region of an antibody half molecule or a first antigen-binding fragment thereof. The first antigen-binding fragment refers to a fragment of the variable region of an antibody half molecule that retains the ability to bind to the first antigen.

[0045] The first antigen can be, for example, a protein expressed in a target cell. The protein is preferably an antigen expressed in an abnormal cell that causes the target disease. The antigen expressed in an abnormal cell is preferably a membrane protein. The membrane protein is preferably its extracellular region.

[0046] The first antigen may be the same as or different from the second antigen described below. Preferably, the first antigen and the second antigen are different. When the first antigen and the second antigen are different, the targeting specificity to abnormal cells by the combination of the first antigen-binding molecule and the second antigen-binding molecule is improved. Preferably, either or both of the first antigen and the second antigen are expressed in abnormal cells but not in normal cells, and more preferably, both of the first antigen and the second antigen are expressed in abnormal cells but not in normal cells.

[0047] The type of antigen is not particularly limited, and any antigen may be used. Examples of antigens include, but are not limited to, receptors or fragments thereof, cancer antigens, MHC antigens, and differentiation antigens.

[0048] Examples of such receptors include receptors belonging to receptor families such as the hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase receptor family, serine / threonine kinase receptor family, TNF receptor family, G protein-coupled receptor family, GPI-anchored receptor family, tyrosine phosphatase receptor family, adhesion factor family, and hormone receptor family. There are many publications regarding the receptors belonging to these receptor families and their characteristics. For example, see Cooke BA., King RJB., van der Molen HJ. eds. New Comprehensive Biochemistry Vol. 18B "Hormones and Their Actions Part II" pp. 1-46 (1988) Elsevier Science Publishers BV., New York, USA; Patthy L. (1990) Cell, 61: 13-14; Ullrich A., et al. (1990) Cell, 61: 203-212; Massagul J. (1992) Cell, 69: 1067-1070; Miyajima A., et al. (1992) Annu. Rev. Immunol., 10: 295-331; Taga T. and Kishimoto T. (1992) FASEB J., 7: 3387-3396, Fantl WI., et al. (1993) Annu. Rev. Biochem., 62: 453-481, Smith CA., et al. (1994) Cell, 76: 959-962, Flower DR. (1999) Biochim. Biophys. Acta, 1422: 207-234, and Miyasaka Masayuki (ed.), Cell Engineering Special Edition Handbook Series "Adhesion Factor Handbook" (1994) (Shujunsha, Tokyo, Japan).Specific examples of receptors belonging to the above receptor families include human or mouse erythropoietin (EPO) receptor, human or mouse granulocyte colony-stimulating factor (G-CSF) receptor, human or mouse thrombopoietin (TPO) receptor, human or mouse insulin receptor, human or mouse Flt-3 ligand receptor, human or mouse platelet-derived growth factor (PDGF) receptor, human or mouse interferon (IFN)-α and β receptors, human or mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL)-10 receptor, human or mouse insulin-like growth factor (IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, and human or mouse ciliary neurotrophic factor (CNTF) receptor (hEPOR: Simon, S. et al. (1990) Blood 76, 31-35.; mEPOR: D'Andrea, A.D. et al. (1989) Cell 57, 107-111). 277-285.; hG-CSFR: Fukunaga, R. et al. (1990) Proc.Natl. Acad. Sci. USA. 87, 8702-8706.; mG-CSFR: Fukunaga, R. et al. (1990) Cell61, 341-350.; hTPOR: Vigon, I. et al. (1992) 89, 5640-5644.; mTPOR: Skoda, RC. Et al. (1993) 12, 2645-2653.; hInsR: Ullrich, A. et al. (1985) Nature 313, 756-761.; hFlt-3: Small, D. et al. (1994) Proc. Natl. Acad. Sci. USA. 91, 459-463.; hPDGFR: Gronwald, RGK. Et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 3435-3439.; hIFNα / βR: Uze, G. et al. (1990) Cell 60, 225-234. and Novick, D. et al. (1994) Cell 77, 391-400.).

[0049] Cancer antigens are antigens that are expressed as cells become malignant and are also called tumor-specific antigens. Abnormal sugar chains that appear on cell surfaces or protein molecules when cells become cancerous also become cancer antigens, specifically called cancer sugar chain antigens. Examples of cancer antigens include CA19-9, CA15-3, and cereal SSEA-1 (SLX).

[0050] MHC antigens are broadly divided into MHC class I antigens and MHC class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, and -H, while MHC class II antigens include HLA-DR, -DQ, and -DP.

[0051] Differentiation antigens include CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, These include CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, and CDw130.

[0052] b. a first polypeptide The first polypeptide comprises either or both of a first CH2 and a first CH3. The first polypeptide preferably comprises a first CH3. In one embodiment, the first polypeptide may further comprise a hinge region portion of an antibody half molecule. In this embodiment, the first polypeptide may comprise an Fc region portion of an antibody half molecule.

[0053] In another embodiment, the first polypeptide may further comprise a CH1 in an antibody half molecule. In this embodiment, the first polypeptide may further comprise a CL in an antibody half molecule. In this embodiment, the first polypeptide may comprise a constant region portion in an antibody half molecule. The constant region portion comprises an Fc region portion.

[0054] When the first polypeptide comprises an Fc region or constant region of an antibody half molecule, the Fc region or constant region may be further modified to improve or reduce their inherent effector functions, specifically, but not limited to, modifications that enhance or reduce binding to FcγR, FcRn, or C1q.

[0055] c. Other parts The first antigen-binding molecule may have a compound other than the above-mentioned first antigen-binding region and first polypeptide. Examples of "compounds other than the first antigen-binding region and first polypeptide" include peptidic or non-peptidic linkers and other compounds. Examples of other compounds include peptidic or non-peptidic cytotoxic agents.

[0056] 2. Second antigen-binding molecule The second antigen-binding molecule has a second antigen-binding region and a second polypeptide.

[0057] a. second antigen-binding region The second antigen-binding region is a region that binds to a second antigen. Preferably, the second antigen-binding region comprises the variable region of an antibody half molecule or a second antigen-binding fragment thereof. The second antigen-binding fragment refers to a fragment of the variable region of an antibody half molecule that retains the ability to bind to a second antigen.

[0058] The second antigen can be, for example, a protein expressed in a target cell. The protein is preferably an antigen expressed in an abnormal cell that causes the target disease. The antigen expressed in an abnormal cell is preferably a membrane protein. The membrane protein is preferably its extracellular region.

[0059] The second antigen may be the same as or different from the first antigen. Preferably, the first antigen and the second antigen are different. When the first antigen and the second antigen are different, the target specificity to abnormal cells by the combination of the first antigen-binding molecule and the second antigen-binding molecule is improved. Preferably, either or both of the first antigen and the second antigen are expressed in abnormal cells but not in normal cells, and more preferably, both of the first antigen and the second antigen are expressed in abnormal cells but not in normal cells.

[0060] b. a second polypeptide The second polypeptide comprises either or both of a second CH2 and a second CH3. The second polypeptide preferably comprises a second CH3. In one embodiment, the second polypeptide may further comprise a hinge region portion of an antibody half molecule. In this embodiment, the second polypeptide may comprise an Fc region portion of an antibody half molecule.

[0061] In another embodiment, the second polypeptide may further comprise a CH1 in an antibody half molecule. In this embodiment, the second polypeptide may further comprise a CL in an antibody half molecule. In this embodiment, the second polypeptide may comprise a constant region portion in an antibody half molecule. The constant region portion comprises an Fc region portion.

[0062] When the second polypeptide comprises an Fc region or constant region of an antibody half molecule, the Fc region or constant region may be further modified to improve or reduce their inherent effector functions. Specific examples include, but are not limited to, modifications that enhance or reduce binding to FcγR, FcRn, or C1q.

[0063] c. Other parts The second antigen-binding molecule may have a compound other than the second antigen-binding region and second polypeptide described above. Examples of "compounds other than the second antigen-binding region and second polypeptide" include peptidic or non-peptidic linkers and other compounds. Examples of other compounds include peptidic or non-peptidic cytotoxic agents.

[0064] 3. Relationship between the first antigen-binding molecule and the second antigen-binding molecule The first antigen-binding molecule and the second antigen-binding molecule are not bound by a covalent bond.In the pharmaceutical composition, the first antigen-binding molecule and the second antigen-binding molecule can interact as long as they are not bound by a covalent bond.The interaction that is not a covalent bond includes hydrogen bond and intermolecular bond.The amount of the interaction is preferably small.The smaller the amount, the more reduced side effects.

[0065] In one embodiment, the molar ratio of the binding amount of the first antigen-binding molecule to the second antigen-binding molecule measured by surface plasmon resonance can be used as an index of the interaction. 2 When the affinity of both antigen-binding molecules was measured using a sensor chip on which 50 pg of the first antigen-binding molecule was immobilized per 1000 μg of the first antigen-binding molecule and a measurement solution containing 2.5 mg / mL of the second antigen-binding molecule, the molar ratio of the binding amount of the second antigen-binding molecule to the first antigen-binding molecule was within the range of 1:0.1 to 1:0.9. The molar ratio may be 1:0.9 or less as the upper limit of the amount of binding of the second antigen-binding molecule, preferably 1:0.8 or less, more preferably 1:0.7 or less, even more preferably 1:0.65 or less, and most preferably 1:0.5 or less. The lower the upper limit, the less likely heterodimers are to be formed in the absence of cells expressing the first and second antigens, and the more reduced side effects will be. On the other hand, the molar ratio may be 1:0.1 or higher as the lower limit of the amount of binding of the second antigen-binding molecule, and is preferably 1:0.14 or higher, more preferably 1:0.17 or higher, even more preferably 1:0.2 or higher, and most preferably 1:0.23 or higher. The higher the lower limit, the more easily heterodimers of the first and second antigen-binding molecules are formed on the surface of cells expressing the first and second antigens, and the higher the effector function.

[0066] An example of an apparatus used for surface plasmon resonance is Biacore (registered trademark) T200 (GE Healthcare). The test solution used in surface plasmon resonance measurements is, for example, HBS-EP+10X (GE Healthcare). HBS-EP+10X is a test solution with a 10x concentration, so it is diluted 1 / 10 before use. The specific composition of the test solution when used is 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20, pH 7.4. The preferred temperature of the test solution during measurement is 25°C.

[0067] In one embodiment, a first antigen-binding molecule and a second antigen-binding molecule are more likely to form heterodimers than homodimers when mixed in a liquid. In this embodiment, a "homodimer" refers to a dimer formed by a non-covalent interaction between a first antigen-binding molecule and a second antigen-binding molecule, or a dimer formed by a non-covalent interaction between a second antigen-binding molecule and a second antigen-binding molecule. A "heterodimer" refers to a dimer formed by a non-covalent interaction between a first antigen-binding molecule and a second antigen-binding molecule. Examples of interactions include hydrogen bonds and intermolecular bonds. From the viewpoint of reducing side effects, the first antigen-binding molecule and the second antigen-binding molecule preferably do not easily interact with each other in a liquid. However, since the interaction between the first antigen-binding molecule and the second antigen-binding molecule is in an equilibrium state, the interaction may occur if the concentrations of the first antigen-binding molecule and the second antigen-binding molecule in the liquid are increased to a level above that suitable for administration to a subject in the pharmaceutical composition. In this case, the higher the concentrations of the first antigen-binding molecule and the second antigen-binding molecule, the greater the amount of interacting first antigen-binding molecule and the second antigen-binding molecule.

[0068] Specific embodiments of the first and second antigen-binding molecules that more easily form heterodimers than homodimers when mixed in a liquid include a first polypeptide that comprises a first CH3 and a second polypeptide that comprises a second CH3, and modifications that make the first CH3 and the second CH3 more likely to form heterodimers than homodimers when mixed in a liquid include at least one of the following modifications (i) to (iii): (i) a modification in which one of the first CH3 and the second CH3 has a positively charged region and the other has a negatively charged region, and the positively charged region interacts with the negatively charged region when the heterodimer is formed; (ii) a modification in which one of the first CH3 and the second CH3 has a convex portion and the other has a concave portion, and the convex portion fits into the concave portion and interacts with the heterodimer when the heterodimer is formed; (iii) The first CH3 and the second CH3 are CH3s of modified IgG, a portion of the modified IgG CH3 is replaced with a portion of IgA CH3, and when the heterodimer is formed, the portion of the IgA CH3 replaced with the first CH3 and the portion of the IgA CH3 replaced with the second CH3 interact with each other.

[0069] Examples of the (i) modification include those disclosed in WO 2006 / 106905, WO 2009 / 089004, WO 2010 / 129304, and WO 2014 / 084607. Specific examples of the method include modifying at least one combination of amino acids with the same charge among the combinations of positions 356 and 439, 357 and 370, and 399 and 409 (EU numbering system) in the amino acid sequence of the heavy chain constant region of a polypeptide having a first antigen-binding activity, and modifying at least one combination of amino acids with the opposite charge to that of the polypeptide having a second antigen-binding activity among the combinations of positions 356 and 439, 357 and 370, and 399 and 409 (EU numbering system) in the heavy chain constant region of a polypeptide having a second antigen-binding activity or no antigen-binding activity. More specifically, for example, in the amino acid sequences of the heavy chain constant regions of a polypeptide having a first antigen-binding activity and a polypeptide having a second antigen-binding activity, a mutation is introduced into one of the polypeptides to substitute Glu at position 356 (EU numbering system) with Lys, and a mutation is introduced into the other polypeptide to substitute Lys at position 439 (EU numbering system) with Glu.

[0070] Examples of the modification (ii) include those disclosed in WO 96 / 027011 and Margaret Merchant et al., Nature Biotechnology 1998, 16, 677-681. Specific examples of the method include a combination of introducing T366Y into CH3 and Y407A into the other CH3, a combination of introducing T366W into one CH3 and Y407A into the other CH3, a combination of introducing F405A into one CH3 and T394W into the other CH3, a combination of introducing Y407T into one CH3 and T366Y into the other CH3, or a combination of introducing T366Y / F405A into one CH3 and T394W / Y407T into the other CH3. or a combination of introducing T366W / F405W into one CH3 and T394S / Y407A into the other CH3; or a combination of introducing F405W / Y407A into one CH3 and T366W / T394S into the other CH3; or a combination of introducing F405W into one CH3 and T394S into the other CH3; or a combination of introducing T366W into one CH3 and T366S / L368A / Y407V into the other CH3. The modification (ii) can also be combined with the modification (i). Examples of such combinations include those disclosed in WO 2012 / 058768.

[0071] The (iii) modification is a technique that efficiently induces interaction between polypeptides with different sequences through complementary interaction of the CH3 domains by using a strand-exchange engineered domain CH3 in which a portion of the CH3 domain of one antibody H chain is replaced with a corresponding IgA-derived sequence and the complementary portion of the CH3 domain of the other H chain is replaced with a corresponding IgA-derived sequence (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently facilitate heterodimer formation. Examples of the (iii) modification include the modification technique disclosed in WO 2007 / 110205.

[0072] In another specific embodiment, the first and second antigen-binding molecules that are more likely to form heterodimers than homodimers when mixed in liquid may be modified in the hinge region, for example, by the modification techniques disclosed in WO 2011 / 143545.

[0073] Either or both of the first CH3 and the second CH3 preferably have a substitution of at least one amino acid residue among the amino acid residues at positions 357, 397, and 409 in the EU numbering system. By adding such a modification, the above-mentioned surface plasmon resonance can be measured at 1 mm 2 When the affinity of both antigen-binding molecules is measured using a sensor chip on which 50 pg of the first antigen-binding molecule is immobilized per molecule and a measurement solution containing 2.5 mg / mL of the second antigen-binding molecule, the amount of the second antigen-binding molecule bound to the first antigen-binding molecule can be easily set within the above-mentioned molar ratio range.

[0074] In one embodiment, it is preferred that the amount of heterodimer formed in the presence of cells expressing the first antigen and the second antigen is greater than in the absence of the cells. Conditions in which the cells are absent include conditions in which cells expressing the first and second antigens are absent, but conditions in which cells expressing the first antigen but not the second antigen, or cells expressing the second antigen but not the first antigen, are present. That is, "the amount of heterodimer formed in the presence of cells expressing the first and second antigens is greater than that in the absence of cells" includes cases in which, when the pharmaceutical composition is administered to a living body, the amount of heterodimer formed on the surface of cells expressing the first and second antigens is greater than the amount of heterodimer formed on the surface of cells expressing the first antigen but not the second antigen, or cells expressing the second antigen but not the first antigen.

[0075] In one embodiment, when a heterodimer is formed on the surface of a cell expressing a first antigen and a second antigen, the FcγR-binding activity of the heterodimer is higher than the FcγR-binding activity of a monomer of the first antigen-binding molecule or a monomer of the second antigen-binding molecule, or the FcγR-binding activity of the homodimer when a homodimer is formed. This means, for example, that when the pharmaceutical composition is administered to a subject having cells expressing the first antigen and the second antigen, the heterodimer formed by the first antigen-binding molecule and the second antigen-binding molecule that has reached the cell surface induces higher FcγR activation and exerts effector function than the monomer simply bound to the cell surface or the homodimer formed on the cell surface. This further reduces side effects. In this embodiment, the FcγR includes, for example, rodent and primate FcγRs, and may be any one of these FcγRs. In this embodiment, the FcγR is preferably rodent or primate FcγR. Rodents are preferably mice and rats. Primates are preferably cynomolgus monkeys and humans. In this embodiment, the FcγR includes human FcγR and its homologs in rodents and non-human primates that are structurally homologous and have similar functions. In this embodiment, FcγR subclasses include human FcγRI, human FcγRII, and human FcγRIII, as well as their rodent and non-human primate homologs. Among these, the FcγR is preferably human FcγRII or human FcγRIII, or their rodent and non-human primate homologs, more preferably human FcγRIII or its rodent and non-human primate homologs. The human FcγR is preferably human FcγRII or human FcγRIII, more preferably human FcγRIII. In this embodiment, human FcγRII is further divided into human FcγRIIA, human FcγRIIB, and human FcγRIIC. Of these, human FcγRII is preferably human FcγRIIB. Human FcγRIII is further divided into human FcγRIIIA and human FcγRIIIB. Of these, human FcγRIII is preferably human FcγRIIIA.

[0076] In one embodiment, the effector function under the condition that cells expressing the first antigen and the second antigen exist is higher than that under the condition that cells expressing the first antigen but not the second antigen exist, or cells expressing the second antigen but not the first antigen exist, thereby further reducing side effects. The effector function is preferably ADCC and CDC, more preferably ADCC.

[0077] In one embodiment, when either or both of the first and second polypeptides comprise a hinge region portion of an antibody half molecule, the hinge region portion in either or both of the first and second polypeptides has a substitution of another amino acid residue at either or both of the cysteine ​​residues at positions 226 and 229 in the EU numbering system. In this embodiment, preferably, the hinge region portions in both of the first and second polypeptides have a substitution of another amino acid residue at either or both of the cysteine ​​residues at positions 226 and 229 in the EU numbering system, or the hinge region portions in either or both of the first and second polypeptides have a substitution of another amino acid residue at both of the cysteine ​​residues at positions 226 and 229 in the EU numbering system. More preferably, the hinge region portions in both of the first and second polypeptides have a substitution of another amino acid residue at both of the cysteine ​​residues at positions 226 and 229 in the EU numbering system. Such substitution can suppress disulfide bonds between H chains, making it easier to prevent the first and second antigen-binding molecules from being bound by a covalent bond. In this embodiment, more preferably, substitution of one or both of the cysteine ​​residues at positions 226 and 229 (EU numbering system) with another amino acid residue is combined with substitution of at least one of the amino acid residues at positions 357, 397, and 409 (EU numbering system) in either or both of the first CH3 and second CH3 with another amino acid residue. This combination of alterations prevents the first and second antigen-binding molecules from being covalently bound and makes them more likely to form heterodimers than homodimers when mixed in liquid.

[0078] 4. Other ingredients The pharmaceutical composition may contain other components in addition to the first and second antigen-binding molecules. Other ingredients include, for example, a pharmaceutically acceptable carrier.

[0079] Pharmaceutical compositions can be formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and mixing them in unit dosage forms required for generally accepted pharmaceutical practice. The amount of active ingredient in these preparations is set so that an appropriate volume within the specified range is obtained.

[0080] Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as distilled water for injection.

[0081] Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80™, HCO-50), may be used in combination.

[0082] Oily liquids include sesame oil and soybean oil, and may contain benzyl benzoate and / or benzyl alcohol as solubilizers. Buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may also be added. The prepared injection solution is usually filled into suitable ampoules.

[0083] In one embodiment, when the target disease of the pharmaceutical composition is a malignant tumor among cell proliferative disorders, preferably both the first and second antigens are cancer antigens, and the pharmaceutical composition contains a cytotoxic agent as another component, which includes immune checkpoint inhibitors in addition to the cytotoxic agents exemplified above.

[0084] 5. Dosage form The pharmaceutical composition is preferably administered parenterally. For example, it can be an injection, a nasal administration, a pulmonary administration, or a transdermal administration composition. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. The first antigen-binding molecule and the second antigen-binding molecule may be formulated in the same formulation or in separate formulations. When the pharmaceutical composition contains a cytotoxic agent as another component, the cytotoxic agent may be formulated in the same formulation as the first antigen-binding molecule or the second antigen-binding molecule or in separate formulations. When formulated in separate formulations, the timing of administration can be determined for each component.

[0085] 6.Target diseases The target disease of the pharmaceutical composition is not particularly limited, but is preferably a disease caused by pathogenic cells expressing a first antigen and a second antigen, i.e., a disease in which it is desirable for the first antigen-binding molecule and the second antigen-binding molecule to form a heterodimer on the cell surface and exert effector function. Specific target diseases include, for example, cell proliferative diseases, immune-enhancing diseases, and infectious diseases. Cell proliferative diseases include tumors. Immune-enhancing diseases include autoimmune diseases. Infectious diseases include bacterial infections and viral infections.

[0086] 7. Manufacturing method The first and second antigen-binding molecules are produced by general methods for obtaining proteins. Antigen-binding molecules are usually obtained by expressing them in host cells using nucleic acids encoding them. Antigen-binding molecules expressed in host cells are usually recovered from the host cells and purified. The first and second antigen-binding molecules may be obtained by co-expression in host cells or by expression in separate host cells. Specific production methods are described below.

[0087] Nucleic acids are usually carried (inserted) into an appropriate vector and introduced into host cells. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, when Escherichia coli is used as the host, a cloning vector such as the pBluescript vector (Stratagene) is preferred, although various commercially available vectors can also be used. When a vector is used for the purpose of producing an antigen-binding molecule, an expression vector is particularly useful. The expression vector is not particularly limited as long as it expresses a polypeptide in a test tube, in Escherichia coli, in cultured cells, or in an individual organism. For example, preferred expression vectors include the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for Escherichia coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) for individual organisms. The DNA of the present invention can be inserted into a vector by conventional methods, for example, by ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology, ed. Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 11.4-11.11).

[0088] The host cells are not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing antigen-binding molecules include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vectors can be introduced into host cells by known methods, such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology, edited by Ausubel et al. (1987) Published by John Wiley & Sons, Sections 9.1-9.9), lipofectamine (GIBCO BRL), and microinjection.

[0089] To secrete an antigen-binding molecule expressed in a host cell into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the antigen-binding molecule of interest. These signals may be endogenous or heterologous to the antigen-binding molecule of interest.

[0090] In the above-mentioned production methods, when the antigen-binding molecule is secreted into the medium, the medium is collected, whereas when the antigen-binding molecule is produced intracellularly, the cells are first lysed and then the antigen-binding molecule is collected.

[0091] Antigen-binding molecules can be recovered and purified from recombinant cell cultures by known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.

[0092] In the production of bispecific antibodies in which the H chains are linked by a covalent bond such as a disulfide bond, a purification step is often required to remove antigen-binding molecules with undesired H and L chain combinations. In contrast, in the production of the first and second antigen-binding molecules of the present invention, such a purification step can be omitted because the first and second antigen-binding molecules are not covalently linked. For example, when the first and second antigen-binding molecules are coexpressed in host cells, it is sufficient to simply collect fractions containing the first and second antigen-binding molecules from the culture medium or cell lysate using various chromatography techniques. When the first and second antigen-binding molecules are expressed in separate host cells, the culture medium and cell lysate may be mixed and then the antigen-binding molecules may be purified. Alternatively, the first and second antigen-binding molecules may be purified separately from the culture medium or cell lysate and then mixed. From the viewpoint of reducing the effort required for the purification step to eliminate antigen-binding molecules comprising undesired combinations of H chains and L chains and reducing the waste of antigen-binding molecules eliminated during this step, it is preferable to express the first antigen-binding molecule and the second antigen-binding molecule in separate host cells.

[0093] When preparing a pharmaceutical composition by mixing the first and second antigen-binding molecules immediately before administering it to a subject, or when sequentially administering a first pharmaceutical composition containing the first antigen-binding molecule but not the second antigen-binding molecule and a third pharmaceutical composition containing the second antigen-binding molecule but not the first antigen-binding molecule, the first and second antigen-binding molecules are preferably expressed in separate cells and purified to produce pharmaceutical compositions containing the first and second antigen-binding molecules separately.

[0094] C. Alternative Embodiments of Antigen-Binding Molecules and Pharmaceutical Compositions 1. Alternative embodiments of the first antigen-binding molecule Another specific embodiment of the first antigen-binding molecule is, for example, a first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising either or both of a first CH2 and a first CH3, which, when mixed in liquid with a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising either or both of a second CH2 and a second CH3, is more likely to form a heterodimer with the second antigen-binding molecule than a homodimer with the first antigen-binding molecule, and in the heterodimer, the first antigen-binding molecule and the second antigen-binding molecule are not covalently bound. In this embodiment, the first antigen-binding region, the first polypeptide, and other portions are the same as those described in "1. First antigen-binding molecule." The second antigen-binding region, the second polypeptide, and other portions are the same as those described in "2. Second antigen-binding molecule." The relationship between the first antigen-binding molecule and the second antigen-binding molecule is the same as that described in "3. Relationship between the first antigen-binding molecule and the second antigen-binding molecule."

[0095] In this embodiment, the first antigen-binding molecule can be produced as a first pharmaceutical composition containing the first antigen-binding molecule but not the second antigen-binding molecule. In this case, a second pharmaceutical composition containing the second antigen-binding molecule is produced separately. The second pharmaceutical composition may or may not contain the first antigen-binding molecule. The second pharmaceutical composition may or may not be produced in the same establishment. The first and second pharmaceutical compositions are used in combination for a subject. In this embodiment, the first pharmaceutical composition is the same as the above-mentioned "1. First antigen-binding molecule," "2. Second antigen-binding molecule," "3. Relationship between the first and second antigen-binding molecules," "4. Other ingredients," "5. Dosage form," "6. Target disease," and "7. Production method," except that it does not contain a second antigen-binding molecule.

[0096] 2. Other aspects of the second antigen-binding molecule Another specific embodiment of the second antigen-binding molecule is, for example, a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising either or both of a second CH2 and a second CH3, which, when mixed in liquid with a first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising either or both of a first CH2 and a first CH3, is more likely to form a heterodimer with the first antigen-binding molecule than a homodimer with the second antigen-binding molecule, and in the heterodimer, the first antigen-binding molecule and the second antigen-binding molecule are not covalently bound. In this embodiment, the first antigen-binding region, the first polypeptide, and other portions are the same as those described in "1. First antigen-binding molecule." The second antigen-binding region, the second polypeptide, and other portions are the same as those described in "2. Second antigen-binding molecule." The relationship between the first antigen-binding molecule and the second antigen-binding molecule is the same as that described in "3. Relationship between the first antigen-binding molecule and the second antigen-binding molecule."

[0097] In this embodiment, the second antigen-binding molecule can be prepared as a third pharmaceutical composition containing the second antigen-binding molecule but not the first antigen-binding molecule. In this case, a fourth pharmaceutical composition containing the first antigen-binding molecule is prepared separately. The fourth pharmaceutical composition may or may not contain the first antigen-binding molecule. These third and fourth pharmaceutical compositions are used in combination. In this embodiment, the third pharmaceutical composition is the same as the above-mentioned "1. First antigen-binding molecule," "2. Second antigen-binding molecule," "3. Relationship between the first and second antigen-binding molecules," "4. Other ingredients," "5. Dosage form," "6. Target disease," and "7. Production method," except that it does not contain the first antigen-binding molecule.

[0098] 3. Alternative Embodiments of Pharmaceutical Compositions Another specific embodiment of the pharmaceutical composition includes, for example, a pharmaceutical composition comprising a first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising a first CH3, and a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising a second CH3, wherein the first antigen-binding molecule and the second antigen-binding molecule are not covalently linked, and the first CH3 and the second CH3 have at least one of the following modifications (iv) to (vi): (iv) a modification in which one of the first CH3 and the second CH3 has a positively charged region and the other has a negatively charged region, and the positively charged region interacts with the negatively charged region when the heterodimer is formed. (v) a modification in which one of the first CH3 and the second CH3 has a convex portion and the other has a concave portion, and the convex portion fits into the concave portion and interacts with each other when the heterodimer is formed; (vi) The first CH3 and the second CH3 are CH3s of modified IgG, a portion of the modified IgG CH3 is replaced with a portion of IgA CH3, and when the heterodimer is formed, the portion of the IgA CH3 replaced with the first CH3 and the portion of the IgA CH3 replaced with the second CH3 interact with each other.

[0099] In this embodiment, the first and second antigen-binding molecules are the same as those described above in "1. First antigen-binding molecule" and "2. Second antigen-binding molecule." Details of the pharmaceutical composition of this embodiment are the same as those described above in "4. Other ingredients," "5. Dosage form," "6. Target disease," and "7. Production method."

[0100] Examples of the modification (iv) above include those disclosed in WO 2006 / 106905, WO 2009 / 089004, WO 2010 / 129304, and WO 2014 / 084607. Specific examples of the method include modifying at least one combination of amino acids with the same charge among the combinations of positions 356 and 439, 357 and 370, and 399 and 409 (EU numbering system) in the amino acid sequence of the heavy chain constant region of the polypeptide having a first antigen-binding activity, and modifying at least one combination of amino acids with the opposite charge to that of the polypeptide having a second antigen-binding activity among the combinations of positions 356 and 439, 357 and 370, and 399 and 409 (EU numbering system) in the heavy chain constant region of the polypeptide having a second antigen-binding activity or no antigen-binding activity. More specifically, for example, in the amino acid sequences of the heavy chain constant regions of a polypeptide having a first antigen-binding activity and a polypeptide having a second antigen-binding activity, a mutation is introduced into one of the polypeptides to substitute Glu at position 356 (EU numbering system) with Lys, and a mutation is introduced into the other polypeptide to substitute Lys at position 439 (EU numbering system) with Glu.

[0101] Examples of the modification (v) include those disclosed in WO 96 / 027011 and Margaret Merchant et al., Nature Biotechnology 1998, 16, 677-681. Specific examples of the method include a combination of introducing T366Y into one CH3 and Y407A into the other CH3, or a combination of introducing T366W into one CH3 and Y407A into the other CH3, or a combination of introducing F405A into one CH3 and T394W into the other CH3, or a combination of introducing Y407T into one CH3 and T366Y into the other CH3, or a combination of introducing T366Y / F405A into one CH3 and T394W / Y407T into the other CH3. or a combination of introducing T366W / F405W into one CH3 and T394S / Y407A into the other CH3; or a combination of introducing F405W / Y407A into one CH3 and T366W / T394S into the other CH3; or a combination of introducing F405W into one CH3 and T394S into the other CH3; or a combination of introducing T366W into one CH3 and T366S / L368A / Y407V into the other CH3. The modification (v) can also be combined with the modification (iv). Examples of such combinations include those disclosed in WO 2012 / 058768.

[0102] The (vi) modification is a technique that efficiently induces interaction between polypeptides with different sequences through complementary interaction of the CH3 domains by using a strand-exchange engineered domain CH3 in which a portion of the CH3 domain of one antibody H chain is replaced with a corresponding IgA-derived sequence and the complementary portion of the CH3 domain of the other H chain is replaced with a corresponding IgA-derived sequence (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently facilitate heterodimer formation. Examples of the (vi) modification include the modification technique disclosed in WO 2007 / 110205.

[0103] In another specific embodiment, the first and second antigen-binding molecules that are more likely to form heterodimers than homodimers when mixed in liquid may be modified in the hinge region, for example, by the modification techniques disclosed in WO 2011 / 143545.

[0104] Either or both of the first CH3 and the second CH3 preferably have a substitution of at least one amino acid residue among the amino acid residues at positions 357, 397, and 409 in the EU numbering system. By adding such a modification, the above-mentioned surface plasmon resonance can be measured at 1 mm 2 When the affinity of both antigen-binding molecules is measured using a sensor chip on which 50 pg of the first antigen-binding molecule is immobilized per molecule and a measurement solution containing 2.5 mg / mL of the second antigen-binding molecule, the amount of the second antigen-binding molecule bound to the first antigen-binding molecule can be easily set within the above-mentioned molar ratio range.

[0105] D. Treatment method In one aspect, the present invention provides a method of treatment comprising administering a first antigen-binding molecule and a second antigen-binding molecule simultaneously or sequentially to a subject having a disease caused by pathogenic cells expressing the first antigen and the second antigen. The first antigen-binding molecule is the same as that in "1. First antigen-binding molecule" in "B. Pharmaceutical compositions" above, or "1. Other embodiments of the first antigen-binding molecule" in "C. Other embodiments of antigen-binding molecules and pharmaceutical compositions." The second antigen-binding molecule is the same as that described above in "2. Second antigen-binding molecule" in "B. Pharmaceutical compositions" or "2. Other embodiments of the second antigen-binding molecule" in "C. Other embodiments of antigen-binding molecules and pharmaceutical compositions." The first and second antigen-binding molecules are not covalently bound to each other before and after administration, but form a heterodimer on the surface of a pathogenic cell to exert an effector function.

[0106] Simultaneous administration includes administration of a pharmaceutical composition containing a first antigen-binding molecule and a second antigen-binding molecule, as well as simultaneous administration of a first pharmaceutical composition containing the first antigen-binding molecule but not the second antigen-binding molecule and a third pharmaceutical composition containing the second antigen-binding molecule but not the first antigen-binding molecule. In sequential administration, a first pharmaceutical composition containing a first antigen-binding molecule but not a second antigen-binding molecule and a third pharmaceutical composition containing a second antigen-binding molecule but not the first antigen-binding molecule are administered at staggered times. The administration interval between the first and second pharmaceutical compositions is set within a range that allows the first and second antigen-binding molecules to form heterodimers on the surface of pathogenic cells and exert their effector functions after administration. "Concurrent or sequential administration" encompasses a combination of concurrent and sequential administration.

[0107] The administration method can be selected appropriately depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing an antibody or a polynucleotide encoding the antibody can be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dosage can be set, for example, in the range of 0.001 to 100,000 mg per patient, although the present invention is not necessarily limited to these numerical values. The dosage and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can determine an appropriate dosage and administration method taking these conditions into consideration.

[0108] E. Screening Methods In one aspect, the present invention provides a screening method for selecting a combination of a first antigen-binding molecule and a second antigen-binding molecule. The screening method is a method for selecting a combination of a first antigen-binding molecule and a second antigen-binding molecule. The combination is selected from a group of variants of a first antigen-binding molecule and a group of variants of a second antigen-binding molecule.

[0109] The group of variants of the first antigen-binding molecule is a collection of variant antigen-binding molecules that have a first antigen-binding region that binds to a first antigen and a first polypeptide that includes either or both of a first CH2 and a first CH3. The 21st group of antigen-binding molecule variants is a collection of antigen-binding molecule variants having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising either or both of a second CH2 and a second CH3.

[0110] Combinations of first and second antigen-binding molecules are selected from these collections so as to satisfy all of the following (a) to (c): (a) The first antigen-binding molecule and the second antigen-binding molecule are not bound by a covalent bond. (b) The first and second antigen-binding molecules more readily form heterodimers between the first and second antigen-binding molecules than homodimers between the first or second antigen-binding molecules. (c) Surface plasmon resonance at 1 mm 2 When the affinity of both antigen-binding molecules was measured using a sensor chip on which 50 pg of the first antigen-binding molecule was immobilized per molecule and a measurement solution containing 2.5 mg / mL of the second antigen-binding molecule, the binding amount of the second antigen-binding molecule to the first antigen-binding molecule was found to be in the molar ratio range of 1:0.1 to 1:0.9.

[0111] The selection methods (a) and (b) include, for example, fractionation based on molecular size, such as size exclusion chromatography. The selection method (c) is carried out, for example, based on whether the molar ratio calculated from the results of surface plasmon resonance is within the above range. [Example]

[0112] [Test Example 1] Construction of an expression vector for antibody half molecules and expression and purification of antibody half molecules Amino acid substitutions were introduced using a QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR, or an Infusion Advantage PCR cloning kit (TAKARA) by methods known to those skilled in the art to construct expression vectors. The nucleotide sequence of the resulting expression vector was determined by methods known to those skilled in the art. The constructed plasmids were transiently introduced into human embryonic kidney carcinoma cell line HEK293H (Invitrogen) or FreeStyle293 cells (Invitrogen) to express antibody half molecules. Antibody half molecules were purified from the resulting culture supernatant using rProtein A Sepharose® Fast Flow (GE Healthcare) by methods known to those skilled in the art. The concentration of purified antibody half molecules was determined by measuring absorbance at 280 nm using a spectrophotometer and calculating the extinction coefficient calculated from the obtained value using the PACE method (Protein Science 1995; 4: 2411-2423).

[0113] [Test Example 2] Analysis of molecular weight of antibody half molecules The molecular weight of the resulting antibody half molecules was determined using HPLC with an Agilent 1260 Infinity (registered trademark) (Agilent Technologies) and a G3000SW column. XL (TOSOH) was used for analysis by a method known to those skilled in the art. The antibody half molecule protein concentration was 0.25 mg / mL, and 80 μL was injected.

[0114] [Test Example 3] Preparation of FcγR and evaluation of binding activity to FcγR The extracellular domain of FcγR was prepared as follows. First, the gene encoding the extracellular domain of FcγR was synthesized by methods known to those skilled in the art. Polymorphisms are known in FcγRIIIa, and the polymorphic sites were identified with reference to J. Clin. Invest., 1997, 100(5):1059-1070.

[0115] The resulting gene fragment was inserted into an animal cell expression vector to prepare an expression vector. The constructed expression vector was transiently transfected into FreeStyle293 cells (Invitrogen), derived from human embryonic renal carcinoma cells, to express the target protein. After culturing, the resulting culture supernatant was collected and filtered through a 0.22 μm filter. The resulting culture supernatant was purified in four steps: the first step was cation exchange column chromatography (SP Sepharose® FF), the second step was His-tag affinity column chromatography (HisTrap HP), the third step was gel filtration column chromatography (Superdex® 200), and the fourth step was sterile filtration. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer, and the concentration of the purified protein was calculated from the absorbance value obtained using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423). The interaction between the target antibody half molecule and FcγR was analyzed using a Biacore® T200. The Biotin CAPture Kit, Series S (GE Healthcare) was used for the measurement, and HBS-EP+10X (GE Healthcare) diluted 1:10 was used as the running buffer. The measurement temperature was 25°C. The sensor chip used was a Series S Sencor Chip CAP (GE Healthcare) on which a pre-biotinylated antigen peptide had been interacted and immobilized. The target antibody half molecule was captured onto these chips, and then allowed to interact with FcγR diluted in the running buffer. The antigen and antibody half molecule captured on the chip were washed according to the instructions provided with the kit, and the chip was regenerated and reused.

[0116] The binding activity of antibody half molecules to FcγR was evaluated mainly using the binding activity to FcγR and the dissociation constant for FcγR as indicators.

[0117] The dissociation constant of each antibody half molecule with FcγR was calculated by kinetic analysis of the Biacore® measurement results. Specifically, the sensorgrams obtained by measurement using Biacore® Evaluation Software were globally fitted using a 1:1 Langmuir binding model to calculate the binding rate constant ka (L / mol / s) and the dissociation rate constant kd (1 / s), and the dissociation constant KD (mol / L) was calculated from these values.

[0118] [Test Example 4] Measurement of ADCC activity of half molecules of each test antibody using FcγRIIIa-V158 Jurkat cells (Promega) as effector cells Using FcγRIIIa-V158 Jurkat cells (hereinafter referred to as Jurkat cells) as effector cells, the ADCC activity of each test antibody half molecule was measured as follows.

[0119] Preparation of Jurkat cells Jurkat cells were collected from the flask and washed once with RPMI 1640 medium (Gibco) containing 4% FBS (hereinafter referred to as Assay Buffer). The cells were then placed in Assay Buffer at a cell density of 3 x 10 6 The cells were suspended at 1000 cells / mL, and the resulting cell suspension was used as a Jurkat cell solution in subsequent experiments.

[0120] (1) Preparation of target cells SK-Hep-1 cells expressing human glypican 3 (SK-pca60), SKE-4B2 cells expressing human epiregulin, or EREG_SK-pca60_#2 cells expressing both human glypican 3 and human epiregulin were detached from the dish and washed once with Assay Buffer. The cells were then placed in Assay Buffer at a cell density of 1 x 10 6 The cells were suspended at 1000 cells / mL, and the resulting cell suspension was used as the target cell solution in subsequent experiments.

[0121] (2) Preparation of luminescent reagent Add 100 mL of Bio-Glo Luciferase Assay Buffer (Promega) to the bottle of Bio-Glo Luciferase Assay Substrate (Promega) and mix by inverting. Protect the bottle from light and freeze at -20°C. This luminescent reagent was used in subsequent experiments.

[0122] (3) ADCC reporter assay (ADCC activity) ADCC activity was evaluated by measuring the fold change in luciferase luminescence. First, 25 μl of the target cells prepared in (2) was placed in each well of a 96-well flat-bottom white plate (2.5 x 10 4 Next, 25 μl of antibody half molecule solutions prepared at various concentrations (0.00003, 0.0003, 0.003, 0.03, 0.3, 3, 30 μg / mL) were added to each well. 25 μl of Jurkat cell solution (7.5 x 10 cells / well) prepared in (1) was added to each well. 4 The plate containing 150 μl of 1000 cells / well was placed in a 5% carbon dioxide incubator at 37°C for 24 hours. The luminescence reagent prepared in (3) was thawed, and 75 μl was added to each well. The plate was then left to stand at room temperature for 10 minutes. The luminescence of luciferase in 150 μl of culture supernatant in each well of the plate was measured using a luminometer. ADCC activity was calculated based on the following formula 1.

[0123] (Formula 1) Fold change=A / B

[0124] In the above formula 1, A represents the average luciferase luminescence of 150 μl of culture supernatant in each well. Furthermore, B represents the average luciferase luminescence of 150 μl of culture supernatant when 25 μl of Assay Buffer was added instead of the antibody half molecule solution in the experiment (3). The test was performed in triplicate, and the average ADCC activity (fold change) in the test, which reflects the ADCC activity of each test antibody half molecule, was calculated.

[0125] [Test Example 5] Measurement of interactions between antibody half molecules The interactions between the resulting antibody half molecules were analyzed using a Biacore® T200. Measurements were performed using a Biotin CAPture Kit, Series S (GE Healthcare), and a running buffer of HBS-EP+10X (GE Healthcare) diluted 1:10. The measurement temperature was 25°C. The sensor chip used was a Series S Sencor Chip CAP (GE Healthcare) with a pre-biotinylated antigen peptide interacted and immobilized. Approximately 50 RU of the target antibody half molecule A was captured onto these chips, and the target antibody half molecule B diluted with the running buffer to 2.5 mg / mL was allowed to interact with the antibody half molecule B at a flow rate of 30 μL / min for 180 seconds. The molar binding ratio was calculated using the following equation (2). The antibody half molecule A captured on the chip was washed according to the instructions provided with the kit, and the chip was regenerated and reused.

[0126] (Formula 2) Molar binding ratio = (binding amount of antibody half molecule B bound as analyte (RU, maximum value) / molecular weight of antibody half molecule B) / (binding amount of captured antibody half molecule A (RU) / molecular weight of antibody half molecule A)

[0127] [Test Example 6] Measurement of blood concentrations of 1D3-Kn125 / Hl076, 1D3-DA303v2, and 1D3-DB220v2 Measurements were performed using electrochemical immunoassay (ECLIA). Plasma samples were added to a MULTI-ARRAY 96-well plate (MSD) coated with human IgG-heavy and light chain antibodies (Bethyl) and allowed to react at room temperature. Subsequently, anti-human IgG (Jackson Immuno Research) was added, followed by SULFO-TAG streptavidin (MSD) and further reaction. Measurements were performed using a SECTOR S 600 (MSD).

[0128] [Test Example 7] Measurement of blood concentration of 1D3-DA303v2 / DB220v2 Measurements were performed by LC-MS. Plasma samples were mixed with Ab-Capcher Mag (ProteNova) and then a mixture containing lysozyme, DTT, and urea was added and allowed to react. Iodoacetamid solution (Sigma-Aldrich) was then added, followed by trypsin solution. The supernatant was then collected, trifluoroacetic acid (Wako) was added, and the mixture was injected into an Acquity UPLC (Waters) column and analyzed using a Xevo TQ-S (Waters).

[0129] [Test Example 8] Measurement of B cells in blood Blood was collected from C57BL / 6NCr Slc mice (male, 7 weeks old, Japan SLC Co., Ltd.) via the dorsal paw vein using a hemacrit capillary tube (Terumo Corporation). 15 μL of collected blood was added to 2 mL of ACK Lynching Buffer (Gibco) and incubated for 5 minutes at room temperature in the dark. After 5 minutes of centrifugation at 500 × g, the supernatant was removed. This process was repeated. A buffer (FACS buffer) was prepared by adding 75 mL of BSA stock solution (Miltenyi Biotec) to 1450 mL of MACS buffer (Miltenyi Biotec). FcR blocking reagent (Miltenyi Biotec) was diluted 10-fold and 20 μL per tube was used to suspend the cells. After incubation at room temperature for 10 minutes, 0.5 μL of BUV395 anti-CD45R / B220 (BD), 0.5 μL of PE-CF594 anti-IgM (BD), and 0.4 μL of Zombie NIR Fixable Viability Kit were added and incubated at 4°C for 20 minutes. After addition of 2 mL of FACS buffer, the cells were centrifuged at 500 × g for 5 minutes, the supernatant was removed, and the cells were resuspended in 400 μL of FACS buffer and analyzed using a BD FACS LSR Fortessa X-20 (BD).

[0130] [Test Example 9] Analysis of the proportion of B cells in live cells Analysis was performed using Flowjo ver. 7.6 (Tomy Digital Biology). After gating on live cells, the CD45R / B220+ IgM+ fraction to be analyzed was designated as B cells. The percentage of B cells among live cells was calculated for each sample.

[0131] [Example 1] Preparation of antibody half molecules The specific amino acid substitution procedure for producing antibody half molecules with selective cytotoxicity against double-positive cells is described below. Table 1 shows the abbreviations and names of the produced and evaluated antibody half molecules, the CH3 modifications, and the sequence numbers of the antibody half molecules. If the H-chain variable region portion of the antibody half molecule is designated VH, the sequence corresponding to the H-chain of the antibody half molecule having CH in its constant region is referred to as VH-CH. If the L-chain variable region portion of the antibody half molecule is designated VL, the sequence corresponding to the L-chain of the antibody half molecule having CL in its constant region is referred to as VL-CL. For example, in the case of a homodimerized antibody in which the expression vector corresponding to the antibody half molecule H-chain used for expression is VH1-CH1 and the expression vector corresponding to the antibody half molecule L-chain is VL1-CL1, the antibody half molecules obtained by purification after expression are designated VH1-CH1 / VL1-CL1. When one of the expression vectors corresponding to the antibody half molecule H chain used to express a four-chain heterodimerized antibody is VH1-CH1, the other antibody half molecule H chain is VH2-CH2, and one of the expression vectors corresponding to the antibody half molecule L chain is VL1-CL1 and the other antibody half molecule L chain is VL2-CL2, the expression vector is represented as VH1-CH1 / VL1-CL1 / / VH2-CH2 / VL2-CL2. Furthermore, when equal amounts of each antibody half molecule are mixed, if antibody half molecule A is VH1-CH1 / VL1-CL1 and antibody half molecule B is VH2-CH2 / VL2-CL2, the expression vector is represented as VH1-CH1 / VL1-CL1+VH2-CH2 / VL2-CL2. For simplicity, the abbreviations for the constant regions may be used alone, such as A1, B1, or A1+B1. Amino acid modifications are indicated, such as D356K. The first letter (corresponding to the D in D356K) represents the alphabetical character of the amino acid residue before modification when expressed in single letter code, the following number (corresponding to the 356 in D356K) represents the EU number of the modification site, and the last letter (corresponding to the K in D356K) represents the alphabetical character of the amino acid residue after modification when expressed in single letter code. Furthermore, a sequence in which the terminal GK of natural IgG1 was deleted was used as a template for modification.

[0132] First, the modifications described in WO 2013 / 002362 were used to enhance ADCC activity upon heterodimerization. L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A were substituted in CH2 of antibody half molecule A, and D270E, K326D, A330M, and K334E were substituted in antibody half molecule B, respectively. To prevent disulfide bond formation between the hinge regions of the antibody, C226S and C229S were used in antibody half molecules A and B, respectively. In addition to these mutations, modifications described in WO 2006 / 106905 were used to suppress homodimer formation and promote heterodimer formation. Specifically, D356K was substituted in antibody half molecule A, and K439E was substituted in antibody half molecule B. However, because the interaction at the CH3 interface is strong, antibody half molecules cannot be obtained with these mutations alone. Therefore, the modifications described in WO 2015 / 046467 were used to weaken the interaction at the CH3 interface. Modifications were introduced into each of the antibody half molecules A and B at the amino acid residues E357, V397, and K409 at the CH3 interface. The desired antibody half molecules A and B were obtained by combining these ADCC-enhancing modifications, hinge region modifications, heterodimerization modifications, and CH3 interface destabilizing modifications. In other words, antibody half molecules A and B do not homodimerize in the blood or on cells expressing each antigen alone; only when they simultaneously bind to cells containing the two antigens do the antibody half molecules heterodimerize and become able to bind to FcγRIIIa.

[0133] [Table 1]

[0134] [Example 2] Evaluation of the molecular weight of antibody half molecules The molecular weights of the produced antibody half molecules A and B were evaluated by size exclusion chromatography. The antibody against the human interleukin-6 receptor disclosed in WO 2009 / 125825 was used as a whole antibody control (peak W in Figure 1a). The heavy chain variable region was MRAH (SEQ ID NO: 11), and the heavy chain constant region used the sequence of natural IgG1 (SEQ ID NO: 12). The light chain variable region was MRAL (SEQ ID NO: 13), and the light chain constant region used the sequence of natural κ chain k0 (SEQ ID NO: 14). As a control for the antibody half molecule state (peak H in Figure 1b), the H-chain variable region used was EGLVH (SEQ ID NO: 15), which is the H-chain variable region portion of the anti-human epiregulin antibody described in WO 2013 / 100120, and the H-chain constant region portion was wtIgG4C4 (SEQ ID NO: 16) having the modifications described in the report by Lu et al. (Shan L, Colazet M, Rosenthal KL, Yu XQ, Bee JS, Ferguson A, Damschroder MM, Wu H, Dall'Acqua WF, Tsui P, Oganesyan V. (2016) Generation and Characterization of an IgG4 Monomeric Fc Platform. PLoS One. 2016 Aug 1;11(8)). The L-chain variable region used was EGLVL (SEQ ID NO: 17), which is the L-chain variable region portion of the anti-human epiregulin antibody described in WO 2013 / 100120, and the L-chain constant region used the sequence of the native κ chain k0. Measurements were performed according to the method described in Test Example 2. In this case, MRAH was used for each H-chain variable region, and MRAL-k0 was used for all L-chain measurements. As a result of the measurements, as shown in Figure 1, when only the antibody half molecules A1, A3, A4, and A5 were present, a mixture of antibody half molecules (peak H) and whole antibody (peak W) was obtained (Figure 1c, e.g.), while for A2, antibody half molecules (peak W) and molecules presumed to be in equilibrium between antibody half molecules and whole antibody (peak E) were obtained (Figure 1d), and when only the antibody half molecules B1, B2, B3, B4, and B5 were present as antibody half molecules (peak H) (Figure 1h-l). Furthermore, mixing antibody half molecules A1 and B1 (Fig. 1m), A2 and B2 (Fig. 1n), A3 and B3 (Fig. 1o), A4 and B4 (Fig. 1p), and A5 and B5 (Fig. 1q) did not increase the formation of whole antibodies (peak W) compared with the mixtures of antibody half molecules A1, A2, A3, A4, and A5 alone, or antibody half molecules B1, B2, B3, B4, and B5 alone. Mixing A1-B1 (Fig. 1m) and A3-B3 (Fig. 1o) resulted in the formation of molecules presumably in equilibrium between antibody half molecules and whole antibodies (peak E).

[0135] [Example 3] Evaluation of antibody half molecule binding ability to FcγRIIIa Next, whether these antibody half molecules remain in the antibody half molecule state in solution and do not exhibit FcγRIIIa-binding ability, but heterodimerize upon localized concentration upon antigen binding, restoring FcγRIIIa binding, was measured according to the method described in Test Example 3. Measurements were performed using MRAH for the H-chain variable region, MRAL for the L-chain variable region, and k0 for the L-chain constant region. The value for native IgG1 used was the value described in the report by Mimoto et al. (Mimoto F, Igawa T, Kuramochi T, Katada H, Kadono S, Kamikawa T, Shida-Kawazoe M, Hattori K. (2013) Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant. MAbs 5(2), 229-236). As a result of the measurement, as shown in Table 2, A1-A5, a series of antibody half molecules A, and B1-B5, a series of antibody half molecules B, did not exhibit FcγRIIIa-binding ability when used alone, but only when the antibody half molecules A1 and B1, A2 and B2, A3 and B3, A4 and B4, and A5 and B5 were mixed, respectively, did they exhibit FcγRIIIa-binding ability.

[0136] [Table 2] *nd stands for not detected and indicates that the value was below the lower limit of measurement and could not be measured.

[0137] [Example 4] Evaluation of ADCC activity of antibody half molecules Whether antibody half molecules containing these constant regions have superior selective cytotoxicity to double-positive cells compared to existing bispecific antibodies was determined according to the method described in Test Example 4. The antigen-expressing cells used were SK-pca60 (described in International Publication No. 2016 / 182064), in which glypican 3 was forcibly expressed in SK-Hep-1, and SKE-4B2 (described in International Publication No. 2014 / 208482), in which epiregulin was forcibly expressed. The glypican 3 and epiregulin double-positive cells used were EREG_SK-pca60_#2, in which epiregulin was forcibly expressed in SK-pca60. For the antibody half molecule A series used in Example 3, the H chain variable region was GH0 (SEQ ID NO: 18), an anti-GPC3 antibody described in WO 2009 / 041062, and the L chain variable region was GL0 (SEQ ID NO: 19), an anti-GPC3 antibody described in WO 2009 / 041062, and the L chain constant region was k0. For antibody half molecule B, EGLVH was used, the L chain variable region was EGLVL, and the L chain constant region was k0. In addition, GH0-Kn125P17 / GL0-k0 / / EGLVH-H1076N17 / EGLVL-k0 (GH0 / EGLVH-BiAb) was used as an existing bispecific antibody. The constant region of the bispecific antibody was substituted with Kn125P17 (SEQ ID NO: 20) for L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A in CH2 described in WO 2013 / 002362, and with Hl076N17 (SEQ ID NO: 21) for D270E, K326D, A330M, and K334E, respectively, to enhance ADCC activity upon heterodimerization. Furthermore, to generate bispecific antibodies utilizing differences in charge, D356K and V397Y described in WO 2015 / 046467 were substituted with Kn125P17, and V397Y and K439E were substituted with Hl076N17, respectively. Furthermore, the sequence of positions 356-358 of the sequence was that of EEM, a native IgG1 allotype.The GH0-Kn125P17 / GL0-k0 and EGLVH-Hl076N17 / EGLVL-k0 antibodies thus prepared were mixed using a method known to those skilled in the art that utilizes the difference in the charge of the constant regions (Proc. Natl. Acad. Sci., 110, 5145-5150, 2013) to produce the desired bispecific antibodies.

[0138] Measurements were performed by mixing antibody half molecules A1 and B1, A2 and B2, A3 and B3, A4 and B4, and A5 and B5. A1 + B1, A4 + B4, and A5 + B5 showed low ADCC activity against SK-pca60 and SKE-4B2, but strong ADCC activity against EREG_SK-pca60_#2. A2 + B2 and A3 + B3 showed low ADCC activity against SKE-4B2, but strong ADCC activity against SK-pca60 and EREG_SK-pca60_#2. When existing bispecific antibodies were used, strong ADCC activity was observed against all SK-pca60, SKE-4B2, and EREG_SK-pca60_#2 cells. From the above, it was revealed that the antibody half molecules A1, A4, A5 and B1, B4, B5 prepared in this study have superior selective cytotoxic activity against double-positive cells to existing bispecific antibodies against both types of cells, and it was also revealed that the antibody half molecules A2, A3 and B2, B3 have superior selective cytotoxic activity against double-positive cells to existing bispecific antibodies against one type of cell.

[0139] [Example 5] Evaluation of interactions between antibody half molecules The interactions between these antibody half molecules A1 and B1, A2 and B2, A3 and B3, A4 and B4, and A5 and B5 were measured using Biacore (registered trademark) according to the method described in Test Example 5. For the antibody half molecules captured here, PF1H (SEQ ID NO: 22), which is the H-chain variable region of the anti-human IL6 receptor antibody described in WO 2009 / 041621, was used as the H-chain variable region; PF1L (SEQ ID NO: 23), which is the L-chain variable region of the anti-human IL6 receptor antibody described in WO 2009 / 041621, was used as the L-chain variable region; and k0 was used as the L-chain constant region. The heavy chain variable region of the antibody half molecule used as the analyte was IC17H (SEQ ID NO: 24), an anti-KLH antibody described in WO 2015 / 174439, and the light chain variable region was IC17L (SEQ ID NO: 25), an anti-KLH antibody described in WO 2015 / 174439, with the light chain constant region being k0. As a result, as shown in Table 3, it was shown that when approximately 50 RU of antibody half molecules A1-A5 were captured, the selective ADCC activity against double-positive cells was exhibited when the molar binding ratios of antibody half molecules B1-B5 were 0.18-0.63, respectively.

[0140] [Table 3]

[0141] [Example 6] In vivo evaluation of antibody half molecules We evaluated whether the antibody half molecules DA303v2 and DB220v2 form dimers and exhibit cytotoxic activity only when bound to antigen in vivo in mice using antibody half molecules containing the variable region of an anti-mouse CD19 antibody.

[0142] 6-1. Preparation of anti-mouse CD19 antibody half molecules The anti-mouse CD19 antibody (Clone: ​​1D3) was prepared by cloning the HB-305 cell line from ATCC using methods known to those skilled in the art. The antibody half molecule H-chain constant region was G1d (SEQ ID NO: 26), which was derived by removing the C-terminal Gly and Lys residues of native human IgG1. The CH2 region contained the ADCC-enhancing modifications L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and the CH3 region contained the D356K / V397Y / K409D modifications to promote antibody half molecule formation and heterodimerization. This resulted in the construction of a gene for an antibody half molecule H-chain variable region of the anti-mouse CD19 antibody. Similarly, a gene for an antibody H chain half molecule containing the H chain variable region of an anti-mouse CD19 antibody was created, which had the constant region portion DB220v2 (sequence number: 28) of G1d, in which the ADCC-enhancing modifications D270E / K326D / A330M / K334E were introduced into CH2 and the modifications V397Y / K409D / K439E were introduced into CH3 to promote antibody half molecule formation and heterodimerization. Furthermore, to create whole antibodies with the same ADCC-enhancing modifications as these antibody half molecules, an antibody H chain gene having the H chain variable region of an anti-mouse CD19 antibody was prepared. The antibody H chain constant region was Kn125 (SEQ ID NO: 29), in which the ADCC-enhancing modifications L234Y / L235Q / G236W / S239M / H268D / D270E / S298A were introduced into CH2 of G1d as antibody H chain constant regions and the heterodimerization modifications Y349C / T366W were introduced into CH3. The antibody H chain constant region was Hl076 (SEQ ID NO: 30), in which the ADCC-enhancing modifications D270E / K326D / A330M / K334E were introduced into CH2 of G1d as antibody H chain constant regions and the heterodimerization modifications D356C / T366S / L368A / Y407V were introduced into CH3. Knobs-into-holes technology (Margaret Merchant et al., Nature Biotechnology 1998, 16, 677-681) was introduced into Kn125 and Hl076, and the H chain genes of these two antibodies were co-expressed with the L chain gene of an anti-mouse CD19 antibody to generate a heterodimerized whole antibody.Similarly, we created an antibody H chain gene containing an anti-mouse CD19 antibody H chain variable region with the constant region F760 (SEQ ID NO: 31) as the H chain constant region, which was modified (L235R / S239K) to attenuate binding to mouse FcγR as described in WO 2013 / 047748. We also created an antibody H chain gene containing an anti-mouse CD19 antibody H chain variable region with the mouse IgG2a constant region (mFa55: SEQ ID NO: 32) with enhanced ADCC activity as described in WO 2015 / 174439. As an antibody that does not bind to mouse CD19, we also created an antibody H chain gene containing the constant region F760nN17 (SEQ ID NO: 33), which has attenuated FcγR binding, and the H chain variable region IC17H (SEQ ID NO: 24), an anti-KLH antibody H chain variable region described in WO 2015 / 174439. For the antibody L chains, a gene for an antibody L chain having a native human kappa constant region (k0: SEQ ID NO: 14) and an anti-mouse CD19 antibody L chain variable region was prepared, as was a gene for an antibody L chain having a native mouse kappa constant region (mk0: SEQ ID NO: 34) and an anti-mouse CD19 antibody L chain variable region. An antibody L chain gene having an anti-KLH antibody L chain variable region IC17L (SEQ ID NO: 25) and a native human kappa constant region (k0) was also prepared. The genes prepared here were expressed in the combinations shown in Table 4, and the desired antibodies were obtained using the method described in Test Example 1.

[0143] [Table 4]

[0144] 6-2. Evaluation of binding of anti-mouse CD19 antibody half molecules or their dimers to mouse FcγR Of the antibody half molecules prepared in Example 6-1, 1D3-mFa55, 1D3-Kn125 / Hl076, 1D3-DA303v2, 1D3-DB220v2, and an equal mixture of 1D3-DA303v2 and 1D3-DB220v2 were assayed for binding to mouse FcγRI, II, III, and IV. For comparison, a gene for an antibody half molecule H chain containing the H chain variable region of an anti-mouse CD19 antibody, G1d, was prepared by removing the C-terminal Gly and Lys from the sequence of native human IgG1, and a gene for an antibody L chain containing the native human kappa constant region k0 and the anti-mouse CD19 antibody L chain variable region were prepared, and the anti-mouse CD19 antibody half molecule, 1D3-G1d, was obtained according to the method of Test Example 1. Mouse FcγRI, II, III, and IV antibodies were prepared according to the method described in International Publication No. 2014 / 030750. Interaction analysis of each antibody half molecule or its dimer with FcγR was performed using a Biacore® T200 (GE Healthcare). HBS-EP+10X (GE Healthcare) diluted 1:10 was used as the running buffer, and the measurement temperature was 25°C. A Series S Sensor Chip CM5 (GE Healthcare) with Protein A / G (PIERCE) immobilized by amine coupling was used. The antibody half molecules of interest were captured on this sensor chip, and measurements were performed by allowing them to interact with FcγR diluted in the running buffer. The antibody half molecules captured on the sensor chip were washed by reacting with 25 mM NaOH and 10 mM glycine-HCl (pH 1.5), and the sensor chip was regenerated and reused. Kinetic analysis to calculate the KD values ​​of each antibody half molecule or its dimer for FcγR was performed according to the following method.First, antibody half molecules of interest were captured on the sensor chip and allowed to interact with mFcγR diluted in running buffer. The measurement results for the obtained sensorgram were globally fitted using a 1:1 Langmuir binding model in Biacore® Evaluation Software to calculate the binding rate constant ka (L / mol / s) and dissociation rate constant kd (1 / s), and the dissociation constant KD (mol / L) was calculated from these values. Furthermore, when the interaction between an antibody half molecule or its dimer and FcγR was determined to be too weak to be accurately analyzed using the above kinetic analysis, the KD for that interaction was calculated using the following 1:1 binding model equation described in the Biacore® T100 Software Handbook BR1006-48 Edition AE:

[0145] The behavior of molecules interacting in a 1:1 binding model on Biacore (registered trademark) can be expressed by the following equation 3.

[0146] (Formula 3) R eq = C R max / (KD+C) + RI R eq : a plot of steady state binding levels against analyte concentration C: concentration RI: bulk refractive index contribution in the sample R max : analyze binding capacity of the surface

[0147] By rearranging this equation, KD can be expressed as the following equation 4.

[0148] (Formula 4) KD = C R max / (Req -RI)-C

[0149] In this equation, R max By substituting the values ​​of RI and C, it is possible to calculate KD. The values ​​of RI and C can be obtained from the sensorgram of the measurement results and the measurement conditions. max The calculation of R was performed according to the following method. For antibodies with sufficiently strong interactions that were evaluated simultaneously during the measurement, the R obtained when performing global fitting using the 1:1 Langmuir binding model was used. max Divide the value by the amount of antibody captured on the sensor chip for comparison, and multiply by the amount of antibody captured by the modified antibody to be evaluated to obtain R max In this measurement, RI = 0, C = 500 nM (FcγRI, FcγRIV) or 4000 nM (FcγRII, FcγRIII). Rmax is the R obtained when global fitting was performed using a 1:1 Langmuir binding model on the sensorgram obtained as a result of the interaction analysis of G1d with each FcγR. max The value was divided by the amount of G1d captured and multiplied by the amount of each antibody captured. This is because the limiting amount that each FcγR can bind to is the same as that of G1d for either antibody half molecule or its dimer, and the R max This calculation is based on the assumption that R is proportional to the amount of antibody bound to the chip at the time of measurement. eq was defined as the amount of binding of each FcγR to the antibody half molecule or its dimer on the sensor chip observed during measurement.

[0150] The KD values ​​for mouse FcγR of each antibody obtained in this manner are shown in Table 5. The grayed-out values ​​in the table were calculated using Equation 4 above because binding of FcγR to antibody half molecules or dimers was weak and could not be accurately analyzed by kinetic analysis. The "KD fold for mouse FcγRs" value is the KD of G1d for each FcγR divided by the KD of the respective antibody, indicating the degree to which the affinity of each antibody for each FcγR is enhanced or weakened compared to G1d. ND indicates that no binding was observed on the sensorgram.

[0151] [Table 5]

[0152] The results in Table 5 show that mFa55, a mouse IgG2a antibody with enhanced FcγR binding, had 44.7-fold enhanced binding to FcγRI, 3.4-fold enhanced binding to FcγRII, 11.7-fold enhanced binding to FcγRIII, and 57.5-fold enhanced binding to FcγRIV compared to G1d. Kn125 / H1076, a human IgG1-type heterodimerized ADCC-enhancing antibody, had 0.4-fold reduced binding to FcγRI, and 5.7-fold enhanced binding to FcγRII, 13.2-fold enhanced binding to FcγRIII, and 253.3-fold enhanced binding to FcγRIV compared to G1d. DA303v2, an antibody half molecule, did not bind to any of the mouse FcγRs. The antibody half molecule DB220v2 also exhibited reduced binding to all mouse FcγRs, with affinities 0.05-fold lower for FcγRI, 0.03-fold lower for FcγRII, and 0.04-fold lower for FcγRIII compared to G1d. It also showed no binding to FcγRIV. 1D3-DA303v2 / 1D3-DB220v2, which was prepared by mixing equal amounts of the two antibody half molecules DA303v2 and DB220v2, showed reduced binding to FcγRI by 0.03-fold, 0.08-fold, and 0.4-fold lower for FcγRIII compared to G1d, but enhanced binding to FcγRIV by 11.5-fold. These results suggest that either antibody half molecule alone almost completely loses its FcγR-binding activity, whereas when the two antibody half molecules are mixed to form a heterodimer, binding to FcγRIV is enhanced, and thus effector activity is expected to be exhibited.

[0153] 6-3. Pharmacokinetic evaluation of single and mixed antibody half molecules in mice The pharmacokinetics of 1D3-Kn125 / Hl076, 1D3-DA303v2, 1D3-DB220v2, and 1D3-DA303v2 / DB220v2, all prepared by the method described in Example 6-1, were evaluated in mice. Administration of 10 mg / kg was performed via the tail vein, and blood was collected periodically from the jugular vein. Plasma concentrations of 1D3-Kn125 / Hl076, 1D3-DA303v2, and 1D3-DB220v2 were measured by electrochemical immunoassay (ECLIA: Test Example 6), and the respective antibody half molecules of 1D3-DA303v2 / DB220v2 were measured by LC-MS (Test Example 7). Compared to the whole antibody 1D3-Kn125 / Hl076, 1D3-DA303v2, 1D3-DB220v2, and 1D3-DA303v2 / DB220v2 were eliminated from the blood very rapidly, decreasing to approximately 1 / 100 of the concentration immediately after administration within 3 days (Figure 3). Furthermore, there was no significant difference in the plasma concentration profiles of 1D3-DA303v2 and 1D3-DB220v2 between single and mixed administration. This suggests that even in mixed administration, the antibody half molecules exist as single molecules without heterodimerization, and heterodimerization only occurs upon antigen binding via the variable region.

[0154] 6-4. Evaluation of B cell depletion in vivo in mice Anti-mouse CD19 antibody half molecules (listed in Table 4), purified and prepared as described in Example 6-1, were intravenously administered to mice to examine whether they exhibited cytotoxic activity. The doses used in the in vivo B cell depletion assay were determined based on the time course of whole antibody and antibody half molecule elimination shown in Example 6-3, so that the trough concentrations in each sample would be similar. Specifically, various antibodies or antibody half molecules were intravenously administered to C57BL / 6NCr Slc mice under the administration conditions shown in Table 6 to maintain the required blood concentration (n = 3 per group). Three days after administration, blood was collected from the dorsal paw vein. B cells in the blood were stained as described in Test Example 8, and then B cells were detected among live cells by FACS. The percentage of B cells in the blood was calculated using the method described in Test Example 9. Compared to KLH-F760nN17, 1D3-F760 showed no cytotoxic activity, while the positive controls, 1D3-mFa55 and 1D3-Kn125 / Hl076, showed significant cytotoxic activity. Under these conditions, the antibody half molecules 1D3-DA303v2 and 1D3-DB220v2, administered alone, did not show cytotoxic activity. However, when administered in equal amounts, they exhibited significant cytotoxic activity compared to KLH-F760nN17 (Figure 4). As shown in Example 6-3, even when administered in a mixed state, antibody half molecules are thought to exist as antibody half molecules unless their variable regions bind to antigen. Therefore, the B cell depletion activity observed here is considered to be the result of each antibody half molecule binding to antigen via its variable region to form a heterodimer, resulting in ADCC activity.

[0155] [Table 6]

[0156] [Test Example 10] Construction of an expression vector for antibody half molecules and expression and purification of antibody half molecules Amino acid substitutions were introduced using a QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR, or an Infusion Advantage PCR cloning kit (TAKARA) by methods known to those skilled in the art to construct expression vectors. The nucleotide sequence of the resulting expression vector was determined by methods known to those skilled in the art. The constructed plasmid was transiently transfected into Expi293 cells (Invitrogen) to express antibody half molecules. Antibody half molecules were purified from the resulting culture supernatant using a MonoSpin ProA 96-well plate type (registered trademark) (GL Science) by methods known to those skilled in the art. The concentration of purified antibody half molecules was determined by measuring absorbance at 280 nm using a spectrophotometer and calculating the extinction coefficient calculated from the obtained value using the PACE method (Protein Science 1995; 4: 2411-2423).

[0157] [Test Example 11] Analysis of molecular weight of antibody half molecules The molecular weight of the obtained antibody half molecules was analyzed by a method known to those skilled in the art using HPLC, ACQUITY UPLC H-Class (registered trademark) (Waters), and a SuperSW3000 (TOSOH) column. The antibody half molecule protein concentration was 0.10 mg / mL, and 10 μL was injected.

[0158] [Example 7] Preparation of antibody half molecules To verify whether homodimer formation can be suppressed without using the heterodimerization modifications used in Example 1, either no heterodimerization modifications were used, or the modifications described in Ha et al.'s report (Ha JH, Kim JE, Kim YS (2016) Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins. Front Immunol. 2016 Oct 6;7:394.) were used. Specifically, half-molecule antibodies A and B were prepared with only ADCC-enhancing modifications, hinge region modifications, and CH3 interface destabilizing modifications, and in addition, antibody half molecule A was substituted with Y349T and T394F, and antibody half molecule B was substituted with S364H and F405A, respectively. The abbreviations and names of the antibody half molecules prepared and evaluated, the CH3 modifications, and the sequence numbers of the antibody half molecules are shown in Table 7.

[0159] [Table 7]

[0160] [Example 8] Evaluation of the molecular weight of antibody half molecules The molecular weights of the produced antibody half molecules A and B were evaluated by size exclusion chromatography. The antibody against the human interleukin-6 receptor disclosed in WO 2009 / 125825 was used as a whole antibody control (peak W in Figure 5-1a). The heavy chain variable region was MRAH (SEQ ID NO: 11), and the heavy chain constant region used the sequence of natural IgG1 (SEQ ID NO: 12). The light chain variable region was MRAL (SEQ ID NO: 13), and the light chain constant region used the sequence of natural κ chain k0 (SEQ ID NO: 14). As a control for the antibody half molecule state (peak H in Figure 5b), the H-chain variable region used was EGLVH (SEQ ID NO: 15), which is the H-chain variable region portion of the anti-human epiregulin antibody described in WO 2013 / 100120, and the H-chain constant region portion was wtIgG4C4 (SEQ ID NO: 16) having the modifications described in the report by Lu et al. (Shan L, Colazet M, Rosenthal KL, Yu XQ, Bee JS, Ferguson A, Damschroder MM, Wu H, Dall'Acqua WF, Tsui P, Oganesyan V. (2016) Generation and Characterization of an IgG4 Monomeric Fc Platform. PLoS One. 2016 Aug 1;11(8)). The L-chain variable region used was EGLVL (SEQ ID NO: 17), which is the L-chain variable region portion of the anti-human epiregulin antibody described in WO 2013 / 100120, and the L-chain constant region used the sequence of the native κ chain k0. Measurements were performed according to the method described in Test Example 11. In this case, MRAH was used for each H-chain variable region, and MRAL-k0 was used for all L-chain measurements. As shown in Figure 5, when only the antibody half molecules A1 and A6 were used, a mixture of antibody half molecules (H peak) and whole antibody (W peak) was obtained (Figures 5c and 5d). When only the antibody half molecules A7, B1, B6, and B7 were used, only antibody half molecules (H peak) were present (Figures 5e-h). Furthermore, mixing the antibody half molecules A1 and B1 (Figure 5i), A6 and B6 (Figure 5j), and A7 and B7 (Figure 5k) did not increase the formation of whole antibody (W peak) compared to the antibody half molecules A1, A6, and A7 alone or the antibody half molecules B1, B6, and B7 alone, respectively.

[0161] [Example 9] Evaluation of ADCC activity of antibody half molecules Whether antibody half molecules containing these constant regions have superior selective cytotoxicity to double-positive cells compared to existing bispecific antibodies was determined according to the method described in Test Example 4. The antigen-expressing cells used were SK-pca60 (described in International Publication No. 2016 / 182064), in which glypican 3 was forcibly expressed in SK-Hep-1, and SKE-4B2 (described in International Publication No. 2014 / 208482), in which epiregulin was forcibly expressed. The glypican 3 and epiregulin double-positive cells used were EREG_SK-pca60_#2, in which epiregulin was forcibly expressed in SK-pca60. In this case, for the antibody half molecule A series, the H chain variable region was GH0 (SEQ ID NO: 18), an anti-GPC3 antibody described in WO 2009 / 041062, and the L chain variable region was GL0 (SEQ ID NO: 19), an anti-GPC3 antibody described in WO 2009 / 041062, and the L chain constant region was k0. For antibody half molecule B, EGLVH was used, the L chain variable region was EGLVL, and the L chain constant region was k0. In addition, GH0-Kn125P17 / GL0-k0 / / EGLVH-H1076N17 / EGLVL-k0 (GH0 / EGLVH-BiAb) was used as an existing bispecific antibody. The constant region of the bispecific antibody was substituted with Kn125P17 (SEQ ID NO: 20) for L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A in CH2 described in WO 2013 / 002362, and with Hl076N17 (SEQ ID NO: 21) for D270E, K326D, A330M, and K334E, respectively, to enhance ADCC activity upon heterodimerization. Furthermore, to generate bispecific antibodies utilizing differences in charge, D356K and V397Y described in WO 2015 / 046467 were substituted with Kn125P17, and V397Y and K439E were substituted with Hl076N17, respectively. Furthermore, the sequence of positions 356-358 of the sequence was that of EEM, a native IgG1 allotype.The GH0-Kn125P17 / GL0-k0 and EGLVH-Hl076N17 / EGLVL-k0 antibodies thus prepared were mixed using a method known to those skilled in the art that utilizes the difference in the charge of the constant regions (Proc. Natl. Acad. Sci., 110, 5145-5150, 2013) to produce the desired bispecific antibodies. The antibody half molecules A1 and B1, A6 and B6, and A7 and B7 were mixed and added. The results are shown in Figure 6. As with A1 + B1, A6 + B6 and A7 + B7 also showed low ADCC activity against SK-pca60 and SKE-4B2, but strong ADCC activity against EREG_SK-pca60_#2. When existing bispecific antibodies were used, strong ADCC activity was observed against all cells, including SK-pca60, SKE-4B2, and EREG_SK-pca60_#2. These results demonstrate that the antibody half molecules A6, A7, and B6, B7 developed in this study have superior selective cytotoxicity against dual-positive cells compared to existing bispecific antibodies.

[0162] [Example 10] Evaluation of interactions between antibody half molecules The interactions between these antibody half molecules A1 and B1, A6 and B6, and A7 and B7 were measured using Biacore (registered trademark) according to the method described in Test Example 5. For the antibody half molecule captured here, PF1H (SEQ ID NO: 22), which is the H-chain variable region of the anti-human IL6 receptor antibody described in WO 2009 / 041621, was used as the H-chain variable region, PF1L (SEQ ID NO: 23), which is the L-chain variable region of the anti-human IL6 receptor antibody described in WO 2009 / 041621, was used as the L-chain variable region, and k0 was used as the L-chain constant region. The heavy chain variable region of the antibody half molecule used as the analyte was IC17H (SEQ ID NO: 24), an anti-KLH antibody described in WO 2015 / 174439, and the light chain variable region was IC17L (SEQ ID NO: 25), an anti-KLH antibody described in WO 2015 / 174439, with the light chain constant region being k0. As a result, as shown in Table 8, when approximately 50 RU of antibody half molecules A6 and A7 were captured, the molar binding ratios of antibody half molecules B6 and B7, respectively, were within the range of 0.18-0.63, demonstrating that selective ADCC activity against double-positive cells was also exhibited. Antibody half molecule B was diluted 18.6-28.0 times with running buffer in Example 5, whereas in this example it was diluted 5.7-9.0 times.

[0163] [Table 8]

Claims

1. The antibody comprises a first antigen-binding molecule having a first antigen-binding region that binds to a first antigen and a first polypeptide comprising a first CH2 and / or a first CH3, and a second antigen-binding molecule having a second antigen-binding region that binds to a second antigen and a second polypeptide comprising a second CH2 and / or a second CH3, Both or either one of the first CH3 and the second CH3 has a substitution of at least one amino acid residue among amino acid residues at positions 357, 397, and 409 according to the EU numbering system, with another amino acid residue; the first antigen-binding molecule and the second antigen-binding molecule form a heterodimer when the first antigen-binding molecule binds to the first antigen and the second antigen-binding molecule binds to the second antigen in the presence of cells expressing the first antigen and the second antigen; both the first polypeptide and the second polypeptide further comprise a hinge region portion of an antibody half molecule; at least one cysteine ​​residue in the hinge region is substituted with another amino acid residue; the at least one cysteine ​​residue is at either or both of positions 226 and 229 according to the EU numbering system; the first antigen and the second antigen are different antigens, Pharmaceutical compositions, except those that share a common light chain.

2. The composition of claim 1, wherein the amount of the second antigen-binding molecule bound to the first antigen-binding molecule is in the range of 1:0.1 to 1:0.9 in molar ratio when the affinity of both antigen-binding molecules is measured by surface plasmon resonance using a sensor chip on which 50 pg of the first antigen-binding molecule is immobilized per mm2 and a measurement solution containing 2.5 mg / mL of the second antigen-binding molecule.

3. The composition of claim 1 or 2, wherein the amount of heterodimer formed in the presence of cells expressing the first antigen and the second antigen is greater than in the absence of the cells.

4. The composition according to any one of claims 1 to 3, wherein the FcγR-binding activity of the heterodimer formed is higher than the FcγR-binding activity of a monomer of the first antigen-binding molecule or a monomer of the second antigen-binding molecule, or the FcγR-binding activity of the homodimer formed between the first antigen-binding molecules or the second antigen-binding molecules.

5. 5. The composition of any one of claims 1 to 4, wherein the first polypeptide comprises the first CH3, the second polypeptide comprises the second CH3, and the first CH3 and the second CH3 have at least one of the following modifications (i) to (iii): (i) a modification in which one of the first CH3 and the second CH3 has a positively charged region and the other has a negatively charged region, and the positively charged region interacts with the negatively charged region when the heterodimer is formed; (ii) a modification in which one of the first CH3 and the second CH3 has a convex portion and the other has a concave portion, and the convex portion fits into the concave portion and interacts with each other when the heterodimer is formed; (iii) The first CH3 and the second CH3 are CH3s of modified IgG, a portion of the modified IgG CH3 is replaced with a portion of IgA CH3, and when the heterodimer is formed, the portion of the IgA CH3 replaced with the first CH3 and the portion of the IgA CH3 replaced with the second CH3 interact with each other.

6. The composition of claim 1 , wherein the first polypeptide and the second polypeptide each comprise a constant region portion of an antibody half molecule.

7. The composition according to any one of claims 1 to 6, wherein the effector function in the presence of cells expressing the first antigen and the second antigen is higher than that in the presence of cells expressing the first antigen but not the second antigen, or cells expressing the second antigen but not the first antigen.

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