Method for producing antibodies

Abstract

A method for producing cells that produce multispecific binding molecules, said method including a step for introducing a first vector and a second vector into cells, wherein the first vector includes the genes of all polypeptide chains constituting the multispecific binding molecules, the second vector includes the gene of a polypeptide chain having low expression, and the multispecific binding molecules may be multispecific antibodies.

Description

Antibody production method

[0001] The present invention relates to a method for producing an antibody.

[0002] When recombinant antibodies useful as medicines are produced using genetic engineering, mammalian cells are often used as host cells for producing recombinant antibodies because they allow for complex post-translational modifications and folding that prokaryotic cells cannot perform. Gene transfer into host cells is carried out by incorporating an antibody-producing gene into a plasmid to prepare an expression vector, and then introducing the expression vector into mammalian cells.

[0003] Antibody drugs are made using the immunoglobulin skeleton. There are five major classes of immunoglobulins: IgG, IgM, IgD, IgE, and IgA. However, nearly all antibody drugs approved to date have sequences derived from IgG. An IgG antibody molecule contains four polypeptide chains (two heavy chains and two light chains). These polypeptide chains form a symmetric Y-shaped structure through a combination of noncovalent interactions and covalent disulfide bonds between the chains. IgG antibody molecules contain heavy chains known as gamma chains. Light chains can be either kappa or lambda.

[0004] When preparing cells for producing a recombinant antibody, one copy of DNA encoding the antibody's H chain and one copy of DNA encoding the antibody's L chain are typically introduced into host cells. In contrast, WO 2009 / 051108 (Patent Document 1) describes a method for producing an antibody using host cells transfected with an expression vector containing one copy of DNA encoding the recombinant antibody's H chain and two copies of DNA encoding the L chain.

[0005] WO 2006 / 060769 (Patent Document 2) describes a method for producing a heterodimeric or heteromultimeric protein using two expression vectors, each encoding a different polypeptide chain. When the protein is an immunoglobulin, the first vector encodes the heavy chain (H chain) and the second vector encodes the light chain (L chain). The method describes the optimal ratio of heavy chain plasmid to light chain plasmid for maximizing antibody productivity.

[0006] The basic structure of an IgG antibody molecule is a monospecific molecule consisting of two identical H chains and two identical L chains, with the same binding specificity on both arms (the tips of the F(ab) region). In contrast, multispecific antibodies have been created in recent years, in which a single antibody molecule is capable of binding to multiple different targets (e.g., WO2006 / 109592 (Patent Document 3)). Multispecific antibodies include bispecific antibodies (BsAbs), which bind to two antigens, and multispecific antibodies, which have even more antigen-binding sites.

[0007] Bispecific antibodies have two different antigen specificities. Typically, IgG-type bispecific antibodies contain two different heavy chains and two different light chains. When these four types of polypeptide chains are expressed in cells, random association of the heavy and light chains generates a variety of different antibody species, resulting in the formation of nine types of impure antibodies in addition to the desired combination. The formation of impure antibodies not only reduces the yield of the desired bispecific antibody, but also poses a major problem in industrial production. Due to the similarity in properties among the 10 types of antibodies formed, the separation and removal of impure antibodies using conventional methods used in antibody pharmaceutical production is difficult.

[0008] When the H chain or L chain of a bispecific antibody is shared, there are three possible combinations of expression products (two types of monospecific antibodies and one type of bispecific antibody), which makes it possible to improve the yield. There is a need for a technique for further improving the yield of the desired bispecific antibody and purifying it to a high purity.

[0009] WO 2016 / 146594 (Patent Document 4) describes a method for purifying a bispecific antibody with a common heavy chain from a mixture of antibodies. This bispecific antibody is constructed from a single heavy chain and two different light chains. Host cells for antibody production are transformed with an expression vector (a three-gene expression vector) containing DNA encoding the common heavy chain, DNA encoding a first light chain, and DNA encoding a second light chain.

[0010] Transient and Stable CHO Expression, Purification and Characterization of Novel Hetero-Dimeric Bispecific IgG Antibodies: Biotechnol. Prog., 2017, Vol. 33, No. 2, pp. 469-477 (Non-Patent Document 1) describes that for producing a specific bispecific antibody in CHO cells using a single plasmid vector, the optimal ratio was 60% common L chain and 20% each of the H chains.

[0011] Optimizing assembly and production of native bispecific antibodies by codon de-optimization MABS 2017, VOL. 9, NO. 2, pp. 231-2397 (Non-Patent Document 2) describes that in the production of bispecific antibodies using a common H chain, increasing the expression level of a poorly expressed arm by codon optimization did not improve the yield of the desired bispecific antibody.

[0012] Tuning Relative Polypeptide Expression to Optimize Assembly, Yield and Downstream Processing of Bispecific Antibodies; Antibodies 2018, 7, 29 (Non-Patent Document 3) describes the production of bispecific antibodies using a common H chain, in which the ratio of L chains was varied in various ways.

[0013] WO 2009 / 051108 (Patent No. 4976502) WO 2006 / 060769 (Patent No. 2008-522589) WO 2006 / 109592 (Patent No. 4917024) WO 2016 / 146594 (Patent No. 6894843)

[0014] Transient and Stable CHO Expression, Purification and Characterization of Novel Hetero-Dimeric Bispecific IgG Antibodies: Biotechnol. Prog., 2017, Vol. 33, No. 2, P469-477Optimizing assembly and production of native bispecific antibodies by codon de-optimization MABS 2017, VOL. 9, NO. 2, P231-P2397Tuning Relative Polypeptide Expression to Optimize Assembly, Yield and Downstream Processing of Bispecific Antibodies; Antibodies 2018, 7, 29

[0015] An object of the present invention is to provide a method for producing bispecific antibodies with high production efficiency.

[0016] The present inventors discovered that the use of two types of expression vectors in the production of bispecific antibodies having a common light chain improves the production yield and selectivity of the desired heterodimeric bispecific antibodies, and thus completed the present invention.

[0017] That is, the present invention relates to constructing a cell line by transforming host cells with two types of recombinant vectors to produce a multispecific binding molecule such as a bispecific antibody, and to producing a multispecific binding molecule composed of a desired combination of polypeptide chains using the obtained cell line. More specifically, the present invention can be expressed as follows.

[0018] (1) A method for producing cells that produce a multispecific binding molecule, comprising the steps of introducing a first vector and a second vector into cells, wherein the first vector contains genes for all polypeptide chains that constitute the multispecific binding molecule, and the second vector contains genes for polypeptide chains that are less expressed.

[0019] (2) The method of (1), wherein the multispecific binding molecule is an antigen-binding molecule or an antibody, and the multispecific antigen-binding molecule or antibody may be a bispecific antibody having a common light chain or a common heavy chain, and the bispecific antibody may be an IgG antibody having a common heavy chain or a common light chain.

[0020] (3) A method for producing cells that produce bispecific antibodies that share a common light chain, comprising the step of introducing a first vector and a second vector into cells, wherein the first vector contains genes for all polypeptide chains that constitute the bispecific antibody, and the second vector contains genes for polypeptide chains that are poorly expressed in the bispecific antibody.

[0021] (4) A method for producing (3), wherein the polypeptide chain with low expression is one of two different types of H chains, and the second vector contains a nucleic acid sequence encoding the H chain and a nucleic acid sequence encoding a common L chain.

[0022] (5) The method of (4), wherein the second vector contains more nucleic acid sequences encoding L chain polypeptides than nucleic acid sequences encoding H chain polypeptides.

[0023] (6) A method for producing (5), wherein the first vector contains a nucleic acid sequence encoding a first H chain, a nucleic acid sequence encoding a second H chain, and a nucleic acid sequence encoding a common L chain in a ratio of 1:1:1 or 1:1:2, and the second vector contains a nucleic acid sequence encoding the H chain with lower expression and a nucleic acid sequence encoding the common L chain in a ratio of 1:2.

[0024] (7) A method for producing (4), in which the first vector contains one copy of a nucleic acid sequence encoding a first H chain, one copy of a nucleic acid sequence encoding a second H chain, and one or two copies of a nucleic acid sequence encoding a common L chain, and the second vector contains one copy of a nucleic acid sequence encoding the H chain with lower expression (either the first or second H chain) and two or more copies of a nucleic acid sequence encoding the common L chain.

[0025] (8) A cell that produces an antigen-binding molecule, the cell comprising a first vector and a second vector, wherein the first vector comprises genes for all polypeptide chains constituting the antigen-binding molecule and the second vector comprises a gene for a polypeptide chain with low expression; the antigen-binding molecule may be multispecific or may be a bispecific antibody in which the H chain or L chain is shared.

[0026] (9) A cell that produces a bispecific antibody having a common light chain, the cell comprising a first vector and a second vector, wherein the first vector comprises genes for all polypeptide chains that constitute the bispecific antibody, and the second vector comprises genes for polypeptide chains that are less expressed in the bispecific antibody.

[0027] (10) The cell of (9), wherein the second vector contains a gene for the H chain with lower expression and further contains a gene for a common L chain, and the second vector may contain a greater number of copies of the gene for the common L chain than the gene for the H chain.

[0028] (11) The cell described in (9), wherein the first vector contains one copy of the gene for the first H chain, one copy of the gene for the second H chain, and one or two copies of the gene for the common L chain, and the second vector contains one copy of the gene for the H chain with lower expression and two or more copies of the gene for the common L chain.

[0029] (12) A method for producing an antigen-binding molecule or a bispecific antibody using the cells of any one of (1) to (11) above.

[0030] (13) The method for producing the above-mentioned cells, cells, or production method, wherein the cells produce an anti-FIX(a) / FX bispecific antibody, for example, emicizumab.

[0031] (14) The method for producing a cell, the cell, or the production method described above, wherein the cell is an animal cell, for example, a mammalian cell, preferably a CHO cell.

[0032] (15) A method for producing a pharmaceutical containing the antigen-binding molecule or bispecific antibody produced by the above method.

[0033] In the above-described cell production method, when the method is a method for producing cells that produce bispecific antibodies having a common L chain, the "lowly expressed polypeptide" is a polypeptide that constitutes a monospecific antibody that is expressed in a lower amount out of two types of monospecific antibodies with homozygous H chains (i.e., impure antibodies).

[0034] The above-mentioned method for producing cells may further include a selection step of selecting clones that highly express the target gene after introducing the first vector and the second vector into the cells.

[0035] The above-mentioned methods for producing antigen-binding molecules or bispecific antibodies include a culture step of culturing clone cells (i.e., strains that produce the antigen-binding molecules or bispecific antibodies of interest) obtained by the above-mentioned cell production method. Furthermore, the methods of producing antigen-binding molecules or bispecific antibodies of the present invention may also include a purification step of purifying the antigen-binding molecules or bispecific antibodies of interest from the culture obtained by the clone cell culture step.

[0036] The present invention also encompasses transformed cells obtained by the above-described cell production method. The transformed cells contain the above-described first and second vectors. For example, cells transformed to produce bispecific antibodies having a common L chain contain a first and second vector, where the first vector is an expression vector containing foreign DNA encoding all of the polypeptide chains that constitute the bispecific antibody, and the second vector is an expression vector containing foreign DNA encoding a polypeptide chain that is less expressed in the bispecific antibody, and the second vector may also contain foreign DNA encoding an additional polypeptide chain.

[0037] According to the present invention, the expression level of a bispecific antibody is increased while the expression levels of two monospecific antibodies, which are impure antibodies, are balanced, making handling during purification easier. Using the present invention, cells suitable for producing bispecific antibodies can be prepared and established as cell lines with high production efficiency for producing antibody pharmaceuticals.

[0038] The diagrams show the strand configurations of the three types of plasmids, and the heterozygous production rates of cell pools transfected with the three types of plasmids.

[0039] 1. Multispecific Binding Molecules Multispecific binding molecules are molecules that can bind to two or more different targets. Multispecific binding molecules contain different polypeptide chains, each containing a target binding site that recognizes a different target. Examples of multispecific binding molecules include protein molecules that contain two, three, four, or more types of polypeptide chains.

[0040] Examples of multispecific binding molecules include antigen-binding molecules. One antigen-binding molecule is composed of multiple polypeptide chains. A multispecific binding molecule can be a multispecific antigen-binding molecule having at least a first antigen-binding site that recognizes a first antigen and a second antigen-binding site that recognizes a second antigen. Preferred multispecific antigen-binding molecules include multispecific antibodies that can specifically bind to at least two different antigens. A multispecific antibody can have a first antigen-binding site, a second antigen-binding site, and optionally a third antigen-binding site.

[0041] In the present invention, "different antigens" do not necessarily mean that the antigens themselves are different, and cases where the epitopes are different are also included in "different antigens." Therefore, for example, different epitopes within a single molecule are also included in "different antigens."

[0042] Preferred multispecific antibodies of the present invention include bispecific antibodies (sometimes called bispecific antibodies) that can specifically bind to two different antigens.

[0043] A bispecific antibody can be, for example, an IgG antibody molecule composed of two different H chains (heavy chains) and two different L chains (light chains), i.e., four types of polypeptide chains. Alternatively, a bispecific antibody can be, for example, an IgG antibody molecule in which the H chain or L chain is shared.

[0044] In one embodiment of the present invention, the L chains of a bispecific antibody may be different, but preferably have a common L chain (referred to as a "common L chain"). A "common L chain" can associate with two or more different H chains and exhibit binding ability to their respective antigens. Here, "different H chains" preferably refers to, but is not limited to, H chains of antibodies against different antigens, and refers to H chains whose amino acid sequences differ from each other. A common L chain can be obtained, for example, according to the method described in WO2006 / 109592 (Patent Document 3).

[0045] Bispecific antibodies are immunoglobulin molecules that have specificity for two different antigens. The origin of the polypeptide chains that make up the antibody is not particularly limited, and they can be derived from human, mouse, rat, etc., and H chains and L chains from different animal species may coexist. Each polypeptide chain may be modified into a chimeric state using genetic engineering techniques, or may be a synthetic immunoglobulin chain. In one embodiment, the bispecific antibody of the present invention is an antibody modified using genetic engineering techniques. In another embodiment, the bispecific antibody of the present invention is a natural antibody. The bispecific antibody of the present invention is preferably a full-length IgG antibody.

[0046] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody. A full length antibody has a heavy chain that includes an Fc region.

[0047] Native antibodies refer to immunoglobulin molecules with various naturally occurring structures. As a common example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical disulfide-bonded light chains (L chains) and two identical heavy chains (H chains). From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ). Naturally occurring IgG antibodies, which are composed of two identical light chains (L chains) and two identical heavy chains (H chains), are monospecific molecules and are not "multispecific binding molecules" according to the present invention.

[0048] 2. Construction of a cell line producing a multispecific binding molecule The present invention provides a method for producing cells that produce a multispecific binding molecule using two vectors, which comprises the step of introducing a first vector and a second vector into a cell, wherein the first vector contains genes for all polypeptide chains that constitute the multispecific binding molecule, and the second vector contains genes for polypeptide chains with lower expression.

[0049] In the present invention, a "lowly expressed polypeptide chain" refers to a polypeptide chain that is expressed less than other polypeptide chains when the genes for multiple polypeptide chains that make up a multispecific binding molecule are introduced into the same cell. For example, in the case of a bispecific antibody having a common L chain, a lowly expressed polypeptide chain can be identified by comparing the amount of homoantibodies formed other than the desired bispecific antibody, which are formed by the association of identical polypeptide chains (i.e., the same H chains and the common L chain). In the case of a bispecific antibody having a common L chain, the second vector is a vector containing genes encoding the lowly expressed H chain and the common L chain.

[0050] The term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term generally includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. A vector can effect expression of a gene nucleic acid to which it is operatively linked. Such vectors are also referred to as "expression vectors."

[0051] In the present invention, a vector containing a gene encoding a polypeptide chain is an expression vector containing a nucleic acid sequence encoding the amino acid sequence of the polypeptide. By contacting such a vector with any suitable host cell, a foreign gene encoding the polypeptide of interest is introduced, and the cell becomes a transformed cell capable of producing the polypeptide.

[0052] Any suitable method can be used to contact cells with vectors so that the cells express the polypeptides encoded by the genes contained in each vector. Methods for contacting cells so that the cells are modified to express a specific polypeptide are well known in the art. Suitable methods for contacting cells include, for example, infection with a viral vector, calcium chloride transfection, transfection using lipofection reagents, cationic polymers, DEAE, or calcium phosphate, and electroporation. By contacting the host cells with the vectors, the vectors are introduced into the host cells.

[0053] In the step of contacting cells with two vectors and introducing these vectors into the cells, the order of contacting the vectors is arbitrary. To contact the cells with the two vectors, the cells may be contacted with the first vector and the second vector sequentially (e.g., contacting the cells with the first vector after the second vector, or contacting the cells with the second vector before the first vector), or the cells may be contacted with the first vector and the second vector simultaneously.

[0054] In the present invention, the origin of cells that are contacted with a vector for the purpose of producing a multispecific binding molecule protein, i.e., cells into which genes encoding the polypeptide chains that constitute the multispecific binding molecule are introduced to become a host cell line that produces the multispecific binding molecule, is not particularly limited. The cells can be adherent cells or suspension cells (i.e., cells that grow in suspension). Cells derived from eukaryotic microorganisms such as bacteria, filamentous fungi, or yeast, or from multicellular organisms (invertebrates and vertebrates), can both be used as host cells. Examples of invertebrate cells include plant and insect cells.

[0055] Vertebrate cells are suitable for use as hosts in the present invention. For example, mammalian cell lines adapted to grow in suspension may be useful. Mammalian cells suitable for constructing cell lines for protein production are known in the art, and include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0056] Examples of suitable host cells for producing antibody pharmaceuticals having a full-length IgG backbone include the above-mentioned human cells or non-human mammalian cells, while prokaryotic cells (e.g., Escherichia coli) are suitable host cells for amplifying or replicating (cloning) recombinant expression vectors.

[0057] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Host cells also include mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected.

[0058] In general, recombinant antibodies can be obtained by cloning the DNA encoding them from antibody-producing cells such as hybridomas or sensitized lymphocytes that produce antibodies, incorporating it into an appropriate vector, and introducing it into host cells for production. IgG-type bispecific antibodies can be produced by introducing into cells the genes for the L chains and H chains that constitute the two IgGs of interest, a total of four genes, and coexpressing them. When bispecific antibodies share a common L chain, bispecific IgG can be expressed by expressing IgG by introducing into cells the genes for this common L chain and both H chains, a total of three genes.

[0059] Assuming that the expression levels of the common L chain and both H chains are equal, when a vector containing DNA encoding a first H chain, DNA encoding a second H chain, and DNA encoding a common L chain in a 1:1:2 ratio is used, the three polypeptide chains expressed at this ratio will associate to produce the desired bispecific IgG (i.e., heterologous H chains) at a ratio of 50% and monospecific IgG (each consisting of identical H chains) at a ratio of 25%. However, there are cases where all polypeptide chains constituting a bispecific antibody are not expressed at the same level. For example, the expression level of one of the two H chains may be lower than that of the other, resulting in different production levels of the two homoantibodies.

[0060] In one aspect, the present invention provides a novel method for producing cells that produce bispecific antibodies with a common light chain. The method comprises the step of introducing a first vector and a second vector into cells. The first vector contains genes for all polypeptide chains that constitute the bispecific antibody. Thus, the first vector contains all three genes encoding the first heavy chain, second heavy chain, and common light chain of the bispecific antibody, respectively. For example, the first vector contains copies of the three genes encoding the first heavy chain, second heavy chain, and common light chain of the bispecific antibody, for example, in a ratio of 1:1:1 or 1:1:2. For example, the first vector contains one copy of the first heavy chain gene, one copy of the second heavy chain gene, and one or two copies of the common light chain gene. The second vector contains genes for polypeptides that are poorly expressed in the bispecific antibody. Among the polypeptides constituting a bispecific antibody having a common L chain, the polypeptide with low expression may be either the first H chain or the second H chain. Thus, the second vector comprises a gene encoding either the first H chain or the second H chain.

[0061] If necessary, the second vector may further contain a gene encoding a common L chain. The second vector may contain more copies of the gene encoding the common L chain than the gene encoding the H chain. For example, the second vector may contain the gene encoding either the first H chain or the second H chain and the gene encoding the common L chain in a 1:1 ratio or a 1:2 ratio. For example, the second vector may contain one copy of the gene for the H chain with the lower expression level and one, two, or more copies of the gene for the common L chain.

[0062] In one embodiment of the present invention, cells that produce bispecific antibodies having a common L chain are transformed with a first vector and a second vector, where the first vector encodes all of the polypeptide chains that constitute the bispecific antibody, and the second vector can contain more nucleic acid sequences encoding L chain polypeptides than nucleic acid sequences encoding H chain polypeptides. For example, the second vector can contain one copy of a gene encoding any of the H chains and two or more copies of a gene encoding the common L chain.

[0063] In the present invention, cells producing bispecific antibodies having a common L chain are preferably cells suitable for producing recombinant proteins to be used as pharmaceuticals. Mammalian cells suitable for constructing cell lines for recombinant protein production have already been described.

[0064] In the present invention, the two vectors (i.e., the first vector and the second vector) used to generate cells that produce multispecific binding molecules or bispecific antibodies can be, for example, plasmid vectors or viral vectors. In the present invention, for example, the vector can contain, as needed, control sequences (depending on the host) such as initiation and termination codons for transcription and translation, an appropriate promoter, a marker gene that enables selection of hosts into which a foreign gene has been introduced, and the like. Furthermore, for example, the vector can contain an antibody signal sequence that promotes secretion of the antibody into the extracellular environment. In the present invention, for example, the vector can be designed for transient or stable expression. Those skilled in the art will be able to appropriately construct recombinant expression vectors using known standard techniques depending on the purpose.

[0065] In the present invention, the first vector and the second vector can be independently any type of vector, and the first vector and the second vector may have the same regulatory sequences, suitable promoters, marker genes, etc., but may differ only in the recombinant polypeptide coding sequences contained therein.

[0066] Vectors that can be used in the present invention will now be described in more detail. For example, when Escherichia coli is used as the host, in order to amplify and prepare a large amount of vectors in Escherichia coli (e.g., JM109, DH5α, HB101, XL1Blue), it is preferable that the vector has an "ori" for amplification in Escherichia coli and further has a selection gene for the transformed Escherichia coli (e.g., a drug resistance gene that can be detected by a drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). Examples of vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, and pCR-Script. In addition to the above vectors, for the purpose of subcloning and excision of cDNA, examples of suitable vectors include pGEM-T, pDIRECT, and pT7.

[0067] Expression vectors are particularly useful when vectors are used to produce multispecific binding molecules or bispecific antibodies. For example, when expression in E. coli is desired, the expression vector preferably has the above-mentioned characteristics that allow the vector to be amplified in E. coli. When the host is E. coli such as JM109, DH5α, HB101, or XL1-Blue, it also preferably has a promoter that allows efficient expression in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or T7 promoter. In addition to the above vectors, such vectors include pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen), pEGFP, and pET (in this case, the host is preferably BL21, which expresses T7 RNA polymerase).

[0068] The vector may contain a signal sequence for polypeptide secretion, such as the pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4379) when the polypeptide is produced in the periplasm of E. coli.

[0069] In addition to the case where E. coli is used as a host, examples of vectors that can be used in the method of the present invention include mammalian-derived expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, pCDM8, and INPEP4 (Biogen-IDEC)), insect cell-derived expression vectors (e.g., the "Bac-to-BAC baculovairus expression system" (GIBCO BRL), pBacPAK8), plant-derived expression vectors (e.g., pMH1, pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (e.g., pZIpneo), yeast-derived expression vectors (e.g., the "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608, pKTH50).

[0070] When the objective is expression in animal cells such as CHO cells, COS cells, or NIH3T3 cells, the vector preferably contains a promoter necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), the CMV promoter (Niwa et al., Gene. (1991) 108, 193), the mouse β-globin promoter (mBGP), or the CAG promoter (Niwa et al., Gene. (1991) 108, 193; Miyazaki et al., Gene (1989) 79, 269). It is even more preferable if the vector contains a gene for selecting transformed cells (for example, a drug resistance gene that can be detected by a drug (neomycin, G418, etc.)). Examples of vectors with such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc. It is known that mRNA containing polyA is stable in cells, and it is preferable that the vector contains a polyA signal necessary for adding polyA to a gene, such as a mouse β-globin polyA signal, a bovine growth hormone polyA signal (rBG-pA), or an SV40 polyA signal.

[0071] In one embodiment of the present invention, a neomycin resistance gene, a CAG promoter, rBG-pA, or the like can be suitably used for expression in CHO cells. Recombinant proteins produced using CHO cells have been confirmed to be safe for use as pharmaceuticals and are now commonly used. A preferred embodiment of the present invention for producing bispecific antibodies as active ingredients of pharmaceuticals is a recombinant protein expression system using CHO cells.

[0072] Means for delivering genes into cells are well known in the art and may be selected appropriately depending on the cells used as a host, and commercially available gene transfer systems may also be used. Methods for introducing vectors into mammalian cells include methods using transfection reagents such as electroporation and lipofection, and methods using viral vectors. Methods for inserting foreign genes into the host genome of mammalian cells include random integration, targeted integration (site-specific gene insertion using recombinase, a sequence-specific recombinase enzyme), transposon vectors, and site-specific nucleases. Site-specific gene transfer methods into specific locations in the host genome are expected to be a method for efficiently obtaining cells with excellent production of target proteins and excellent passage stability.

[0073] In the cells of the present invention, antigen-binding molecules (antibodies) may be expressed in a transient expression system or a stable expression system, with expression in a stable expression system being preferred.

[0074] Transient expression systems involve introducing circular plasmids into cells using techniques such as calcium phosphate, electroporation, and lipofection, resulting in expression. Circular plasmids are less efficiently integrated into chromosomes, and the target gene often resides extrachromosomally. This makes it difficult to maintain long-term expression of the target gene from a circular plasmid.

[0075] A constitutive expression system is a method in which a linear plasmid prepared by restriction enzyme treatment or the like is incorporated into cells and expressed using calcium phosphate, electroporation, lipofection, or other methods. Linear plasmids are more efficiently inserted into chromosomes than circular plasmids, and the target gene is more efficiently maintained on the chromosome. This makes it possible to maintain the expression of the target gene for a long period of time. Furthermore, the introduction of a drug resistance gene into the plasmid enables drug selection, allowing the efficient selection of cells in which the target gene is maintained on the chromosome. Animal cells used in constitutive expression systems include CHO cells, NS0 cells, and SP2 / 0 cells, with CHO cells being preferred.

[0076] Furthermore, to achieve stable gene expression and increase the intracellular copy number of a gene, one method involves introducing a vector (e.g., pCHOI) containing a complementary DHFR gene into CHO cells lacking a nucleic acid synthesis pathway and amplifying the gene with methotrexate (MTX). To achieve transient gene expression, one method involves transforming COS cells carrying a gene expressing SV40 T antigen on their chromosomes with a vector (e.g., pcD) containing an SV40 replication origin. Replication origins derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can also be used. Furthermore, to increase the gene copy number in a host cell system, the expression vector can contain a selectable marker, such as the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene.

[0077] 3. Production of antigen-binding molecules (culturing step) The present invention further provides methods for producing antigen-binding molecules of interest using the above-described cells that produce multispecific binding molecules or cells that produce bispecific antibodies having a common L chain.

[0078] Antigen-binding molecules can be produced by culturing the above-mentioned cells under conditions suitable for antibody production. Cell culture methods are known in the art. The conditions under which cells are cultured vary depending on the cell type. These conditions include the temperature of the environment, the culture vessel containing the cells, and various gases (e.g., CO) that constitute the cell culture atmosphere or environment. ２ These parameters include the composition of the medium, the medium, the cell density, and the schedule for replacing the medium with fresh medium. These parameters are known in the art or can be determined empirically. For example, any method can be used for culturing cells in a medium such that the cells express (and, in some cases, secrete) the polypeptide encoded by the vector contacted to the cells.

[0079] Cell culture can be performed using media commonly used in cell (preferably animal cell) culture. For example, DMEM, MEM, RPMI 1640, and IMDM can be used as animal cell culture media. Commercially available animal cell culture media, such as D-MEM (Dulbecco's Modified Eagle Medium), D-MEM / F-12 1:1 Mixture (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), RPMI 1640, CHO-S-SFM II (Invitrogen), CHO-SF (Sigma-Aldrich), EX-CELL 301 (JRH biosciences), CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), and PF-ACF-CHO (Sigma-Aldrich), can also be used. Serum supplements such as fetal calf serum (FCS) can be added to these media, or serum-free culture can be performed.

[0080] The medium usually contains amino acids, vitamins, lipid factors, an energy source, an osmotic pressure adjusting agent, an iron source, a pH buffer, and optionally, for example, trace metal elements, surfactants, growth cofactors, nucleosides, and the like. The contents of these components are typically within the ranges of 0.05-1500 mg / L for amino acids, 0.001-10 mg / L for vitamins, 0-200 mg / L for lipid factors, 1-20 g / L for energy sources, 0.1-10,000 mg / L for osmotic pressure regulators, 0.1-500 mg / L for iron sources, 1-10,000 mg / L for pH buffers, 0.00001-200 mg / L for trace metal elements, 0-5,000 mg / L for surfactants, 0.05-10,000 μg / L for growth cofactors, and 0.001-50 mg / L for nucleosides, but are not limited to these and can be determined appropriately depending on the type of cells to be cultured, the type of antigen-binding molecule of interest, etc.

[0081] More specifically, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, etc., preferably L-alanine, L-arginine, L- Amino acids such as asparagine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p-aminobenzoic acid, calcium pantothenate, and pyridine hydrochloride. lipid factors such as choline chloride, choline tartrate, linoleic acid, oleic acid, cholesterol, etc., preferably choline chloride; energy sources such as glucose, galactose, mannose, fructose, etc., preferably glucose; osmotic regulators such as sodium chloride, potassium chloride, potassium nitrate, etc., preferably sodium chloride; iron sources such as ferric EDTA, ferric citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate, etc., preferably ferric chloride, ferric EDTA, ferric citrate; and pH buffers such as sodium bicarbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, MOPS, etc., preferably sodium bicarbonate.

[0082] In addition to the above components, the medium may contain trace metal elements such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride, sodium silicate, etc., preferably copper sulfate, zinc sulfate, magnesium sulfate, etc.; surfactants such as Tween 80 and Pluronic (registered trademark) F68; growth cofactors such as recombinant insulin, recombinant IGF-1, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-α, ethanolamine hydrochloride, sodium selenite, retinoic acid, putrescine hydrochloride, etc., preferably sodium selenite, ethanolamine hydrochloride, recombinant IGF-1, putrescine hydrochloride, etc.; nucleosides such as deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine, uridine, etc. In addition, suitable examples of the above medium may contain antibiotics such as streptomycin, penicillin G potassium, and gentamicin, and pH indicators such as phenol red.

[0083] The pH of the medium varies depending on the cells being cultured, but is preferably about 6 to 8, generally 6.8 to 7.6, and in many cases 7.0 to 7.4. Culture is usually carried out at about 30 to 40°C for about 15 to 200 hours, with medium replacement, aeration, and agitation as necessary.

[0084] When the cells are CHO cells, they can be cultured using methods known to those skilled in the art. For example, they can usually be cultured in an atmosphere with a CO2 concentration in the gas phase of 0-40%, preferably 2-10%, at 30-39°C, preferably about 37°C. The culture period for cells suitable for producing the desired antigen-binding molecule (antibody or fragment thereof) is usually 1 day to 3 months, preferably 1 day to 2 months, and more preferably 1 day to 1 month.

[0085] Examples of various culture apparatuses that can be used for culturing animal cells include fermenter-type tank culture apparatuses, airlift-type culture apparatuses, culture flask-type culture apparatuses, spinner flask-type culture apparatuses, microcarrier-type culture apparatuses, fluidized bed-type culture apparatuses, hollow fiber-type culture apparatuses, roller bottle-type culture apparatuses, and packed tank-type culture apparatuses.

[0086] The culture may be any of batch culture, fed-batch culture, continuous culture, etc., but fed-batch culture or continuous culture is preferred, with fed-batch culture being more preferred.

[0087] When producing a bispecific antibody as an active ingredient of a pharmaceutical, it is desirable that the bispecific antibody of interest is secreted into the medium by culturing cells, and that the association of the L chain and H chain proceeds naturally in the culture of antibody-producing cells.

[0088] The heavy and light chain polypeptides that make up antibody molecules assemble with the support of BiP (immunoglobulin heavy chain binding protein), and then fold to complete the complete antibody structure. This assembly process is dependent on the light chain polypeptide (Molecular Biology of the Cell, 1999, 10, 2209). Therefore, increasing the ratio of light chain genes and increasing the proportion of light chain polypeptides is thought to promote the assembly of heavy and light chain polypeptides, thereby increasing production yields.

[0089] 4. Production of antigen-binding molecules (purification process) The culture supernatant of animal cells contains impurities such as components secreted by the producing cells in addition to the target antigen-binding molecules (antibodies in the narrow sense). Therefore, purification is carried out to remove the impurities and extract the target protein. The highly pure protein obtained through these culture and purification processes is called a "drug substance," which then goes through formulation processes (addition of additives, sterile filtration, etc.) and filling and packaging processes to become a finished pharmaceutical product.

[0090] Purification can be carried out at any time after culturing the cells. Methods for purifying proteins from culture media or culture supernatants are known in the art. Suitable purification methods include, for example, chromatography, electrophoresis, etc.

[0091] Antigen-binding molecules such as antibodies can be separated and purified using methods commonly used for polypeptides. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as affinity chromatography columns, filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). The concentration of the antibody obtained above can be measured by absorbance measurement or enzyme-linked immunosorbent assay (ELISA), etc.

[0092] Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, columns using protein A columns include Hyper D, POROS, and Sepharose FF (Pharmacia).

[0093] Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographies can be performed using liquid-phase chromatography such as HPLC and FPLC. Purification using these chromatographies can yield antibody drug substances.

[0094] Before or after purification, the polypeptide can be treated with an appropriate polypeptide-modifying enzyme, such as trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, or glucosidase, to optionally modify or partially remove peptides.

[0095] By using the cells of the present invention, the ratio of the amounts of antibody-type molecules produced is constant, facilitating the separation and removal of impure antibodies, allowing highly purified antigen-binding molecules (antibodies) with desired multispecificity to be obtained. A typical example of the antigen-binding molecule of interest is an IgG-type antibody molecule having a common L chain, which contains three types of polypeptide chains (i.e., a common L chain and two types of H chains that are different). A combination in which the two types of H chains are identical is an impure antibody.

[0096] 5. Specific Bispecific Antibodies Emicizumab (also known as Ace910; or Hemlibra®) is a recombinant humanized bispecific monoclonal antibody directed against activated blood coagulation factor IX (F IX(a)) and blood coagulation factor X (FX). It is used as a therapeutic agent to replace activated blood coagulation factor VIII (FVIIIa), which is deficient or dysfunctional in hemophilia A, by binding to F IX(a) with one arm and FX with the other, thereby forming a precise cross-link between F IX(a) and FX (Sampei, et al. PLoS ONE 2013; 8(2): e57479; Kitazawa, et al. Nature Medicine 2012; 18(10): 1570).

[0097] Emicizumab is an anti-FIX(a) / FX bispecific antibody with a common light chain. Emicizumab contains an H chain polypeptide containing an antigen-binding site that recognizes FIX(a), an H chain polypeptide containing an antigen-binding site that recognizes FX, and a common light chain polypeptide. The sequences of these polypeptides are publicly known (SEQ ID NO: 20 of WO2012 / 067176 is the heavy chain on the FIX side, SEQ ID NO: 25 is the heavy chain on the FX side, and SEQ ID NO: 32 is the common light chain).

[0098] The present inventors have previously succeeded in increasing the production rate of heteroantibodies (anti-FIX(a) / FX bispecific antibodies) by controlling the charge of each antibody chain and using a common L chain. However, when a plasmid encoding an F IX-recognizing H chain, an FX-recognizing H chain, and a common L chain is used, the F IX homoantibody (anti-F IX antibody) is abundant and the FX homoantibody (anti-FX antibody) is low.

[0099] Recently, in order to obtain a cell line with improved heterodimer production in the production of a WCB (working cell bank) during manufacturing, we conducted various studies and discovered a cell line construction method that achieves both high production and heterodimer selectivity by constructing the line using a plasmid that introduces the FIX H chain: FX H chain: common L chain in a 1:1:2 ratio plus a secondary plasmid that introduces the H chain of the side (anti-FX antibody) that produces less homodimer: common L chain in a 1:2 ratio.

[0100] Therefore, a preferred embodiment of the present invention provides a method for producing cells that produce emicizumab, comprising the step of introducing a first vector and a second vector into cells. In this method, the first vector contains all of the polypeptide chains that constitute emicizumab, i.e., the genes for the FIX heavy chain, FX heavy chain, and common light chain, in a 1:1:1 or 1:1:2 copy ratio, respectively, and the second vector may contain the FX heavy chain gene and, optionally, the common light chain gene. For example, the second vector may contain the FX heavy chain gene and the common light chain gene in a 1:1 or 1:2 copy ratio. In a specific embodiment, the first vector contains one copy of the FIX heavy chain gene, one copy of the FX heavy chain gene, and two copies of the common light chain gene, and the second vector contains one copy of the FX heavy chain gene and two or more copies of the common light chain gene.

[0101] 6. Production of Pharmaceuticals When an antigen-binding molecule or bispecific antibody produced by the method of the present invention has biological activity that allows it to be used as a pharmaceutical, a pharmaceutical can be produced by mixing the antigen-binding molecule or bispecific antibody with a pharmaceutically acceptable carrier or additive and formulating the mixture.

[0102] Examples of pharmaceutically acceptable carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, surfactants acceptable as pharmaceutical additives, and the like.

[0103] The actual additives are selected from the above alone or in appropriate combination depending on the dosage form of the pharmaceutical, but are of course not limited to these. For example, when used as an injectable preparation, the purified polypeptide can be dissolved in a solvent such as physiological saline, buffer solution, glucose solution, etc., and an adsorption inhibitor such as Tween 80, Tween 20, gelatin, human serum albumin, etc. can be added to the solution. Alternatively, the polypeptide can be lyophilized to form a dosage form that can be dissolved and reconstituted before use, and sugar alcohols and saccharides such as mannitol and glucose can be used as excipients for lyophilization.

[0104] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0105] Example 1: Gene transfer using one type of plasmid each. A CAG promoter was attached upstream of the structural genes for the emicizumab FIX heavy chain, FX heavy chain, and common light chain, and rBG-pA was attached downstream to create the FIX heavy chain expression unit, FX heavy chain expression unit, and common light chain expression unit. The FIX heavy chain expression unit, FX heavy chain expression unit, and common light chain expression unit were ligated to pBluescriptII incorporating a neomycin resistance gene to create the IX1X1L1 plasmid, consisting of one copy of the FIX heavy chain, one copy of the FX heavy chain, and one copy of the common light chain; the L1IX1X1 plasmid, in which the light chain positions are swapped; and the IX1X1L2 plasmid, consisting of one copy of the FIX heavy chain, one copy of the FX heavy chain, and two copies of the common light chain (Figure 1). These plasmids were then introduced into CHO DXB11-derived host cells by electroporation. Electroporation was performed using a Nucleofector®. Subsequently, the cells were cultured in the presence of 15 nmol / L MTX to select cells into which the expression plasmid had been introduced.

[0106] We obtained IX1X1L1 plasmid-transfected cell pools, L1IX1X1 plasmid-transfected cell pools, and IX1X1L2 plasmid-transfected cell pools and compared them in fed-batch cultures in 24-well plates. Cultures were performed in 0.8 mL cultures at 37°C and 160 rpm. On day 14, antibody concentrations in the culture medium were measured, and the percentage of desired heterodimers was evaluated by IEC. The results showed that the antibody production yield and percentage of desired heterodimers in the IX1X1L2 plasmid-transfected cell pool were higher than those in the IX1X1L1 or L1IX1X1 plasmid-transfected cell pools (Figure 2). These results suggest that expression of a single copy of the common light chain gene is insufficient, and that increasing the copy number may have a beneficial effect on antibody production, balancing the expression of each antibody chain through the gene dosage effect.

[0107] Example 2: Gene transfer using a secondary plasmid (X1L2) Since almost no homozygous peaks for the FX H chain were observed in Figure 2 of Example 1, it is possible that the expression level of the FX H chain was insufficient. It is also possible that there is still room for improvement in the expression level of the common L chain. Therefore, we next created an X1L2 plasmid consisting of one copy of the FX H chain and two copies of the common L chain, and transferred this to cells transfected with the IX1X1L2 plasmid. The results were compared with cells transfected with only the IX1X1L2 plasmid.

[0108] First, the IX1X1L2 plasmid was electroporated into CHO cell DXB11-derived host cells, and a cell pool exhibiting 15 nmol / L MTX resistance was obtained by culturing the cells in the presence of 15 nmol / L MTX. The newly constructed X1L2 plasmid was then electroporated into this cell pool, and the cells were cultured in the presence of 15 nmol / L MTX and 200 μg / mL hygromycin. Finally, a cell pool exhibiting 100 nmol / L MTX resistance was obtained by culturing the cells in the presence of 100 nmol / L MTX.

[0109] Two cell pools (A01 and A07) transfected with only the IX1X1L2 plasmid were compared with the two cell pools transfected with the X1L2 plasmid by fed-batch culture. Fed-batch culture was performed in a 96-well plate at 80 μL of culture medium, 37°C, and 250 rpm. The percentage of heterozygotes produced in the culture medium on day 14 was assessed by IEC. The percentage of heterozygotes produced in the cell pools transfected with the X1L2 plasmid in addition to the IX1X1L2 plasmid was higher than that of the cell pool transfected with only the IX1X1L2 plasmid. Specifically, when the X1L2 plasmid was transfected into cell pool A01, 15 of the 17 cell pools obtained by transfection showed a higher percentage of heterozygotes than A01. Similarly, when the X1L2 plasmid was added to cell pool A07, the percentage of the desired heterodimers produced in all 10 cell pools obtained by the addition was higher than that of A07. This result suggests that the addition of the FX heavy chain and common light chain genes improves the expression balance of each antibody gene.

[0110] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for producing a cell that produces a multispecific binding molecule, the method comprising the step of introducing into the cell a first vector and a second vector, wherein the first vector contains genes for all polypeptide chains constituting the multispecific binding molecule and the second vector contains genes for polypeptide chains with lower expression, and the multispecific binding molecule may be a multispecific antibody.

2. The method of claim 1, wherein the multispecific binding molecule is a bispecific antibody, the bispecific antibody being an IgG type antibody in which the heavy or light chain is shared.

3. A method for producing a cell that produces a bispecific antibody having a common L chain, comprising the step of introducing a first vector and a second vector into a cell, wherein the first vector contains genes for all of the polypeptide chains that constitute the bispecific antibody, and the second vector contains a gene for a polypeptide chain that is lowly expressed in the bispecific antibody.

4. The method of claim 3, wherein the under-expressed polypeptide chain is one of two different heavy chains, the second vector comprises a nucleic acid sequence encoding said heavy chain, and the second vector further comprises a nucleic acid sequence encoding a common light chain.

5. The method of claim 4, wherein the second vector contains more nucleic acid sequences encoding a common light chain than nucleic acid sequences encoding a heavy chain.

6. The method of claim 5, wherein the first vector contains one copy of a nucleic acid sequence encoding a first H chain, one copy of a nucleic acid sequence encoding a second H chain, and one or two copies of a nucleic acid sequence encoding a common L chain, and the second vector contains one copy of a nucleic acid sequence encoding a lower-expressing H chain and two or more copies of a nucleic acid sequence encoding a common L chain.

7. A cell that produces an antigen-binding molecule, the cell comprising a first vector and a second vector, the first vector comprising genes for all polypeptide chains constituting the antigen-binding molecule and the second vector comprising a gene for a polypeptide chain with low expression, the antigen-binding molecule may be multispecific, and the multispecific antigen-binding molecule may be a bispecific antibody in which the H chain or L chain is shared.

8. A cell that produces a bispecific antibody having a common L chain, the cell comprising a first vector and a second vector, the first vector comprising genes for all of the polypeptide chains that constitute the bispecific antibody, and the second vector comprising genes for the lower-expressing H chain and the common L chain.

9. The cell of claim 8, wherein the second vector contains a gene for a common light chain in greater copy number than a gene for a heavy chain.

10. The cell described in claim 9, wherein the first vector contains one copy of a first H chain gene, one copy of a second H chain gene, and one or two copies of a common L chain gene, and the second vector contains one copy of a gene for a lower-expressing H chain and two or more copies of the common L chain gene.

11. A method for producing an antigen-binding molecule, comprising producing an antigen-binding molecule using the cell of claim 7, or a method for producing a bispecific antibody, comprising producing a bispecific antibody using the cell of claim 8.

12. A method for producing a cell according to any one of claims 1 to 6 or a method for producing a cell according to claim 11, wherein the cell is a mammalian cell.

13. A method for producing a cell according to any one of claims 3 to 6 or a method for producing a cell according to claim 11, wherein the bispecific antibody is an anti-FIX(a) / FX bispecific antibody.

14. The method of claim 13, wherein the bispecific antibody is emicizumab.

15. A method for producing a pharmaceutical comprising an antigen-binding molecule or a bispecific antibody produced by the method of claim 11.

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