Method for modifying the isoelectric point of an antibody by amino acid substitution of CDR

Abstract

To provide a method for purifying a multispecific polypeptide comprising a first polypeptide and a second polypeptide each having an antibody variable region.SOLUTION: A method for purifying a multispecific polypeptide comprising a first polypeptide and a second polypeptide having a variable region of an antibody comprises modifying a nucleic acid encoding a polypeptide comprising an amino acid residue so that the charge of at least one amino acid residue which may be exposed on a surface of a CDR region of the polypeptide concerned is modified; modifying both or either of the nucleic acid encoding the amino acid residue of the first polypeptide and the nucleic acid encoding the amino acid residue of the second polypeptide so that a difference of an isoelectric point between the first polypeptide and the second polypeptide increases as compared to that before modifying; culturing a host cell comprising the nucleic acid in which either of the nucleic acid is modified so that the nucleic acid expresses; and purifying the multispecific polypeptide from a culture by chromatography.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for modifying the isoelectric point while retaining the binding activity of an antibody to an antigen by amino acid substitution of CDR, a method for controlling the pharmacokinetics (blood kinetics) of an antibody in plasma, a pharmaceutical composition containing an antibody with a modified isoelectric point as an active ingredient, and a method for producing the same. Further, the present invention relates to a method for controlling the plasma half-life of an anti-IL-6 receptor antibody, an anti-glypican 3 antibody, and an anti-IL-31 receptor antibody by modifying the amino acid residues exposed on the surface of the CDR region of these antibodies, an antibody (anti-IL-6 receptor antibody, anti-glypican 3 antibody, and anti-IL-31 receptor antibody) whose plasma half-life is controlled by modifying amino acid residues, a pharmaceutical composition containing the antibody as an active ingredient, and a method for producing these pharmaceutical compositions. Furthermore, the present invention relates to a pharmaceutical composition containing an anti-IL-6 receptor antibody as an active ingredient and a method for producing the same.

[0002]

Background Art

[0002] Antibodies have attracted attention as pharmaceuticals because of their long plasma half-life and few side effects. Among them, many IgG-type antibody drugs have been marketed, and numerous antibody drugs are currently being developed. (Non-Patent Document 1, Non-Patent Document 2). Most of the currently marketed antibody drugs are chimeric antibodies, humanized antibodies, and human antibodies. However, many antibody drugs with improved efficacy, convenience, and cost and more excellent characteristics are currently being developed by improving humanized antibodies or human antibodies. Various technologies applicable to these antibody drugs have been developed, and technologies for improving effector function, antigen-binding ability, pharmacokinetics, stability, or reducing immunogenicity risk have been reported. As a method for enhancing efficacy or reducing the dosage, technologies for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC activity) and complement-dependent cytotoxicity (CDC activity) by amino acid substitution in the Fc region of IgG antibodies have been reported (Non-Patent Documents 3 and 4). Also, as a technology for improving antigen-binding ability and antigen-neutralizing ability, affinity maturation technology (Non-Patent Document 5) has been reported, and it is possible to improve the binding activity to an antigen by introducing mutations into amino acids such as the CDR region of the variable region.

[0003] Among the problems of current antibody drugs, high manufacturing costs due to a very large amount of administered protein are cited. Regarding the administration form, in the case of chronic autoimmune diseases, a subcutaneous administration preparation is desirable, but generally, a subcutaneous administration preparation needs to be a high-concentration preparation. In the case of an IgG-type antibody preparation, it is generally considered that a preparation of about 100 mg / mL is the limit from the viewpoint of stability and the like (Non-Patent Document 6). By increasing the plasma half-life of the antibody so as to exhibit a sustained therapeutic effect, it is possible to reduce the amount of administered protein, enable subcutaneous administration at long dosing intervals, and provide an antibody drug having excellent characteristics of low cost and high convenience.

[0004] FcRn is highly involved in the long plasma half-life of antibodies. Regarding the differences in plasma half-life among antibody isotypes, IgG1 and IgG2 are known to have the longest plasma half-life, while IgG3 and IgG4 are inferior to them (Non-Patent Document 7). As a method for further extending the plasma half-life of IgG1 and IgG2 antibodies with excellent plasma half-life, amino acid substitutions in the constant region that enhance binding to FcRn have been reported (Non-Patent Documents 8, 9, and 10). However, introducing artificial amino acid mutations into the constant region has problems from the perspective of immunogenicity. In contrast, recently, a method for improving the pharmacokinetics of antibodies by introducing mutations into the amino acids of the variable region of antibodies has been reported (Patent Document 1).

[0005] According to Patent Document 1, it is possible to control the pharmacokinetics of IgG by changing the isoelectric point and lower the isoelectric point of the antibody and lengthen the plasma half-life without attenuating the binding activity of the antibody to the antigen by introducing amino acid substitutions into the framework of the antibody variable region. Specifically, for example, it is possible to lower the isoelectric point of the antibody without attenuating the binding activity of the antibody to the antigen by introducing amino acid substitutions at H10, H12, H23, H39, H43, and H105 in Kabat numbering. Furthermore, it is also possible to introduce amino acid mutations into other framework sequences without attenuating the binding activity, but in order to significantly lower the isoelectric point, it was considered that introducing only amino acid substitutions into the framework sequence might be insufficient. This is because the framework sequence after amino acid substitution generally uses a human antibody sequence to reduce immunogenicity, but the human antibody framework sequence is highly conserved and has little diversity, so the degree of freedom for amino acid substitution is small. Therefore, when it is insufficient to lower the isoelectric point of the antibody only by introducing amino acid substitutions into the framework, it has been difficult to further lower the isoelectric point.

[0006] On the other hand, CDR sequences have enormous diversity due to somatic mutations and acquire binding to antigens Due to its high diversity, the degree of freedom of amino acid substitution is significantly greater compared to the framework. However, the CDR sequences are the most important elements for exhibiting strong binding activity to antigens. Generally, amino acid substitutions in the CDR sequences are known to affect the binding activity of antibodies to antigens. Therefore, it is difficult to lower the isoelectric point of antibodies without significantly attenuating the binding activity of antibodies to antigens by amino acid substitution in the CDR sequences. In addition, since the CDR sequences vary greatly depending on the type of antigen, it is extremely difficult to substitute the amino acids in the CDR sequences of antibodies without significantly attenuating the binding activity of antibodies to antigens, regardless of the type of antibody. In fact, this can be easily inferred from many events shown below. This can be easily inferred from many events shown below.

[0007] When humanizing antibodies of non-human animal species, CDR grafting, which generally transplants the CDR sequences of non-human animal species into human framework sequences, is commonly used. When the humanized antibody obtained by CDR grafting does not show the same binding activity as the chimeric antibody, it is possible to restore the binding activity by substituting amino acids in a part of the framework sequence that determines the structure of the CDR with the framework sequence of the antibody from the non-human animal species from which the antibody is derived (Non-Patent Document 11). Thus, the sequence and structure of the CDR are extremely important for the binding activity and specificity of the antibody to the antigen it has. Also, due to the change of antibody CDR residues caused by the isomerization reaction of aspartic acid residues, the deamidation reaction of asparagine residues, and the oxidation reaction of methionine residues in the antibody CDR, the attenuation of the binding activity of the antibody to the antigen is widely known (Non-Patent Document 12), indicating that the CDR sequence is extremely important for the binding activity of the antibody to the antigen. Furthermore, amino acids in the H-chain CDR2 sequence of the antibody Since it is widely known that the attenuation of the binding activity of antibodies to antigens due to changes in antibody CDR residues caused by the isomerization reaction of aspartic acid residues, the deamidation reaction of asparagine residues, and the oxidation reaction of methionine residues in the antibody CDR (Non-Patent Document 12), the CDR sequence is extremely important for the binding activity of the antibody to the antigen. Moreover, amino acids in the H-chain CDR2 sequence of the antibody When amino acid substitutions are introduced, it has often been reported that antigen-binding activity is significantly attenuated and furthermore the antibody expression level also decreases (Non-Patent Documents 13 to 15). In particular, when amino acid substitutions are introduced into H51, it has been found that the antibody expression level significantly decreases (Non-Patent Document 16). Also, when mutations are introduced into the H-chain CDR3 sequence of an antibody, it has often been reported that antigen-binding activity is significantly attenuated in most cases (Non-Patent Documents 17 and 18). Also, when alanine scanning of the CDR sequences of an antibody is performed, by substituting the amino acids present in the CDR with alanine, in most cases, the binding activity of that antibody to the antigen is significantly attenuated (Non-Patent Documents 19 to 23), and it is considered that the effect on the binding activity to the antigen when substituting with alanine varies depending on the type of antibody. That is, generally, it is considered that amino acid substitutions in the CDR sequences of an antibody attenuate the binding activity to the antigen, and there has been no report so far of amino acid substitution sites that do not significantly attenuate the binding activity of the antibody to the antigen, independent of the type of antibody. In antibody engineering for creating antibody molecules having more excellent properties, amino acid substitutions in the CDR sequences of an antibody

[0008] are mostly carried out for the purpose of affinity maturation. Affinity maturation generally involves presenting an antibody library having randomized CDR sequences on phage or ribosome against the CDR sequences of a certain antibody molecule, and obtaining an antibody with improved binding activity to the antigen by panning against the antigen. This is a method, and by this method, it is possible to find amino acid substitutions in the CDR sequences of an antibody that improve the binding activity to the antigen (Non-Patent Documents 5, 24 to 26). However, since the amino acid substitutions that improve the binding activity to the antigen obtained by this method vary depending on the type of antibody, amino acid substitutions in the CDR sequences that improve the binding activity to the antigen, independent of the type of antibody, cannot be found. ​There has been no report on the substitution site so far. Except for affinity maturation, a method of improving the expression level of an antibody in mammalian cells by substituting amino acids in the CDR sequence at a specific site has been reported (Patent Document 2). According to Patent Document 2, by substituting the amino acids in the CDR sequence at a specific site with a specific sequence, it is possible to improve the expression level of an antibody in mammalian cells regardless of the type of antibody. Also, de-immunization to avoid T-cell epitopes present in the CDR sequence of an antibody to attenuate the immunogenicity of the antibody has been reported, but a method of amino acid substitution to remove T-cell epitopes present in the CDR sequence without attenuating the binding activity of the antibody regardless of the type of antibody has not been reported so far (Non-Patent Documents 27 and 28). As described above, since the CDR sequence of an antibody is deeply involved in the binding to an antigen, attenuation of the binding activity due to amino acid substitution in the CDR sequence is common, and the influence on the binding activity to the antigen by amino acid substitution in the CDR sequence varies depending on the type of antibody. In Patent Document 1, an example of controlling the isoelectric point by amino acid substitution in the CDR is shown, but depending on the type of antibody, it is considered possible to attenuate the binding activity to the antigen. Also, although a method of improving the expression of an antibody by common amino acid substitution regardless of the type of antibody has been reported, a method of improving the binding activity of an antibody to an antigen and a method of removing T-cell epitopes without significantly attenuating the binding activity of the antibody to the antigen have not been reported so far. Moreover, there has been no report at all on the CDR sequence of an antibody in which amino acids can be substituted without significantly attenuating the binding activity of the antibody to the antigen regardless of the type of antibody. In addition, a method of improving the expression level of an antibody in mammalian cells by substituting the amino acids in the CDR sequence at a specific site has been reported (Patent Document 2). According to Patent Document 2, by substituting the amino acids in the CDR sequence at a specific site with a specific sequence, it is possible to improve the expression level of an antibody in mammalian cells regardless of the type of antibody. Also, de-immunization to avoid T-cell epitopes present in the CDR sequence of an antibody to attenuate the immunogenicity of the antibody has been reported, but a method of amino acid substitution to remove T-cell epitopes present in the CDR sequence without attenuating the binding activity of the antibody regardless of the type of antibody has not been reported so far (Non-Patent Documents 27 and 28). There has been no report on the substitution site so far. Except for affinity maturation, a method of improving the expression level of an antibody in mammalian cells by substituting amino acids in the CDR sequence at a specific site has been reported (Patent Document 2). According to Patent Document 2, by substituting the amino acids in the CDR sequence at a specific site with a specific sequence, it is possible to improve the expression level of an antibody in mammalian cells regardless of the type of antibody. Also, de-immunization to avoid T-cell epitopes present in the CDR sequence of an antibody to attenuate the immunogenicity of the antibody has been reported, but a method of amino acid substitution to remove T-cell epitopes present in the CDR sequence without attenuating the binding activity of the antibody regardless of the type of antibody has not been reported so far (Non-Patent Documents 27 and 28).

[0009] Thus, since the CDR sequence of an antibody is deeply involved in the binding to an antigen, attenuation of the binding activity due to amino acid substitution in the CDR sequence is common, and the influence on the binding activity to the antigen by amino acid substitution in the CDR sequence varies depending on the type of antibody. In Patent Document 1, an example of controlling the isoelectric point by amino acid substitution in the CDR is shown, but depending on the type of antibody, it is considered possible to attenuate the binding activity to the antigen. Also, although a method of improving the expression of an antibody by common amino acid substitution regardless of the type of antibody has been reported, a method of improving the binding activity of an antibody to an antigen and a method of removing T-cell epitopes without significantly attenuating the binding activity of the antibody to the antigen have not been reported so far. Moreover, there has been no report at all on the CDR sequence of an antibody in which amino acids can be substituted without significantly attenuating the binding activity of the antibody to the antigen regardless of the type of antibody. In Patent Document 1, an example of controlling the isoelectric point by amino acid substitution in the CDR is shown, but depending on the type of antibody, it is considered possible to attenuate the binding activity to the antigen. Also, although a method of improving the expression of an antibody by common amino acid substitution regardless of the type of antibody has been reported, a method of improving the binding activity of an antibody to an antigen and a method of removing T-cell epitopes without significantly attenuating the binding activity of the antibody to the antigen have not been reported so far. Moreover, there has been no report at all on the CDR sequence of an antibody in which amino acids can be substituted without significantly attenuating the binding activity of the antibody to the antigen regardless of the type of antibody. In addition, a method of improving the expression level of an antibody in mammalian cells by substituting the amino acids in the CDR sequence at a specific site has been reported (Patent Document 2). According to Patent Document 2, by substituting the amino acids in the CDR sequence at a specific site with a specific sequence, it is possible to improve the expression level of an antibody in mammalian cells regardless of the type of antibody. Also, de-immunization to avoid T-cell epitopes present in the CDR sequence of an antibody to attenuate the immunogenicity of the antibody has been reported, but a method of amino acid substitution to remove T-cell epitopes present in the CDR sequence without attenuating the binding activity of the antibody regardless of the type of antibody has not been reported so far (Non-Patent Documents 27 and 28). There has been no report on the substitution site so far. Except for affinity maturation, a method of improving the expression level of an antibody in mammalian cells by substituting amino acids in the CDR sequence at a specific site has been reported (Patent Document 2). According to Patent Document 2, by substituting the amino acids in the CDR sequence at a specific site with a specific sequence, it is possible to improve the expression level of an antibody in mammalian cells regardless of the type of antibody. Also, de-immunization to avoid T-cell epitopes present in the CDR sequence of an antibody to attenuate the immunogenicity of the antibody has been reported, but a method of amino acid substitution to remove T-cell epitopes present in the CDR sequence without attenuating the binding activity of the antibody regardless of the type of antibody has not been reported so far (Non-Patent Documents 27 and 28).

[0010] The prior art documents of the present invention are shown below.

Prior Art Documents

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Non-Patent Document 27

Non-Patent Document 28

Patent Document

[0012]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0013] The present invention has been made in view of such circumstances, and an object thereof is to provide a method for modifying the isoelectric point while retaining the binding activity of a polypeptide containing a variable region of an antibody to an antigen, a method for controlling the plasma half-life of an antibody, a pharmaceutical composition containing an antibody having a controlled plasma half-life as an active ingredient, and a method for producing the antibody and the pharmaceutical composition containing the antibody as an active ingredient. In addition, the present invention provides a method for controlling the plasma half-life of an anti-IL-6 receptor antibody, an anti-glypican 3 antibody, and an anti-IL-31 receptor antibody by modifying amino acid residues exposed on the surface of the CDR region of the antibody, an anti-IL-6 receptor antibody, an anti-glypican 3 antibody, and an anti-IL-31 receptor antibody having a controlled plasma half-life by modifying amino acid residues, a method for producing the antibody, and a pharmaceutical composition containing the antibody as an active ingredient. - antibody, and a pharmaceutical composition containing the antibody as an active ingredient. Furthermore, the present invention aims to provide a pharmaceutical composition comprising a second-generation molecule superior to tocilizumab, which enhances antigen neutralizing ability, reduces the dosing frequency by improving plasma retention, continuously exhibits a therapeutic effect, and improves immunogenicity, safety, and physical properties by modifying the amino acid sequences of the variable and constant regions of tocilizumab, a humanized anti-IL-6 receptor IgG1 antibody, and a method for producing these pharmaceutical compositions. - antibody, and a pharmaceutical composition containing the antibody as an active ingredient.

Means for Solving the Problems

[0014] The inventors of the present invention intensively studied a method for modifying the isoelectric point of a polypeptide containing a variable region of an antibody while retaining the binding activity of the variable region to an antigen. As a result, the inventors found specific positions on the CDR amino acid sequence that can modify the isoelectric point while retaining the binding activity of the variable region to an antigen among the amino acid residues constituting the complementarity-determining region (CDR) of the variable region of the antibody. In addition, the variable region of the antibody amino acid residues, and found specific positions on the CDR amino acid sequence that can modify the isoelectric point while retaining the binding activity of the variable region to the antigen. Further, the variable region of the antibody By controlling the isoelectric point of a polypeptide containing a domain, the plasma half-life of the polypeptide can be controlled. Furthermore, by utilizing the difference in isoelectric points, it has been found that a polypeptide containing the variable region of an antibody, which consists of a heteromultimer, can be efficiently produced. Specifically, among the amino acid residues in the amino acid sequence constituting the variable region of the antibody, on a specific CDR amino acid sequence that can regulate the charge on the surface of the antibody molecule without affecting functions of the antibody such as the binding activity of the antibody variable region to the antigen and its structure the position was identified. Furthermore, the inventors modified the isoelectric point by regulating the surface charge of the antibody, confirmed that the plasma half-life of the polypeptide containing the variable region of the antibody can be controlled, and confirmed that the antibody thus controlled in plasma half-life actually retains the binding activity to the antigen. Furthermore, the inventors completed the present invention by confirming that by controlling the plasma half-life of the antibody, the tumor growth inhibitory effect on cancer cells exhibited by the antibody exhibiting cytotoxic activity including the antibody is increased. Also, by regulating the charge of the CDR the isoelectric point was modified, and it was confirmed that a heterodimer composed of antibodies binding to two or more different antigens can be separated and purified.

[0015] Furthermore, the inventors modified the amino acid sequences of the variable region and the constant region of tocilizumab, which is a first-generation humanized anti-IL-6 receptor IgG1 antibody to enhance the drug efficacy, improve the plasma retention by improving the plasma retention, reduce the administration frequency, continuously exert the therapeutic effect, and improve the immunogenicity, safety, and physical properties (stability and uniformity), and conducted intensive research towards the creation of a second-generation molecule superior to tocilizumab. As a result, the inventors found multiple CDR mutations that improve the antigen-binding ability (affinity) in the variable region of tocilizumab and They succeeded in significantly improving the affinity by that combination. The inventors also succeeded in improving the retention in plasma by introducing a modification that reduces the isoelectric point of the variable region sequence. The inventors also reduced the number of T-cell epitope peptides predicted in silico in the mouse-derived sequences remaining in the framework of TOCILIZUMAB and in the variable region, and succeeded in reducing the immunogenicity risk. At the same time, they also succeeded in improving the stability at high concentrations. Furthermore, in the constant region of TOCILIZUMAB, while minimizing the appearance of new T-cell epitope peptides, they succeeded in finding a novel constant region sequence that does not show binding to Fcγ receptor, has stability under acidic conditions, heterogeneity derived from the disulfide in the hinge region, heterogeneity derived from the C-terminus of the H chain, and improved stability in high-concentration formulations. By combining these modifications of the CDR region amino acid sequence, modification of the variable region amino acid sequence, and modification of the constant region amino acid sequence, they succeeded in creating a second-generation molecule superior to TOCILIZUMAB. The number of mouse-derived sequences and T-cell epitope peptides predicted in silico in the variable region was reduced, and the immunogenicity risk was reduced. They also succeeded in improving the stability at high concentrations. Furthermore, the inventors in the constant region of TOCILIZUMAB, while minimizing the appearance of new T-cell epitope peptides, did not show binding to Fcγ receptor, had stability under acidic conditions, heterogeneity derived from the disulfide in the hinge region, heterogeneity derived from the C-terminus of the H chain, and improved stability in high-concentration formulations, and succeeded in finding a novel constant region sequence. By combining these modifications of the CDR region amino acid sequence, modification of the variable region amino acid sequence, and modification of the constant region amino acid sequence, they succeeded in creating a second-generation molecule superior to TOCILIZUMAB.

[0016] More specifically, the following [1] to

[44] are provided. [1] A method for modifying the isoelectric point while retaining the binding activity of a variable region of a polypeptide containing the variable region of an antibody to an antigen, the method comprising modifying the charge of at least one amino acid residue that can be exposed on the surface of the complementarity-determining region (CDR) of the polypeptide. , [2] The method according to [1], wherein the polypeptide containing the variable region of the antibody further contains an FcRn binding region. [3] The method according to [1], wherein the polypeptide containing the variable region of the antibody is an IgG antibody. [4] The method according to [1], wherein the polypeptide containing the variable region of the antibody is a chimeric antibody, a humanized antibody or a human antibody. The method according to [1], wherein the polypeptide comprising the variable region of the antibody is a multispecific polypeptide that binds to at least two types of antigens. The method according to [1], wherein the modification of the charge of the amino acid residue is an amino acid substitution. The method according to [1], wherein the modification of the charge of the amino acid residue is a modification that changes the theoretical isoelectric point by 1.0 or more. The method according to [1]. The amino acid residue that can be exposed on the surface of the CDR region is selected from the amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region or the amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region. The method according to [1], which is at least one amino acid residue. A polypeptide comprising the variable region of an antibody with a modified isoelectric point, obtained by the method according to any one of [1] to [8]. A method for controlling the pharmacokinetics of a polypeptide in plasma, which comprises modifying the isoelectric point of a polypeptide comprising the variable region of an antibody by the method according to any one of [1] to [8]. The control of the pharmacokinetics is an increase or decrease in any parameter of plasma clearance (CL), area under the concentration curve (AUC), mean plasma residence time, and plasma half-life (t1 / 2). The method according to

[10] . A polypeptide comprising the variable region of an antibody with controlled pharmacokinetics in plasma, obtained by the method according to

[10] . A method for producing a polypeptide comprising an antibody variable region with a modified isoelectric point, comprising: (a) Modifying the nucleic acid encoding the polypeptide comprising the amino acid residue so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide is modified; (b) culturing a host cell so that the nucleic acid is expressed; and (c) recovering the polypeptide comprising the antibody variable region from the host cell culture. The method comprising: The method according to [1]. 〔14〕The method according to 〔13〕, wherein the polypeptide comprising the variable region of the antibody further comprises an FcRn binding region. 〔15〕The method according to 〔13〕, wherein the polypeptide comprising the variable region of the antibody is an IgG antibody. Method 〔16〕The method according to 〔13〕, wherein the polypeptide comprising the variable region of the antibody is a chimeric antibody, a humanized antibody or a human antibody. 〔17〕The method according to 〔13〕, wherein the polypeptide comprising the variable region of the antibody is a multispecific polypeptide that binds to at least two antigens. 〔18〕The method according to 〔13〕, wherein the modification of the charge of the amino acid residue is an amino acid substitution. 〔19〕The method according to 〔13〕, wherein the modification of the charge of the amino acid residue is a modification that changes the theoretical isoelectric point by 1.0 or more. 〔13〕The method according to 〔13〕. 〔20〕The amino acid residues that can be exposed on the surface of the CDR region are the amino acid residues at positions 31, 61, 62, 64 and 65 according to Kabat numbering in the heavy chain variable region or the amino acid residues at positions 24, 27, 53, 54 and 55 according to Kabat numbering in the light chain variable region Selected, at least one amino acid residue, the method according to 〔13〕. 〔21〕A polypeptide comprising the variable region of an antibody with a modified isoelectric point, obtained by the method according to any one of 〔13〕 to 〔20〕. 〔22〕A method for producing a polypeptide comprising the variable region of an antibody with controlled plasma pharmacokinetics, comprising modifying the isoelectric point of the polypeptide comprising the variable region of the antibody by the method according to any one of 〔13〕 to 〔20〕. 〔23〕The control of the pharmacokinetics is an increase or decrease in any parameter of plasma clearance (CL), area under the concentration curve (AUC), mean Plasma residence time, plasma half-life (t1 / 2), the method according to 〔22〕. 〔24〕A polypeptide comprising the variable region of an antibody with controlled plasma pharmacokinetics, produced by the method according to 〔22〕. A method for producing a multispecific polypeptide comprising a first polypeptide and a second polypeptide having variable regions of an antibody, comprising: (a) modifying a nucleic acid encoding a polypeptide containing the amino acid residue so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide is modified, and the modification of the nucleic acid is such that the difference in isoelectric point between the first polypeptide and the second polypeptide is increased compared to before the modification, and modifying both or either of the nucleic acid encoding the amino acid residue of the first polypeptide and the nucleic acid encoding the amino acid residue of the second polypeptide, (b) culturing a host cell so that the nucleic acid is expressed, (c) recovering the multispecific antibody from the host cell culture A method comprising: 〔26〕The method according to 〔25〕, wherein the step of recovering the multispecific polypeptide comprising the first polypeptide and the second polypeptide from the host cell culture is performed by standard chromatography. 〔27〕The method according to 〔25〕, wherein in the modification of the nucleic acid, the nucleic acid is modified so that the peaks obtained by analysis using standard chromatography of the homomultimer of the first polypeptide, the homomultimer of the second polypeptide, and the heteromultimer of the first polypeptide and the second polypeptide are more separated peaks compared to before the modification. 〔28〕The method according to 〔25〕, wherein the multispecific polypeptide is a multispecific antibody. 〔29〕A multispecific antibody produced by the method according to 〔27〕. 〔30〕The multispecific antibody according to 〔29〕, wherein the multispecific antibody is a bispecific antibody. 〔31〕An antibody comprising a CDR selected from the group consisting of a human-derived CDR, a non-human animal-derived CDR, and a synthetic CDR, a human-derived framework region (FR), and a human constant region. and at least one amino acid residue that can be exposed on the surface of the CDR has a charge different from that of the amino acid residue at the corresponding position of the wild-type CDR, and an antibody in which the isoelectric point is modified while maintaining the binding activity to the antigen as compared with the antibody before modification, 〔32〕The antibody according to 〔31〕, wherein the human constant region contains the human Fc region, 〔33〕The antibody according to 〔31〕, wherein the pharmacokinetics in plasma is controlled by modification of the isoelectric point, 〔34〕An IgG antibody in which the charge of at least one amino acid residue selected from the amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region, or the amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region is modified, and the isoelectric point is modified as compared with before the modification of the amino acid residue, , 〔35〕The antibody according to 〔34〕, wherein the modified amino acid residue is selected from the amino acid residues included in any one of the following (a) or (b); (a) Glutamic acid (E), aspartic acid (D); (b) Lysine (K), arginine (R), histidine (H), 〔36〕A multispecific antibody comprising a first polypeptide and a second polypeptide, wherein at least one amino acid residue selected from the amino acid residues at positions 31, 61, 62, , 64, and 65 according to Kabat numbering in the heavy chain variable region of the first polypeptide, or the amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region of the first polypeptide has a charge, and the isoelectric points of the first polypeptide and the second polypeptide are different from each other, 〔37〕The amino acid residues at positions 31, 61, 〔37〕The amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region of the second polypeptide, or the Kabat numbering in the light chain variable region at positions 62, 64, and 65, or the amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region The charge of at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54, and 55 by barring has a charge opposite to or no charge compared to the charge of the amino acid residue selected in the first polypeptide, the antibody according to

[36] ,

[38] the combination of the amino acid residue having the charge and the amino acid residue having a charge opposite to the amino acid residue is each selected from the amino acid residues included in any of the following groups (a) or (b); the antibody according to

[36] ; (c) Glutamic acid (E), aspartic acid (D); (d) Lysine (K), arginine (R), histidine (H),

[39] A multispecific antibody comprising the first polypeptide and the second polypeptide, wherein the homomultimer of the first polypeptide and the homomultimer of the second polypeptide are peaks separated by analysis using standard chromatography; the antibody according to

[36] ;

[40] A composition comprising the antibody according to

[31] to

[39] and a pharmaceutically acceptable carrier,

[41] A nucleic acid encoding a polypeptide constituting the antibody according to

[31] to

[39] ;

[42] A host cell having the nucleic acid according to

[41] ;

[43] A method for producing the antibody according to

[31] to

[39] , comprising culturing the host cell according to

[42] and recovering the polypeptide from the cell culture;

[44] In a polypeptide containing the variable region of an antibody, a method of substituting an amino acid residue that can be exposed on the surface of the complementarity determining region (CDR) of the polypeptide while maintaining the binding activity of the polypeptide to the antigen, at least in the Kabat numbering in the heavy chain variable region Substituting at least one amino acid residue selected from the amino acid residues at positions 31, 61, 62, 64, and 65, or the amino acid residues at positions 24, 27, 53, 54, and 55 by Kabat numbering in the light chain variable region Or a method of substituting at least one amino acid residue selected therefrom.

[0017] The present invention also provides the following [1] to

[38] . [1] A method for producing an antibody to glypican 3 with controlled blood kinetics, comprising the steps of: (a) modifying a nucleic acid encoding at least one amino acid residue that results in a modification of the charge of at least one amino acid residue that may be exposed on the surface of an anti-glypican 3 antibody; (b) culturing a host cell carrying the nucleic acid such that the nucleic acid is expressed; (c) recovering the glypican 3 antibody from the culture of the host cell. [2] The method according to [1], wherein the control of blood dynamics is an extension or reduction of any of the parameters of blood half-life, mean blood residence time, and blood clearance. [3] The method according to [1], wherein the modification of the charge of the amino acid residue in step (a) is performed by amino acid substitution. [4] The method according to [1], wherein the amino acid residues that can be exposed on the surface of the anti-glypican 3 antibody are located in a region other than the FcRn-binding region of the anti-glypican 3 antibody. [5] The method according to [4], wherein the FcRn-binding region comprises an Fc region. [6] The method according to [1], wherein the anti-glypican 3 antibody is an IgG antibody. [7] The amino acid residue whose charge is to be modified is a heavy chain variable region or a light chain variable region of an IgG antibody. The method according to claim 6, wherein the amino acid residue is in the region [8] The glypican 3 antibody comprises a complementarity determining region (CDR), a human-derived framework region (FR) and a human constant region, wherein the amino acid sequence in step (a) is The modification of the charge of the residues is carried out by modifying a small number of residues that may be exposed on the antibody surface in the CDR or FR of the antibody to be modified. The method according to [7], wherein at least one amino acid residue is modified to an amino acid residue having a charge different from that of the amino acid residue. [9] The method according to [8], wherein the anti-glypican 3 antibody has a reduced content of fucose bound to its Fc region.

[10] An antibody to glypican 3, produced by the method according to any one of [1] to [9]. 〔11〕A method for stabilizing a glypican 3 antibody, which comprises at least one amino acid residue modification of the glypican 3 antibody, resulting in an increase in the Tm value of the complementarity-determining region (CDR), human-derived framework region (FR), and human constant region-containing glypican 3 antibody; A method for stabilizing a glypican 3 antibody, characterized by the modification of at least one amino acid residue constituting the antibody; (a) Modifying a nucleic acid encoding at least one amino acid residue that results in an increase in the Tm value of the glypican 3 antibody to be modified, (b) Culturing a host cell retaining the nucleic acid so that the nucleic acid is expressed, (c) A method comprising recovering the antibody from the culture of the host cell; 〔12〕The method according to

[11] , characterized in that the amino acid residue in step (a) is present in the FR1 region and / or the FR2 region of its H chain or L chain; The method according to

[11] , characterized in that the amino acid residue in the FR2 region of the H chain according to

[12] is substituted with the amino acid residue in the FR2 region of the VH4 subclass; The method according to

[12] , characterized in that the amino acid residue in the FR2 region of the L chain according to

[12] is substituted with the amino acid residue in the FR2 region of the VK3 subclass; 〔14〕A method for controlling the cytotoxic activity of an antibody, comprising the following steps; 〔15〕A method for controlling the cytotoxic activity of an antibody, comprising: (a) Modifying a nucleic acid encoding at least one amino acid residue that results in a change in the charge of at least one amino acid residue that can be exposed on the surface of an antibody having cytotoxic activity, (b) Culturing a host cell retaining the nucleic acid so that the nucleic acid is expressed, (c) A method comprising recovering the antibody from the culture of the host cell; 〔16〕The method according to

[15] , wherein the control of the pharmacokinetics is the control of any parameter of the blood half-life, average blood residence time, and blood clearance; 〔17〕The method according to

[15] , wherein the change in the charge of the amino acid residue in step (a) is by amino acid substitution; 〔18〕The method according to

[15] , wherein the amino acid residue that can be exposed on the surface of the antibody is in a region other than the FcRn binding region in the antibody. 〔19〕The method according to

[18] , wherein the FcRn binding region consists of an Fc region. 〔20〕The method according to

[15] , wherein the glypican 3 antibody is an IgG antibody. 〔21〕The amino acid residue whose charge is modified is an amino acid residue in the heavy chain variable region or the light chain variable region of the IgG antibody. The method according to

[20] . 〔22〕The antibody comprises a complementarity determining region (CDR) derived from an animal other than human, a framework region (FR) derived from human, and a human constant region. The modification of the charge of the amino acid residue in step (a) is a modification from at least one amino acid residue that can be exposed on the surface of the antibody in the CDR or FR of the antibody to be modified to an amino acid residue having a charge different from that of the amino acid residue. The method according to

[21] . 〔23〕The method according to

[22] , wherein the antibody is an antibody having a reduced fucose content bound to the Fc region of the antibody. 〔24〕An antibody produced by the method according to

[15] to

[23] . 〔25〕The antibody according to

[24] , wherein the antibody is a glypican 3 antibody. 〔26〕Any one or more of the following substitutions for the amino acid residues constituting the H chain V region represented by SEQ ID NO: 195; 〔27〕(a) Substitution of K, the 19th amino acid residue, with T. 〔28〕(b) Substitution of Q, the 43rd amino acid residue, with E. 〔29〕(c) Substitution of K, the 63rd amino acid residue, with S. 〔30〕(d) Substitution of K, the 65th amino acid residue, with Q. 〔31〕(e) Substitution of G, the 66th amino acid residue, with D. 〔32〕The H chain V region subjected to the substitution, and 〔33〕Any one or more of the following substitutions for the amino acid residues constituting the L chain V region represented by SEQ ID NO: 201; 〔34〕(a) Substitution of K, the 19th amino acid residue, with T. (f) Substitution of Q, the 27th amino acid residue, with E, (g) Substitution of K, the 79th amino acid residue, with T, (h) Substitution of R, the 82nd amino acid residue, with S, The L-chain V region subjected to the above, An antibody comprising the same,

[27] The antibody according to

[26] , comprising the H chain represented by SEQ ID NO: 197 and the L chain represented by SEQ ID NO: 203,

[28] The antibody according to

[26] , comprising the H chain represented by SEQ ID NO: 198 and the L chain represented by SEQ ID NO: 204,

[29] Any one or more of the following substitutions to the amino acid residues constituting the H-chain V region represented by SEQ ID NO: 195; (a) Substitution of Q, the 43rd amino acid residue, with K, (b) Substitution of D, the 52nd amino acid residue, with N, (c) Substitution of Q, the 107th amino acid residue, with R, The H-chain V region subjected to the above, and, Any one or more of the following substitutions to the amino acid residues constituting the L-chain V region represented by SEQ ID NO: 201; (d) Substitution of E, the 17th amino acid residue, with Q, (e) Substitution of Q, the 27th amino acid residue, with R, (f) Substitution of Q, the 105th amino acid residue, with R, The L-chain V region subjected to the above, An antibody comprising the same,

[30] The H-chain variable region represented by SEQ ID NO: 198 and the L-chain variable region represented by SEQ ID NO: 204, the antibody according to

[29] comprising the same,

[31] The H-chain variable region represented by SEQ ID NO: 199 and the L-chain variable region represented by SEQ ID NO: 205, the antibody according to

[29] comprising the same,

[32] The antibody according to

[26] to

[31] , having the C region of a human antibody,

[33] The antibody according to

[32] , and a composition comprising a pharmaceutically acceptable carrier, A cancer therapeutic agent containing the antibody described in 〔34〕〔32〕 as an active ingredient, 〔35〕The cancer therapeutic agent described in 〔34〕, wherein the cancer is liver cancer, 〔36〕A nucleic acid encoding a polypeptide constituting the antibody described in 〔26〕 to 〔31〕, 〔37〕A host cell retaining the nucleic acid described in 〔36〕, 〔38〕A method for producing the antibody described in 〔26〕 to 〔31〕, comprising the steps of culturing the host cell described in 〔37〕 and recovering the polypeptide from the cell culture.

[0018] The present invention also provides the following 〔1〕 to 〔41〕. 〔1〕An anti-IL-6 receptor antibody described in any one of the following (a) to (y); (a) An antibody comprising a heavy chain variable region having a CDR1 in which the 1st Ser in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with another amino acid, (b) An antibody comprising a heavy chain variable region having a CDR1 in which the 5th Trp in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with another amino acid, (c) An antibody comprising a heavy chain variable region having a CDR2 in which the 1st Tyr in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with another amino acid, (d) An antibody comprising a heavy chain variable region having a CDR2 in which the 8th Thr in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with another amino acid, (e) An antibody comprising a heavy chain variable region having a CDR2 in which the 9th Thr in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with another amino acid, (f) An antibody comprising a heavy chain variable region having a CDR3 in which the 1st Ser in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with another amino acid, (g) An antibody comprising a heavy chain variable region having a CDR3 in which the 2nd Leu in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with another amino acid, ​​​​​​(h) An antibody comprising a heavy chain variable region having a CDR3 in which Thr at the 5th position in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with another amino acid and having a CDR3 in which Thr at the 5th position in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with another amino acid (i) An antibody comprising a heavy chain variable region having a CDR3 in which Ala at the 7th position in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with another amino acid and having a CDR3 in which Ala at the 7th position in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with another amino acid (j) An antibody comprising a heavy chain variable region having a CDR3 in which Met at the 8th position in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with another amino acid and having a CDR3 in which Met at the 8th position in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with another amino acid (k) An antibody comprising a heavy chain variable region having a CDR3 in which Ser at the 1st position and Thr at the 5th position in the amino acid sequence set forth in SEQ ID NO: 3 are substituted with other amino acids (l) An antibody comprising a heavy chain variable region having a CDR3 in which Leu at the 2nd position, Ala at the 7th position and Met at the 8th position in the amino acid sequence set forth in SEQ ID NO: 3 are substituted with other amino acids (m) An antibody comprising a light chain variable region having a CDR1 in which Arg at the 1st position in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid and having a CDR1 in which Arg at the 1st position in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid (n) An antibody comprising a light chain variable region having a CDR1 in which Gln at the 4th position in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid and having a CDR1 in which Gln at the 4th position in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid (o) An antibody comprising a light chain variable region having a CDR1 in which Tyr at the 9th position in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid and having a CDR1 in which Tyr at the 9th position in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid (p) An antibody comprising a light chain variable region having a CDR1 in which Asn at the 11th position in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid and having a CDR1 in which Asn at the 11th position in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid (q) An antibody comprising a light chain variable region having a CDR2 in which Thr at the 2nd position in the amino acid sequence set forth in SEQ ID NO: 5 is substituted with another amino acid and having a CDR2 in which Thr at the 2nd position in the amino acid sequence set forth in SEQ ID NO: 5 is substituted with another amino acid (r) An antibody comprising a light chain variable region having a CDR2 in which Gln at the 1st position in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with another amino acid An antibody comprising a light chain variable region having a CDR3 as described (s) An antibody comprising a light chain variable region having a CDR3 in which the 3rd Gly in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with another amino acid An antibody comprising a light chain variable region having a CDR3 as described (t) An antibody comprising a light chain variable region having a CDR3 in which the 9th Tyr in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid An antibody comprising a CDR1 as described and a light chain variable region having a CDR3 in which the 3rd Gly in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with another amino acid An antibody comprising a light chain variable region having a CDR3 in which the 3rd Gly in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with another amino acid (u) An antibody comprising a light chain variable region having a CDR3 in which the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with another amino acid (v) An antibody comprising a light chain variable region having a CDR3 in which the 1st Gln and the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 6 are substituted with another amino acid (w) An antibody comprising a CDR2 in which the 9th Thr in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with another amino acid And a heavy chain variable region having a CDR3 in which the 1st Ser and the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 3 are substituted with another amino acid An antibody comprising a heavy chain variable region having a CDR3 in which the 1st Ser and the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 3 are substituted with another amino acid (x) An antibody comprising the heavy chain variable region described in (k) and the light chain variable region described in (v), or (y) The antibody described in (x) further comprising the CDR2 of (e) [2] An anti-IL-6 receptor antibody comprising a light chain variable region having a CDR2 in which the 2nd Thr in the amino acid sequence set forth in SEQ ID NO: 5 is substituted with another amino acid An anti-IL-6 receptor antibody comprising a light chain variable region having a CDR2 in which the 2nd Thr in the amino acid sequence set forth in SEQ ID NO: 5 is substituted with another amino acid [3] An anti-IL-6 receptor antibody according to any one of the following (a) to (y); (a) An antibody comprising a heavy chain variable region having an FR1 in which the 13th Arg in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with another amino acid An antibody comprising a heavy chain variable region having an FR1 in which the 13th Arg in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with another amino acid (b) An antibody comprising a heavy chain variable region having an FR1 in which the 16th Gln in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with another amino acid An antibody comprising a heavy chain variable region having the recited FR1, (c) An antibody comprising a heavy chain variable region having the recited FR1 in which Thr at position 23 is substituted with another amino acid in the amino acid cleavage described in SEQ ID NO: 7 An antibody comprising a heavy chain variable region having the recited FR1, (d) An antibody comprising a heavy chain variable region having the recited FR1 in which Thr at position 30 is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 7 An antibody comprising a heavy chain variable region having the recited FR1, (e) An antibody comprising a heavy chain variable region having the recited FR1 in which Arg at position 13, Gln at position 16, Thr at position 23 and Thr at position 30 are substituted with other amino acids in the amino acid sequence described in SEQ ID NO: 7 and including, (f) An antibody comprising a heavy chain variable region having the recited FR2 in which Arg at position 8 is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 8 and including, (g) An antibody comprising a heavy chain variable region having the recited FR3 in which Met at position 4 is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 9 and including, (h) An antibody comprising a heavy chain variable region having the recited FR3 in which Leu at position 5 is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 9 and including, (i) An antibody comprising a heavy chain variable region having the recited FR3 in which Arg at position 16 is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 9 and including, (j) An antibody comprising a heavy chain variable region having the recited FR3 in which Val at position 27 is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 9 and including, (k) An antibody comprising a heavy chain variable region having the recited FR3 in which Met at position 4, Leu at position 5, Arg at position 16 and Val at position 27 are substituted with other amino acids in the amino acid sequence described in SEQ ID NO: 9 and including, (l) An antibody comprising a heavy chain variable region having the recited FR4 in which Gln at position 3 is substituted with another amino acid in the amino acid sequence described in SEQ ID NO: 10 and including, (m) An antibody comprising a light chain variable region having an FR1 in which Arg at position 18 in the amino acid sequence set forth in SEQ ID NO: 11 is substituted with another amino acid and having an FR1 in which Arg at position 18 in the amino acid sequence set forth in SEQ ID NO: 11 is substituted with another amino acid (n) An antibody comprising a light chain variable region having an FR2 in which Lys at position 11 in the amino acid sequence set forth in SEQ ID NO: 12 is substituted with another amino acid and having an FR2 in which Lys at position 11 in the amino acid sequence set forth in SEQ ID NO: 12 is substituted with another amino acid (o) An antibody comprising a light chain variable region having an FR3 in which Gln at position 23 in the amino acid sequence set forth in SEQ ID NO: 13 is substituted with another amino acid and having an FR3 in which Gln at position 23 in the amino acid sequence set forth in SEQ ID NO: 13 is substituted with another amino acid (p) An antibody comprising a light chain variable region having an FR3 in which Pro at position 24 in the amino acid sequence set forth in SEQ ID NO: 13 is substituted with another amino acid and having an FR3 in which Pro at position 24 in the amino acid sequence set forth in SEQ ID NO: 13 is substituted with another amino acid (q) An antibody comprising a light chain variable region having an FR3 in which Ile at position 27 in the amino acid sequence set forth in SEQ ID NO: 13 is substituted with another amino acid and having an FR3 in which Ile at position 27 in the amino acid sequence set forth in SEQ ID NO: 13 is substituted with another amino acid (r) An antibody comprising a light chain variable region having an FR3 in which Gln at position 23, Pro at position 24, and Ile at position 27 in the amino acid sequence set forth in SEQ ID NO: 13 are substituted with other amino acids (s) An antibody comprising a light chain variable region having an FR4 in which Lys at position 10 in the amino acid sequence set forth in SEQ ID NO: 14 is substituted with another amino acid and having an FR4 in which Lys at position 10 in the amino acid sequence set forth in SEQ ID NO: 14 is substituted with another amino acid (t) An antibody comprising a heavy chain variable region having an FR4 in which Ser at position 5 in the amino acid sequence set forth in SEQ ID NO: 10 is substituted with another amino acid and having an FR4 in which Ser at position 5 in the amino acid sequence set forth in SEQ ID NO: 10 is substituted with another amino acid (u) An antibody comprising a heavy chain variable region having an FR4 in which Gln at position 3 and Ser at position 5 in the amino acid sequence set forth in SEQ ID NO: 10 are substituted with other amino acids (v) An antibody comprising a heavy chain variable region having an FR3 having the amino acid sequence set forth in SEQ ID NO: 184 and having an FR3 having the amino acid sequence set forth in SEQ ID NO: 184 (w) An antibody comprising a heavy chain variable region comprising the FR1 described in (e), the FR2 described in (f), the FR3 described in (k), and the FR4 described in (l) or (u) and having a heavy chain variable region comprising the FR1 described in (e), the FR2 described in (f), the FR3 described in (k), and the FR4 described in (l) or (u) An antibody comprising a light chain variable region comprising FR1 described in (x) (m), FR2 described in (n), FR3 described in (r), and FR4 described in (s), or (y) An antibody comprising a heavy chain variable region described in (w) and a light chain variable region described in (x), [4] An anti-IL-6 receptor antibody described in any one of the following (a) to (l); (a) An antibody comprising a heavy chain variable region having a CDR1 in which the 1st Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with another amino acid (b) An antibody comprising a heavy chain variable region having a CDR2 in which the 9th Thr in the amino acid sequence described in SEQ ID NO: 2 is substituted with another amino acid (c) An antibody comprising a heavy chain variable region having a CDR2 in which the 16th Ser in the amino acid sequence described in SEQ ID NO: 2 is substituted with another amino acid (d) An antibody comprising a heavy chain variable region having a CDR2 in which the 9th Thr and the 16th Ser in the amino acid sequence described in SEQ ID NO: 2 are substituted with other amino acids (e) An antibody comprising a light chain variable region having a CDR1 in which the 1st Arg in the amino acid sequence described in SEQ ID NO: 4 is substituted with another amino acid (f) An antibody comprising a light chain variable region having a CDR2 in which the 2nd Thr in the amino acid sequence described in SEQ ID NO: 5 is substituted with another amino acid (g) An antibody comprising a light chain variable region having a CDR2 in which the 4th Arg in the amino acid sequence described in SEQ ID NO: 5 is substituted with another amino acid (h) An antibody comprising a light chain variable region having a CDR2 in which the 2nd Thr and the 4th Arg in the amino acid sequence described in SEQ ID NO: 5 are substituted with other amino acids (i) An antibody comprising a light chain variable region having a CDR3 in which the 5th Thr in the amino acid sequence described in SEQ ID NO: 6 is substituted with another amino acid (j) An antibody comprising a heavy chain variable region containing CDR1 as described in (a), CDR2 as described in (d), and CDR3 having the amino acid sequence as described in SEQ ID NO: 3. (k) An antibody comprising a light chain variable region containing CDR1 as described in (e), CDR2 as described in (h), and CDR3 as described in (i). Or (l) An antibody comprising the heavy chain variable region as described in (j) and the light chain variable region as described in (k). [5] An anti-IL-6 receptor antibody as described in any one of the following (a) to (f); (a) CDR1 in which the 1st Ser in the amino acid sequence as described in SEQ ID NO: 1 is substituted with another amino acid sequence, CDR2 in which the 9th Thr and 16th Ser in the amino acid sequence as described in SEQ ID NO: 2 are substituted with other amino acids, and CDR3 in which the 1st Ser and 5th Thr in the amino acid sequence as described in SEQ ID NO: 3 are substituted with other amino acids, and an antibody comprising a heavy chain variable region containing the same. (b) CDR1 in which the 1st Arg in the amino acid sequence as described in SEQ ID NO: 4 is substituted with another amino acid, CDR2 in which the 2nd Thr and 4th Arg in the amino acid sequence as described in SEQ ID NO: 5 are substituted with other amino acids, and CDR3 in which the 1st Gln and 5th Thr in the amino acid sequence as described in SEQ ID NO: 6 are substituted with other amino acids, and an antibody comprising a light chain variable region containing the same. (c) An antibody comprising a heavy chain variable region having the amino acid sequence as described in SEQ ID NO: 22. (d) An antibody comprising a light chain variable region having the amino acid sequence as described in SEQ ID NO: 23. (e) An antibody comprising the heavy chain variable region as described in (a) and the light chain variable region as described in (b). (f) An antibody comprising the heavy chain variable region as described in (c) and the light chain variable region as described in (d). (g) A human antibody constant region as described in any one of the following (a) to (c). (h) In the amino acid sequence as described in SEQ ID NO: 19, at position 329 (EU numbering 446). Or (i) An antibody comprising the heavy chain variable region as described in (j) and the light chain variable region as described in (k). [6] A human antibody constant region as described in any one of the following (a) to (c); (a) In the amino acid sequence as described in SEQ ID NO: 19, at position 329 (EU numbering 446). characterized in that both the Gly at position 330 (EU numbering 447) and the Lys at position 330 (EU numbering 447) are deleted, a human antibody constant region, (b) In the amino acid sequence set forth in SEQ ID NO: 20, the Gly at position 325 (EU numbering 446) and the Lys at position 326 (EU numbering 447) are both deleted, characterized in that, a human antibody constant region, (c) In the amino acid sequence set forth in SEQ ID NO: 21, the Gly at position 326 (EU numbering 446) and the Lys at position 327 (EU numbering 447) are both deleted, characterized in that, a human antibody constant region, [7] In the amino acid sequence set forth in SEQ ID NO: 20, the IgG2 constant region in which the amino acids at positions 209 (EU numbering 330), 210 (EU numbering 331), and 218 (EU numbering 339) are substituted with other amino acids, [8] In the amino acid sequence set forth in SEQ ID NO: 20, the IgG2 constant region in which the amino acid at position 276 (EU numbering 397) is substituted with other amino acids, [9] In the amino acid sequence set forth in SEQ ID NO: 20, the IgG2 constant region in which the amino acids at positions 14 (EU numbering 131), 102 (EU numbering 219), and / or 16 (EU numbering 133) are substituted with other amino acids,

[10] The IgG2 constant region according to [9], characterized in that the amino acids at positions 20 (EU numbering 137) and 21 (EU numbering 138) in the amino acid sequence set forth in SEQ ID NO: 20 are further substituted with other amino acids, ​​​​​​​​​〔11〕In the amino acid sequence set forth in SEQ ID NO: 20, the IgG2 constant region in which His at position 147 (position 268 in EU numbering), Arg at position 234 (position 355 in EU numbering) and / or Gln at position 298 (position 419 in EU numbering) are replaced with other amino acids region, 〔12〕In the amino acid sequence set forth in SEQ ID NO: 20, positions 209 (position 330 in EU numbering), 210 (position 331 in EU numbering), 218 (position 339 in EU numbering ), 276 (position 397 in EU numbering), 14 (position 131 in EU numbering), 16 (position 133 in EU numbering), 102 (position 219 in EU numbering), 20 (position 137 in EU numbering) and 21 (position 138 in EU numbering) of the amino acids are replaced with other amino acids, and the IgG2 constant region having the amino acid sequence 〔13〕In the IgG2 constant region described in 〔12〕, the IgG2 constant region having an amino acid sequence in which Gly at position 325 (position 446 in EU numbering) and Lys at position 326 (position 447 in EU numbering) are further deleted 〔14〕In the amino acid sequence set forth in SEQ ID NO: 20, positions 276 (position 397 in EU numbering), 14 (position 131 in EU numbering), 16 (position 133 in EU numbering), 102 (position 219 in EU numbering), 20 (position 137 in EU numbering) and 21 (position 138 in EU numbering) of the amino acids are replaced with other amino acids and the amino acid sequence having the IgG2 constant region 〔15〕In the IgG2 constant region described in 〔14〕, the IgG2 constant region having an amino acid sequence in which Gly at position 325 (position 446 in EU numbering) and Lys at position 326 (position 447 in EU numbering) are further deleted 〔16〕In the amino acid sequence set forth in SEQ ID NO: 20, Cys at the 14th position (EU numbering 131st position), Arg at the 16th position (EU numbering 133rd position), Cys at the 102nd position (EU numbering 219th position), Glu at the 20th position (EU numbering 137th position), Ser at the 21st position (EU numbering 138th position), His at the 147th position (EU numbering 268th position), Arg at the 234th position (EU numbering 355th position), and Gln at the 298th position (EU numbering 419th th position) are replaced with other amino acids, and an IgG2 constant region having the amino acid sequence, 〔17〕In the IgG2 constant region described in 〔16〕, an IgG2 constant region having an amino acid sequence in which Gly at the 325th position (EU numbering 446th position) and Lys at the 326th position (EU numbering 447th position) are further deleted, 〔18〕In the amino acid sequence set forth in SEQ ID NO: 20, Cys at the 14th position (EU numbering 131st position), Arg at the 16th position (EU numbering 133rd position), Cys at the 102nd position (EU numbering 219th position), Glu at the 20th position (EU numbering 137th position), Ser at the 21st position (EU numbering 138th position), His at the 147th position (EU numbering 268th position), Arg at the 234th position (EU numbering 355th position), Gln at the 298th position (EU numbering 419th position) and Asn at the 313th position (EU numbering 434th position) are replaced with other amino acids, and an IgG2 constant region having the amino acid sequence, 〔19〕In the IgG2 constant region described in 〔18〕, an IgG2 constant region having an amino acid sequence in which Gly at the 325th position (EU numbering 446th position) and Lys at the 326th position (EU numbering 447th position) are further deleted, 〔20〕An IgG4 constant region, characterized in that the amino acid at the 289th position (EU numbering 409th position) in the amino acid sequence set forth in SEQ ID NO: 21 is replaced with other amino acids, 〔21〕In the amino acid sequence set forth in SEQ ID NO: 21, at the 289th position (EU numbering 409th position), 14th position, 16th position, 20th position, 21st position, 97th position, 100th position, 102nd An IgG4 constant region having an amino acid sequence in which the amino acids at positions 103, 104, and 105 (EU numbering positions 131, 133, 137, 138, 214, 217, 219, 220, 221, 222), 113, 114, and 115 (EU numbering positions 233, 234, 235) are substituted with other amino acids and the amino acid at position 116 (EU numbering position 236) is deleted, In the IgG4 constant region described in

[22]

[21] , an IgG4 constant region in which Gly at position 326 (EU numbering position 446) and Lys at position 327 (EU numbering position 447) are further deleted, An IgG2 constant region having an amino acid sequence in which Ala at position 209 (EU numbering position 330), Pro at position 210 (EU numbering position 331), Thr at position 218 (EU numbering position 339), Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), and Ser at position 21 (EU numbering position 138) in the amino acid sequence described in SEQ ID NO: 20 are substituted with other amino acids, In the IgG2 constant region described in

[24]

[23] , an IgG2 constant region having an amino acid sequence in which Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are further deleted, An IgG2 constant region having an amino acid sequence in which Cys at position 14 (EU numbering position 131), Arg at position 16 (EU numbering position 133), Cys at position 102 (EU numbering position 219), Glu at position 20 (EU numbering position 137), and Ser at position 21 (EU numbering position 138) in the amino acid sequence described in SEQ ID NO: 20 are substituted with other amino acids, In the IgG2 constant region described in

[26]

[25] , an IgG2 constant region having an amino acid sequence in which Gly at position 325 (EU numbering position 446) and Lys at position 326 (EU numbering position 447) are further deleted, 〔27〕Constant region having the amino acid sequence set forth in SEQ ID NO: 24, 〔28〕Constant region having the amino acid sequence set forth in SEQ ID NO: 118, 〔29〕Constant region having the amino acid sequence set forth in SEQ ID NO: 25, 〔30〕Constant region having the amino acid sequence set forth in SEQ ID NO: 151, 〔31〕Constant region having the amino acid sequence set forth in SEQ ID NO: 152, 〔32〕Constant region having the amino acid sequence set forth in SEQ ID NO: 153, 〔33〕Constant region having the amino acid sequence set forth in SEQ ID NO: 164, 〔34〕Human antibody constant region having the amino acid sequence set forth in SEQ ID NO: 194 (M40ΔGK) , 〔35〕Human antibody constant region having the amino acid sequence set forth in SEQ ID NO: 192 (M86ΔGK) , 〔36〕An antibody having the constant region according to any one of 〔6〕 to 〔35〕, 〔37〕The antibody according to 〔36〕, which is characterized by binding to the IL-6 receptor 〔38〕An anti-human IL-6 receptor antibody having a binding activity to the IL-6 receptor of 1 nM or less 〔39〕An anti-human IL-6 receptor antibody having an actually measured isoelectric point of the full-length antibody of 7.0 or less or a theoretical isoelectric point of the variable region of 5.0 or less 〔40〕Under the condition that the antibody concentration is 100 mg / mL in a buffer of 20 mM Histidine-HCl, 150 mM NaCl, pH 6.5 to 7.0, the increase in the aggregate ratio of the antibody after 1 month at 25°C is 0.3% or less An anti-IL-6 receptor antibody characterized by 〔41〕A pharmaceutical composition comprising the antibody according to any one of 〔36〕 to 〔40〕.

Brief Description of Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] 〔Embodiment of the Invention〕 The present invention provides a method for modifying the isoelectric point while retaining the binding activity of a variable region of an antibody to an antigen in a polypeptide containing the variable region of the antibody, the method comprising converting the charge of at least one amino acid residue that can be exposed on the surface of the complementarity determining region (CDR) of the polypeptide. The present invention also provides a polypeptide containing a variable region of an antibody with a modified isoelectric point obtained by the method (for example, an antibody containing a CDR selected from the group consisting of a human-derived CDR, a CDR derived from an animal other than human, and a synthetic CDR, a human-derived framework region (FR), and a human constant region, wherein at least one amino acid residue that can be exposed on the surface of the CDR has a charge different from that of the amino acid residue at the corresponding position of the wild-type CDR, and retains the binding activity to the antigen and has a modified isoelectric point as compared to the antibody before modification).

[0021] As a preferred embodiment of the method of the present invention, there is provided a method including modifying the charge of at least one amino acid residue that can be exposed on the surface of an antibody. That is, by modifying the charge of the amino acid residue of the antibody and changing its isoelectric point (pI), the pharmacokinetics (blood kinetics) of the antibody in plasma can be controlled. As a result, an antibody with controlled plasma pharmacokinetics can exhibit, for example, better antitumor activity against cancer cells than an antibody without such control.

[0022] In the method of the present invention, maintaining the binding activity to an antigen means having an activity of at least 80% or more, preferably 85% or more, particularly preferably 90% or more, compared to the binding activity of the peptide before modification. Also, when the antibody binds to the antigen, it is sufficient that the binding activity is maintained to such an extent that the function of the antibody is maintained. For example, under physiological conditions, the affinity measured at 37°C may be 100 nM or less, preferably 50 nM or less, more preferably 10 nM or less, and even more preferably 1 nM or less. Whether a polypeptide containing the variable region of an antibody with a modified isoelectric point obtained by the method of the present invention retains the binding activity to an antigen can be determined by known methods, such as BIACORE (molecular interaction analysis), cell proliferation assay, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radio immunoassay), or fluorescence immunoassay.

[0023] Examples of the "polypeptide containing the variable region of an antibody" of the present invention include, but are not limited to, antibodies, antibody fragments with reduced molecular weight, and Scaffold proteins. In the present invention, a Scaffold protein can be used as long as it is a three-dimensionally stable peptide that can bind to at least one antigen. Examples of such peptides include antibody variable region fragments, fibronectin, Protein A domain, and LDL receptor A domain. , in addition to lipocalin and others, molecules described by Nygren et al. (Current Opinion in Structural Biology, 7:463-469 (1997), Journal of Immunol Methods, 290:3-28 (2004)), Binz et al. (Nature Biotech 23:1257-1266 (2005)), Hosse et al. (Protein Science 15:14-27 (2006)) are included.

[0024] In the present invention, the term "antibody" is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, antibody variants (chimeric antibodies, humanized antibodies, minimized antibodies (including antibody fragments), multispecific antibodies, etc.) as long as they exhibit the desired biological activity. In the present invention, when obtaining (producing) these antibodies, the antibody modification method of the present invention can be preferably used.

[0025] The "antibody" in the present invention includes antibodies in which the amino acid sequence has been modified by substitution, deletion, addition and / or insertion of amino acids, etc., with respect to the antibody in which the charge of amino acid residues has been modified as described above. Also included are antibodies in which the charge of amino acid residues has been further modified with respect to antibodies in which the amino acid sequence has been modified by substitution, deletion, addition and / or insertion of amino acids, or by chimerization or humanization, etc. That is, it may be modified simultaneously with the process of humanizing a mouse antibody, or alternatively, a humanized antibody may be further modified.

[0026] Substitution, deletion, addition and / or insertion of amino acids, and modification of the amino acid sequence such as humanization and chimerization can be carried out by methods known to those skilled in the art. Similarly, the variable region and constant region of the antibody used when producing the antibody in the present invention as a recombinant antibody may also have its amino acid sequence modified by substitution, deletion, addition and / or insertion of amino acids, or by chimerization or humanization, etc.

[0027] The antibodies in the present invention may be antibodies derived from any animal, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, etc. Furthermore, for example, modified antibodies with substituted amino acid sequences such as chimeric antibodies, especially humanized antibodies, may also be used. Also, any antibody such as an antibody conjugate with various molecules, an antibody fragment, or a small molecule antibody may be used.

[0028] A "chimeric antibody" is an antibody produced by combining sequences derived from different animals. For example, an antibody consisting of the variable (V) regions of the heavy and light chains of a mouse antibody and the constant (C) regions of the heavy and light chains of a human antibody can be exemplified. The production of chimeric antibodies is known. For example, DNA encoding the antibody V region is ligated to DNA encoding the human antibody C region, and this is incorporated into an expression vector

[0029] Also, the small molecule antibodies in the present invention are not particularly limited in their structure, production method, etc., as long as they have the ability to bind to an antigen. Among small molecule antibodies, there are also antibodies having higher activity than full-length antibodies (Orita et al., Blood (2005) 105: 562-566). In this specification, a "small molecule antibody" refers to any part of a full-length antibody (whole antibody, for example, whole IgG, etc.) without particular limitation Although it is not required, it preferably contains a heavy chain variable region (VH) or a light chain variable region (VL). Examples of preferred antibody fragments include, for example, Fab, F(ab')2, Fab', Fv, and the like. The amino acid sequence of VH or VL in the antibody fragment may be modified by substitution, deletion, addition, and / or insertion. Furthermore, as long as the ability to bind to the antigen is retained, a part of VH and VL may be deleted. For example, among the aforementioned antibody fragments, "Fv" is the smallest antibody fragment containing a complete antigen recognition site and a binding site. "Fv" is a dimer (VH-VL dimer) in which one VH and one VL are strongly bound by non-covalent bonds. The antigen binding site is formed on the surface of the VH-VL dimer by the three complementarity determining regions (CDRs) of each variable region. Six CDRs confer the antigen binding site to the antibody. However, even one variable region (or half of the Fv containing only the three CDRs specific for the antigen), although it has a lower affinity than the whole binding site, has the ability to recognize and bind the antigen. Therefore, molecules smaller than such Fv are also included in the low-molecular-weight antibody in the present invention. Also, the variable region of the low-molecular-weight antibody may be chimerized or humanized.

[0030] The low-molecular-weight antibody preferably contains both VH and VL. Examples of low-molecular-weight antibodies include antibody fragments such as Fab, Fab', F(ab')2, and Fv, and scFv (single chain Fv) that can be produced using the antibody fragment (Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; Pluckthun, "The Pharmacology of Monoclonal Antibodies", Vol. 113, edited by Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)), Diabody (Holliger et al., Proc. Natl. Acad. Sci. USA (1993) 90: 6444-8; EP404097; WO93 / 11161; Johnson et al., Method in Enzymology (1991) 203: 88-98; Holliger et al., Protein Engineering (1996) 9: 299-305; Perisic et al., Structure (1994) 2: 1217-26; John et al., Protein Engineering (1999) 12(7): 597-604; Atwell et al., Mol. Immunol. (1996) 33: 1301-12), sc(Fv)2 (Hudson et al., J Immunol. Methods (1999) 231: 177-89; Orita et al., Blood (2005) 105: 562-566), Triabody (Journal of Immunological Methods (1999) 231: 177-89), and Tandem Diabody (Cancer Research (2000) 60: 4336-41), etc. can be mentioned.

[0031] Antibody fragments can be obtained by treating an antibody with an enzyme, such as a protease like papain, pepsin, etc. (see Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al., Science (1985) 229: 81). Also, based on the amino acid sequence of the antibody fragment, it can also be produced by genetic recombination.

[0032] The low-molecular-weight antibody having a modified structure of the antibody fragment can be constructed by using an antibody fragment obtained by enzymatic treatment or genetic recombination. Alternatively, a gene encoding the entire low-molecular-weight antibody can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (see, for example, Co et al., J. Immunol. (1994) 152: 2968-76; Better and Horwitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178: 497-515; Lamoyi, Methods Enzymol. (1986) 121: 652-63; Rousseaux et al., Methods Enzymol. (1986) 121: 663-9; Bird and Walker, Trends Biotechnol. (1991) 9: 132-7).

[0033] Also, the above-mentioned "scFv" is a single-chain polypeptide in which two variable regions are linked via a linker or the like as necessary. The two variable regions contained in scFv are usually one VH and one VL, but may also be two VHs or two VLs. Generally, the scFv polypeptide contains a linker between the VH and VL domains, whereby the VH and VL paired portions necessary for antigen binding are It is formed. Generally, in order to form complementary parts between VH and VL within the same molecule, a linker for linking VH and VL is generally a peptide linker with a length of 10 amino acids or more. However, the linker of the scFv in the present invention is not limited to such a peptide linker as long as it does not prevent the formation of scFv. As a general review of scFv, reference can be made to Pluckthun, "The Pharmacology of Monoclonal Antibody", Vol. 113 (Rosenburg and Moore ed., Springer Verlag, NY, pp. 269-315 (1994)).

[0034] In addition, "diabody (Db)" refers to a bivalent antibody fragment constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404,097, WO93 / 11161, etc.). A diabody is a dimer composed of two polypeptide chains, and each polypeptide chain has a light chain variable region (VL) and a heavy chain variable region (VH) in the same chain, which are linked by a short linker, for example, a linker of about 5 residues, so that they cannot bind to each other. Since VL and VH encoded on the same polypeptide chain cannot form a single-chain V region fragment due to the short linker between them and form a dimer, a diabody has two antigen-binding sites. At this time, when VL and VH against two different epitopes (a, b) are linked with a linker of about 5 residues in a combination of VLa-VHb and VLb-VHa and simultaneously expressed, they are secreted as bispecific Db.

[0035] Since a diabody contains two molecules of scFv, it contains four variable regions and, as a result, has two antigen-binding sites. Different from the case of scFv that does not form a dimer, the form of a diabody When aiming for [a certain result], usually, when the linker connecting VH and VL within each scFv molecule is a peptide linker, it is about 5 amino acids long. However, the linker of the scFv forming a Diabody is not limited to such a peptide linker as long as it does not interfere with the expression of the scFv and the formation of the Diabody. is not limited to such peptide linkers.

[0036] Among the multiple isotypes of antibodies, since the IgG antibody has a sufficiently large molecular weight, its main metabolic pathway is not the renal excretion pathway. IgG antibodies having the Fc region as a part of their molecule are known to have a long in vivo half-life by being recycled through the salvage pathway of the neonatal Fc receptor (FcRn) expressed on endothelial cells such as blood vessels. It is considered that IgG antibodies are mainly metabolized by the metabolic pathway in endothelial cells (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL. Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin. J Immunol. (1998),160(2),1029 - 35). That is, it is considered that IgG antibodies are recycled when the IgG antibodies non-specifically taken up by endothelial cells bind to FcRn, while the IgG antibodies that cannot bind are metabolized. The plasma half-life of an IgG antibody whose Fc portion is modified so that its binding activity to FcRn is reduced becomes shorter is shortened. Conversely, to enhance the binding activity to FcRn, the amino acid residues constituting the Fc region of the IgG antibody are modified to increase the binding activity to FcRn. By doing so, the plasma half-life of the IgG antibody can be extended (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL. Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin. J Immunol. (1998), 160(2), 1029-35). As described above, the conventional method for controlling the plasma pharmacokinetics of IgG antibodies has been carried out by modifying the binding activity to FcRn by modifying the amino acid residues constituting the Fc region. However, as shown in the following examples, in the present invention, it has been clarified that the plasma half-life of the antibody depends highly on the pI. That is, it has been shown that it is possible to control the plasma half-life of the antibody without modifying the amino acid sequence constituting Fc, which may lead to the acquisition of immunogenicity.

[0037] Although not intending to be bound by a particular theory, the inventors currently think as follows. The rate of uptake of non-specific IgG antibodies by endothelial cells is thought to depend on the physicochemical Coulomb interaction between the negatively charged cell surface and the IgG antibody. Therefore, by decreasing (increasing) the pI of the IgG antibody, the Coulomb interaction is reduced (increased). It is considered that the non-specific uptake into endothelial cells is decreased (increased) by the addition of the ionizing agent, and the metabolism in endothelial cells is decreased (increased), thereby controlling the pharmacokinetics in plasma. Since the Coulombic interaction between endothelial cells and the negative charges on the cell surface is a physicochemical interaction, it is considered that this interaction does not depend primarily on the amino acid sequence constituting the antibody. Therefore, the method for controlling the pharmacokinetics in plasma found in the present invention is not only applicable to a specific antibody, but is widely applicable to any polypeptide containing the variable region of an antibody. As such a peptide, a peptide having a molecular weight of 50,000 or more is preferable, a peptide having a molecular weight of 100,000 or more is more preferable, and a peptide having a molecular weight of 140,000 or more is further preferable. With such a peptide, the main metabolic pathway is not renal excretion, and it is possible to fully obtain the effect of controlling the pharmacokinetics in plasma of the present invention. In the present invention, the reduction (increase) of the Coulombic interaction means an increase (decrease) of the Coulombic force represented by repulsion.

[0038] The polypeptide comprising the FcRn binding domain in the present invention is not limited to IgG antibodies, but may be any Fc receptor. Any protein may be used as long as it is capable of binding (having binding activity or affinity) to the FcRn receptor (FcRn). Preferably, the polypeptide comprising an FcRn-binding region in the present invention is, without particular limitation, a protein comprising an antibody Fc region or an Fc-like region. A modified Fc region can also be used as the Fc region, for example, the modified Fc region described in J Immunol. (1998), 160(2), 1029-35. An example of the polypeptide comprising an FcRn-binding region in the present invention is an IgG antibody. Modified versions of these antibodies (proteins) can also be used. In the present invention, the polypeptide comprising an FcRn-binding region of the present invention includes any protein capable of binding to FcRn, regardless of whether it is a protein of interest or not. In the present invention, the most preferred example of the polypeptide comprising an FcRn-binding region of the present invention is an IgG antibody.

[0039] When using an IgG antibody as the antibody of the present invention, any subtype may be used as long as it is an IgG-type antibody molecule, and it may also be a multi-specific (e.g., bispecific) IgG antibody. This bispecific antibody is an antibody that has specificity for two different epitopes, and includes antibodies that recognize different antigens as well as antibodies that recognize different epitopes on the same antigen. Also, even in the case of antibody molecules, low-molecular-weight antibodies such as scFv and Fab, for which renal excretion is the main metabolic pathway, cannot control plasma pharmacokinetics by pI as described above. However, the present invention is applicable to any antibody molecular form as long as it is a polypeptide containing the variable region of an antibody for which renal excretion is not the main metabolic pathway. For example, scFv-Fc, dAb-Fc, Fc fusion proteins, etc. can be mentioned. Since renal excretion of these molecules is not the main metabolic pathway, it is possible to control plasma pharmacokinetics by changing the pI by the method found in the present invention. The antibody molecule to which the present invention can be applied may also be an antibody-like molecule. An antibody-like molecule is a molecule that exhibits a function by binding to a target molecule (Binz HK, Amstutz P, Pluckthun A., Engineering novel binding proteins from nonimmunoglobulin domains., Nat Biotechnol. 2005 Oct;23(10):1257-68.), and examples include DARPins, Affibody, Avimer, etc. In addition, when the antibody of the present invention is, for example, a bispecific anti-glypican 3 antibody, the antibody can also specifically bind to epitopes of glypican 3 and antigens other than glypican 3. For example, as antigens other than glypican 3, surface antigens that specifically bind to these cells can be preferably used to recruit NK cells, cytotoxic T cells, LAK cells

[0040] etc. An antibody MUSE11 that recognizes MUC1, an adenocarcinoma-related antigen, and an antibody that recognizes the LAK cell surface antigen can be used. Using a bispecific antibody prepared from the antibody OKT3, it has been shown that cytotoxic activity by LAK cells against cholangiocarcinoma is exerted (Katayose Y, Kudo T, Suzuki M, Shinoda M, Saijyo S, Sakurai N, Saeki H, Fukuhara K, Imai K, Matsuno S. MUC1-specific targeting immunotherapy with bispecific antibodies: inhibition of xenografted human bile duct carcinoma growth. Cancer Res. (1996) 56(18), 4205-12). Instead of the antibody MUSE11 that recognizes the above MUC1, the glypican 3 antibody with improved plasma pharmacokinetics provided by the present invention can be preferably used. Further, as the bispecific glypican 3 antibody provided by the present invention, antibodies that recognize different epitopes of the glypican 3 molecule can also be preferably utilized.

[0041] Note that the above "bispecific antibody" may be, for example, an antibody having a structure in which the heavy chain variable region and the light chain variable region are linked as a single chain (for example, sc(Fv)2). Also, an antibody-like molecule (for example, scFv-Fc) in which an scFv (or sc(Fv)2) in which the heavy chain variable region (VH) and the light chain variable region (VL) are linked is bound to an Fc region (a constant region lacking the CH1 domain) may be used. The multispecific antibody formed from scFv-Fc has a structure of the (scFv)2-Fc type in which the first polypeptide is VH1-linker-VL1-Fc and the second polypeptide is composed of VH2-linker-VL2-Fc. Alternatively, it may be an antibody-like molecule in which a single domain antibody is bound to the Fc region (Curr Opin Drug Discov Devel. 2006, 9(2), 184-93).

[0042] ​​​ In the present invention, the modification of the charge of an amino acid residue can be carried out through amino acid substitution. The amino acid substitution can be carried out by the method described below. In the present invention, the amino acid residue that can be exposed on the surface of the CDR region to be substituted is From the viewpoint of maintaining the binding activity to an antigen, the 31st , 61st, 62nd, 64th and 65th amino acid residues according to Kabat numbering in the heavy chain variable region or the Kabat number It is preferably at least one amino acid residue selected from the 24th, 27th, 53rd, 54th and 55th amino acid residues according to ring in the light chain variable region. The substitution of these amino acid residues is useful in that it retains the function (such as the binding activity to an antigen) that the polypeptide containing the variable region of the antibody had before the substitution of the amino acid residue, and in that the substitution is independent of the type of antibody.

[0043] The present invention also provides a method for controlling the pharmacokinetics of a polypeptide containing the variable region of an antibody by modifying the isoelectric point of the polypeptide. Further included in the present invention is a polypeptide containing the variable region of an antibody whose pharmacokinetics are controlled by the method.

[0044] In the present invention, "the pharmacokinetics in plasma are controlled" means that the pharmacokinetics in plasma of the antibody before and after the modification of the amino acids constituting the antibody are compared, and the pharmacokinetics in plasma are modified in a desired direction. That is, when it is desired to extend the half-life of the drug (in plasma) of the antibody, "control of the pharmacokinetics in plasma" means that the half-life in plasma of the antibody is extended. When it is desired to shorten the half-life in plasma of the antibody, "control of the pharmacokinetics in plasma" means that the half-life in plasma of the antibody is shortened.

[0045] In the present invention, whether the pharmacokinetics of an antibody in plasma has been modified in a desired direction, that is, whether the pharmacokinetics in plasma is controlled as desired, can be appropriately evaluated, for example, by conducting a pharmacokinetic study using mice, rats, rabbits, dogs, monkeys, etc. Further, the "elongation of plasma half-life" or "shortening of plasma half-life" in the present invention, more specifically, in addition to the parameter of plasma half-life (t1 / 2), can be grasped by any parameter such as mean plasma residence time, plasma clearance (CL), area under the concentration curve (AUC), plasma half-life, etc. ("Understanding through Pharmacokinetics Exercises" (Nanzando)). For example, by performing non-compartmental analysis according to the procedure manual attached to the pharmacokinetic analysis software WinNonlin (Pharsight), the "control of plasma kinetics" provided by the present invention can be appropriately evaluated using these parameters. Also, it is possible to sustain the function of an antibody by controlling its pharmacokinetics in plasma. For example, if the method of the present invention is applied to an antibody having cytotoxic activity, its function can be sustained, and it becomes possible to adjust the duration of the function of the polypeptide before modification, such as cytotoxic activity effect, antagonist activity, agonist activity, etc. In the present invention, the "amino acid residue that can be exposed on the surface" usually refers to the amino acid residue on the surface of the polypeptide constituting the antibody. The "amino acid residue on the surface of the polypeptide" refers to an amino acid residue whose side chain can contact a solvent molecule (usually water molecules in many cases). It is not necessary that all of its side chains contact the solvent molecule. When even a part of its side chain contacts the solvent molecule, that amino acid residue is defined as an amino acid on the surface. By homology modeling using commercially available software, those skilled in the art can create a homology model of a polypeptide or an antibody. Based on the homology model, the amino acid residues on the surface of the polypeptide constituting an appropriate antibody can be preferably selected as the "amino acid residues on the surface of the polypeptide".

[0046] Also, it is possible to sustain the function of an antibody by controlling its pharmacokinetics in plasma. For example, if the method of the present invention is applied to an antibody having cytotoxic activity, its function can be sustained, and it becomes possible to adjust the duration of the function of the polypeptide before modification, such as cytotoxic activity effect, antagonist activity, agonist activity, etc.

[0047] In the present invention, the "amino acid residue that can be exposed on the surface" usually refers to the amino acid residue on the surface of the polypeptide constituting the antibody. The "amino acid residue on the surface of the polypeptide" refers to an amino acid residue whose side chain can contact a solvent molecule (usually water molecules in many cases). It is not necessary that all of its side chains contact the solvent molecule. When even a part of its side chain contacts the solvent molecule, that amino acid residue is defined as an amino acid on the surface. By homology modeling using commercially available software, those skilled in the art can create a homology model of a polypeptide or an antibody. Based on the homology model, the amino acid residues on the surface of the polypeptide constituting an appropriate antibody can be preferably selected as the "amino acid residues on the surface of the polypeptide".

[0048] In the present invention, the "amino acid residue that can be exposed on the surface" is not particularly limited, but is preferably an amino acid residue outside the FcRn binding region in the antibody. Examples of this FcRn binding region include, for example, the Fc region.

[0049] In the antibody of the present invention, the amino acid residue whose charge is to be modified is preferably an amino acid residue constituting the heavy chain variable region or the light chain variable region of the antibody. Specific examples of the variable region include the complementarity determining region (CDR) and the framework region (FR).

[0050] A person skilled in the art can appropriately select the surface amino acid residues in the antibody variable region using a homology model prepared by homology modeling or the like. That is, among the amino acid residues based on the Kabat numbering of the H chain variable region, H1, H3, H5, H8, H10, H12, H13, H15, H16, H19, H23, H25, H26, H31, H39, H42, H43, H44, H46, H61, H62, H64, H65, H68, H71, H72, H73, H75, H76, H81, H82b, H83, H85, H86, H105, H108, H110, H112, the surface amino acid residues in the antibody variable region can be appropriately selected. For example, in the H chain FR region of the humanized glypican 3 antibody represented by SEQ ID NO: 195, the 1st, 3rd, 5th, 8th, 10th, 12th, 13th, 15th, 16th, 19th, 23rd, 25th, 26th, 39th, 42nd, 43rd, 44th, 46th, 69th, 72nd, 73rd, 74th, 76th, 77th, 82nd, 85th, 87th, 89th, 90th, 107th, 110th, 112th, 114th amino acid residues can be exemplified as surface amino acids, but the present invention is not limited thereto. Also, the surface amino acid residues in the H chain CDR groups can be selected by a similar homology model. That is, H97, which is an amino acid residue based on the Kabat numbering is exposed on the surface in almost all antibodies. For example, the sequence The 101st serine in the H-chain CDR of the humanized glypican 3 antibody represented by SEQ ID NO: 195 corresponds to the amino acid residue. Other amino acid residues in the H-chain CDR of the humanized glypican 3 antibody represented by SEQ ID NO: 195 include the 52nd, 54th, 62nd, 63rd, 65th, and 66th amino acid residues are preferably mentioned.

[0051] In the light chain variable region, surface amino acid residues in the antibody variable region can be appropriately selected from L1, L3, L7, L8, L9, L11, L12, L16, L17, L18, L20, L22, L24, L27, L38, L39, L41, L42, L43, L45, L46, L49, L53, L54, L55, L57, L60, L63, L65, L66, L68, L69, L70, L74, L76, L77, L79, L80, L81, L85, L100, L103, L105, L106, L107 based on Kabat numbering. For example, the 1st, 3rd, 7th, 8th, 9th, 11th, 12th, 16th, 17th, 18th, 20th, 22nd, 43rd, 44th, 45th, 46th, 48th, 49th , 50th, 54th, 62nd, 65th, 68th, 70th, 71st, 73rd, 74th, 75th, 79th, 81st, 82nd, 84th, 85th, 86th, 90th, 105th, 108th, 110th, 111th, 112th of the humanized glypican 3 antibody represented by SEQ ID NO: 195 can be exemplified as surface amino acids, but the present invention is not limited thereto. Also, the surface amino acid residues in the light chain CDR can be selected by a homology model similar to the homology model by which the surface amino acid residues in the heavy chain CDR are determined thereby. The amino acid residues in the light chain CDR of the humanized glypican 3 antibody represented by SEQ ID NO: 201 preferably include the 24th, 27th, 33rd, 55th, and 59th amino acid residues.

[0052] The "modification" of the amino acid residue in the method provided by the present invention specifically refers to substituting the original amino acid residue with another amino acid residue, deleting the original amino acid residue, and adding new The term "alteration of the charge of an amino acid residue" refers to, for example, adding an amino acid residue, but preferably refers to substituting an original amino acid residue with another amino acid residue. That is, in the present invention, "alteration of the charge of an amino acid residue" preferably includes amino acid substitution.

[0053] In order to "alter the charge of amino acid residues" as described above, the glypican 3 antibody of the present invention may be modified by, for example, modifying the charge of the H-chain variable region constituting the humanized glypican 3 antibody shown in SEQ ID NO: 195. At least one of the amino acid residues at positions 19, 43, 52, 54, 62, 63, 65, 66, and 107 in the Preferably, the charge of at least one amino acid residue selected from the 17th, 24th, 27th, 33rd, 55th, 59th, 79th, 82nd, and 105th amino acid residues in the L-chain variable region constituting the humanized glypican 3 antibody shown in SEQ ID NO: 201 is modified. Of the above amino acid residues, amino acid residues other than the amino acid residue whose charge has been modified do not need to be modified as long as the desired effect of controlling pharmacokinetics in plasma is obtained, but may be modified appropriately to have the same type of charge as the modified amino acid residue or to have no charge.

[0054] In the CDRs of the anti-human IL-6 receptor antibody (6R_a_H1L1) provided by the present invention, in order to perform the above-mentioned "modification of the charge of amino acid residues" while maintaining the binding activity to the antigen, for example, at least one amino acid residue selected from the 31st, 64th, and 65th amino acid residues according to Kabat numbering in the heavy chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 221 has its charge preferably modified. Also, for example, at least one amino acid residue selected from the 24th, 27th, 53rd, and 55th amino acid residues according to Kabat numbering in the light chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 224 has its charge preferably modified. Among the above-mentioned amino acid residues, the amino acid residues other than the amino acid residues whose charges have been modified do not need to be modified as long as the control effect of the target plasma pharmacokinetics is obtained, but can be appropriately modified to have the same type of charge as the modified amino acid residue or no charge.

[0055] In the CDRs of the anti-human IL-6 receptor antibody (6R_b_H1L1) provided by the present invention, in order to perform the above-mentioned "modification of the charge of amino acid residues" while maintaining the binding activity to the antigen, for example, at least one amino acid residue selected from the 31st amino acid residue according to Kabat numbering in the heavy chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 227 has its charge preferably modified. Also, for example, at least one amino acid residue selected from the 24th, 53rd, 54th, and 55th amino acid residues according to Kabat numbering in the light chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 229 has its charge preferably modified. Among the above-mentioned amino acid residues, the amino acid residues other than the amino acid residues whose charges have been modified do not need to be modified as long as the control effect of the target plasma pharmacokinetics is obtained, but can be appropriately modified to have the same type of charge as the modified amino acid residue or no charge.

[0056] In the CDRs of the anti-human GPC3 antibody provided by the present invention, while maintaining the binding activity to the antigen , in order to perform the above-mentioned "modification of the charge of amino acid residues", for example, selected from the 61st, 62nd, 64th, and 65th amino acid residues according to Kabat numbering in the heavy chain variable region constituting the anti-human GPC3 antibody represented by SEQ ID NO: 233, the charge of at least one amino acid residue is preferably modified. Also, for example, selected from the 24th and 27th amino acid residues according to Kabat numbering in the light chain variable region constituting the anti-human GPC3 antibody represented by SEQ ID NO: 236, the charge of at least one amino acid residue is preferably modified. Among the above-mentioned amino acid residues, amino acid residues other than the amino acid residue whose charge has been modified do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is obtained, but may be appropriately modified to have the same type of charge as the modified amino acid residue or to have no charge. In the CDR of the anti-human IL-31 receptor antibody provided by the present invention, in order to perform the above-mentioned "modification of the charge of amino acid residues" while maintaining the binding activity to the antigen, for example, selected from the 61st, 62nd, 64th, and 65th amino acid residues according to Kabat numbering in the heavy chain variable region constituting the anti-human IL-31 receptor antibody represented by SEQ ID NO: 23 9, the charge of at least one amino acid residue is preferably modified. Also, for example, selected from the 24th and 54th amino acid residues according to Kabat numbering in the light chain variable region constituting the anti-human IL-31 receptor antibody represented by SEQ ID NO: 242, the charge of at least one amino acid residue is preferably modified. Among the above-mentioned amino acid residues, amino acid residues other than the amino acid residue whose charge has been modified do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is obtained, but may be appropriately modified to have the same type of charge as the modified amino acid residue or to have no charge.

[0057] In the CDR of the anti-human IL-31 receptor antibody provided by the present invention, in order to perform the above-mentioned "modification of the charge of amino acid residues" while maintaining the binding activity to the antigen, for example, selected from the 61st, 62nd, 64th, and 65th amino acid residues according to Kabat numbering in the heavy chain variable region constituting the anti-human IL-31 receptor antibody represented by SEQ ID NO: 23 9, the charge of at least one amino acid residue is preferably modified. Also, for example, selected from the 24th and 54th amino acid residues according to Kabat numbering in the light chain variable region constituting the anti-human IL-31 receptor antibody represented by SEQ ID NO: 242, the charge of at least one amino acid residue is preferably modified. Among the above-mentioned amino acid residues, amino acid residues other than the amino acid residue whose charge has been modified do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is obtained, but may be appropriately modified to have the same type of charge as the modified amino acid residue or to have no charge. In the CDR of the anti-human IL-31 receptor antibody provided by the present invention, in order to perform the above-mentioned "modification of the charge of amino acid residues" while maintaining the binding activity to the antigen, for example, selected from the 61st, 62nd, 64th, and 65th amino acid residues according to Kabat numbering in the heavy chain variable region constituting the anti-human IL-31 receptor antibody represented by SEQ ID NO: 23 9, the charge of at least one amino acid residue is preferably modified. Also, for example, selected from the 24th and 54th amino acid residues according to Kabat numbering in the light chain variable region constituting the anti-human IL-31 receptor antibody represented by SEQ ID NO: 242, the charge of at least one amino acid residue is preferably modified. Among the above-mentioned amino acid residues, amino acid residues other than the amino acid residue whose charge has been modified do not need to be modified as long as the desired effect of controlling plasma pharmacokinetics is obtained, but may be appropriately modified to have the same type of charge as the modified amino acid residue or to have no charge.

[0058] It is known that some amino acids carry a charge. Generally, lysine (K), arginine (R), and histidine (H) are known as positively charged amino acids (cationic amino acids). As negatively charged amino acids (anionic amino acids), aspartic acid (D), glutamic acid (E), etc. are known. Amino acids other than these are known as amino acids without a charge.

[0059] The above-mentioned "modified amino acid residue" is preferably appropriately selected from amino acid residues included in any one of the following groups (a) or (b), but is not particularly limited to these amino acids. (a) Glutamic acid (E), aspartic acid (D) (b) Lysine (K), arginine (R), histidine (H)

[0060] In addition, when the original (unmodified) amino acid residue already has a charge, modifying it to an amino acid residue without a charge is also one of the preferred embodiments of the present invention. That is, the modifications in the present invention include (1) substitution of an amino acid with a charge to an amino acid without a charge, (2) substitution of an amino acid with a charge to an amino acid with the opposite charge to that amino acid, and (3) substitution of an amino acid without a charge to an amino acid with a charge.

[0061] In the present invention, it is preferable that the amino acid residues constituting the antibody are modified so that the isoelectric point (pI) of the antibody changes. Further, when there are a plurality of amino acid residues to be modified, a small number of amino acid residues without a charge may be included among the amino acid residues to be subjected to the modification.

[0062] Preferable examples of the "modification of the charge of amino acid residues" in the glypican 3 antibody provided by the present invention include the following. As modifications for increasing the pI value, for example, at least one substitution of Q43K, D52N, and Q107R can be made in the heavy chain variable region constituting the humanized glypican 3 antibody represented by SEQ ID NO: 195, and particularly preferably, it is modified to the amino acid sequence represented by SEQ ID NO: 198. Also, for example, at least one substitution of E17Q, Q27R, and Q105R can be made in the light chain variable region constituting the humanized glypican 3 antibody represented by SEQ ID NO: 201, and particularly preferably, it is modified to the amino acid sequence represented by SEQ ID NO: 204. On the other hand, as modifications for decreasing the pI value, at least one substitution of K19T, Q43E, K63S, K65Q, and G66D can be made in the heavy chain variable region constituting the humanized glypican 3 antibody represented by SEQ ID NO: 195 to be made, and particularly preferably, it is modified to the amino acid sequence represented by SEQ ID NO: 197. Also, for example, at least one substitution of Q27E, K79T, and R82S can be made in the light chain variable region constituting the humanized glypican 3 antibody represented by SEQ ID NO: 201, and particularly preferably, it is modified to the amino acid sequence represented by SEQ ID NO: 203.

[0063] Preferable examples of the "modification of the charge of amino acid residues" in the anti-human IL-6 receptor antibody (6R_a_H1L1) provided by the present invention include at least one amino acid substitution among the amino acid substitutions described in Table 20.

[0064] Preferable examples of the "modification of the charge of amino acid residues" in the anti-human IL-6 receptor antibody (6R_b_H1L1) provided by the present invention include at least one amino acid substitution among the amino acid substitutions described in Table 22.

[0065] Preferable examples of the "modification of the charge of amino acid residues" in the anti-human GPC3 antibody provided by the present invention include at least one amino acid substitution among the amino acid substitutions described in Table 24. ​​

[0066] "Modification of the charge of amino acid residues" in the anti-human IL-31 receptor antibody provided by the present invention Preferable examples thereof include at least one amino acid substitution among the amino acid substitutions described in Table 27.

[0067] The number of amino acid residues to be modified in the present invention is not particularly limited. For example, when modifying the variable region of an antibody, in order not to reduce the binding activity to an antigen and not to increase immunogenicity, it is preferable that the minimum number of amino acid residues necessary to achieve the desired controlled plasma pharmacokinetics be modified. In addition, modifications of amino acid residues that result in an increase in the binding activity to an antigen and modifications of amino acid residues that result in a decrease in immunogenicity can also be preferably implemented in appropriate combinations.

[0068] Known means can be used to measure the antigen-binding activity of an antibody. For example, ELISA( enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), or fluorescence immunoassay can be used. The above methods are described in the general textbook "Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988".

[0069] As a method for measuring the binding activity of an antibody to cells, for example, the methods described on pages 359-420 of Antibodies A Laboratory Manual. (Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988) can be mentioned. That is, BIACORE using cells as antigens, cell proliferation assay, ELISA, and FACS (fluorescence activated cell sorting) principle can be used for evaluation. In the ELISA format, the binding activity of the antibody to cells is the enzyme It is quantitatively evaluated by comparing the signal levels generated by the reaction. That is, the test antibody is added to an ELISA plate on which each forced expression cell is immobilized, and an enzyme-labeled antibody that recognizes the test antibody is used to detect the antibody bound to the cell. Alternatively, in FACS, a dilution series of the test antibody is prepared, and the antibody binding titer to each forced expression cell is determined so that the binding activity to the cell can be compared. The binding between an antigen expressed on the cell surface suspended in a buffer or the like, which is not bound to a carrier such as an ELISA plate, and an antibody against the antigen can be measured by the FACS format. Examples of flow cytometers used for such measurements include FACSCanto

[0070] II, FACSAria , FACSArray TM , FACSVantage TM SE, FACSCalibur TM TM TM (all of the above, BD Biosciences), EPICS ALTRA HyPerSort, Cytomics FC 500, EPICS XL-MCL ADC, EPICS XL ADC, Cell Lab Quanta / Cell Lab Quanta SC (all of the above, Beckman Coulter), etc. TM (all of the above, BD Biosciences) and EPICS ALTRA HyPerSort, Cytomics FC 500, EPICS XL-MCL ADC, EPICS XL ADC, Cell Lab Quanta / Cell Lab Quanta SC (all of the above, Beckman Coulter), etc.

[0071] As an example of a preferred method for measuring the binding activity of an antibody to an antigen, a cell expressing the antigen is reacted with the test antibody, the cell is stained with a FITC-labeled secondary antibody that recognizes the test antibody, and then measurement is performed using FACSCalibur (BD), and the fluorescence intensity is analyzed using CELL QUEST Software (BD). According to this method, the antigen on the cell surface expressing the antigen When measuring with FACSCalibur after staining with an FITC-labeled secondary antibody that specifically recognizes the test antibody conjugated thereto, the value of the Geometric Mean (test Geo-Mean value) obtained by a method of analyzing the fluorescence intensity using CELL QUEST Software can be determined by comparing it with the control Geo-Mean value obtained using a control antibody. The calculation formula for obtaining the Geo-Mean value (Geometric Mean) is described in the CELLQUEST Software User's Guide (BD biosciences).

[0072] In addition, in order not to increase the immunogenicity in the human body to which the antibody is administered, it is preferable that the modified amino acid sequence is a human sequence (a sequence found in natural antibodies derived from humans), but the present invention is not limited thereto. Furthermore, mutations can be preferably introduced at positions other than those where modifications are introduced to change the isoelectric point so that each of the plurality of FRs (FR1, FR2, FR3, FR4) becomes a human sequence after modification. A method of replacing each FR with a human sequence in this way has been reported in a non-patent document (Ono K, Ohtomo T, Yoshida K, Yoshimura Y, Kawai S, Koishihara Y, Ozaki S, Kosaka M, Tsuchiya M., The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity., Mol Immunol. (1999) 36(6), 387-395). Also, in order to change the isoelectric point of the antibody, it may be modified with other human FRs so that the charge of each FR changes (for example, FR3 may be exchanged with other human FRs so that the isoelectric point of the antibody decreases). Such a humanization method has been reported in a non-patent document (Dall'Acqua WF, Damschroder MM, Zhang J, Woods RM, Widjaja L, Yu J, Wu H.., Antibody humanization by framework shuffling., Methods. (2005) 36(1), 43-60) 。

[0073] In addition, when the desired controlled plasma pharmacokinetics are not achieved by slight modification of the surface charge, the desired antibody showing the desired controlled plasma pharmacokinetics can be preferably obtained by repeating the evaluation of the surface charge modification and plasma pharmacokinetics.

[0074] Non-patent literature (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, Klingbeil C, Co MS, Berg EL. Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin. J Immunol. (1998), 160(2), 1029-35) compared the plasma pharmacokinetics of the chimeric antibody (IgG4) chimeric EP5C7.g4 and the humanized antibody (IgG4) HuEP5C7.g4, which are anti-E, P-Selectin antibodies, in rhesus monkeys, and showed that the plasma pharmacokinetics of both were equivalent. In addition, the non-patent literature (Gobburu JV, Tenhoor C, Rogge MC, Frazier DE Jr, Thomas D, Benjamin C, Hess DM, Jusko WJ. Pharmacokinetics / dynamics of 5c8, a monoclonal antibody to CD154 (CD40 ligand) suppression of an immune response in monkeys. J Pharmacol Exp Ther. (1998) 286(2), 925-30) compared the plasma pharmacokinetics of the chimeric antibody ch5d8 and the humanized antibody Hu5c8, which are anti-CD154 antibodies, in cynomolgus monkeys, and showed that the plasma pharmacokinetics of both were equivalent. The non-patent literature (Kashmiri SV, Shu L, Padlan EA, Milenic DE, Schlom J, Hand PH., Generation, characterization, and in vivo studies of humanized anticarcinoma antibody CC49., Hybridoma. (1995) 14(5), 461-73) showed that the plasma pharmacokinetics of the chimeric antibody cCC49 and the humanized antibody HuCC49 in mice were equivalent.In addition, non-patent literature (Graves SS, Goshorn SC, Stone DM, Axworthy DB, Reno JM, Bottino B, Searle S, Henry A, Pedersen J, Rees AR, Libby RT., Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody., Clin Cancer Res. (1999) 5(4), 899-908) and non-patent literature (Couto JR, Blank EW, Peterson JA, Ceriani RL., Anti-BA46 monoclonal antibody Mc3: humanization using a novel positional consensus and in vivo and in vitro characterization., Cancer Res. (1995) 55(8), 1717-22) show that. , in the evaluation in mice, it has been shown that the plasma pharmacokinetics and distribution of mouse antibodies and humanized antibodies are equivalent. Since both mouse Fc and human Fc cross the mouse FcRn, it is considered that the plasma pharmacokinetics and distribution of the chimeric antibody and the humanized antibody are equivalent. This As shown in these examples, the plasma drug pharmacokinetics are equivalent between chimeric antibodies and humanized antibodies with the same CDRs. That is, when humanized by known methods such as those shown in non-patent literature (Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. (1997) 15(7), 637-40), the plasma pharmacokinetics are equivalent compared to chimeric antibodies, and it is not possible to produce a humanized antibody with controlled plasma pharmacokinetics by known methods.

[0075] In contrast, by using the method found in the present invention, when humanizing a chimeric antibody, modifications are made to the amino acid residues that can be exposed on the surface of the chimeric antibody, thereby modifying the pI of the antibody, and a humanized antibody with controlled pharmacokinetics in plasma (i.e., an extended plasma half-life or a shortened plasma half-life) compared to the chimeric antibody can be produced. Modifications to the amino acids that can be exposed on the surface of the humanized antibody for controlling pharmacokinetics in plasma may be carried out simultaneously with the humanization of the antibody, or the pI of the humanized antibody may be further modified by modifying the amino acid residues that can be exposed on its surface using the humanized antibody as a starting material. The isoelectric point value in the present invention can be measured by isoelectric focusing electrophoresis known to those skilled in the art. Also, the theoretical isoelectric point value can be calculated using gene and amino acid sequence analysis software (such as Genetyx). In the present invention, for example, it is useful when it is necessary to significantly change the isoelectric point for sufficient control of pharmacokinetics in plasma. In particular, it is preferably the case when it is necessary to change the isoelectric point value by 1.0 or more, more preferably 3.0 or more, based on the theoretical isoelectric point value.

[0076] Non-patent document (Adams CW, Allison DE, Flagella K, Presta L, Clarke J, Dybdal N, McKeever K, Sliwkowski MX. Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab., Cancer Immunol Immunother. (2006) 55(6), 717-27) describes 3 cases of humanization using the FR sequences of the same human antibody. The plasma pharmacokinetics of the humanized antibodies trastuzumab, bevacizumab, and pertuzumab of the same type are described to be almost equivalent. That is, when humanization is performed using the same FR sequence, the plasma pharmacokinetics are almost equivalent. According to the method found in the present invention, in addition to the humanization process as described above, by modifying the amino acid residues that can be exposed on the surface of the antibody to modify the pI of the antibody, it becomes possible to control the drug (in plasma) concentration.

[0077] Furthermore, the method of the present invention can also be applied to human antibodies. By modifying the amino acid residues that can be exposed on the surface of a human antibody prepared from a human antibody library or a human antibody-producing mouse, etc., and modifying the pI of the human antibody, a human antibody can be prepared whose plasma pharmacokinetics are controlled (i.e., its plasma half-life is extended, or its plasma pharmacokinetics are shortened) compared to the plasma pharmacokinetics of the initially prepared human antibody.

[0078] A decrease in the pI value of an antibody extends the plasma half-life of the antibody. Conversely, an increase in the pI value of an antibody shortens the plasma half-life and improves the tissue migration of the antibody (Vaisitti T, Deaglio S, Malavasi F., Cationization of monoclonal antibodies: another step towards the "magic bullet"?, J Biol Regul HomeostAgents. (2005) 19(3-4), 105-12, Pardridge WM, Buciak J, Yang J, Wu D. Enhanced endocytosis in cultured human breast carcinoma cells and in vivo biodistribution in rats of a humanized monoclonal antibody after cationization of the protein. (1998) 286(1), 548-54). However, the antibody has increased immunogenicity and also increased internalization activity into cells, so that the antibody requires internalization into cells to exert its activities such as ADCC and CDC activity. Further improvements have been required to apply the antibody to cancer treatment that exerts an effect through a mechanism of cytotoxic activity, which is inhibited by the cytotoxic activity. For antibodies that exert their effects on cancer therapy through mechanisms such as cytotoxic activity, the activity of internalization into cells being an inhibitor of their activity, it was not known whether an increase or decrease in pI value would enhance the tumor-suppressing effect. In the present invention, modified antibodies of humanized antibodies with reduced pI values ​​and modified antibodies of humanized antibodies with increased pI values ​​were produced, and the antitumor effects of both were compared to verify which modification would have a higher tumor-suppressing effect. As a result, it was surprisingly shown that the humanized antibody with reduced pI value exhibited a superior effect against liver cancer.

[0079] In the present invention, the "antibody" includes antibodies in which the amino acid sequence is further modified by substitution, deletion, addition and / or insertion of amino acid residues constituting the antibody, using as a starting material an antibody in which the charge of the amino acid residue has been modified as described above. Further, the "antibody" in the present invention also includes antibodies in which the charge of the amino acid residue constituting the antibody is further modified, using as a starting material an antibody in which the amino acid sequence has been modified by substitution, deletion, addition and / or insertion of amino acid residues, or by chimerization, humanization, etc.

[0080] As an example of the modification for the purpose of improving the characteristics of the antibody provided by the present invention, the modification aimed at enhancing the stability of the antibody (hereinafter referred to as the stability modification) is preferably mentioned. The antibody in an aqueous solution is equilibrated between two states, the native state and the inactive denatured state. As the stability of the native state is represented by the second law of thermodynamics (ΔG = ΔH - TΔS), it depends on the balance of the change in the Gibbs free energy ΔG of the system and its breakdown, the change in enthalpy ΔH (due to changes in hydrophobic interactions and hydrogen bonds in the polypeptide chain, etc.) and the change in entropy ΔS (due to changes in solvation and the degree of freedom of the three-dimensional structure). A positive value of ΔG means that the native state of the protein is more stable than the denatured state of the protein, and when ΔG shows a larger positive value, the stability of the native state of the protein further increases. In order for the protein to denature, it is necessary to break the forces contributing to this stabilization. For example, by exposing the protein solution to a high temperature, the degree of freedom of the three-dimensional structure increases, and the factors contributing to the stabilization of the protein are weakened, thereby causing thermal denaturation of the protein. In this case, the -TΔS term dominates the denaturation. The ΔH of the unfolding due to the thermal denaturation of the protein can be directly measured using DSC (differential scanning calorimetry) as specifically described in the examples described herein. The DSC curve in the process of thermal denaturation of the protein has an endotherm sandwiching a temperature specific to the test protein called the denaturation midpoint (Tm). A positive value of ΔG means that the native state of the protein is more stable than the denatured state of the protein, and when ΔG shows a larger positive value, the stability of the native state of the protein further increases. In order for the protein to denature, it is necessary to break the forces contributing to this stabilization. For example, by exposing the protein solution to a high temperature, the degree of freedom of the three-dimensional structure increases, and the factors contributing to the stabilization of the protein are weakened, thereby causing thermal denaturation of the protein. In this case, the -TΔS term dominates the denaturation. The ΔH of the unfolding due to the thermal denaturation of the protein can be directly measured using DSC (differential scanning calorimetry) as specifically described in the examples described herein. The DSC curve in the process of thermal denaturation of the protein has an endotherm sandwiching a temperature specific to the test protein called the denaturation midpoint (Tm). A positive value of ΔG means that the native state of the protein is more stable than the denatured state of the protein, and when ΔG shows a larger positive value, the stability of the native state of the protein further increases. In order for the protein to denature, it is necessary to break the forces contributing to this stabilization. For example, by exposing the protein solution to a high temperature, the degree of freedom of the three-dimensional structure increases, and the factors contributing to the stabilization of the protein are weakened, thereby causing thermal denaturation of the protein. In this case, the -TΔS term dominates the denaturation. The ΔH of the unfolding due to the thermal denaturation of the protein can be directly measured using DSC (differential scanning calorimetry) as specifically described in the examples described herein. The DSC curve in the process of thermal denaturation of the protein has an endotherm sandwiching a temperature specific to the test protein called the denaturation midpoint (Tm). A positive value of ΔG means that the native state of the protein is more stable than the denatured state of the protein, and when ΔG shows a larger positive value, the stability of the native state of the protein further increases. In order for the protein to denature, it is necessary to break the forces contributing to this stabilization. For example, by exposing the protein solution to a high temperature, the degree of freedom of the three-dimensional structure increases, and the factors contributing to the stabilization of the protein are weakened, thereby causing thermal denaturation of the protein. In this case, the -TΔS term dominates the denaturation. The ΔH of the unfolding due to the thermal denaturation of the protein can be directly measured using DSC (differential scanning calorimetry) as specifically described in the examples described herein. The DSC curve in the process of thermal denaturation of the protein has an endotherm sandwiching a temperature specific to the test protein called the denaturation midpoint (Tm). A positive value of ΔG means that the native state of the protein is more stable than the denatured state of the protein, and when ΔG shows a larger positive value, the stability of the native state of the protein further increases. In order for the protein to denature, it is necessary to break the forces contributing to this stabilization. For example, by exposing the protein solution to a high temperature, the degree of freedom of the three-dimensional structure increases, and the factors contributing to the stabilization of the protein are weakened, thereby causing thermal denaturation of the protein. In this case, the -TΔS term dominates the denaturation. The ΔH of the unfolding due to the thermal denaturation of the protein can be directly measured using DSC (differential scanning calorimetry) as specifically described in the examples described herein. The DSC curve in the process of thermal denaturation of the protein has an endotherm sandwiching a temperature specific to the test protein called the denaturation midpoint (Tm). A peak is formed. By integrating the peak, the denaturation enthalpy change can be obtained. Generally, the value of Tm is an indicator of thermal stability. When a protein undergoes thermal denaturation by DSC the heat capacity change (ΔCp) can also be measured. The heat capacity change accompanying thermal denaturation is mainly due to the hydration of amino acid residues that are not exposed on the molecular surface when the protein exists in the native state and are exposed to solvent molecules as the protein denatures.

[0081] As described above, the "modification" of amino acid residues in the method provided by the present invention specifically refers to substituting the original amino acid residue with another amino acid residue, deleting the original amino acid residue, adding a new amino acid residue, etc., but preferably refers to substituting the original amino acid residue with another amino acid residue. That is, for modifying the stability of the antibody in the present invention, preferably, modification by amino acid substitution is used. As a result of modifying the stability of the amino acid residues constituting the antibody, the Tm value of the antibody increases. That is, the Tm value is preferably used as an indicator of the modification of the stability of the antibody.

[0082] The glypican 3 antibody provided by the present invention, in order to perform the above "modification of stability", for example, 37, 40 in the H-chain variable region constituting the humanized glypican 3 antibody represented by SEQ ID NO: 195 , at least one amino acid residue selected from the 48th and 51st amino acid residues is preferably modified. Also, for example, at least one amino acid residue selected from the 2nd, 25th, 42nd, 48th, 50th, 83rd, and 84th amino acid residues in the L-chain variable region constituting the humanized glypican 3 antibody represented by SEQ ID NO: 201 one amino acid residue is preferably modified. Among the above amino acid residues, amino acid residues other than the amino acid residues subjected to the modification of stability do not need to be modified as long as the desired Tm value is obtained, but can be appropriately modified so as to have a Tm value equal to or higher than that of the humanized glypican 3 antibody used for the modification.

[0083] The modification of stability can be carried out by randomly modifying each amino acid residue constituting the humanized antibody to be modified. Also, a part of the amino acid sequence constituting the humanized antibody to be modified can be replaced with an amino acid sequence constituting an existing antibody having a high Tm value and corresponding in terms of the three-dimensional structure correlation of the antibody with a part of the amino acid sequence of the humanized antibody to be modified. The position of the amino acid residue to be replaced is not limited, but the amino acid residues in the FR region can be preferably modified. Also, the amino acid residues in the CDR region can be appropriately modified as long as the reduction in the binding activity to the antigen is not accompanied. Also, the number of amino acid residues to be modified is not particularly limited, and it can also be carried out by replacing a specific segment of the FR region with a desired segment. The segment can be all of the segments of FR1, FR2, FR3, and FR4 in the FR region, or can also be carried out by a combination of modifications of one or more of each segment. Even the amino acid residues in the CDR region can be appropriately modified as long as the reduction in the binding activity to the antigen is not accompanied. Also, the number of amino acid residues to be modified is not particularly limited, and it can also be carried out by replacing a specific segment of the FR region with a desired segment.

[0084] When modifying the segment of the FR region, the FR2 region of the H chain or L chain can be cited as a preferred example. For example, the modification of the amino acid residues that modify the FR2 of the H chain of the humanized glypican 3 antibody having the VH1b subclass represented by SEQ ID NO: 195 to the VH4 subclass, that is, V37I which replaces the 37th valine with isoleucine, similarly A40P, M48I, L51I modifications are cited as preferred specific examples. Also, for example, the modification of the L chain FR2 region of the humanized glypican 3 antibody having the VK2 subclass represented by SEQ ID NO: 201 to the VK3 subclass, that is, L42Q, S48A, Q50R modifications, and further the V2I modification corresponding to the modification to the germline sequence of FR1 are cited as preferred specific examples.

[0085] Substitutions, deletions, additions and / or insertions of amino acid residues constituting the antibody, as well as modifications of the amino acid sequence such as humanization and chimerization, can all be suitably performed by methods known to those skilled in the art. Similarly, when producing the antibody provided by the present invention as a recombinant antibody, substitutions, deletions, additions and / or insertions of amino acid residues constituting the variable region and constant region of the antibody can be suitably performed.

[0086] The antibodies in the present invention can be suitably used with antibodies derived from any animal, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, etc. Furthermore, modified antibodies with substituted amino acid sequences, such as chimeric antibodies, especially humanized antibodies, can also be suitably used. In addition, antibody modifications conjugated with various molecules can also be suitably used.

[0087] A "chimeric antibody" is an antibody produced by combining sequences from different animals. For example, an antibody composed of the variable (V) regions of the H chain and L chain of a mouse antibody and the constant (C) regions of the H chain and L chain of a human antibody can be suitably exemplified. Methods for producing chimeric antibodies are known. For example, a recombinant DNA in which DNA encoding the antibody V region and DNA encoding the human antibody C region are fused in-frame is incorporated into a commonly used expression vector. The chimeric antibody can be appropriately obtained or isolated from the culture solution by culturing the host cell into which the vector has been introduced.

[0088] A humanized antibody, also referred to as a reshaped human antibody, is an antibody in which the complementary determining regions (CDRs) of an antibody isolated from a non-human mammal, such as a mouse, etc., are linked to the framework regions (FRs) of a human antibody is. The DNA sequence encoding the humanized antibody can be synthesized by an overlap PCR reaction using a plurality of oligonucleotides as templates. The materials for the overlap PCR reaction, and The implementation method is described in WO98 / 13388 and the like. The DNA encoding the variable region of the humanized antibody of the present invention was prepared to have a plurality of oligonucleotides obtained by overlap PCR, which is ligated to the DNA encoding the human antibody constant region so that a codon sequence is formed in-frame. As described above The ligated DNA is then inserted into an expression vector so that the DNA is expressed and introduced into a host. The method for identifying CDRs is known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342, 877). Also, the general genetic recombination techniques are known (see European Patent Application Publication No. EP 125023, WO 96 / 02576). By using these known

[0089] methods, for example, after the CDRs of an antibody obtained from a non-human animal such as a mouse antibody are determined, DNA encoding a recombinant antibody in which the CDR is linked to the FR of a human antibody is constructed. The FR of the human antibody linked via the CDR is selected so that the CDR forms a good antigen-binding site. If necessary, the amino acid residues of the FR in the variable region of the antibody can be appropriately modified so that the CDR of the reconstructed human antibody forms an appropriate antigen-binding site (Sato et al., Cancer Res. (1993) 53, 851-6). The amino acid residues in the FR to be modified are residues that bind directly to the antigen by non-covalent bonds (Amit et al., Science (1986) 233, 747-53) These include residues that affect or act on the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196, 901-17) and residues involved in VH-VL interactions (EP239400 Patent Publication).

[0090] Transformation or transduction with a commonly used expression vector into which the DNA is inserted The humanized antibody encoded by the DNA produced by the host cell is then cultured. The cells are then isolated from the culture medium.

[0091] When the antibody provided by the present invention is an antibody, a humanized antibody, or a human antibody, the C region of the antibody is preferably a C region derived from a human antibody. For example, Cγ1, Cγ2, Cγ3, and Cγ4 can be preferably used as the H chain C region, and Cκ and Cλ can be preferably used as the L chain C region. Furthermore, the human antibody C region can be appropriately modified to improve the stability of the antibody or its production. The chimeric antibody provided by the present invention is a chimeric antibody that is a mixture of the V region of an antibody obtained from a mammal other than a human and a human V region of an antibody obtained from a mammal other than a human. On the other hand, humanized antibodies are preferably composed of the C region of a mammal other than a human. It is preferably composed of the CDR of an antibody obtained from a mammal and the FR and C region of a human antibody. Moreover, a human antibody is preferably composed of the CDR of an antibody obtained from a human and the FR and C region of a human antibody. The C region of a human antibody is composed of a unique amino acid sequence corresponding to an isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. As the C region of a humanized antibody provided by the present invention, the C region of an antibody belonging to any isotype is preferably used. Preferably, the C region of human IgG is used, but is not limited thereto. Moreover, the FR of a human antibody used as the FR of a humanized antibody or human antibody is not particularly limited, and the FR of an antibody belonging to any isotype is preferably used.

[0092] For the purpose of reducing immunogenicity, a method similar to the method described in the non-patent literature (Ono K, Ohtomo T, Yoshida K, Yoshimura Y, Kawai S, Koishihara Y, Ozaki S, Kosaka M, Tsuchiya M., The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity., Mol Immunol. (1999) 36(6), 387-395) can also be applied to replace all or part of the amino acid residues constituting the FR region with the germline sequence. Based on the reasonable prediction that the germline sequence would have low immunogenicity, the amino acid sequence constituting the FR region of the humanized antibody is compared by aligning it with the amino acid sequence of the germline (Abhinandan K. R. and Martin C. R., J. Mol. Biol., (2007) 369, 852-862). As long as the binding activity to the antigen is not lost in the range, the amino acid residues constituting the FR region of different humanized antibodies in this comparison can be replaced with the amino acid residues in the germline sequence. Specific examples include modifications such as replacing L at position 70 with I, T at position 87 with R, and T at position 97 with A among the amino acid residues constituting the H-chain variable region represented by SEQ ID NO: 195. Also, modifications such as replacing S at position 25 with A among the amino acid residues constituting the L-chain variable region represented by SEQ ID NO: 201 are included.

[0093] As long as the variable region and constant region of the modified chimeric antibody, humanized antibody, and human antibody provided by the present invention exhibit binding specificity to the antigen, deletion, substitution, insertion, and / or addition of one or more amino acids constituting the variable region and constant region of the antibody subjected to the modification are suitable to be carried out.

[0094] Chimeric antibodies, humanized antibodies, and human antibodies using human-derived sequences are considered useful when used as therapeutic antibodies administered to humans for therapeutic purposes, etc., because their immunogenicity in the human body is reduced.

[0095] As the sequence of the gene encoding the H chain or L chain of the antibody before mutagenesis in the method of the present invention, known sequences can be used, and in addition, novel sequences of antibody genes can be obtained by methods known to those skilled in the art. The gene can be suitably obtained, for example, from an antibody library. Furthermore, the gene can also be obtained by cloning using a known technique such as RT-PCR using the mRNA of a hybridoma producing a monoclonal antibody as a template.

[0096] Regarding antibody libraries, many antibody libraries are already known. Also, since methods for preparing antibody libraries are also known, those skilled in the art can appropriately obtain or prepare antibody libraries. For example, as suitable antibody libraries, antibody phage libraries disclosed by documents such as Clackson et al., Nature (1991) 352, 624-8, Marks et al., J. Mol. Biol. (1991) 222, 581-97, Waterhouse et al., Nucleic Acids Res. (1993) 21, 2265-6, Griffiths et al., EMBO J. (1994) 13, 3245-60, Vaughan et al., Nature Biotechnology (1996) 14, 309-14, and Japanese Patent Publication No. 20-504970 are exemplified. In addition, known methods such as a method for preparing a library in eukaryotic cells (WO95 / 15393 pamphlet) and ribosome display method are preferably used. Furthermore, techniques for obtaining human antibodies by panning using a human antibody library as a starting material are known to those skilled in the art. That is, a single-chain antibody (scFv) in which the variable regions of the H chain and L chain of a human antibody are fused in-frame is expressed on the surface of phage by the phage display method. By selecting the phage that binds to the antigen, the gene encoding the scFv that binds to the antigen is isolated from the phage. By identifying the sequence of the gene, the sequences of the DNAs encoding the variable regions of the H chain and L chain of the antibody that binds to the antigen can be determined. The antibody gene having the said sequence is appropriately inserted into an expression vector and expressed in an appropriate host cell as described below, whereby human antibodies are appropriately obtained. These methods are already well-known, and the methods disclosed in WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388 are exemplified.

[0097] As a method for obtaining a gene encoding an antibody from a hybridoma producing a monoclonal antibody, basically, known techniques can be employed. Although described in detail below, briefly, after an animal is immunized with a desired sensitizing antigen according to a normal immunization method, immune cells obtained from the animal are subjected to cell fusion with known parent cells by a normal cell fusion method. According to a normal screening method, monoclonal antibody-producing cells (hybridomas) are screened, and cDNA of the variable region (V region) of the antibody is synthesized by reverse transcriptase using mRNA obtained from the selected hybridoma as a template. By fusing the cDNA in-frame with DNA encoding the desired antibody constant region (C region), the antibody gene is preferably appropriately obtained.

[0098] More specifically, the following examples are preferably cited, but the present invention is not limited to these examples. The sensitizing antigen used to obtain the antibody provided by the present invention may be a complete antigen having immunogenicity or an incomplete antigen containing a hapten or the like that does not exhibit immunogenicity. For example, a full-length protein, or a partial polypeptide or peptide thereof, etc. can be preferably used. The soluble GPC3 core polypeptide represented by SEQ ID NO: 207 is cited as a preferred specific example thereof. In addition, substances composed of polysaccharides, nucleic acids, lipids, etc. are also known to act as antigens, and the antigen to which the antibody of the present invention binds is not particularly limited to the above-described substance forms. The preparation of the antigen is preferably carried out by a method known to those skilled in the art. For example, a method using baculovirus (for example, WO98 / 46777, etc.) can be preferably used. When the immunogenicity of the antigen is low, the animal can be preferably immunized with the antigen bound to a macromolecule having immunogenicity such as albumin. Further, when the sensitizing antigen is a molecule that penetrates the cell membrane, if necessary, a polypeptide fragment of the extracellular region of the molecule is preferably used as the sensitizing antigen. Alternatively, a cell expressing the molecule on the cell surface can be preferably used as the sensitizing antigen. Furthermore, when the sensitizing antigen is an insoluble molecule, the molecule is solubilized by binding it to another water-soluble molecule, and the solubilized binding molecule is preferably used as the sensitizing antigen.

[0099] Antibody-producing cells can be preferably obtained by immunizing an animal with the appropriate sensitizing antigen described above. Alternatively, antibody-producing cells can be obtained by immunizing lymphocytes capable of producing antibodies in vitro. As the animal to be immunized, various vertebrates and mammals can be used. In particular, animals of the order Rodentia, Lagomorpha, and Primates are generally used as the animals to be immunized. Examples include animals of the order Rodentia such as mice, rats, and hamsters, animals of the order Lagomorpha such as rabbits, and animals of the order Primates such as cynomolgus monkeys, rhesus monkeys, baboons, and chimpanzees. In addition, transgenic animals that retain the repertoire of human antibody genes on their genomes are also known, and by using such animals, human antibodies can be preferably obtained (see WO96 / 34096; Mendez et al., Nat. Genet. (1997) 15, 146-56). Instead of using such transgenic animals, for example, after human lymphocytes are sensitized in vitro with a desired antigen or cells expressing a desired antigen, they are cell-fused with human myeloma cells such as U266, and a desired human antibody having binding activity to the antigen can be preferably obtained (see Japanese Patent Publication No. 1-59878). Also, all repertoires of human antibody genes are retained on their genomes, and by immunizing transgenic animals with a desired antigen (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO96 / 34096, WO96 / 33735), the desired human antibody can be preferably obtained.

[0100] Immunization of the animal is carried out, for example, by appropriately diluting and suspending the sensitizing antigen with Phosphate-Buffered Saline (PBS) or physiological saline, emulsifying it by mixing with an adjuvant if necessary, and then injecting the sensitizing antigen into the abdominal cavity or subcutaneously of the animal. Thereafter, preferably, the sensitizing antigen mixed with Freund's incomplete adjuvant is injected several times every 4 to 21 days It is administered. Confirmation of the production of antibodies against the sensitizing antigen in the immunized animal is carried out by measuring the antibody titer in the serum of the animal against the sensitizing antigen by a conventional method, for example, a known analytical method such as enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS), etc. It can be carried out.

[0101] Hybridomas can be prepared by fusing antibody-producing cells obtained from an animal immunized with the desired sensitizing antigen or lymphocytes with myeloma cells using a fusion agent (for example, polyethylene glycol) commonly used for cell fusion (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 59-103). The preparation of hybridomas can be suitably carried out, for example, according to the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. (1981) 73, 3-46), etc. By culturing and growing the hybridoma cells prepared by the above method, monoclonal antibodies that specifically bind to the antigen protein produced by the hybridoma are obtained. The binding specificity of the monoclonal antibody to the antigen protein can be appropriately measured by known analytical methods such as immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS), etc. Then, if necessary, hybridomas that produce antibodies with the desired specificity, binding activity, or activity measured are preferably subcloned by a technique such as the limiting dilution method, and the monoclonal antibodies produced by the hybridomas can be isolated. nzymol. (1981) 73, 3-46), etc. nzymol. (1981) 73, 3-46), etc. can be suitably carried out. By culturing and growing the hybridoma cells prepared by the above method, monoclonal antibodies that specifically bind to the antigen protein produced by the hybridoma are obtained. The binding specificity of the monoclonal antibody to the antigen protein can be appropriately measured by known analytical methods such as immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS), etc. Then, if necessary, hybridomas that produce antibodies with the desired specificity, binding activity, or activity measured are preferably subcloned by a technique such as the limiting dilution method, and the monoclonal antibodies produced by the hybridomas can be isolated.

[0102] Subsequently, the gene encoding the selected antibody can be cloned from the above-described hybridoma or antibody-producing cells (sensitized lymphocytes, etc.) using a probe that can specifically bind to the gene (for example, an oligonucleotide complementary to the sequence encoding the antibody constant region, etc.). It can also be cloned by the RT-PCR method using mRNA obtained from hybridomas or antibody-producing cells (sensitized lymphocytes, etc.) as a template. Immunoglobulins are classified into five different classes, IgA, IgD, IgE, IgG, and IgM, based on differences in their structure and function. Furthermore, each class is classified into several isotypes (for example, IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2, etc.). The antibodies provided by the present invention may be derived from antibodies belonging to any of these classes and subclasses, and are not particularly limited to any class and subclass, but antibodies belonging to the IgG class are particularly preferred. can be mentioned as.

[0103] The genes encoding the amino acid sequences constituting the H chain and L chain of the antibody can be appropriately modified by genetic engineering techniques. For example, by modifying the nucleic acid residues encoding the amino acid sequences constituting antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies, camel antibodies, etc., artificial modifications are made for the purpose of reducing heterologous antigenicity against humans, etc. Recombinant antibodies, such as chimeric antibodies and humanized antibodies, can be appropriately produced. A chimeric antibody is an antibody composed of the variable regions of the H chain and L chain of an antibody derived from a non-human mammal, for example, a mouse, and the constant regions of the H chain and L chain of a human antibody. For example, DNA encoding the variable region of an antibody derived from a mouse is ligated to DNA encoding the constant region of a human antibody, and after introducing a recombinant vector incorporating this into a host, it can be obtained by expression. A humanized antibody, also referred to as a reshaped human antibody, is formed by the codon sequences of the complementary determining regions (CDRs) of an antibody isolated from a non-human mammal, for example, a mouse, and the framework region of a human antibody being in-frame. an antibody linked as described below. The DNA sequence encoding this humanized antibody can be synthesized by an overlap PCR reaction using a plurality of oligonucleotides as templates. Materials for the overlap PCR reaction and methods for performing the same are described in WO98 / 13388 and the like.

[0104] The DNA encoding the variable region of the recombinant antibody of the present invention is obtained by overlap PCR from a plurality of oligonucleotides prepared to have nucleotide sequences that overlap with each other, and this is ligated so that a codon sequence is formed in-frame with the DNA encoding the human antibody constant region. The DNA ligated as described above is then inserted into an expression vector so that the DNA is expressed and introduced into a host. The antibody encoded by the DNA is expressed by culturing the host. The expressed antibody is obtained by appropriately purifying it from the culture solution of the host etc. (see EP239400; WO96 / 02576). The FRs of the humanized antibody linked via the CDRs are selected such that the complementarity-determining regions form a good antigen-binding site for the antigen. If necessary, the amino acid residues constituting the FRs of the variable region of the antibody selected such that the complementarity-determining regions of the recombinant human antibody form an appropriate antigen-binding site for the antigen can be appropriately substituted (K. Sato et al., Cancer Res. (1993) 53, 851-856).

[0105] In addition to the modifications related to the above-mentioned humanization, for example, modifications for improving biological properties of the antibody such as the binding activity to the antigen recognized by the antibody can be carried out. The modifications in the present invention can be preferably performed by methods such as site-directed mutagenesis (see, for example, Kunkel, Proc. Natl. Acad. Sci. USA (1985) 82, 488), PCR mutagenesis, cassette mutagenesis, etc. Generally, the amino acid sequence constituting the modified antibody with improved biological properties has an identity and / or similarity of 70% or more, more preferably 80% or more, still more preferably 90% or more (for example, 95% or more, 97%, 98%, 99%, etc.) with respect to the amino acid sequence constituting the antibody to be modified (that is, the antibody that is the basis of the modified antibody). In the present specification, sequence identity and / or similarity means the ratio of amino acid residues that are identical (the same residue) or similar (amino acid residues classified into the same group based on the properties of the side chains of common amino acids) to the amino acid residues constituting the antibody that is the basis of the modified antibody after aligning the sequences and introducing gaps as necessary so that the sequence identity takes the maximum value. Usually, natural amino acid residues are classified into groups based on the nature of their side chains: (1) hydrophobic: alanine, isoleucine, valine, methionine, and leucine; (2) neutral hydrophilic: asparagine, glutamine, cysteine, threonine, and serine; (3) acidic: aspartic acid and glutamic acid; (4) basic: arginine, histidine, and lysine; (5) residues affecting the orientation of the chain: glycine and proline; and (6) aromatic: tyrosine, tryptophan, and phenylalanine. with respect to the amino acid sequence constituting the antibody to be modified (that is, the antibody that is the basis of the modified antibody). In the present specification, sequence identity and / or similarity means the ratio of amino acid residues that are identical (the same residue) or similar (amino acid residues classified into the same group based on the properties of the side chains of common amino acids) to the amino acid residues constituting the antibody that is the basis of the modified antibody after aligning the sequences and introducing gaps as necessary so that the sequence identity takes the maximum value. Usually, natural amino acid residues are classified into groups based on the nature of their side chains: (1) hydrophobic: alanine, isoleucine, valine, methionine, and leucine; (2) neutral hydrophilic: asparagine, glutamine, cysteine, threonine, and serine; (3) acidic: aspartic acid and glutamic acid; (4) basic: arginine, histidine, and lysine; (5) residues affecting the orientation of the chain: glycine and proline; and (6) aromatic: tyrosine, tryptophan, and phenylalanine. identity is maximized by aligning the sequences and introducing gaps as necessary, and then the ratio of amino acid residues that are identical (the same residue) or similar (amino acid residues classified into the same group based on the properties of the side chains of common amino acids) to the amino acid residues constituting the antibody that is the basis of the modified antibody. Usually, natural amino acid residues are classified into groups based on the nature of their side chains: (1) hydrophobic: alanine, isoleucine, valine, methionine, and leucine; (2) neutral hydrophilic: asparagine, glutamine, cysteine, threonine, and serine; (3) acidic: aspartic acid and glutamic acid; (4) basic: arginine, histidine, and lysine; (5) residues affecting the orientation of the chain: glycine and proline; and (6) aromatic: tyrosine, tryptophan, and phenylalanine. (1) hydrophobic: alanine, isoleucine, valine, methionine, and leucine; (2) neutral hydrophilic: asparagine, glutamine, cysteine, threonine, and serine; (3) acidic: aspartic acid and glutamic acid; (4) basic: arginine, histidine, and lysine; (5) residues affecting the orientation of the chain: glycine and proline; and (6) aromatic: tyrosine, tryptophan, and phenylalanine. (1) hydrophobic: alanine, isoleucine, valine, methionine, and leucine; (2) neutral hydrophilic: asparagine, glutamine, cysteine, threonine, and serine; (3) acidic: aspartic acid and glutamic acid; (4) basic: arginine, histidine, and lysine; (5) residues affecting the orientation of the chain: glycine and proline; and (6) aromatic: tyrosine, tryptophan, and phenylalanine. acid and glutamic acid; (4) basic: arginine, histidine, and lysine; (5) residues affecting the orientation of the chain: glycine and proline; and (6) aromatic: tyrosine, tryptophan, and phenylalanine. (4) basic: arginine, histidine, and lysine; (5) residues affecting the orientation of the chain: glycine and proline; and (6) aromatic: tyrosine, tryptophan, and phenylalanine.

[0106] In addition, as a modification aimed at enhancing the function of an antibody, for example, improvement of the cytotoxic activity exhibited by an antibody such as a humanized antibody is also preferably cited as a specific embodiment. Examples of cytotoxic activity include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, etc. In the present invention, CDC activity refers to cytotoxic activity by the complement system. On the other hand, ADCC activity refers to the activity in which when a specific antibody adheres to the cell surface antigen of a target cell, an Fcγ receptor-bearing cell (immune cell, etc.) binds to the Fc portion thereof via the Fcγ receptor and damages the target cell. Whether the test antibody has ADCC activity or has CDC activity can be measured by known methods (for example, Current protocols in Immunology, Chapter7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc.,(1993), etc.).

[0107] Specifically, first, preparation of effector cells, complement solution, and target cells is carried out. (1) Preparation of effector cells The spleen is removed from CBA / N mice or the like, and spleen cells are separated in RPMI1640 medium (Invitrogen). After washing with the same medium containing 10% fetal bovine serum (FBS, HyClone), the effector cells can be prepared by adjusting the cell concentration to 5x10 6 cells / ml. (2) Preparation of complement solution Baby Rabbit Complement (CEDARLANE) is diluted 10-fold with a 10% FBS-containing medium (Invitrogen), and the complement solution can be prepared. (3) Preparation of target cells (3) Preparation of target cells Cells expressing the antigen protein to which the test antibody binds can be radiolabeled by culturing them with 0.2 mCi of sodium 51Cr chromate (GE Healthcare Biosciences) in DMEM medium containing 10% FBS at 37°C for 1 hour. As cells expressing the antigen protein to which the test antibody binds, cells transformed with the gene encoding the antigen protein to which the test antibody binds, ovarian cancer, prostate cancer, breast cancer, uterine cancer, liver cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, and colorectal cancer cells, etc. can be used. After radiolabeling, the cells are washed three times with RPMI1640 medium containing 10% FBS, and the cell concentration is adjusted to 2x10 5 cells / ml, whereby the target cells can be prepared.

[0108] The ADCC activity, or CDC activity can be measured by the method described below. For the measurement of ADCC activity, in a 96-well U-bottom plate (Becton Dickinson), 50 μl each of the target cells and the test antibody are added, and a reaction is carried out on ice for 15 minutes. Then, 100 μl of effector cells are added, and the reaction mixture is incubated in a carbon dioxide incubator for 4 hours. The final concentration of the test antibody can be appropriately used within the range of 0 to 10 μg / ml. After the incubation, 100 μl of the supernatant is collected, and the radioactivity of the supernatant is measured with a gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, Packard Instrument Company). The cytotoxic activity (%) can be calculated based on the formula (A-C) / (B-C) x 100 using the value of the radioactivity obtained. A is the radioactivity (cpm) when using the sample of each test antibody, B is the radioactivity (cpm) when using the sample added with 1% NP-40 (nacalai tesque), and C is the radioactivity (cpm) when using the sample containing only the target cells. After the incubation, 100 μl of the supernatant is collected, and the radioactivity of the supernatant is measured with a gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, Packard Instrument Company). The cytotoxic activity (%) can be calculated based on the formula (A-C) / (B-C) x 100 using the value of the radioactivity obtained. A is the radioactivity (cpm) when using the sample of each test antibody, B is the radioactivity (cpm) when using the sample added with 1% NP-40 (nacalai tesque), and C is the radioactivity (cpm) when using the sample containing only the target cells.

[0109] ​​On the other hand, in the case of measuring CDC activity, in a 96-well flat-bottom plate (Becton Dickinson), target cells and the test antibody are each added in an amount of 50 μl, and a reaction is carried out on ice for 15 minutes. Then, 100 μl of the complement solution is added to the reaction mixture, and the mixture is incubated in a carbon dioxide incubator for 4 hours. The final concentration of the test antibody can be appropriately used within the range of 0 to 3 μg / ml. After culturing, 100 μl of the supernatant is collected, and the radioactivity of the supernatant is measured with a gamma counter. The cytotoxic activity can be calculated in the same manner as the measurement of ADCC activity.

[0110] On the other hand, in the case of measuring cytotoxic activity with an antibody conjugate, in a 96-well flat-bottom plate (Becton Dickinson), target cells and the test antibody conjugate are each added in an amount of 50 μl, and a reaction is carried out on ice for 15 minutes. The plate is incubated in a carbon dioxide incubator for 1 to 4 hours. The final concentration of the antibody can be appropriately used within the range of 0 to 3 μg / ml. After culturing, 100 μl of the supernatant is collected, and the radio activity of the supernatant is measured. The cytotoxic activity can be calculated in the same manner as the measurement of ADCC activity.

[0111] The variable regions of the H chain and L chain of the antibody are usually composed of three CDRs and four FRs as described above. In a preferred embodiment of the present invention, the amino acid residues to be "modified" can be appropriately selected from among the amino acid residues constituting the CDR or FR, for example. Also, those skilled in the art can suitably obtain the amino acid sequence constituting the FR of the variable region of the antibody, which is a sequence existing in organisms such as humans or mice, by using public databases such as Kabat.

[0112]

[0113] ​​In a preferred embodiment of the present invention, a humanized antibody with controlled plasma pharmacokinetics is provided by the method of the present invention. The humanized antibody is, for example, a humanized antibody comprising a complementarity-determining region (CDR) derived from an animal other than human, a framework region (FR) derived from human, and a human C region, wherein at least one amino acid residue that can be exposed on the antibody surface in the CDR or FR has a charge different from that of the amino acid residue at the corresponding position in the CDR or FR of the original antibody, and it is a humanized antibody with controlled plasma pharmacokinetics compared to a chimeric antibody having the same C region. and is an amino acid residue having a charge different from that of the amino acid residue at the corresponding position in the CDR or FR of the original antibody. It is a humanized antibody with controlled plasma pharmacokinetics compared to a chimeric antibody having the same C region.

[0114] Furthermore, in a preferred embodiment of the present invention, a human antibody with controlled plasma pharmacokinetics is provided by the method of the present invention. The human antibody is, for example, a human antibody comprising a complementarity-determining region (CDR ) derived from human, a framework region (FR) derived from human, and a human C region, wherein at least one amino acid residue that can be exposed on the antibody surface in the CDR or FR has a charge different from that of the amino acid residue at the corresponding position in the CDR or FR of the original antibody, and it is a human antibody with controlled plasma pharmacokinetics compared to a chimeric antibody having the same C region.

[0115] The above-mentioned human constant region preferably refers to a region containing a wild-type human Fc region, but a modified Fc can also be suitably used. The "modified Fc" may include those in which the amino acid residues constituting the Fc are modified, and those in which the modifications applied to the Fc portion are modified. Modifying the mode of sugar chain modification added to the Fc portion is preferably cited as a specific example of the modification of the modification. In this specification, the "antibody with reduced fucose content bound to the Fc region of the antibody" specifically disclosed as a reference example is cited as such a preferred specific example.

[0116] ​​"An antibody with a reduced fucose content bound to the Fc region of the antibody" refers to an antibody that has a significantly lower amount of bound fucose, preferably undetectable, when compared to a control antibody. Usually, fucose is added to the N-glycosidically linked sugar chains bound to two sugar chain binding sites present in the Fc regions of the two H chains that make up one molecule of the antibody. In the present invention, "an antibody with a reduced fucose content bound to the Fc region of the antibody" refers to an antibody that has a fucose content of 50% or less, preferably 25% or less, more preferably 10% or less, and particularly preferably 0% or less of the total sugar chain content of the control antibody when compared with such a normal antibody as a control. The fucose content can be measured using the analysis method specifically shown as a reference example below. The method for producing an antibody with a reduced fucose content, in addition to being described as a reference example of the present invention, for example, a method of producing by animal cells lacking fucosyltransferase (Biotechnol Bioeng. (2004), 87(5), 614-22), a method of producing by animal cells with modified complex branched sugar chain modification (Biotechnol Bioeng. (2006) 93(5), 851-61), etc. can be preferably exemplified. Further, as a method of producing using cells other than animal cells as host cells, a method of producing by plant cells (Nature Biotechnology (2006) 24, 1591-7) or a method of producing by yeast cells (Nature Biotechnology (2006) 24, 210-5), etc. can also be preferably mentioned. Fucose is added to the N-glycosidically linked sugar chains bound to . In the present invention, "an antibody with a reduced fucose content bound to the Fc region of the antibody" refers to an antibody that has a fucose content of 50% or less, preferably 25% or less, more preferably 10% or less, and particularly preferably 0% or less of the total sugar chain content of the control antibody when compared with such a normal antibody as a control. The fucose content can be measured using the analysis method specifically shown as a reference example below. The method for producing an antibody with a reduced fucose content, in addition to being described as a reference example of the present invention, for example, a method of producing by animal cells lacking fucosyltransferase (Biotechnol Bioeng. (2004), 87(5), 614-22), a method of producing by animal cells with modified complex branched sugar chain modification (Biotechnol Bioeng. (2006) 93(5), 851-61), etc. can be preferably exemplified. Further, as a method of producing using cells other than animal cells as host cells, a method of producing by plant cells (Nature Biotechnology (2006) 24, 1591-7) or a method of producing by yeast cells (Nature Biotechnology (2006) 24, 210-5), etc. can also be preferably mentioned. as examples. Further, as a method of producing using cells other than animal cells as host cells, a method of producing by plant cells (Nature Biotechnology (2006) 24, 1591-7) or a method of producing by yeast cells (Nature Biotechnology (2006) 24, 210-5), etc. can also be preferably mentioned. can be preferably mentioned.

[0117] A preferred embodiment of the production method of the present invention is a method for producing a polypeptide containing an antibody variable region with a modified isoelectric point, comprising (a) modifying the nucleic acid encoding the polypeptide containing the amino acid residue so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide is converted, and (b) culturing the host cell so that the nucleic acid is expressed, (c) A method comprising recovering a polypeptide comprising an antibody variable region from a host cell culture. Also, as a preferred embodiment of the production method of the present invention, there is provided a method for producing a polypeptide comprising an antibody variable region with controlled pharmacokinetics in plasma, comprising: (a) Modifying a nucleic acid encoding a polypeptide containing at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide so that the charge of the amino acid residue is converted; (b) Culturing a host cell so that the nucleic acid is expressed; (c) A method comprising recovering a polypeptide comprising an antibody variable region from a host cell culture. (c) A method comprising recovering a polypeptide comprising an antibody variable region from a host cell culture. Furthermore, the present invention also includes a polypeptide comprising a variable region of an antibody with controlled pharmacokinetics in plasma produced by the above method.

[0118] The present invention also provides a method for producing a multispecific polypeptide comprising a first polypeptide and a second polypeptide having antibody variable regions. Furthermore, the present invention provides a multispecific polypeptide produced by the method. As a preferred embodiment of the production method of the present invention, there is provided a method comprising modifying either or both of a nucleic acid encoding an amino acid residue of a first polypeptide and a nucleic acid encoding an amino acid residue of a second polypeptide so that the difference in isoelectric points between the first polypeptide and the second polypeptide is increased. That is, by changing the charges of the amino acid residues of the first polypeptide and the second polypeptide, the difference in isoelectric point (pI) of the polypeptide is increased, and a multispecific antibody can be produced using the difference in isoelectric point. Specifically, it is a production method including the following steps (a) to (c). (a) At least modifying a nucleic acid encoding a polypeptide containing the amino acid residue so that the charge of one amino acid residue is modified, wherein the modification of the nucleic acid increases the difference in isoelectric point between the first polypeptide and the second polypeptide compared to before the modification, by modifying both or either one of the nucleic acid encoding the amino acid residue of the first polypeptide and the nucleic acid encoding the amino acid residue of the second polypeptide, (b) culturing a host cell so that the nucleic acid is expressed, (c) recovering the multispecific antibody from the host cell culture

[0119] The polypeptide in the present invention generally refers to a peptide having a length of about 10 amino acids or more and a protein. It is usually a polypeptide derived from an organism, but is not particularly limited. For example, it may be a polypeptide consisting of an artificially designed sequence. It may be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. Furthermore, fragments of the above polypeptides are also included in the polypeptides of the present invention. In the present invention, the "multispecific polypeptide comprising a first polypeptide and a second polypeptide having variable regions of an antibody" is a polypeptide comprising variable regions of an antibody that binds to at least two or more different antigens or different epitopes within the same antigen. For the polypeptide comprising the variable region of the antibody, as described above, for example, an antibody, a miniaturized antibody, a Scaffold protein, etc. are included.

[0120] In the present invention, "the difference in isoelectric point of the polypeptide increases" means that in two or more polypeptides, by modifying the charge of the surface amino acids, the isoelectric points of each other do not become equal, or the isoelectric point difference between two or more polypeptides is made larger. The difference in isoelectric point can be observed, for example, by using a method such as isoelectric focusing electrophoresis. In the present invention, it is preferable to control the isoelectric point while maintaining the structure and function (activity) of the polypeptide.

[0121] That is, the present invention relates to a method for producing a multispecific polypeptide comprising a first polypeptide and a second polypeptide having variable regions of an antibody, comprising: (a) Modifying both or either of the nucleic acid encoding the amino acid residues of the first polypeptide and the nucleic acid encoding the amino acid residues of the second polypeptide so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region is modified such that the difference in isoelectric point between the first polypeptide and the second polypeptide is 1.0 or more, preferably 1.2 or more, more preferably 1.5 or more; (b) Culturing a host cell so that the nucleic acid is expressed; (c) Recovering the multispecific antibody from the host cell culture, and provides a method for producing a multispecific antibody.

[0122] The present invention also provides a method for modifying a multispecific polypeptide for purifying a multispecific polypeptide comprising a first polypeptide and a second polypeptide having variable regions of an antibody. As a preferred embodiment of the purification method of the present invention, it includes modifying both or either of the nucleic acid encoding the amino acid residues of the first polypeptide and the nucleic acid encoding the amino acid residues of the second polypeptide so that the difference in isoelectric point between the first polypeptide and the second polypeptide increases. That is, by changing the charge of the amino acid residues of the first polypeptide and the second polypeptide, a difference in isoelectric point (pI) is introduced into the polypeptide, and the multispecific antibody can be purified using the difference in isoelectric point. Specifically, it is a purification method including the following steps (a) to (c). (a) Modifying the nucleic acid encoding the polypeptide containing the amino acid residue so that the charge of at least one amino acid residue that can be exposed on the surface of the CDR region of the first polypeptide and the second polypeptide is modified, and the modification of the nucleic acid is such that the difference in isoelectric point between the first polypeptide and the second polypeptide increases compared to before the modification, and modifying both or either of the nucleic acid encoding the amino acid residues of the first polypeptide and the nucleic acid encoding the amino acid residues of the second polypeptide; Also including (b) Culturing a host cell so that the nucleic acid is expressed; (b) Culture the host cell so that the nucleic acid is expressed, (c) Purify the multispecific antibody from the host cell culture by standard chromatography

[0123] In addition, the method for producing a multispecific antibody including the step of purifying by the above purification method is also included in the present invention.

[0124] In the above method of the present invention, "modifying a nucleic acid" means modifying the nucleic acid sequence so as to be a codon corresponding to the amino acid residue introduced by the "modification" in the present invention. More specifically, it means modifying the nucleic acid constituting the codon to be subjected to modification so that the codon corresponding to the amino acid residue before modification becomes the codon of the amino acid residue introduced by the modification. Usually, it means performing a genetic manipulation or mutation treatment such as substituting at least one base of the nucleic acid constituting the codon so as to be a codon encoding the target amino acid residue. That is, the codon encoding the amino acid residue to be subjected to modification is replaced by the codon encoding the amino acid residue introduced by the modification. Such modification of the nucleic acid can be appropriately carried out by those skilled in the art using known techniques such as site-directed mutagenesis and PCR mutagenesis. It is possible to carry out.

[0125] Also, the nucleic acid in the present invention is usually held (inserted) into an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it can stably hold the inserted nucleic acid. For example, if Escherichia coli is used as the host, as a cloning vector, pBluescript vector (Stratagene) etc. are preferable, but various commercially available vectors can be used. That is. When a vector is used to produce the polypeptide of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it can express the polypeptide in vitro, in Escherichia coli, in cultured cells, or in an organism. For example, for in vitro expression, the pBEST vector (Promega) can be used; for Escherichia coli, the pET vector (Invitrogen) can be used; for cultured cells, the pME18S-FL3 vector (GenBank Accession No. AB009864) can be used; and for an organism, the pME18S vector (Mol Cell Biol. (1988) 8, 466-472) is preferred. Yes. Insertion of the DNA of the present invention into the vector can be preferably carried out by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0126] The host cell is not particularly limited, and various host cells can be used according to the purpose. Examples of cells for expressing the polypeptide 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. Introduction of the vector into the host cell can be carried out by known methods such as the calcium phosphate precipitation method, the electroporation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), the lipofection method, and the microinjection method.

[0127] For secreting a polypeptide (antibody) expressed in a host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, an appropriate secretion signal can be suitably incorporated into the antibody of interest. These signals can preferably utilize endogenous signals specific to the polypeptide (antibody) of interest or heterologous signals.

[0128] In the above production method, when the polypeptide (antibody) is secreted into the medium, the recovery of the polypeptide (antibody) is carried out by recovering the medium. When the antibody of the present invention is produced intracellularly, the cells are first lysed, and then the antibody is recovered.

[0129] For the purification of the antibody of the present invention recovered from recombinant cell cultures, 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 can be preferably used.

[0130] In the present invention, the polypeptide that modifies nucleic acid is preferably a homomultimer of a first polypeptide, a homomultimer of a second polypeptide, and a heteromultimer of the first polypeptide and the second polypeptide. Examples of the homomultimer of the first polypeptide, the homomultimer of the second polypeptide, and the heteromultimer of the first polypeptide and the second polypeptide include, but are not limited to, those described in the examples.

[0131] Examples of the standard chromatography in the present invention include, but are not limited to, cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, hydrophobic charge interaction chromatography, chromatofocusing, etc.

[0132] In the above method of the present invention, it is preferable that the first polypeptide and the second polypeptide contain a heavy chain variable region (VH). The variable region may include, for example, a complementarity determining region (CDR) and a framework region (FR).

[0133] Furthermore, in the above method of the present invention, it is preferable that the variable region of the multispecific polypeptide contains a light chain variable region.

[0134] Furthermore, in the above method of the present invention, it is preferable that the first polypeptide and the second polypeptide contain a heavy chain constant region. As the heavy chain constant region, those that cause a pI difference between the first polypeptide and the second polypeptide are more preferable. Such heavy chain constant regions include the heavy chain constant regions of antibodies having a pI difference, and the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, which originally have a difference in pI, can be used to introduce a pI difference into the first and second polypeptides. Alternatively, a non-wild-type human constant region can be prepared by simultaneously modifying only the amino acids in the heavy chain constant regions of the first and second polypeptides that are due to the isoelectric point differences between these subclasses, or the adjacent amino acids that do not affect their isoelectric points, to introduce a pI difference into the two constant regions. Examples of the sites for modification to introduce a pI difference into the constant region include, for example, the H chain In the EU numbering of the constant region, positions 137, 196, 203, 214, 217, 233 rd, 268th, 274th, 276th, 297th, 355th, 392nd, 419th, 435th. In addition, since a pI difference is caused by removing the sugar chain of the heavy chain constant region, position 297 of the sugar chain addition site is also mentioned as a site for modification to introduce a pI chain. The present invention also includes a method in which the above-described first polypeptide and second polypeptide include a heavy chain constant region, a method in which the above-described first polypeptide and second polypeptide include a heavy chain variable region, and / or a method in which the multispecific antibody includes a third polypeptide including a light chain variable region, and the first polypeptide and the second polypeptide each form a multimer with the third polypeptide.

[0135] The present invention also includes a multispecific polypeptide produced by the above method.

[0136] Furthermore, when the first polypeptide in the multispecific antibody provided by the present invention includes a heavy chain variable region, in order to "increase the difference in isoelectric points", for example, positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region Examples of embodiments include making at least one amino acid residue selected from the amino acid residues have a charge. Also, when it includes a light chain variable region, in order to "increase the difference in isoelectric points", for example, according to Kabat numbering in the light chain variable region Examples of embodiments include making at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54, and 55 have a charge. Among the amino acid residues of the first polypeptide indicated by the above numbering, the amino acid residues other than the amino acid residues having the charge may have the same charge as the amino acid residues having the charge, may not have a charge, or may have an opposite charge as long as there is a difference in the isoelectric points between the first polypeptide and the second polypeptide.

[0137] The above-mentioned multispecific antibody of the present invention is preferably characterized in that the second polypeptide has a charge opposite to that of the amino acid residue having the charge of the first polypeptide or has no charge. Specifically, the second polypeptide contains a heavy chain variable region, and at least one amino acid residue selected from the amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region has a charge opposite to that of the amino acid residue selected in the variable region contained in the first polypeptide or has no charge, and is a multispecific antibody. When it contains a light chain variable region, at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region has a charge opposite to that of the amino acid residue selected in the variable region contained in the first polypeptide or has no charge, and is a multispecific antibody. Among the amino acid residues of the second polypeptide indicated by the above numbering, amino acid residues other than the amino acid residues having the charge may have the same charge as the amino acid residues having the charge, may have no charge, or may have an opposite charge as long as there is a difference in the isoelectric point between the first polypeptide and the second polypeptide. Among the amino acid residues selected from the amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region of the second polypeptide, at least one amino acid residue has a charge opposite to that of the amino acid residue selected in the variable region contained in the first polypeptide or has no charge, and is a multispecific antibody. When it contains a light chain variable region, among the amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region, at least one amino acid residue has a charge opposite to that of the amino acid residue selected in the variable region contained in the first polypeptide or has no charge, and is a multispecific antibody. Among the amino acid residues selected from the amino acid residues at positions 31, 61, 62, 64, and 65 according to Kabat numbering in the heavy chain variable region of the second polypeptide, at least one amino acid residue has a charge opposite to that of the amino acid residue selected in the variable region contained in the first polypeptide or has no charge, and is a multispecific antibody. When it contains a light chain variable region, among the amino acid residues at positions 24, 27, 53, 54, and 55 according to Kabat numbering in the light chain variable region, at least one amino acid residue has a charge opposite to that of the amino acid residue selected in the variable region contained in the first polypeptide or has no charge, and is a multispecific antibody. Among the amino acid residues of the second polypeptide indicated by the above numbering, amino acid residues other than the amino acid residues having the charge may have the same charge as the amino acid residues having the charge, may have no charge, or may have an opposite charge as long as there is a difference in the isoelectric point between the first polypeptide and the second polypeptide.

[0138] Furthermore, when the multispecific antibody contains a constant region of an antibody, in order to lower its isoelectric point, for example, the sequence at position 137 is that of IgG2 or IgG4, the sequence at position 196 is that of IgG1 or IgG2 or IgG4, the sequence at position 203 is that of IgG2 or IgG4, the sequence at position 214 is that of IgG2, the sequence at position 217 is that of IgG1 or IgG3 or IgG4, the sequence at position 233 is that of IgG1 or IgG3 or IgG4, the sequence at position 268 is that of IgG4, the sequence at position 274 is that of IgG2 or IgG3 or IgG4, the sequence at position 276 is that of IgG1 or IgG2 or IgG4, the sequence at position 355 is that of IgG4, the sequence at position 392 is that of IgG3, the sequence at position 419 is that of IgG4, the sequence at position 435 is that of IgG1 or IgG2 or IgG4 It is desirable to apply the following columns. Also, in order to increase the isoelectric point, for example, at position 137, the sequence of IgG1 or IgG3, at position 196, the sequence of IgG3, at position 203, the sequence of IgG1 or IgG3, at position 214, the sequence of IgG1 or IgG3 or IgG4, at position 217, the sequence of IgG2, at position 233, the sequence of IgG2, at position 268, the sequence of IgG1 or IgG2 or IgG3, at position 274, the sequence of IgG1, at position 276, the sequence of IgG3, at position 355, the sequence of IgG1 or IgG2 or IgG3, at position 392, the sequence of IgG1 or IgG2 or IgG4, at position 419, the sequence of IgG1 or IgG2 or IgG3, at position 435, the sequence of IgG3, it is desirable to apply. The application of these sequences only needs to result in a sufficient difference in isoelectric point between both heavy chains, and it is not necessarily necessary to apply all the sequences.

[0139] In the above antibody, "having the same charge" means that, for example, any of the amino acid residues according to the Kabat numbering in the heavy chain variable region, or the amino acid residues according to the EU numbering in the heavy chain constant region has an amino acid residue included in any one of the following groups (a) or (b). (a) Glutamic acid (E), Aspartic acid (D) (b) Lysine (K), Arginine (R), Histidine (H)

[0140] Also, "having opposite charges" means that, for example, when at least one of the amino acid residues according to the Kabat numbering or the EU numbering in a second polypeptide having a heavy chain variable region and / or a heavy chain constant region is an amino acid residue at the corresponding position in the heavy chain variable region and / or the heavy chain constant region included in the first polypeptide, and the amino acid residue has an amino acid residue included in any one of the above groups (a) or (b), the remaining amino acid residues have amino acid residues included in a different group.

[0141] That is, in the present invention, there is provided a multispecific antibody in which the amino acid residues having the same kind of charge are selected from the amino acid residues included in any one of the groups (a) or (b) above.

[0142] When the original (unmodified) amino acid residue already has a charge, modification to an amino acid residue having no charge is also one of the preferred embodiments of the present invention.

[0143] In the present invention, it is preferable that the amino acid residues are modified so that the difference in isoelectric point (pI) between the first polypeptide and the second polypeptide increases. Further, when there are a plurality of amino acid residues introduced by the modification, a small number of amino acid residues having no charge may be included among these amino acid residues.

[0144] The present invention also relates to a composition (drug) containing a polypeptide (for example, an antibody such as an IgG antibody) whose pharmacokinetics in plasma is controlled by the method of the present invention, and a pharmaceutically acceptable carrier.

[0145] In the present invention, the pharmaceutical composition generally refers to a drug for the treatment or prevention of diseases, or for examination / diagnosis.

[0146] The pharmaceutical composition of the present invention can be preferably formulated by a method known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution or suspension injection with water or other pharmaceutically acceptable liquids. For example, it can be preferably formulated by appropriately combining a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dosage form required for generally recognized pharmaceutical practice. The amount of the active ingredient in these formulations is set so as to obtain an appropriate dosage within the indicated range.

[0147] The sterile composition for injection can be preferably formulated according to ordinary pharmaceutical practice using a vehicle such as distilled water for injection.

[0148] Examples of the aqueous injection solution include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable solubilizing agents, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80 (TM), HCO-50), can be preferably used in combination.

[0149] Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol can be preferably used in combination as a solubilizing agent. In addition, buffer agents (e.g., phosphate buffer solution and sodium acetate buffer solution), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants can be preferably formulated. The injection solution prepared as described above is usually filled into appropriate ampoules.

[0150] The pharmaceutical composition of the present invention can preferably be administered by parenteral administration. For example, it can be appropriately prepared as an injection formulation , a nasal administration formulation, a pulmonary administration formulation, or a transdermal administration formulation. For example, it can be appropriately administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0151] The administration method can be appropriately selected according to the age and symptoms of the patient. The dosage of the pharmaceutical composition containing an antibody or a polynucleotide encoding the antibody can be appropriately set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, for example, the dosage can be set or adjusted to 0.001 to 100000 mg per patient, but the present invention is not necessarily limited to these numerical values. The dosage and administration method vary depending on the body weight, age, symptoms, etc. of the patient, but those skilled in the art can set appropriate dosage and administration methods considering these conditions.

[0152] The present invention also provides a nucleic acid encoding an antibody (for example, a humanized glypican 3 antibody, etc.) whose pharmacokinetics in plasma are controlled by the method of the present invention. Further, a vector carrying the nucleic acid is also included in the present invention.

[0153] Furthermore, the present invention provides a host cell containing the above nucleic acid. The type of the host cell is not particularly limited, and examples include bacterial cells such as Escherichia coli and various animal cells. The host cell can be suitably used as a production system for the production and expression of the antibody of the present invention. That is, the present invention provides a production system used for the production of an antibody using the host cell. As the production system, in vitro and in vivo production systems can be suitably used. In the in vitro production system As the host cell used, eukaryotic cells and prokaryotic cells are preferably used.

[0154] Examples of eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, etc., amphibian cells such as Xenopus laevis oocytes (Valle et al., Nature (1981) 291, 338 - 340), and insect cells such as Sf9, Sf21, Tn5 are preferably exemplified. In the expression of the antibody of the present invention, CHO - DG44, CHO - DX11B, COS7 cells, HEK293 cells, and BHK cells are preferably used. When aiming for large - scale expression by animal cells, CHO cells are particularly preferred as host cells For example, methods such as the calcium phosphate method, the DEAE - dextran method, the method using cationic ribosome DOTAP (Boehringer Mannheim), the electroporation method, and lipofection are preferably used to introduce a recombinant vector or the like into the host cell.

[0155] Examples of plant cells include cells derived from Nicotiana tabacum and Lemna minor are known as protein production systems, and the antibodies of the present invention can be produced by culturing these cells with a virus. Examples of fungal cells include yeast, such as cells of the genus Saccharomyces (Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc.), and filamentous fungi, such as cells of the genus Aspergillus (Aspergillus niger, etc.). Protein expression systems using these are known and can be used as host cells for producing the antibodies of the present invention.

[0156] When prokaryotic cells are used, a production system using bacterial cells is preferably used. Examples of bacterial cells include, in addition to the above-mentioned E. coli, a production system using Bacillus subtilis is known, and any of these bacterial cells can be preferably used for producing the antibodies of the present invention.

[0157] To produce an antibody using the host cell of the present invention, a host cell transformed with an expression vector containing a polynucleotide encoding the antibody of the present invention is cultured, and the polynucleotide encoding the antibody is expressed in the culture. The culture can be preferably performed according to a known method. For example, when animal cells are used as the host, culture media such as DMEM, MEM, RPMI1640, and IMDM can be preferably used. At this time, serum supplements such as FBS and fetal bovine serum (FCS) are preferably used in combination. Also, cells can be cultured by serum-free culture. Depending on the host cell, it can be preferably cultured under conditions of pH about 6 to 8 during culture. The culture is usually performed at about 30 to 40°C for about 15 to 200 hours, and medium replacement, aeration, and stirring are added as necessary.

[0158] On the other hand, as a system for producing the antibody of the present invention in vivo, for example, a production system using animals or a production system using plants can be preferably used. That is, a polynucleotide encoding the antibody of the present invention is introduced into these animals or plants, and the glypican 3 antibody is produced and recovered in the body of the animal or plant. The "hosts" in the present invention include these animals and plants. When an animal is used as the host, production systems using mammals or insects are available. As mammals, goats, pigs, sheep, mice, cows, etc. are preferably used (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). When a mammal is used, a transgenic animal is used.

[0159]

[0160] For example, the polynucleotide encoding the antibody of the present invention is prepared as a fusion gene with a gene encoding a polypeptide specifically produced in milk, such as goat β-casein. Then, a polynucleotide fragment containing this fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. The target antibody can be obtained from the milk produced by the transgenic goat or its offspring born from the goat that received the embryo. In order to increase the amount of milk containing the antibody produced by the transgenic goat, a hormone is appropriately administered to the transgenic goat (Ebert et al., Bio / Technology (1994) 12, 699-702).

[0161] As the insect producing the antibody of the present invention, for example, silkworms can be used. When silkworms are used, a baculovirus into which a polynucleotide encoding the target antibody is inserted on its viral genome is used in the infection of silkworms. The target glypican 3 antibody can be obtained from the body fluid of the infected silkworms (Susumu et al., Nature (1985) 315, 592-4).

[0162] ​Furthermore, when a plant is used for the production of the antibody of the present invention, for example, tobacco can be used as the plant. When tobacco is used, a recombinant vector obtained by inserting a polynucleotide encoding the target antibody into a plant expression vector, for example, pMON 530, can be introduced into a bacterium such as Agrobacterium tumefaciens. The bacterium can be used for infection of tobacco, for example, Nicotiana tabacum (Ma et al., Eur. J. Immunol. (1994) 24, 131-8). ) and the desired glypican 3 antibody can be obtained from the leaves of the infected tobacco. Similarly, the bacterium can be used for infection of Lemna minor, and the desired glypican 3 antibody can be obtained from the cloned infected Lemna minor cells (Cox KM et al. Nat. Biotechnol. (2006) 24(12), 1591-7). The antibody of the present invention thus obtained can be isolated from inside or outside the host cell (such as medium, milk, etc.) and purified as a substantially pure and homogeneous antibody. For the separation and purification of the antibody, separation and purification methods usually used for the purification of polypeptides can be preferably used, but are not limited thereto. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, dialysis, recrystallization, etc. can be appropriately selected and combined so that the antibody can be preferably separated and purified.

[0163]

[0164] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshaket al. (1996) Cold Spring Harbor Laboratory Press). These chromatographies can be performed using liquid chromatography, such as HPLC, FPLC and other liquid chromatographies. Examples of columns used for affinity chromatography include protein A columns and protein G columns. For example, columns such as Hyper D, POROS, Sepharose F.F. (manufactured by Pharmacia), etc.

[0165] A method for producing the antibody of the present invention with controlled plasma pharmacokinetics, which includes culturing the host cell of the present invention as described above and recovering the glypican 3 antibody from the culture of the cell, is also one of the preferred embodiments of the present invention.

[0166] The present invention modifies the amino acid sequences of the variable region and the constant region of tocilizumab, a humanized anti-IL-6 receptor IgG1 antibody, to enhance the drug efficacy, improve the plasma retention to reduce the dosing frequency and continuously exert the therapeutic effect, and improve the immunogenicity, safety and physical properties, to provide a pharmaceutical composition composed of a second-generation molecule superior to tocilizumab, and a method for producing these pharmaceutical compositions. Further, the present invention provides an antibody constant region suitable for use as a pharmaceutical.

[0167] The present invention relates to an anti-IL-6 receptor antibody having excellent antigen-binding activity, neutralizing activity, plasma retention, stability and / or homogeneity, and reducing the immunogenicity risk. Preferably, the anti-IL-6 receptor antibody is a humanized PM-1 antibody (Tocilizumab). More specifically, the present invention provides a humanized PM-1 antibody with enhanced antigen-binding activity by amino acid substitution, a humanized PM-1 antibody with enhanced neutralizing activity, a humanized PM-1 antibody with improved plasma retention, a humanized PM-1 antibody with reduced immunogenicity risk, a humanized PM-1 antibody with improved stability, and a humanized PM-1 antibody with improved homogeneity. Specifically, the present invention provides a humanized PM-1 antibody with enhanced antigen-binding activity by amino acid substitution, a humanized PM-1 antibody with enhanced neutralizing activity, a humanized PM-1 antibody with improved plasma retention, a humanized PM-1 antibody with reduced immunogenicity risk, a humanized PM-1 antibody with improved stability, and a humanized PM-1 antibody with improved homogeneity.

[0168] The humanized PM-1 antibody binds to the human IL-6 receptor and inhibits the binding of human IL-6 to the human IL-6 receptor. In this specification, the correspondence between the amino acid sequence of the humanized PM-1 antibody and the sequence numbers in the sequence listing is as follows. Heavy chain amino acid sequence SEQ ID NO: 15 Light chain amino acid sequence SEQ ID NO: 16 Amino acid sequence of the heavy chain variable region SEQ ID NO: 17 Amino acid sequence of the light chain variable region SEQ ID NO: 18 Amino acid sequence of the heavy chain CDR1 (HCDR1) SEQ ID NO: 1 Amino acid sequence of the heavy chain CDR2 (HCDR2) SEQ ID NO: 2 Amino acid sequence of the heavy chain CDR3 (HCDR3) SEQ ID NO: 3 Amino acid sequence of the heavy chain FR1 (HFR1) SEQ ID NO: 7 Amino acid sequence of the heavy chain FR2 (HFR2) SEQ ID NO: 8 Amino acid sequence of the heavy chain FR3 (HFR3) SEQ ID NO: 9 Amino acid sequence of the heavy chain FR4 (HFR4) SEQ ID NO: 10 The amino acid sequence of the light chain CDR1 (LCDR1) is SEQ ID NO: 4 The amino acid sequence of the light chain CDR2 (LCDR2) is SEQ ID NO: 5 The amino acid sequence of the light chain CDR3 (LCDR3) is SEQ ID NO: 6 The amino acid sequence of the light chain FR1 (LFR1) is SEQ ID NO: 11 The amino acid sequence of the light chain FR2 (LFR2) is SEQ ID NO: 12 The amino acid sequence of the light chain FR3 (LFR3) is SEQ ID NO: 13 The amino acid sequence of light chain FR4 (LFR4) is SEQ ID NO: 14

[0169] <Affinity-neutralizing activity enhanced antibody> The present invention provides an anti-human IL-6 receptor antibody having high binding activity and / or neutralizing activity against the human IL-6 receptor. More specifically, the present invention provides the antibodies described in the following (a) to (y), and a method for producing the antibodies. (a) An anti-human IL-6 receptor antibody having a heavy chain CDR1 in which the first Ser in the amino acid sequence (HCDR1) described in SEQ ID NO: 1 is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Trp (RD_68), Thr (RD_37), Asp (RD_8) , Asn (RD_11), Arg (RD_31), Val (RD_32), Phe (RD_33), Ala (RD_34), Gln (RD_35), Tyr (RD_36), Leu (RD_38), His (RD_42), Glu (RD_45) or Cys (RD_46) is preferred. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Trp is shown in SEQ ID NO: 26. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Thr is shown in SEQ ID NO: 27. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Asp is shown in SEQ ID NO: 28. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Asn is shown in SEQ ID NO: 29. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Arg is shown in SEQ ID NO: 30. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Val is shown in SEQ ID NO: 31. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Phe is shown in SEQ ID NO: 32. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Ala is shown as SEQ ID NO: 33. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Gln is shown as SEQ ID NO: 34. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Tyr is shown as SEQ ID NO: 35. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Leu is shown as SEQ ID NO: 36. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with His is shown as SEQ ID NO: 37. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Glu is shown as SEQ ID NO: 38. The sequence in which the first Ser in the amino acid sequence described in SEQ ID NO: 1 is substituted with Cys is shown as SEQ ID NO: 39.

[0170] (b) An anti-human IL-6 receptor antibody having a heavy chain CDR1 in which the fifth Trp in the amino acid sequence (HCDR1) described in SEQ ID NO: 1 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution with Ile (RD_9) or Val (RD_30) is preferred. The sequence in which the fifth Trp in the amino acid sequence described in SEQ ID NO: 1 is substituted with Ile is shown as SEQ ID NO: 40. The sequence in which the fifth Trp in the amino acid sequence described in SEQ ID NO: 1 is substituted with Val is shown as SEQ ID NO: 41.

[0171] (c) An anti-human IL-6 receptor antibody having a heavy chain CDR2 in which the first Tyr in the amino acid sequence (HCDR2) described in SEQ ID NO: 2 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution with Phe (RD_82) is preferred. The sequence in which the first Tyr in the amino acid sequence described in SEQ ID NO: 2 is substituted with Phe is shown as SEQ ID NO: 42.

[0172] (d) An anti-human IL-6 receptor antibody having a heavy chain CDR2 in which the 8th Thr in the amino acid sequence (HCDR2) set forth in SEQ ID NO: 2 is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Arg (RD_79) is preferred. The sequence in which the 8th Thr in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with Arg is shown in SEQ ID NO: 43.

[0173] (e) An anti-human IL-6 receptor antibody having a heavy chain CDR2 in which the 9th Thr in the amino acid sequence (HCDR2) set forth in SEQ ID NO: 2 is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Ser (RD_12) or Asn (RD_61) is preferred. The sequence in which the 9th Thr in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with Ser is shown in SEQ ID NO: 44. The sequence in which the 9th Thr in the amino acid sequence set forth in SEQ ID NO: 2 is substituted with Asn is shown in SEQ ID NO: 45.

[0174] (f) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the 1st Ser in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3 is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Ile (RD_2), Val (RD_4), Thr (RD_80) or Leu (RD_5) is preferred. The sequence in which the 1st Ser in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Ile is shown in SEQ ID NO: 46. The sequence in which the 1st Ser in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Val is shown in SEQ ID NO: 47. The sequence in which the 1st Ser in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Thr is shown in SEQ ID NO: 48. The sequence in which the 1st Ser in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Leu is shown in SEQ ID NO: 49.

[0175] (g) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the second Leu in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution with Thr (RD_84) is preferred. The sequence in which the second Leu in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Thr is shown in SEQ ID NO: 50.

[0176] (h) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the fifth Thr in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution with Ala (RD_3) or Ile (RD_83) is preferred. Other preferred substitutions include substitution of the fifth Thr with Ser (RDC_14H). The sequence in which the fifth Thr in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Ala is shown in SEQ ID NO: 51. The sequence in which the fifth Thr in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Ile is shown in SEQ ID NO: 52. The sequence in which the fifth Thr in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Ser is shown in SEQ ID NO: 53.

[0177] (i) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the seventh Ala in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3 is substituted with another amino acid. The substituted amino acid sequence is not particularly limited, but substitution with Ser (RD_81) or Val (PF_3H) is preferred. The sequence in which the seventh Ala in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Ser is shown in SEQ ID NO: 54. The sequence in which the seventh Ala in the amino acid sequence set forth in SEQ ID NO: 3 is substituted with Val is shown in SEQ ID NO: 55.

[0178] (j) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which Met at position 8 in the amino acid sequence set forth in SEQ ID NO: 3 (HCDR3) has been substituted with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Leu(PF_4H) is preferred. The amino acid sequence set forth in SEQ ID NO: 3 in which Met at position 8 has been replaced with Leu is shown in SEQ ID NO: 56.

[0179] (k) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the 1st Ser and the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 3 have been substituted with other amino acids. The amino acids after substitution are not particularly limited, but the first Ser is replaced with Leu and the fifth Thr is replaced with Ala. Other preferred substitutions include substitution of the first Ser with Val and substitution of the fifth Thr with Ala (RDC_2H), substitution of the first Ser with Ile and substitution of the fifth Thr with Ala (RDC_3H), substitution of the first Ser with Thr and substitution of the fifth Thr with Ala (RDC_4H), Substitution of 1st Ser by Val and 5th Thr by Ile (RDC_5H), 1st Ser by Ile and the 5th Thr replaced by Ile (RDC_6H), the 1st Ser replaced by Thr and the 5th Thr replaced by Ile (RDC_7H), or the 1st Ser replaced by Leu and the 5th Thr replaced by Ile Examples of such conversions include (RDC_8H). In the amino acid sequence of SEQ ID NO: 3, the first Ser is changed to Leu and the fifth Thr is changed to Ala. The substituted sequence is shown in SEQ ID NO:57. In the amino acid sequence of SEQ ID NO: 3, the first Ser is changed to Val and the fifth Thr is changed to Ala. The substituted sequence is shown in SEQ ID NO:58. In the amino acid sequence set forth in SEQ ID NO: 3, the first Ser is replaced with Ile and the fifth Thr is replaced with Ala. The resulting sequence is shown as SEQ ID NO: 59. In the amino acid sequence set forth in SEQ ID NO: 3, the first Ser is replaced with Thr and the fifth Thr is replaced with Ala. The resulting sequence is shown as SEQ ID NO: 60. In the amino acid sequence set forth in SEQ ID NO: 3, the first Ser is replaced with Val and the fifth Thr is replaced with Ile. The resulting sequence is shown as SEQ ID NO: 61. In the amino acid sequence set forth in SEQ ID NO: 3, the first Ser is replaced with Ile and the fifth Thr is replaced with Ile. The resulting sequence is shown as SEQ ID NO: 62. In the amino acid sequence set forth in SEQ ID NO: 3, the first Ser is replaced with Thr and the fifth Thr is replaced with Ile. The resulting sequence is shown as SEQ ID NO: 63. In the amino acid sequence set forth in SEQ ID NO: 3, the first Ser is replaced with Leu and the fifth Thr is replaced with Ile. The resulting sequence is shown as SEQ ID NO: 64.

[0180] (l) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the second Leu, seventh Ala, and eighth Met in the amino acid sequence (HCDR3) set forth in SEQ ID NO: 3 are replaced with other amino acids. The amino acids after substitution are not particularly limited, but it is preferable to replace the second Leu with Thr, the seventh Ala with Val, and the eighth Met with Leu (RD_78). Antibody. The amino acid sequence in which the second Leu is replaced with Thr, the seventh Ala is replaced with Val, and the eighth Met is replaced with Leu in the amino acid sequence set forth in SEQ ID NO: 3 is shown as SEQ ID NO: 65. Antibody. The amino acid sequence in which the second Leu is replaced with Thr, the seventh Ala is replaced with Val, and the eighth Met is replaced with Leu in the amino acid sequence set forth in SEQ ID NO: 3 is shown as SEQ ID NO: 65.

[0181] (m) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the first Arg in the amino acid sequence (LCDR1) set forth in SEQ ID NO: 4 is replaced with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Phe (RD_18) is preferred. The sequence in which the first Arg in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with Phe is shown in SEQ ID NO: 66.

[0182] (n) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the fourth Gln in the amino acid sequence (LCDR1) set forth in SEQ ID NO: 4 is substituted with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Arg (RD_26) or Thr (RD_20) is preferred. The sequence in which the fourth Gln in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with Arg is shown in SEQ ID NO: 67. The sequence in which the fourth Gln in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with Thr is shown in SEQ ID NO: 68.

[0183] (o) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the ninth Tyr in the amino acid sequence (LCDR1) set forth in SEQ ID NO: 4 is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Phe (RD_73) is preferred. The sequence in which the ninth Tyr in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with Phe is shown in SEQ ID NO: 69.

[0184] (p) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the eleventh Asn in the amino acid sequence (LCDR1) set forth in SEQ ID NO: 4 is substituted with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Ser (RD_27) is preferred. The sequence in which the eleventh Asn in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with Ser is shown in SEQ ID NO: 70.

[0185] (q) An anti-human IL-6 receptor antibody having a light chain CDR2 in which the second Thr in the amino acid sequence (LCDR2) described in SEQ ID NO: 5 is substituted with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Gly is preferred. The sequence in which the second Thr in the amino acid sequence described in SEQ ID NO: 5 is substituted with Gly is shown in SEQ ID NO: 71.

[0186] (r) An anti-human IL-6 receptor antibody having a light chain CDR3 in which the first Gln in the amino acid sequence (LCDR3) described in SEQ ID NO: 6 is substituted with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Gly (RD_28), Asn (RD_29) or Ser (RDC_15L) is preferred. The sequence in which the first Gln in the amino acid sequence described in SEQ ID NO: 6 is substituted with Gly is shown in SEQ ID NO: 72. The sequence in which the first Gln in the amino acid sequence described in SEQ ID NO: 6 is substituted with Asn is shown in SEQ ID NO: 73. The sequence in which the first Gln in the amino acid sequence described in SEQ ID NO: 6 is substituted with Ser is shown in SEQ ID NO: 74.

[0187] (s) An anti-human IL-6 receptor antibody having a light chain CDR3 in which the third Gly in the amino acid sequence described in SEQ ID NO: 6 is substituted with another amino acid and having a light chain CDR3. The amino acid sequence after substitution is not particularly limited, but substitution with Ser is preferred. The sequence in which the third Gly in the amino acid sequence described in SEQ ID NO: 6 is substituted with Ser is shown in SEQ ID NO: 75.

[0188] (t) A light chain CDR1 in which the ninth Tyr in the amino acid sequence (LCDR1) described in SEQ ID NO: 4 is substituted with another amino acid, and a light chain CDR3 in which the third Gly in the amino acid sequence (LCDR3) described in SEQ ID NO: 6 is substituted with another amino acid, and an anti-human IL-6 receptor antibody having a light chain CDR3. The amino acid after substitution is not particularly limited, but preferably, the 9th Tyr in the amino acid sequence set forth in SEQ ID NO: 4 (LCDR1 ) is substituted with Phe, and preferably, the 3rd Gly in the amino acid sequence set forth in SEQ ID NO: 6 (LCDR3) is substituted with Ser (RD_72).

[0189] (u) An anti-human IL-6 receptor antibody having a light chain CDR3 in which the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 6 (LCDR3) is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Arg (RD_23) or Ser is preferred. The sequence in which the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with Arg is shown as SEQ ID NO: 76. The sequence in which the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with Ser is shown as SEQ ID NO: 77.

[0190] (v) An anti-IL-6 receptor antibody having a light chain CDR3 in which the 1st Gln and the 5th Thr in the amino acid sequence set forth in SEQ ID NO: 6 (LCDR3) are substituted with other amino acids. The amino acid after substitution is not particularly limited, but preferably, the 1st Gln is substituted with Gly and the 5th Thr is substituted with Ser (RD_22). Further, other preferred substitutions include substitution of the 1st Gln with Gly and substitution of the 5th Thr with Arg (RDC_11L). The sequence in which the 1st Gln in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with Gly and the 5th Thr is substituted with Ser is shown as SEQ ID NO: 78. The sequence in which the 1st Gln in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with Gly and the 5th Thr is substituted with Arg is shown as SEQ ID NO: 79.

[0191] ​​An anti-IL-6 receptor antibody comprising a heavy chain CDR2 in which Thr at the 9th position in the amino acid sequence (HCDR2) described in SEQ ID NO: 2 is substituted with another amino acid, and a heavy chain CDR3 in which Ser at the 1st position and Thr at the 5th position in the amino acid sequence (HCDR3) described in SEQ ID NO: 3 are substituted with other amino acids. Preferably, Thr at the 9th position in the amino acid sequence (HCDR2) described in SEQ ID NO: 2 is substituted with Asn. Also, as preferred combinations of amino acids after substitution of Ser at the 1st position and Thr at the 5th position in the amino acid sequence (HCDR3) described in SEQ ID NO: 3, Leu and Ala (RDC_27H), Val and Ala (RDC_28H), Ile and Ala (RDC_30H), Thr and Ala (RDC_4H), Val and Ile (RDC_29H), Ile and Ile (RDC_32H), Thr and Ile (RDC_7H), Leu and Ile (RDC_8H) can be mentioned.

[0192] (x) An antibody comprising a variable region having the heavy chain CDR3 described in (k) and a variable region having the light chain CDR3 described in (v). (y) The antibody described in (x) further comprising the heavy chain CDR2 described in (e).

[0193] The present invention provides an antibody comprising at least the amino acid substitutions described in any of (a) to (y) above and a method for producing the antibody. Therefore, the antibodies of the present invention include antibodies containing amino acid substitutions other than those described in any of (a) to (y) above in addition to the amino acid substitutions described in any of (a) to (y) above. The antibodies of the present invention also include antibodies in which a plurality of the amino acid substitutions described in any of (a) to (y) above are combined. Examples of the amino acid substitutions described in (a) to (y) above include substitutions of other amino acids for the above-described CDR amino acid sequences. The amino acid substitutions other than those described in (a) to (y) above include, for example, substitutions of amino acid sequences in other CDR portions, Deletions, additions and / or insertions, etc. may be involved. Further, substitutions, deletions, additions and / or insertions, etc. of the amino acid sequence of FR may be involved. Further, substitutions, deletions, additions and / or insertions, etc. of the amino acid sequence of the constant region may be involved.

[0194] In addition, the antibodies of the present invention include antibodies in which the high-affinity CDRs found in the present invention are grafted into any framework other than the humanized PM-1 antibody In addition, the antibodies of the present invention include antibodies in which the high-affinity CDRs found in the present invention are grafted into a framework other than the humanized PM-1 antibody. In the case of antibodies in which the resulting affinity has decreased, antibodies in which mutations are introduced into the framework region (see, for example, Curr Opin Biotechnol. 1994 Aug;5(4):428-33) to obtain the original affinity antibody, and mutations are introduced into the CDR region to obtain the original affinity antibody (see, for example, US2006 / 0122377) Antibodies into which are introduced are included.

[0195] In the present invention, it is preferable to perform the amino acid substitutions described in any of (a) to (y) above on the humanized PM-1 antibody. In the humanized PM-1 antibody, an antibody in which the amino acid substitution described in any of (a) to (y) above is performed has high neutralizing activity against the IL-6 receptor. In the humanized PM-1 antibody, an antibody in which the amino acid substitution described in any of (a) to (y) above is performed is effective as a therapeutic agent for inflammatory diseases such as rheumatoid arthritis related to IL-6.

[0196] Note that antibodies containing the amino acid substitutions described in any of (a) to (y) above can also be expressed, for example, as in the following (1) or (2). Here, the antibody of (a) is described as an example, but the antibodies of (b) to (y) can be expressed in the same manner. (1) An antibody containing a heavy chain variable region having a CDR1 having an amino acid sequence in which the first Ser in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with another amino acid is replaced with another amino acid (2) As CDR1, an antibody comprising an H chain having an amino acid sequence in which the first Ser in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with another amino acid Antibody having an amino acid sequence in which the first Ser in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with another amino acid

[0197] <Antibody with enhanced binding activity> The present invention further provides an anti-IL-6 receptor antibody having a high binding activity to the IL-6 receptor. The anti-IL-6 receptor antibody having a high binding activity to the IL-6 receptor in the present invention is usually an antibody having an affinity of 1 nM or less measured at 37 °C under physiological conditions, preferably An antibody having an affinity of 0.1 nM or less, more preferably an antibody having an affinity of 0.04 nM or less. Such an anti-IL-6 receptor antibody having a high binding activity to the IL-6 receptor is considered to have an improved ability to neutralize the biological action of the antigen. In the anti-IL-6 receptor antibody having a high binding activity to the IL-6 receptor of the present invention, the introduced amino acid substitution is not particularly limited, and examples thereof include the above-mentioned amino acid substitution. The IL-6 receptor is not particularly limited, but the human IL-6 receptor is preferred. The measurement of the binding activity can be carried out by a method known to those skilled in the art, and for example, it can be measured by Biacore (BIACORE) using SPR.

[0198] <Antibody with reduced immunogenic risk of CDR sequence> The present invention also provides an anti-IL-6 receptor antibody with reduced immunogenicity, particularly a humanized PM-1 antibody. It is considered that the immunogenicity of an antibody is high when there is a T-cell epitope that binds to HLA in the antibody sequence. Therefore, by substituting the antibody sequence to remove the T-cell epitope present in the antibody sequence, the immunogenic risk of the antibody can be reduced.

[0199] The present invention substitutes the amino acids in the amino acid sequence of the antibody, particularly in the CDR sequence, with other amino acids​​ Provided is a humanized anti-human IL-6 receptor antibody in which a T-cell epitope is removed by [description omitted] and immunogenicity is reduced, particularly a humanized PM-1 light chain variable region. The present invention also provides an antibody comprising the light chain variable region. More specifically, the present invention provides a light chain CDR2 in which the second Thr in the amino acid sequence (LCDR2) set forth in SEQ ID NO: 5 is substituted with another amino acid. The present invention also provides a light chain variable region comprising the light chain CDR2. The present invention also provides an anti-IL-6 receptor antibody comprising the light chain variable region. The amino acid sequence after substitution is not particularly limited, but substitution with Gly is preferred. The sequence in which the second Thr in the amino acid sequence set forth in SEQ ID NO: 5 is substituted with Gly is shown in SEQ ID NO: 71. The amino acid substitution is preferably carried out in the light chain variable region of the humanized PM-1 antibody. - - -

[0200] <FR and CDR of H53 / L28> The present invention also provides an anti-human IL-6 receptor antibody with improved plasma pharmacokinetics, stability and / or immunogenicity. In IgG, it has been found that the plasma half-life of IgG having the same Fc region correlates with a high correlation coefficient with pI. Therefore, when attempting to modify the pI of the variable region in two types of antibodies against different antigens, it was successful in controlling the plasma half-life without modifying the Fc region regardless of the type of antigen. The rate of non-specific uptake of the antibody into endothelial cells is thought to depend on the physicochemical Coulomb interaction between the negatively charged cell surface and IgG. By reducing the pI of IgG, the Coulomb interaction is reduced, non-specific uptake into endothelial cells is decreased, and as a result, it becomes possible to increase plasma retention by reducing metabolism in endothelial cells. - - -

[0201] That is, the present invention provides an anti-human IL-6 receptor antibody with a reduced isoelectric point and increased plasma retention by substituting the amino acid sequence of an anti-IL-6 receptor antibody, particularly a humanized PM-1 antibody. Specifically, in Kabat numbering (Kabat EA et al., 1991 Sequences of Proteins of Immunological Interest. NIH), H13 (the 13th amino acid of SEQ ID NO: 7), H16 (the 16th amino acid of SEQ ID NO: 7), H43 (the 8th amino acid of SEQ ID NO: 8), H81 (the 16th amino acid of SEQ ID NO: 9), H105 (the 3rd amino acid of SEQ ID NO: 10 ), L18 (the 18th amino acid of SEQ ID NO: 11), L45 (the 11th amino acid of SEQ ID NO: 12), L79 (the 23rd amino acid of SEQ ID NO: 13), L107 (the 10th amino acid of SEQ ID NO: 14), H31 (the 1st amino acid of SEQ ID NO: 1), L24 (the 1st amino acid of SEQ ID NO: 4) and / or L53 (the 4th amino acid of SEQ ID NO: 5) of humanized PM-1 are substituted with other amino acids that reduce the isoelectric point. By this, it is possible to reduce the isoelectric point without affecting the binding activity and stability of humanized PM-1. In addition, in the humanized PM-1 antibody, when humanizing the mouse sequence, several amino acid residues are left as the mouse sequence in order to maintain the binding activity. Specifically, in the above-mentioned Kabat numbering, H27 (the 27th amino acid of SEQ ID NO: 7), H28 (the 28th amino acid of SEQ ID NO: 7), H29 (the 29th amino acid of SEQ ID NO: 7), H30 (the 30th amino acid of SEQ ID NO: 7) and H71 in the humanized PM-1 antibody use the mouse sequence as it is. Regarding HFR1, it is possible to convert it to a human sequence as HFR1 by substituting H13, H16, H23, H30, and it is considered possible to produce an antibody with a lower immunogenic risk than the humanized antibody PM-1. Furthermore, since humanized PM-1 is an antibody humanized by CDR grafting ​​​​, it is considered that there is room for improvement in terms of stability. For example, it is considered possible to stabilize the antibody by substituting the amino acid residues exposed on the surface in the variable region of the antibody with hydrophilic amino acids. Furthermore, modifying the CDR sequence to a consensus sequence can also stabilize the antibody. In the humanized PM-1 antibody, in the above-mentioned Kabat numbering, substitution of Met at H69 (the 4th amino acid of SEQ ID NO: 9) with Ile (stabilization of the hydrophobic core structure), Leu at H70 (the 5th amino acid of SEQ ID NO: 9) with Ser substitution (hydrophilic modification of surface-exposed residues), Thr at H58 (the 9th amino acid of SEQ ID NO: 2) with Asn substitution (modification to the consensus sequence of heavy chain CDR2), Ser at H65 (the 16th amino acid of SEQ ID NO: 2) with Gly substitution (substitution with Gly in the β-turn part, modification to the consensus sequence of heavy chain CDR2), or Thr at L93 (the 5th amino acid of SEQ ID NO: 6) with Ser substitution (hydrophilic modification of surface-exposed residues) can stabilize the antibody. Also, by substituting Thr at L51, which is the 2nd amino acid of the above-mentioned LCDR2 (SEQ ID NO: 5), with Gly, the T-cell epitope predicted in silico can be removed without affecting the binding activity and stability , thereby reducing the immunogenicity risk. By combining these amino acid substitutions, it is possible to obtain an anti-IL-6 receptor antibody with improved plasma pharmacokinetics, immunogenicity, and stability of the antibody . Examples of such antibodies include the antibodies described in any of the following (1) to (37).

[0202] An antibody comprising a heavy chain variable region having an FR1 in which Arg at the 13th position in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with another amino acid. (1) The amino acid sequence after substitution is not particularly limited, but substitution with Lys is preferred. The sequence in which the 13th Arg is substituted with Lys in the amino acid sequence set forth in SEQ ID NO: 7 is shown as SEQ ID NO: 80. (2) An antibody comprising a heavy chain variable region having an FR1 in which the 16th Gln in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with another amino acid The amino acid sequence after substitution is not particularly limited, but substitution with Glu is preferred. The sequence in which the 16th Gln in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with Glu is shown as SEQ ID NO: 81. (3) An antibody comprising a heavy chain variable region having an FR1 in which the 23rd Thr in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with another amino acid The amino acid sequence after substitution is not particularly limited, but substitution with Ala is preferred. The sequence in which the 23rd Thr in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with Ala is shown as SEQ ID NO: 82. (4) An antibody comprising a heavy chain variable region having an FR1 in which the 30th Thr in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with another amino acid The amino acid sequence after substitution is not particularly limited, but substitution with Ser is preferred. The sequence in which the 30th Thr in the amino acid sequence set forth in SEQ ID NO: 7 is substituted with Ser is shown as SEQ ID NO: 83. (5) An antibody comprising a heavy chain variable region having an FR1 in which the 13th Arg, 16th Gln, 23rd Thr, and 30th Thr in the amino acid sequence set forth in SEQ ID NO: 7 are substituted with other amino acids The amino acids after substitution are not particularly limited, but preferably, the 13th Arg is substituted with Lys, the 16th Gln is substituted with Glu, the 23rd Thr is substituted with Ala, and the 30th Thr is substituted with Ser. ​​​​The sequence in which Arg at the 13th position, Gln at the 16th position, Thr at the 23rd position, and Thr at the 30th position are replaced with Lys, Glu, Ala, and Ser, respectively, in the amino acid sequence set forth in SEQ ID NO:7 is shown in SEQ ID NO:84. (6) An antibody comprising a heavy chain variable region having an FR2 in which Arg at the 8th position in the amino acid sequence set forth in SEQ ID NO:8 is replaced with another amino acid. An antibody comprising a heavy chain variable region having an FR2 in which Arg at the 8th position in the amino acid sequence set forth in SEQ ID NO:8 is replaced with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. The sequence in which Arg at the 8th position in the amino acid sequence set forth in SEQ ID NO:8 is replaced with Glu is shown in SEQ ID NO:85. (7) An antibody comprising a heavy chain variable region having an FR3 in which Met at the 4th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with another amino acid. An antibody comprising a heavy chain variable region having an FR3 in which Met at the 4th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Ile is preferred. The sequence in which Met at the 4th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with Ile is shown in SEQ ID NO:86. (8) An antibody comprising a heavy chain variable region having an FR3 in which Leu at the 5th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with another amino acid. An antibody comprising a heavy chain variable region having an FR3 in which Leu at the 5th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Ser is preferred. The sequence in which Leu at the 5th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with Ser is shown in SEQ ID NO:87. (9) An antibody comprising a heavy chain variable region having an FR3 in which Arg at the 16th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with another amino acid. An antibody comprising a heavy chain variable region having an FR3 in which Arg at the 16th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Lys is preferred. The sequence in which Arg at the 16th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with Lys is shown in SEQ ID NO:88. The sequence in which Arg at the 16th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with Lys is shown in SEQ ID NO:88. (10) An antibody comprising a heavy chain variable region having an FR3 in which Val at the 27th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with another amino acid. An antibody comprising a heavy chain variable region having an FR3 in which Val at the 27th position in the amino acid sequence set forth in SEQ ID NO:9 is replaced with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Ala is preferred. The sequence in which Val at position 27 in the amino acid sequence set forth in SEQ ID NO: 9 is substituted with Ala is shown as SEQ ID NO: 89. (11) A heavy chain variable region-containing antibody having FR3 in which Met at position 4, Leu at position 5, Arg at position 16, and Val at position 27 in the amino acid sequence set forth in SEQ ID NO: 9 are substituted with other amino acids. The amino acid after substitution is not particularly limited, but substitution of Met at position 4 with Ile, Leu at position 5 with Ser, Arg at position 16 with Lys, and Val at position 27 with Ala is preferred. The sequence in which Met at position 4, Leu at position 5, Arg at position 16, and Val at position 27 in the amino acid sequence set forth in SEQ ID NO: 9 are substituted with Ile, Ser, Lys, and Ala, respectively, is shown as SEQ ID NO: 90. (12) An antibody comprising a heavy chain variable region having FR4 in which Gln at position 3 in the amino acid sequence set forth in SEQ ID NO: 10 (HFR4) is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. The sequence in which Gln at position 3 in the amino acid sequence set forth in SEQ ID NO: 10 is substituted with Glu is shown as SEQ ID NO: 91. (13) An antibody comprising a light chain variable region having FR1 in which Arg at position 18 in the amino acid sequence set forth in SEQ ID NO: 11 (LFR1) is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Ser is preferred. The sequence in which Arg at position 18 in the amino acid sequence set forth in SEQ ID NO: 11 is substituted with Ser is shown as SEQ ID NO: 92. (14) An antibody comprising a light chain variable region having FR2 in which Lys at position 11 in the amino acid sequence set forth in SEQ ID NO: 12 (LFR2) is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. The sequence in which Lys at the 11th position in the amino acid sequence described in SEQ ID NO: 12 is substituted with Glu is shown as SEQ ID NO: 93. (15) An antibody comprising a light chain variable region having an FR3 in which Gln at the 23rd position in the amino acid sequence described in SEQ ID NO: 13 is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. The sequence in which Gln at the 23rd position in the amino acid sequence described in SEQ ID NO: 13 is substituted with Glu is shown as SEQ ID NO: 94. (16) An antibody comprising a light chain variable region having an FR3 in which Pro at the 24th position in the amino acid sequence described in SEQ ID NO: 13 is substituted with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Ala is preferred. The sequence in which Pro at the 24th position in the amino acid sequence described in SEQ ID NO: 13 is substituted with Ala is shown as SEQ ID NO: 95. (17) An antibody comprising a light chain variable region having an FR3 in which Ile at the 27th position in the amino acid sequence described in SEQ ID NO: 13 is substituted with another amino acid. The amino acid sequence after substitution is not particularly limited, but substitution with Ala is preferred. The sequence in which Ile at the 27th position in the amino acid sequence described in SEQ ID NO: 13 is substituted with Ala is shown as SEQ ID NO: 96. (18) An antibody comprising a light chain variable region having an FR3 in which Gln at the 23rd position, Pro at the 24th position, and Ile at the 27th position in the amino acid sequence described in SEQ ID NO: 13 (LFR3) are substituted with other amino acids. The amino acid after substitution is not particularly limited, but substitution of Gln at the 23rd position with Glu, Pro at the 24th position with Ala, and Ile at the 27th position with Ala is preferred. The sequence in which Gln at the 23rd position, Pro at the 24th position, and Ile at the 27th position in the amino acid sequence described in SEQ ID NO: 13 are substituted with Glu, Ala, and Ala, respectively, is shown as SEQ ID NO: 97. (19) An antibody comprising a light chain variable region having an FR4 in which Lys at the 10th position in the amino acid sequence described in SEQ ID NO: 14 is replaced with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. The sequence in which Lys at the 10th position in the amino acid sequence described in SEQ ID NO: 14 is replaced with Glu is shown in SEQ ID NO: 98. (20) An antibody comprising a heavy chain variable region having an FR4 in which Ser at the 5th position in the amino acid sequence described in SEQ ID NO: 10 is replaced with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Thr is preferred. The sequence in which Ser at the 5th position in the amino acid sequence described in SEQ ID NO: 10 is replaced with Thr is shown in SEQ ID NO: 132. (21) An antibody comprising a heavy chain variable region having an FR4 in which Gln at the 3rd position and Ser at the 5th position in the amino acid sequence described in SEQ ID NO: 10 are replaced with another amino acid. The amino acid after substitution is not particularly limited, but Gln at the 3rd position is preferably replaced with Glu, and Ser at the 5th position is preferably replaced with Thr. The sequence in which Gln at the 3rd position in the amino acid sequence described in SEQ ID NO: 10 is replaced with Glu and Ser at the 5th position is replaced with Thr is shown in SEQ ID NO: 133. (22) An antibody comprising a humanized PM-1 heavy chain variable region in which the amino acid substitutions described in (5), (6), (11) and (21) are made. (23) An antibody comprising a humanized PM-1 light chain variable region in which the amino acid substitutions described in (13), (14), (18) and (19) are made. (24) An antibody comprising the heavy chain variable region described in (22) and the light chain variable region described in (23). (25) An antibody comprising a heavy chain variable region having a CDR1 in which Ser at the 1st position in the amino acid sequence described in SEQ ID NO: 1 is replaced with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Asp is preferred. (25) An antibody comprising a heavy chain variable region having a CDR1 in which Ser at the 1st position in the amino acid sequence described in SEQ ID NO: 1 is replaced with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Asp is preferred. (25) An antibody comprising a heavy chain variable region having a CDR1 in which Ser at the 1st position in the amino acid sequence described in SEQ ID NO: 1 is replaced with another amino acid. The sequence in which the first Ser is substituted with Asp in the amino acid sequence described in SEQ ID NO: 1 is shown as SEQ ID NO: 28. (26) An antibody comprising a heavy chain variable region having a CDR2 in which the 16th Ser in the amino acid sequence described in SEQ ID NO: 2 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution with Gly is preferred. The sequence in which the 16th Ser in the amino acid sequence described in SEQ ID NO: 2 is substituted with Gly is shown as SEQ ID NO: 99. (27) An antibody comprising a heavy chain variable region having a CDR2 in which the 9th Thr and the 16th Ser in the amino acid sequence of (HCDR2) described in SEQ ID NO: 2 are substituted with other amino acids. The substituted amino acid is not particularly limited, but preferably the 9th Thr is substituted with Asn and the 16th Ser is substituted with Gly. The sequence in which the 9th Thr in the amino acid sequence described in SEQ ID NO: 2 is substituted with Asn and the 16th Ser is substituted with Gly is shown as SEQ ID NO: 100. (28) An antibody comprising a light chain variable region having a CDR1 in which the first Arg in the amino acid sequence of (LCDR1) described in SEQ ID NO: 4 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution with Gln is preferred. The sequence in which the first Arg in the amino acid sequence described in SEQ ID NO: 4 is substituted with Gln is shown as SEQ ID NO: 101. (29) An antibody comprising a light chain variable region having a CDR2 in which the 4th Arg in the amino acid sequence described in SEQ ID NO: 5 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. The sequence in which the 4th Arg in the amino acid sequence described in SEQ ID NO: 5 is substituted with Glu is shown as SEQ ID NO: 102. (30) An antibody comprising a light chain variable region having a CDR2 in which the 2nd Thr and the 4th Arg in the amino acid sequence of (LCDR2) described in SEQ ID NO: 5 are substituted with other amino acids. The amino acid after substitution is not particularly limited, but substitution of the second Thr with Gly and the fourth Arg with Glu is preferred. The sequence in which the second Thr is substituted with Gly and the fourth Arg is substituted with Glu in the amino acid sequence set forth in SEQ ID NO: 5 is shown in SEQ ID NO: 103. (31) An antibody comprising a light chain variable region having a CDR3 in which the fifth Thr in the amino acid sequence set forth in SEQ ID NO: 6 (LCDR3) is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Ser is preferred. The sequence in which the fifth Thr in the amino acid sequence set forth in SEQ ID NO: 6 is substituted with Ser is shown in SEQ ID NO: 77. (32) An antibody comprising a heavy chain variable region in which the amino acid substitutions described in (25) and (27) are made. body. (33) An antibody comprising a light chain variable region in which the amino acid substitutions described in (28), (30) and (31) are made. (34) An antibody comprising the heavy chain variable region described in (32) and the light chain variable region described in (33). (35) An antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 104 (VH of H53 / L28). body. (36) An antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 105 (VL of H53 / L28). body. (37) An antibody having the heavy chain variable region described in (35) and the light chain variable region described in (36).

[0203] The amino acid substitutions described in any of the above (1) to (37) are preferably performed on the humanized PM-1 antibody. The present invention provides an antibody comprising at least the amino acid substitutions described in any of the above (1) to (37) and a method for producing the antibody. Therefore, the antibodies of the present invention include antibodies containing amino acid substitutions other than those described in the above (1) to (37) in addition to the amino acid substitutions described in any of the above (1) to (37). The antibodies of the present invention also include antibodies in which a plurality of the amino acid substitutions described in any of the above (1) to (37) are combined. Examples of the amino acid substitutions described in the above (1) to (37) include substitutions of the amino acid sequences of the above FR and CDR. The amino acids Substitutions other than those described above include substitutions, deletions, additions, and / or insertions of FR and CDR sequences other than those described above. Further, substitutions, deletions, additions, and / or insertions of the amino acid sequence of the constant region are also included. Furthermore, as modifications for lowering the isoelectric point without reducing the activity of the anti-IL-6 receptor antibody other than the above amino acid modifications, for example, modifications in which Lys at position 15 and / or Ser at position 16 in the amino acid sequence set forth in SEQ ID NO: 2 are substituted with other amino acids can be mentioned. The amino acids after substitution are not particularly limited, but Lys at position 15 is preferably substituted with Gln, and Ser at position 16 is preferably substituted with Asp. The sequence in which Lys at position 15 and Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 are substituted with Gln and Asp, respectively, is shown in SEQ ID NO: 121. Such amino acid substitutions may also be performed on the amino acid sequence set forth in SEQ ID NO: 100. The sequence in which Lys at position 15 and Gly at position 16 in the amino acid sequence of SEQ ID NO: 100 are substituted with Gln and Asp, respectively, is shown in SEQ ID NO: 122. Therefore, the present invention provides an antibody comprising a heavy chain variable region having a CDR2 in which Lys at position 15 and / or Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 100 is substituted with other amino acids. ​Furthermore, as another modification for lowering the isoelectric point, there can be mentioned a modification in which the 4th Gln in the amino acid sequence set forth in SEQ ID NO: 4 is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. In the amino acid sequence of SEQ ID NO: 4, the 4th Gln is substituted with Glu, and the amino acid sequence is shown as SEQ ID NO: 123. Also, this amino acid substitution may be performed on the amino acid sequence of SEQ ID NO: 101. The amino acid sequence in which the 4th Gln in the amino acid sequence of SEQ ID NO: 101 is substituted with Glu is shown as SEQ ID NO: 124. Therefore, the present invention provides an antibody comprising a light chain variable region having a CDR1 in which the 4th Gln in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 101 is substituted with another amino acid. Furthermore, as another modification for lowering the isoelectric point, there can be mentioned a modification in which the 6th His in the amino acid sequence set forth in SEQ ID NO: 5 is substituted with another amino acid. The amino acid after substitution is not particularly limited, but substitution with Glu is preferred. In the amino acid sequence of SEQ ID NO: 5, the 6th His is substituted with Glu, and the amino acid sequence is shown as SEQ ID NO: 125. Also, this amino acid substitution may be performed on the amino acid sequence of SEQ ID NO: 103. The amino acid sequence in which the 6th His in the amino acid sequence of SEQ ID NO: 103 is substituted with Glu is shown as SEQ ID NO: 126. Therefore, the present invention provides an antibody comprising a light chain variable region having a CDR2 in which the 6th His in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103 is substituted with another amino acid. Furthermore, in the amino acid sequence of the heavy chain FR3 set forth in SEQ ID NO: 90, as a modification for reducing the immunogenicity risk, there can be mentioned a modification in which the 27th Ala (Kabat numbering H89) is substituted with Val. The amino acid sequence in which the 27th Ala in the amino acid sequence set forth in SEQ ID NO: 90 is substituted with Val is shown as SEQ ID NO: 127. Therefore, the present invention provides an antibody comprising a heavy chain variable region having an FR3 in which the 27th Ala in the amino acid sequence of SEQ ID NO: 90 is substituted with Val. ​Also, with respect to the Arg at the 6th position (Kabat numbering H71), which is the only remaining mouse sequence in the amino acid sequence of the heavy chain FR3 described in SEQ ID NO: 9 or SEQ ID NO: 90, by using the human sequence of the human VH1 subclass (SEQ ID NO: 128) or the human VH3 subclass (SEQ ID NO: 129) in which H71 is conserved as the FR3 sequence, it is considered possible to produce an anti-human IL-6 receptor antibody that is completely a human sequence as a framework. Therefore, the present invention provides an antibody comprising a heavy chain variable region having the FR3 described in SEQ ID NO: 128 or SEQ ID NO: 129. Regarding the Arg at the 6th position (Kabat numbering H71), which is the only remaining mouse sequence in the amino acid sequence of the heavy chain FR3 described in SEQ ID NO: 9 or SEQ ID NO: 90, by using the human sequence of the human VH1 subclass (SEQ ID NO: 128) or the human VH3 subclass (SEQ ID NO: 129) in which H71 is conserved as the FR3 sequence, an anti-human IL-6 receptor antibody that is completely a human sequence as a framework can be produced. Therefore, the present invention provides an antibody comprising a heavy chain variable region having the FR3 described in SEQ ID NO: 128 or SEQ ID NO: 129. Furthermore, as a modification for improving stability in the amino acid sequence of the heavy chain FR4 described in SEQ ID NO: 10, a modification of substituting Ser at the 5th position (Kabat numbering H107) with Ile can be mentioned. The amino acid sequence in which Ser at the 5th position in the amino acid sequence described in SEQ ID NO: 10 is substituted with Ile is shown in SEQ ID NO: 130. Also, this amino acid sequence may be performed on the amino acid sequence of SEQ ID NO: 91. The amino acid sequence in which Ser at the 5th position in the amino acid sequence described in SEQ ID NO: 91 is substituted with Ile is shown in SEQ ID NO: 131. Therefore, the present invention provides an antibody comprising a heavy chain variable region having an FR4 in which Ser at the 5th position is substituted with Ile in the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 91. Such amino acid substitutions are for humanized PM-1 antibody, H53 / L28 (an antibody comprising the heavy chain variable region of SEQ ID NO: 104 and the light chain variable region of SEQ ID NO: 105), or PF1 antibody (SEQ ID NO: 22) ​​It is preferably carried out against an antibody comprising the heavy chain variable region of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 23. The present invention provides an antibody comprising at least such amino acid substitutions and a method for producing the antibody. Therefore, the antibodies of the present invention include, in addition to such amino acid substitutions, antibodies comprising the amino acid substitutions described in (1) to (37) above and / or amino acid substitutions other than those described in (1) to (37) above. Examples of amino acid substitutions other than those described in (1) to (37) above include substitutions, deletions, additions and / or insertions of FR and CDR sequences other than those described above. Further, substitutions, deletions, additions and / or insertions of the amino acid sequence of the constant region are exemplified. In addition, substitutions, deletions, additions and / or insertions of the amino acid sequence of the constant region are exemplified.

[0204] <Anti-human IL-6 receptor antibody with a low isoelectric point> The present invention further provides an anti-IL-6 receptor antibody with a low isoelectric point. The antibodies with a low isoelectric point of the present invention include antibodies with a low measured isoelectric point of the full-length antibody and antibodies with a low theoretical isoelectric point of the variable region (VH / VL). Antibodies are included. In the present invention, an anti-IL-6 receptor antibody with a low measured isoelectric point of the full-length antibody is usually an antibody with a measured isoelectric point of 7.5 or less, preferably an antibody with a measured isoelectric point of 7.0 or less, and more preferably an antibody with a measured isoelectric point of 6.0 or less. The measured isoelectric point can be measured by a method known to those skilled in the art. This is possible, for example, by methods such as non-denaturing gel isoelectric focusing or capillary isoelectric focusing. In the present invention, an anti-IL-6 receptor antibody with a low theoretical isoelectric point of the variable region is usually an antibody with a theoretical isoelectric point of 5.5 or less, preferably an antibody with a theoretical isoelectric point of 5.0 or less, and more preferably an antibody with a theoretical isoelectric point of 4.0 or less. The theoretical isoelectric point can be calculated by a method known to those skilled in the art. This is possible, for example, by using software such as GENETYX (GENETYX CORPORATION) to calculate the theoretical isoelectric points of VH and VL of the variable region. This is possible, for example, by using software such as GENETYX (GENETYX CORPORATION) to calculate the theoretical isoelectric points of VH and VL of the variable region. In the anti-IL-6 receptor antibody with a low isoelectric point of the present invention, the amino acid substitutions to be introduced are not particularly limited, and examples thereof include the above-described amino acid substitutions. Such an anti-IL-6 receptor antibody with a low isoelectric point is considered to have improved retention in plasma. The IL-6 receptor is not particularly limited, but a human IL-6 receptor is preferred.

[0205] <Anti-human IL-6 receptor antibody stable at high concentrations> Furthermore, the present invention provides an anti-IL-6 receptor antibody that is stable at high concentrations. In the present invention, "stable at high concentrations" means that in appropriately selected buffer conditions (for example, 20 mM histidine-HCl, 150 mM NaCl) within the range of pH 6.5 to 7.0 suitable for subcutaneous administration, the aggregation ratio per month at 25°C of a high-concentration antibody solution of the anti-IL-6 receptor antibody at 100 mg / mL (gel The increase in the aggregation ratio (aggregate peak area / total peak area × 100) on filtration chromatography is 0.3% or less, preferably 0.2% or less, more preferably 0.1% or less. The concentration of the anti-IL-6 receptor antibody may be 100 mg / mL or more, and may be, for example, 200 mg / mL or 300 mg / mL. The anti-IL-6 receptor antibody that is stable at high concentrations of the present invention is not particularly limited, and can be produced, for example, by the above-described amino acid substitutions. The IL-6 receptor is not particularly limited, but a human IL-6 receptor is preferred.

[0206] The present invention also provides an antibody obtained by further performing an amino acid substitution that improves the binding activity and / or neutralizing activity described in any of the above (a) to (y) on the humanized PM-1 antibody having the amino acid substitution described in any of the above (1) to (37). As one aspect of such an antibody, it has a heavy chain variable region having the amino acid sequence described in SEQ ID NO: 22 (PF1_H), and the sequence Examples include, but are not limited to, an antibody (PF1) having a light chain variable region with the amino acid sequence described in No. 23 (PF1_L).

[0207] Furthermore, the present invention provides an anti-IL-6 receptor antibody as described in any one of (A) to (I) of the following antibodies. (A) CDR1 having the amino acid sequence described in SEQ ID NO: 165 (CDR1 of VH5-M83), SEQ ID NO : CDR2 having the amino acid sequence described in SEQ ID NO: 166 (CDR2 of VH5-M83), and a heavy chain variable region having CDR3 having the amino acid sequence described in SEQ ID NO: 167 (CDR3 of VH5-M83). (B) CDR1 having the amino acid sequence described in SEQ ID NO: 101 (CDR1 of VL5), SEQ ID NO: 1 : CDR2 having the amino acid sequence described in SEQ ID NO: 168 (CDR2 of VL5), and a light chain variable region having CDR3 having the amino acid sequence described in SEQ ID NO: 79 (CDR3 of VL5). (C) An antibody comprising the heavy chain variable region of (A) and the light chain variable region of (B). (D) CDR1 having the amino acid sequence described in SEQ ID NO: 169 (CDR1 of VH3-M73), SEQ ID NO : CDR2 having the amino acid sequence described in SEQ ID NO: 170 (CDR2 of VH3-M73), and a heavy chain variable region having CDR3 having the amino acid sequence described in SEQ ID NO: 171 (CDR3 of VH3-M73). (E) CDR1 having the amino acid sequence described in SEQ ID NO: 172 (CDR1 of VL3), SEQ ID NO: 1 : CDR2 having the amino acid sequence described in SEQ ID NO: 173 (CDR2 of VL3), and a light chain variable region having CDR3 having the amino acid sequence described in SEQ ID NO: 79 (CDR3 of VL3). (F) An antibody comprising the heavy chain variable region of (D) and the light chain variable region of (E). (G) CDR1 having the amino acid sequence described in SEQ ID NO: 169 (CDR1 of VH4-M73), SEQ ID NO :174, a CDR2 having the amino acid sequence (CDR2 of VH4-M73), and a heavy chain variable region having a CDR3 having the amino acid sequence (CDR3 of VH4-M73) described in SEQ ID NO: 171 (H) A CDR1 having the amino acid sequence (CDR1 of VL1) described in SEQ ID NO: 175, SEQ ID NO: 1 73, a CDR2 having the amino acid sequence (CDR2 of VL1), and SEQ ID NO: 79, the amino acid, a light chain variable region having a CDR3 having the acid sequence (CDR3 of VL1) (I) An antibody comprising the heavy chain variable region of (G) and the light chain variable region of (H).

[0208] Furthermore, the present invention provides an anti-IL-6 receptor antibody described in any one of the following (a) to (q). (a) An antibody comprising a heavy chain variable region having the amino acid sequence (H96-IgG1 variable region) described in SEQ ID NO: 159 (b) In the amino acid sequence (H96-IgG1 variable region) described in SEQ ID NO: 159, Trp at position 35 , Tyr at position 51, Ser at position 63, Lys at position 65, Gly at position 66, Val at position 99, Ile at position 103 , an antibody comprising a heavy chain variable region having an amino acid sequence in which at least one amino acid of Tyr at position 108, Glu at position 111, and Thr at position 113 is substituted with another amino acid (c) In the amino acid sequence (H96-IgG1 variable region) described in SEQ ID NO: 159, Lys at position 65 , an antibody comprising a heavy chain variable region having an amino acid sequence in which Gly at position 66, Val at position 99, Ile at position 103, Glu at position 111, and Thr at positio...

Claims

1. 1. A bispecific antibody comprising a first polypeptide having a heavy chain variable region and a light chain variable region and a second polypeptide having a heavy chain variable region and a light chain variable region, At least one amino acid residue selected from the group consisting of the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide and the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the first polypeptide, is negatively charged and is selected from amino acid residues included in the following group (a): (a) Glutamic acid (E), Aspartic acid (D) at least one amino acid residue selected from the group consisting of the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the second polypeptide and the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the second polypeptide, is positively charged or uncharged; The theoretical isoelectric points of the variable regions of the first polypeptide and the second polypeptide are different from each other; The difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 0.43 or more. Bispecific antibodies.

2. the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the first polypeptide is glutamic acid (E) or aspartic acid (D); the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the second polypeptide is positively charged or uncharged; The bispecific antibody of claim 1.

3. the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is glutamic acid (E) or aspartic acid (D); the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the second polypeptide is positively charged or uncharged; The bispecific antibody of claim 2.

4. the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the first polypeptide is glutamic acid (E), and the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide is aspartic acid (D); the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the second polypeptide and the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the second polypeptide are uncharged; The bispecific antibody of claim 3.

5. 1. A bispecific antibody comprising a first polypeptide having a heavy chain variable region and a light chain variable region and a second polypeptide having a heavy chain variable region and a light chain variable region, At least one amino acid residue selected from the group consisting of amino acid residues at positions 61 and 64, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide, and amino acid residues at positions 24, 53, and 54, according to the Kabat numbering, in the light chain variable region of the first polypeptide, is positively charged and is selected from amino acid residues included in the following group (b): (b) Lysine (K), Arginine (R), Histidine (H) at least one amino acid residue selected from the group consisting of amino acid residues at positions 61 and 64, according to the Kabat numbering, in the heavy chain variable region of the second polypeptide, and amino acid residues at positions 24, 53, and 54, according to the Kabat numbering, in the light chain variable region of the second polypeptide, is negatively charged or uncharged; The theoretical isoelectric points of the variable regions of the first polypeptide and the second polypeptide are different from each other; The difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 0.43 or more. Bispecific antibodies.

6. the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is lysine (K); the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the second polypeptide is negatively charged or uncharged; The bispecific antibody of claim 5.

7. the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; The bispecific antibody of claim 6.

8. the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is lysine (K), and the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the second polypeptide and the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide are uncharged; The bispecific antibody of claim 7.

9. the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; The bispecific antibody of claim 5.

10. the amino acid residue at position 54 according to the Kabat numbering in the light chain variable region of the first polypeptide is arginine (R); the amino acid residue at position 54 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; The bispecific antibody of claim 5.

11. The bispecific antibody according to any one of claims 1 to 10, wherein the selected amino acid residue in the first polypeptide is an amino acid residue that may be exposed on the surface of a complementarity determining region (CDR).

12. 12. The bispecific antibody of claim 1, wherein the bispecific antibody is analyzed by standard chromatography to give a peak separate from homodimers of the first polypeptide and homodimers of the second polypeptide.

13. The bispecific antibody according to any one of claims 1 to 12, wherein the difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 0.59 or more.

14. A bispecific antibody described in claim 13, wherein the difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 1.56 or more.

15. 15. The bispecific antibody of any one of claims 1 to 14, which is a humanized antibody or a human antibody.

16. 16. The bispecific antibody of claim 15, comprising a Cκ or Cλ light chain constant region.

17. 17. The bispecific antibody of claim 16, comprising a Cκ light chain constant region.

18. 18. The bispecific antibody according to any one of claims 15 to 17, which is a human antibody comprising the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4.

19. 19. The bispecific antibody of claim 18, which is a human antibody comprising an IgG4 constant region.

20. The bispecific antibody according to any one of claims 1 to 19, which is an anti-Factor IX antibody / anti-Factor X antibody heterodimer.

21. A composition comprising the bispecific antibody of any one of claims 1 to 20 and a pharma- ceutically acceptable carrier.

22. A nucleic acid encoding a polypeptide constituting the bispecific antibody according to any one of claims 1 to 20.

23. A host cell comprising the nucleic acid of claim 22.

24. 24. The host cell of claim 23, wherein the host cell is a CHO cell.

25. A method for producing a bispecific antibody according to any one of claims 1 to 20, comprising culturing a host cell according to claim 23 or 24, and recovering the polypeptide from the cell culture.

26. 1. A method for producing a bispecific antibody comprising a first polypeptide having a heavy chain variable region and a light chain variable region and a second polypeptide having a heavy chain variable region and a light chain variable region, comprising: purifying said bispecific antibody from a mixture containing said bispecific antibody together with homodimers of said first polypeptide and homodimers of said second polypeptide by standard chromatography; At least one amino acid residue selected from the group consisting of the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide and the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the first polypeptide, is negatively charged and is selected from amino acid residues included in the following group (a): (a) Glutamic acid (E), Aspartic acid (D) at least one amino acid residue selected from the group consisting of the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the second polypeptide and the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the second polypeptide, is positively charged or uncharged; The theoretical isoelectric points of the variable regions of the first polypeptide and the second polypeptide are different from each other; The difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 0.43 or more. method.

27. the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the first polypeptide is glutamic acid (E) or aspartic acid (D); the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the second polypeptide is positively charged or uncharged; 27. The method of claim 26.

28. the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is glutamic acid (E) or aspartic acid (D); the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the second polypeptide is positively charged or uncharged; 28. The method of claim 27.

29. the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the first polypeptide is glutamic acid (E), and the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide is aspartic acid (D); the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the second polypeptide and the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the second polypeptide are uncharged; 29. The method of claim 28.

30. 1. A method for producing a bispecific antibody comprising a first polypeptide having a heavy chain variable region and a light chain variable region and a second polypeptide having a heavy chain variable region and a light chain variable region, comprising: purifying said bispecific antibody from a mixture containing said bispecific antibody together with homodimers of said first polypeptide and homodimers of said second polypeptide by standard chromatography; At least one amino acid residue selected from the group consisting of amino acid residues at positions 61 and 64, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide, and amino acid residues at positions 24, 53, and 54, according to the Kabat numbering, in the light chain variable region of the first polypeptide, is positively charged and is selected from amino acid residues included in the following group (b): (b) Lysine (K), Arginine (R), Histidine (H) at least one amino acid residue selected from the group consisting of amino acid residues at positions 61 and 64, according to the Kabat numbering, in the heavy chain variable region of the second polypeptide, and amino acid residues at positions 24, 53, and 54, according to the Kabat numbering, in the light chain variable region of the second polypeptide, is negatively charged or uncharged; The theoretical isoelectric points of the variable regions of the first polypeptide and the second polypeptide are different from each other; The difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 0.43 or more. method.

31. the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is lysine (K); the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the second polypeptide is negatively charged or uncharged; The method of claim 30.

32. the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; 32. The method of claim 31 .

33. the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is lysine (K), and the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the second polypeptide and the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide are uncharged; 33. The method of claim 32.

34. the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; The method of claim 30.

35. the amino acid residue at position 54 according to the Kabat numbering in the light chain variable region of the first polypeptide is arginine (R); the amino acid residue at position 54 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; The method of claim 30.

36. The method according to any one of claims 26 to 35, wherein the selected amino acid residues in the first polypeptide are amino acid residues that may be exposed on the surface of a complementarity determining region (CDR).

37. 37. The method of any one of claims 26 to 36, wherein the bispecific antibody is analyzed using standard chromatography to yield a peak separate from homodimers of the first polypeptide and homodimers of the second polypeptide.

38. The method according to any one of claims 26 to 37, wherein the difference in theoretical isoelectric point between the variable region of the first polypeptide and the variable region of the second polypeptide is 0.59 or more.

39. The method described in claim 38, wherein the difference in theoretical isoelectric point of the variable region of the first polypeptide and the second polypeptide is 1.56 or more.

40. The method according to any one of claims 26 to 39, wherein the antibody is a humanized antibody or a human antibody.

41. The method of claim 40, wherein the light chain constant region comprises Cκ or Cλ.

42. The method of claim 41, comprising a Cκ light chain constant region.

43. The method according to any one of claims 40 to 42, wherein the antibody is a human antibody comprising a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4.

44. The method of claim 43, wherein the antibody is a human antibody comprising an IgG4 constant region.

45. The method according to any one of claims 26 to 44, wherein the antibody is an anti-Factor IX antibody / anti-Factor X antibody heterodimer.

46. 1. A method for purifying a bispecific antibody comprising a first polypeptide having a heavy chain variable region and a light chain variable region and a second polypeptide having a heavy chain variable region and a light chain variable region, comprising: purifying said bispecific antibody from a mixture containing said bispecific antibody together with homodimers of said first polypeptide and homodimers of said second polypeptide by standard chromatography; At least one amino acid residue selected from the group consisting of the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide and the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the first polypeptide, is negatively charged and is selected from amino acid residues included in the following group (a): (a) Glutamic acid (E), Aspartic acid (D) at least one amino acid residue selected from the group consisting of the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the second polypeptide and the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the second polypeptide, is positively charged or uncharged; The theoretical isoelectric points of the variable regions of the first polypeptide and the second polypeptide are different from each other; The difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 0.43 or more. method.

47. the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the first polypeptide is glutamic acid (E) or aspartic acid (D); the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the second polypeptide is positively charged or uncharged; 47. The method of claim 46.

48. the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is glutamic acid (E) or aspartic acid (D); the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the second polypeptide is positively charged or uncharged; 48. The method of claim 47.

49. the amino acid residue at position 55, according to the Kabat numbering, in the light chain variable region of the first polypeptide is glutamic acid (E), and the amino acid residue at position 31, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide is aspartic acid (D); the amino acid residue at position 55 according to the Kabat numbering in the light chain variable region of the second polypeptide and the amino acid residue at position 31 according to the Kabat numbering in the heavy chain variable region of the second polypeptide are uncharged; 49. The method of claim 48.

50. 1. A method for purifying a bispecific antibody comprising a first polypeptide having a heavy chain variable region and a light chain variable region and a second polypeptide having a heavy chain variable region and a light chain variable region, comprising: purifying said bispecific antibody from a mixture containing said bispecific antibody together with homodimers of said first polypeptide and homodimers of said second polypeptide by standard chromatography; At least one amino acid residue selected from the group consisting of amino acid residues at positions 61 and 64, according to the Kabat numbering, in the heavy chain variable region of the first polypeptide, and amino acid residues at positions 24, 53, and 54, according to the Kabat numbering, in the light chain variable region of the first polypeptide, is positively charged and is selected from amino acid residues included in the following group (b): (b) Lysine (K), Arginine (R), Histidine (H) at least one amino acid residue selected from the group consisting of amino acid residues at positions 61 and 64, according to the Kabat numbering, in the heavy chain variable region of the second polypeptide, and amino acid residues at positions 24, 53, and 54, according to the Kabat numbering, in the light chain variable region of the second polypeptide, is negatively charged or uncharged; The theoretical isoelectric points of the variable regions of the first polypeptide and the second polypeptide are different from each other; The difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 0.43 or more. method.

51. the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is lysine (K); the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the second polypeptide is negatively charged or uncharged; 51. The method of claim 50.

52. the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; 52. The method of claim 51.

53. the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the first polypeptide is lysine (K), and the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 61 or 64 according to the Kabat numbering in the heavy chain variable region of the second polypeptide and the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide are uncharged; 53. The method of claim 52.

54. the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the first polypeptide is lysine (K) or arginine (R); the amino acid residue at position 53 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; 51. The method of claim 50.

55. the amino acid residue at position 54 according to the Kabat numbering in the light chain variable region of the first polypeptide is arginine (R); the amino acid residue at position 54 according to the Kabat numbering in the light chain variable region of the second polypeptide is negatively charged or uncharged; 51. The method of claim 50.

56. The method according to any one of claims 46 to 55, wherein the selected amino acid residues in the first polypeptide are amino acid residues that may be exposed on the surface of a complementarity determining region (CDR).

57. 57. The method of any one of claims 46 to 56, wherein the bispecific antibody is analyzed using standard chromatography to yield a peak separate from homodimers of the first polypeptide and homodimers of the second polypeptide.

58. The method according to any one of claims 46 to 57, wherein the difference in theoretical isoelectric point between the variable region of the first polypeptide and the second polypeptide is 0.59 or more.

59. The method described in claim 58, wherein the difference in theoretical isoelectric point between the variable regions of the first polypeptide and the second polypeptide is 1.56 or more.

60. The method of any one of claims 46 to 59, wherein the antibody is a humanized antibody or a human antibody.

61. The method of claim 60, wherein the light chain constant region comprises Cκ or Cλ.

62. The method of claim 61, comprising a Cκ light chain constant region.

63. The method according to any one of claims 60 to 62, wherein the antibody is a human antibody comprising a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4.

64. The method of claim 63, wherein the antibody is a human antibody comprising an IgG4 constant region.

65. The method according to any one of claims 46 to 64, wherein the antibody is an anti-Factor IX antibody / anti-Factor X antibody heterodimer.

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