A method for modifying the isoelectric point of an antibody by amino acid substitution in CDRs.

Abstract

The present invention provides a method for purifying a multispecific polypeptide comprising a first polypeptide and a second polypeptide having a variable region of an antibody. [Solution] A method for purifying a multispecific polypeptide comprising a first polypeptide and a second polypeptide having a variable region of an antibody, wherein the surface of the CDR region of the polypeptide is exposed A method for purifying a multispecific polypeptide by chromatography, comprising: modifying a nucleic acid encoding a polypeptide containing at least one amino acid residue such that the charge of said amino acid residue is altered; modifying both or either of the nucleic acids encoding the amino acid residue of the first polypeptide and the nucleic acid encoding the amino acid residue of the second polypeptide such that the difference in isoelectric points between the first polypeptide and the second polypeptide increases compared to before the modification; culturing a host cell containing the nucleic acid in which either of the above nucleic acids has been modified, so as to express the nucleic acid; and purifying a multispecific polypeptide from the culture by chromatography.

Description

[Technical Field]

[0001] This invention provides an isoelectric effect by amino acid substitution of CDRs, which maintains the antibody's binding activity to the antigen while maintaining its isoelectric activity. The present invention relates to a method for modifying the isoelectric point, a method for controlling the plasma pharmacokinetics (blood pharmacokinetics) of an antibody, a pharmaceutical composition containing an antibody with a modified isoelectric point as an active ingredient, and a method for producing the same. Furthermore, the present invention relates to 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 antibody's CDR region, the antibody's anti-IL-6 receptor - For controlling the plasma half-lives of antibodies, anti-glypican 3 antibodies, and anti-IL-31 receptor antibodies. Antibodies whose plasma half-life is controlled by modification of amino acid residues (anti-IL-6 receptor antibody, anti-glypican-3 antibody, and anti-IL-31 receptor antibody), and pharmaceuticals containing said antibodies as active ingredients. This invention relates to pharmaceutical compositions and methods for producing such 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. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their long half-life in plasma and low incidence of side effects. Among them, IgG-type antibody drugs have been launched in large numbers, and many more antibody drugs are currently under development. (Non-patent Literature 1, Non-patent Literature 2). Most antibody drugs currently on the market are chimeric antibodies, humanized antibodies, or human antibodies. However, many antibody drugs with superior properties are currently being developed by improving humanized antibodies or human antibodies to enhance efficacy, convenience, and cost. Various technologies are being developed that can be applied to these antibody drugs, and technologies that improve effector function, antigen binding ability, pharmacokinetics, and stability, or reduce immunogenicity risk have been reported. As a method to enhance efficacy or reduce dosage, amino acid substitutions in the Fc region of IgG antibodies can be used to affect antibody-dependent cell-mediated cytotoxicity (ADCC activity) and complement-dependent activity. Techniques to enhance cytotoxic activity (CDC activity) have been reported (Non-patent documents 3 and 4). Furthermore, affinity maturation technology (Non-Patent Literature 5) has been reported as a technique to improve antigen binding and antigen neutralization ability, which involves inducing mutations in amino acids such as the CDR region of the variable region. By adding it, it is possible to improve the binding activity to the antigen.

[0003] One of the current problems with antibody drugs is the high manufacturing cost due to the very large amount of protein administered. Furthermore, regarding the form of administration, subcutaneous administration is preferable for chronic autoimmune diseases, but generally, subcutaneous administration requires a high concentration, and IgG... In the case of Ip antibody preparations, it is generally considered that a formulation of around 100 mg / mL is the limit due to stability and other factors (Non-Patent Literature 6). By extending the plasma half-life of the antibody to exert a sustained therapeutic effect, the amount of protein administered can be reduced, enabling subcutaneous administration at long intervals, and making it possible to provide an antibody drug with excellent characteristics that is low-cost and convenient.

[0004] FcRn plays a significant role in the long plasma half-life of antibodies, and it is known that IgG1 and IgG2 have the longest plasma half-lives among antibody isotypes, while IgG3 and IgG4 have shorter half-lives (Non-Patent Document 7). As a method to further extend the plasma half-life of IgG1 and IgG2 antibodies, which have superior plasma half-lives, amino acid substitutions in the constant region that enhance binding to FcRn have been reported (Non-Patent Documents 8, 9, 10). However, introducing artificial amino acid mutations into the constant region presents challenges from the perspective of immunogenicity. In contrast, a method to improve the pharmacokinetics of antibodies by introducing mutations into the amino acids of the variable region of the antibody has recently been reported (Patent Document 1).

[0005] According to Patent Document 1, it is possible to control the pharmacokinetics of IgG by changing its isoelectric point. Therefore, by introducing amino acid substitutions into the framework of the antibody variable region, the antibody can bind to the antigen. It has been reported that it is possible to lower the isoelectric point of an antibody and lengthen its plasma half-life without reducing its activity. Specifically, for example, by introducing amino acid substitutions to H10, H12, H23, H39, H43, and H105 in Kabat numbering, it is possible to lower the isoelectric point of the antibody without reducing its binding activity to the antigen. Furthermore, it is also possible to introduce amino acid mutations to other framework sequences without reducing binding activity, but it was considered that introducing amino acid substitutions to the framework sequence alone may not be sufficient to significantly lower the isoelectric point. This is because, although human antibody sequences are generally used for the framework sequence after amino acid substitution to reduce immunogenicity, human antibody framework sequences are highly conserved and have little diversity, resulting in little freedom in amino acid substitution. Therefore, when lowering the isoelectric point of the antibody by introducing amino acid substitutions to the framework alone is insufficient, it was 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 the 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 exerting strong binding activity to antigens. Generally, amino acid substitutions in the CDR sequences are known to affect the binding activity of the antibody to the antigen. Therefore, it is difficult to lower the isoelectric point of the antibody without significantly reducing the binding activity of the antibody to the antigen by amino acid substitution in the CDR sequences. Also, since the CDR sequences vary greatly depending on the type of antigen, it has been considered extremely difficult to substitute the amino acids in the CDR sequences of the antibody without significantly reducing the binding activity of the antibody to the antigen, 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 involves transplanting the CDR sequences of non-human animal species into human framework sequences, is used. When the humanized antibody obtained by CDR grafting does not show binding activity equivalent to that of the chimeric antibody, it is possible to restore the binding activity by amino acid substitution of 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, since it is widely known that changes in antibody CDR residues due to the isomerization reaction of aspartic acid residues, the deamidation reaction of asparagine residues, and the oxidation reaction of methionine residues in the antibody CDR reduce the binding activity of the antibody to the antigen (Non-Patent Document 12), the CDR sequences are extremely important for the binding activity of the antibody to the antigen. Furthermore, amino acids in the H-chain CDR2 sequence of the antibody Regarding the H-chain CDR2 sequence of the antibody It has been reported that when an amino acid substitution is introduced, antigen-binding activity is often significantly reduced, and antibody expression levels also decrease (Non-Patent Documents 13-15). In particular, when an amino acid substitution is introduced at H51... It has been found that introducing mutations significantly reduces antibody expression (Non-Patent Literature 16). Furthermore, it has been reported that introducing mutations into the H chain CDR3 sequence of antibodies significantly reduces antigen-binding activity in most cases (Non-Patent Literature 17, 18). Additionally, the introduction of mutations into the antibody CDR sequence... When nin scanning is performed, the amino acids present in the CDR are replaced with alanine. In many cases, the binding activity of the antibody to the antigen is significantly reduced (Non-Patent Documents 19-23), and the effect of alanine substitution on the binding activity to the antigen is thought to vary depending on the type of antibody. In other words, generally, amino acid substitution in the CDR sequence of an antibody reduces its binding activity to the antigen. It is believed that the binding activity to the antigen is weakened, and no amino acid substitution sites have been reported to date that do not significantly weaken the binding activity of the antibody to the antigen, regardless of the type of antibody.

[0008] In antibody engineering for creating antibody molecules with superior properties, the CDR sequence of the antibody Amino acid substitutions are almost always performed for the purpose of affinity maturation. Affinity maturation is generally performed on the CDR sequence of an antibody molecule. Presentation of an antibody library containing randomized CDR sequences on phages or ribosomes. A method for obtaining antibodies with improved antigen-binding activity through panning to the antigen. This method involves adding amino acids to the CDR sequence of an antibody to improve its binding activity to the antigen. It is possible to find substitutions (Non-Patent Document 5, 24-26). However, the amino acid substitutions that improve antigen binding activity obtained by this method differ depending on the type of antibody. Therefore, it is necessary to find amino acid substitutions in the CDR sequence that improve antigen binding activity regardless of the type of antibody. No substitution sites have been reported to date. Aside from affinity maturation, antibody expression levels in mammalian cells can be improved by substituting amino acids in the CDR sequence at specific locations. A method for doing so has been reported (Patent Document 2). According to Patent Document 2, the distribution of a CDR at a specific location By substituting amino acids in a sequence with a specific sequence, it is possible to improve the expression level of antibodies in mammalian cells, regardless of the type of antibody. Furthermore, while de-immunization methods have been reported to avoid T-cell epitopes present in the antibody's CDR sequence to reduce its immunogenicity, it is possible to improve the CDR sequence independently of the antibody type and without reducing antibody binding activity. No amino acid substitution method has been reported to remove the T-cell epitopes present within the molecule (Non-patent documents 27, 28).

[0009] Thus, since the CDR sequence of an antibody is deeply involved in binding to the antigen, a reduction in binding activity due to amino acid substitutions in the CDR sequence is common. The effect on synergistic activity varies depending on the type of antibody. Patent Document 1 describes the amino acids in CDR. While examples of isoelectric point control by substitution have been shown, it is possible that this may reduce the antigen-binding activity depending on the type of antibody. Furthermore, although methods to improve antibody expression through common amino acid substitutions independent of antibody type have been reported, there are no reports to date of methods to improve the antibody's antigen-binding activity or to remove T-cell epitopes without significantly reducing the antibody's antigen-binding activity. Moreover, there is a need for antibody CDR sequences that allow for amino acid substitutions independent of antibody type and without significantly reducing the antibody's antigen-binding activity. No reports have been made regarding this matter.

[0010] The prior art documents for this invention are listed below. [Prior art documents] [Non-patent literature]

[0011]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Outdoor Tools 16

Outdoor Track 17

Outdoor Tools 18

Outdoor Tools 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Optional Trademark23

Optional Trademark24

Direct Entries 25

[0012] [Patent Document 1] WO / 2007 / 114319 [Patent Document 2] US / 2006 / 0019342 [Overview of the Initiative] [Problems that the invention aims to solve]

[0013] The present invention has been made in view of the above circumstances, and its object is to provide a method for modifying the isoelectric point of a polypeptide including a variable region of an antibody while maintaining its binding activity to an antigen, a method for controlling the plasma half-life of an antibody, a pharmaceutical composition containing an antibody with a controlled plasma half-life as an active ingredient, and a method for producing the antibody and a pharmaceutical composition containing the antibody as an active ingredient. Furthermore, the present invention relates to an anti-IL-6 receptor antibody, an anti-glypican 3 antibody, and an anti-IL-31 receptor antibody. - Modification of amino acid residues exposed on the surface of the antibody's CDR region to change the half-life of the antibody in plasma. A method for controlling this, an anti-IL-6 receptor antibody, an anti-glypican-3 antibody, and an anti-IL-31 receptor antibody whose plasma decay period is controlled by modification of amino acid residues, a method for producing said antibodies, and said The objective is to provide a pharmaceutical composition containing the antibody as an active ingredient. Furthermore, the present invention relates to the variable region of TOCILIZUMAB, a humanized anti-IL-6 receptor IgG1 antibody, and It is also an objective to provide a pharmaceutical composition consisting of a second-generation molecule superior to TOCILIZUMAB, which enhances antigen neutralization ability and improves plasma retention by modifying the amino acid sequence of the constant region, thereby reducing the frequency of administration and exerting a sustained therapeutic effect, while also improving immunogenicity, safety, and physical properties, as well as a method for producing such pharmaceutical compositions. [Means for solving the problem]

[0014] The inventors have diligently researched a method for modifying the isoelectric point of a polypeptide containing an antibody variable region while maintaining the binding activity of the variable region to the antigen. As a result, the inventors have identified a complementarity-determining region (CDR) of the antibody variable region. We identified specific positions on the CDR amino acid sequence among amino acid residues that can alter the isoelectric point while maintaining the binding activity to the antigen in question. We discovered that the plasma half-life of a polypeptide containing the variable region can be controlled by controlling its isoelectric point, and furthermore, by utilizing the difference in isoelectric points, polypeptides containing the variable region of an antibody, consisting of heteromultimers, can be efficiently produced. Specifically, among the amino acid residues in the amino acid sequence constituting the variable region of the antibody, we found that a particular CDR amino acid sequence can regulate the charge on the surface of the antibody molecule without affecting the antibody's function, such as the antigen-binding activity of the antibody variable region, or its structure. The location was identified. Furthermore, the inventors determined that by adjusting the surface charge of the antibody, isoelectricity could be controlled. By modifying the points, it was confirmed that the plasma half-life of the polypeptide containing the variable region of the antibody can be controlled, and that antibodies with thus controlled plasma half-lives actually retain their binding activity to the antigen. Furthermore, the inventors completed the present invention by confirming that controlling the plasma half-life of antibodies increases the tumor growth inhibitory effect on cancer cells of antibodies and other cytotoxic antibodies. In addition, the charge of the CDR is adjusted. By altering the isoelectric point, a heterodiform antibody consisting of antibodies that bind to two or more different antigens is created. We confirmed that the mar can be isolated and purified.

[0015] Furthermore, the inventors have developed TOCILIZUMAB, a first-generation humanized anti-IL-6 receptor IgG1 antibody. By modifying the amino acid sequences of the variable and constant regions, we have diligently conducted research toward creating a second-generation molecule superior to TOCILIZUMAB, which enhances pharmacological efficacy, improves plasma retention to reduce administration frequency and sustain therapeutic effects, and improves immunogenicity, safety, and physical properties (stability and uniformity). As a result, the inventors have found that TOCILIZUMAB's capabilities We identified multiple CDR mutations in the variant region that improve antigen binding affinity. This combination successfully improved affinity significantly. Furthermore, the inventors succeeded in improving plasma retention by introducing a modification that lowers the isoelectric point of the variable region sequence. In addition, the inventors succeeded in improving the retention of mice remaining in the TOCILIZUMAB framework. In silico predictions of T-cell epitope peptides in the originating sequence and variable region We succeeded in reducing the number of molecules and thereby reducing the risk of immunogenicity. At the same time, we also succeeded in improving stability at high concentrations. Furthermore, the inventors have found that TOCILIZUMAB has a steady-state range... In this study, we succeeded in discovering a novel constant region sequence that minimizes the emergence of new T-cell epitope peptides, does not show binding to the Fcγ receptor, and improves stability under acidic conditions, heterogeneity derived from the disulfide of the hinge region, heterogeneity derived from the C-terminus of the H chain, and stability in high-concentration formulations. Modification of these CDR region amino acid sequences is possible. By combining modifications to the amino acid sequence of the variable region and the amino acid sequence of the constant region, we succeeded in creating a second-generation molecule superior to TOCILIZUMAB.

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

[44] . [1] A method for modifying the isoelectric point of a polypeptide containing a variable region of an antibody while maintaining the binding activity of the variable region to an antigen, 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 comprising the variable region of the antibody further comprises 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. [5] The method according to [1], wherein the polypeptide containing the variable region of the antibody is a multispecific polypeptide that binds to at least two antigens. [6] The method according to [1], wherein the modification of the charge of the amino acid residue is an amino acid substitution. [7] 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 described in (1), [8] Amino acid residues that can be exposed on the surface of the CDR region are selected from the amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region according to Kabat numbering, or from the amino acid residues at positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat numbering. The method described in [1], which is to be detected, is to detect at least one amino acid residue. [9] A polypeptide containing a variable region of an antibody with an altered isoelectric point, obtained by any of the methods described in [1] to [8]. A method for controlling the plasma pharmacokinetics of a polypeptide, comprising modifying the isoelectric point of the polypeptide containing an antibody variable region by any of the methods described in [1] to [8],

[11] Control of the pharmacokinetics is such that plasma clearance (CL), area under the concentration curve (AUC), mean The method described in

[10] , which is an extension or decrease in either the plasma residence time or the plasma half-life (t1 / 2). Polypeptides containing a variable region of an antibody with controlled plasma pharmacokinetics, obtained by the method described in

[12] and

[10] ,

[13] A method for producing a polypeptide containing an antibody variable region with an altered isoelectric point, (a) At least one amino acid residue that may be exposed on the surface of the CDR region of the polypeptide (b) Modify the nucleic acid encoding a polypeptide containing the amino acid residue so that the load is altered, and (b) culture host cells so that the nucleic acid is expressed. (c) Recovering polypeptides containing antibody variable regions from host cell cultures. Methods including

[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 described in

[13] in which the polypeptide containing the variable region of the antibody is an IgG antibody. law,

[16] The method according to

[13] , wherein the polypeptide containing 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 containing 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 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 described in

[13] ,

[20] Amino acid residues that can be exposed on the surface of the CDR region are from the amino acid residues at positions 31, 61, 62, 64 and 65 in the heavy chain variable region according to Kabat numbering, or from the amino acid residues at positions 24, 27, 53, 54 and 55 in the light chain variable region according to Kabat numbering. The method described in

[13] , wherein at least one amino acid residue is selected. A polypeptide containing a variable region of an antibody with an altered isoelectric point, obtained by any of the methods described in

[21] ,

[13] , to

[20] . A method for producing a polypeptide containing an antibody variable region with controlled plasma pharmacokinetics, comprising modifying the isoelectric point of the polypeptide containing the antibody variable region by any of the methods described in

[22] ,

[13] , to

[20] ,

[23] Control of the pharmacokinetics is such that plasma clearance (CL), area under the concentration curve (AUC), mean The method described in

[22] , which is an extension or decrease in either the plasma residence time or the plasma half-life (t1 / 2). Polypeptides containing a variable region of an antibody with controlled plasma pharmacokinetics, manufactured by the method described in

[24] and

[22] .

[25] A method for producing a multispecific polypeptide comprising a first polypeptide and a second polypeptide having a variable region of an antibody, (a) at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide The method involves modifying a nucleic acid encoding a polypeptide containing the amino acid residue such that its charge is altered, wherein the modification of the nucleic acid involves modifying both or either 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 such that the difference in isoelectric points between the first polypeptide and the second polypeptide increases compared to before the modification. (b) Culture host cells so that the nucleic acid is expressed, (c) Recovering multispecific antibodies from host cell cultures. Methods including

[26] The method according to

[25] , wherein the step of recovering a multispecific polypeptide containing a first polypeptide and a second polypeptide from a host cell culture is performed by standard chromatography.

[27] The method according to

[25] , wherein the nucleic acid is modified such that the peaks obtained by analysis using standard chromatography of the homopolymer of the first polypeptide, the homopolymer of the second polypeptide, and the heteropolymer of the first polypeptide and the second polypeptide are more separated than the peaks obtained before modification.

[28] The method according to

[25] , wherein the multispecific polypeptide is a multispecific antibody. Multispecific antibodies produced by the method described in

[29] and

[27] ,

[30] A multispecific antibody as described in

[29] , wherein the multispecific antibody is a bispecific antibody;

[31] A selection from the group consisting of human-derived CDRs, non-human animal-derived CDRs, and synthetic CDRs. The antibody contained a CDR, a human-derived framework region (FR), and a human constant region. The antibody is characterized by having at least one amino acid residue exposed on the surface of the CDR having a different charge from the amino acid residue at the corresponding position of the wild-type CDR, while retaining its binding activity to the antigen compared to the unmodified antibody, and having a modified isoelectric point.

[32] The antibody according to

[31] , wherein the human constant region includes a human Fc region.

[33] The antibody described in

[31] , whose plasma pharmacokinetics are controlled by altering the isoelectric point.

[34] An IgG antibody in which the charge of at least one amino acid residue is modified, selected from the amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region according to Kabat numbering, or from the amino acid residues at positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat numbering, and the isoelectric point is modified compared to before the modification of the amino acid residue. ,

[35] The antibody according to

[34] wherein the modified amino acid residue is selected from amino acid residues belonging to either group (a) or (b) below; (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 the Kabat numbering of the heavy chain variable region of the first polypeptide is at positions 31, 61, and 62 , amino acid residues at positions 64 and 65, or Kabat numbering in the light chain variable region A multispecific antibody in which at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54, and 55 has an electric charge, and the isoelectric points of the first polypeptide and the second polypeptide are different from each other.

[37] Kabat numbering of positions 31 and 61 in the heavy chain variable region of the second polypeptide The amino acid residues at positions 62, 64, and 65, or Kabatan in the light chain variable region. The antibody according to

[36] , wherein the charge of at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54, and 55 by baring is opposite to the charge of the amino acid residue selected in the first polypeptide, or has no charge;

[38] The antibody according to

[36] , wherein the combination of the amino acid residue having the charge and the amino acid residue having the opposite charge is selected from amino acid residues belonging to either group (a) or (b) below; (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 homopolymer of the first polypeptide and the homopolymer of the second polypeptide are separated peaks by analysis using standard chromatography, as described in

[36] . A composition comprising the antibody described in

[40]

[31] to

[39] and a pharmaceutically acceptable carrier, a nucleic acid encoding the polypeptide constituting the antibody described in

[41]

[31] to

[39] , Host cells having nucleic acids as described in

[42] and

[41] , A method for producing antibodies according to

[31] to

[39] , comprising the steps of culturing host cells as described in

[43] and

[42] , and recovering polypeptides from the cell culture,

[44] A polypeptide containing a variable region of an antibody, which can be exposed on the surface of the complementarity-determining region (CDR) of the polypeptide while retaining its binding activity to the antigen. A method for substituting amino acid residues, wherein the Kabat number in at least the heavy chain variable region The amino acid residues at positions 31, 61, 62, 64, and 65 by Kabat numbering, or the amino acid residues at positions 24, 27, 53, 54, and 55 in the light chain variable region by Kabat numbering. A method for substituting at least one amino acid residue, selected from the above.

[0017] Furthermore, the present invention provides the following [1] to

[38] . [1] A method for producing a glypican-3 antibody with controlled pharmacokinetics, comprising the following steps; (a) Modify the 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 may be exposed on the surface of the glypican 3 antibody, (b) Culture host cells that contain the nucleic acid so that the nucleic acid is expressed, (c) A method comprising recovering a glypican 3 antibody from a culture of the host cells, [2] The method according to [1], wherein the control of blood dynamics is the extension or reduction of any of the parameters of blood half-life, mean blood residence time, or blood clearance. [3] Modification of the charge of amino acid residues in step (a) is the method described in [1] by amino acid substitution. [4] The method according to [1], wherein the amino acid residues that can be exposed on the surface of the glypican 3 antibody are located in a region other than the FcRn binding region in the glypican 3 antibody. [5] The method according to [4] wherein the FcRn binding region consists of an Fc region, [6] The method according to [1], wherein the glypican-3 antibody is an IgG antibody. [7] The amino acid residue whose charge is modified is the heavy chain variable region or light chain variable region of the IgG antibody. The method described in [6], which is an amino acid residue in the region. [8] The glypican-3 antibody is a glypican-3 antibody comprising a complementation-determining region (CDR), a human-derived framework region (FR), and a human constant region, wherein the amino acid in step (a) Modification of the residue charge may expose a small amount of the antibody surface in the CDR or FR of the antibody being modified. The method according to [7], characterized in that it involves modifying at least one amino acid residue to an amino acid residue having a different charge from that amino acid residue. [9] The method according to [8], wherein the glypican 3 antibody is an antibody in which the fucose content bound to its Fc region is reduced. Glypican 3 antibody produced by the methods described in

[10] [1] to [9],

[11] A glypican 3 antibody that provides an increase in Tm value, comprising the following steps: complementarity-determining region (CDR), human-derived framework region (FR), and human constant region. A method for stabilizing a glypican-3 antibody characterized by modification of at least one amino acid residue constituting the antibody; (a) Modify the nucleic acid encoding at least one amino acid residue that results in an increase in the Tm value of the glypican 3 antibody subjected to modification, (b) Culture host cells that contain the nucleic acid so that the nucleic acid is expressed, (c) A method comprising recovering antibodies from a culture of the host cells,

[12] The amino acid residue in step (a) is the FR1 region of its H chain or L chain or / The method according to

[11] , characterized in that it is present in the FR2 region. The method according to

[12] , characterized by substituting amino acid residues in the FR2 region of the H chain described in

[13] and

[12] with amino acid residues in the FR2 region of the VH4 subclass, The method according to

[12] , characterized by substituting amino acid residues in the FR2 region of the L chain described in

[14] and

[12] with amino acid residues in the FR2 region of the VK3 subclass,

[15] A method for controlling the cytotoxic activity of an antibody, consisting of the following steps; (a) Modify 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) Culture host cells that contain the nucleic acid so that the nucleic acid is expressed, (c) A method comprising recovering antibodies from a culture of the host cells,

[16] The method according to

[15] , wherein the control of hemodynamics is the control of one of the parameters: blood half-life, mean blood residence time, or blood clearance.

[17] Modification of the charge of the amino acid residue in step (a) is the method described in

[15] by amino acid substitution.

[18] The method according to

[15] , wherein the amino acid residues that can be exposed on the surface of the antibody are located 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 the heavy chain variable region or light chain variable region of the IgG antibody. The method described in

[20] , which is an amino acid residue in the region.

[22] The antibody is derived from a complementation-determining region (CDR) of an animal other than a human, and a human-derived frame. An antibody comprising a work region (FR) and a human constant region, wherein the modification of the charge of amino acid residues in step (a) is such that a small number of residues can be exposed on the antibody surface in the CDR or FR of the antibody subjected to modification. The method according to

[21] , characterized in that it involves modifying at least one amino acid residue to an amino acid residue having a different charge from that amino acid residue.

[23] The method according to

[22] , wherein the antibody has a reduced fucose content bound to the Fc region of the antibody. Antibodies produced by the methods described in

[24] ,

[15] to

[23] ,

[25] The antibody described in

[24] , wherein the antibody is a glypican-3 antibody.

[26] Substitution of one or more of the following amino acid residues in the H chain V region represented by Sequence ID: 195; (a) Substitution of the 19th amino acid residue K with T, (b) Substitution of the 43rd amino acid residue Q with E, (c) Substitution of K, the 63rd amino acid residue, with S. (d) Substitution of the 65th amino acid residue K with Q, (e) Substitution of the 66th amino acid residue G with D, The H chain V region that has been treated, and Substitutions of one or more of the following amino acid residues in the L-chain V region represented by Sequence ID: 201; (f) Substitution of the 27th amino acid residue Q with E, (g) Substitution of K, the 79th amino acid residue, with T. (h) Substitution of R to S at the 82nd amino acid residue, The L chain V region that has been treated, Antibodies containing,

[27] The antibody described in

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

[28] The antibody described in

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

[29] Substitution of one or more of the following amino acid residues in the H chain V region represented by Sequence ID: 195; (a) Substitution of the 43rd amino acid residue Q with K, (b) Substitution of the 52nd amino acid residue, D, with N. (c) Substitution of the 107th amino acid residue Q with R, The H chain V region that has been treated, and Substitutions of one or more of the following amino acid residues in the L-chain V region represented by Sequence ID: 201; (d) Substitution of the 17th amino acid residue E with Q, (e) Substitution of the 27th amino acid residue Q with R, (f) Substitution of the 105th amino acid residue Q with R, The L chain V region that has been treated, Antibodies containing,

[30] The H chain variable region represented by SEQ ID NO: 198 and the L chain represented by SEQ ID NO: 204 The antibody described in

[29] , including a variable region

[31] The antibody according to

[29] , comprising the H chain variable region represented by SEQ ID NO: 199 and the L chain variable region represented by SEQ ID NO: 205.

[32] The antibodies described in

[26] to

[31] that have the C region of a human antibody, A composition comprising the antibody described in

[33] and

[32] , and a pharmaceutically acceptable carrier. Cancer treatment agents containing the antibodies described in

[34] and

[32] as active ingredients,

[35] The cancer is liver cancer, the cancer treatment agent described in

[34] Nucleic acids encoding polypeptides that constitute the antibodies described in

[36] ,

[26] to

[31] , host cells that hold the nucleic acids described in

[37] ,

[36] A method for producing an antibody according to

[26] to

[31] , comprising the steps of culturing the host cells described in

[38] and

[37] , and recovering polypeptides from the cell culture.

[0018] Furthermore, the present invention provides the following [1] to

[41] . [1] An anti-IL-6 receptor antibody as described in any of (a) to (y) below; (a) In the amino acid sequence described in Sequence ID No. 1, the first Ser is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR1, (b) An antibody comprising a heavy chain variable region having a CDR1 in which the 5th Trp in the amino acid sequence described in SEQ ID NO: 1 is substituted with another amino acid, (c) In the amino acid sequence described in Sequence ID No. 2, the first Tyr is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR2, (d) In the amino acid sequence described in Sequence ID No. 2, the 8th Thr is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR2, (e) In the amino acid sequence described in Sequence ID No. 2, the 9th Thr is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR2, (f) In the amino acid sequence described in Sequence ID No. 3, the first Ser is replaced with another amino acid. An antibody containing a heavy chain variable region having CDR3, (g) In the amino acid sequence described in Sequence ID No. 3, the second Leu is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR3, (h) In the amino acid sequence described in Sequence ID No. 3, the 5th Thr is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR3, (i) In the amino acid sequence described in Sequence ID No. 3, the 7th Ala is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR3, (j) In the amino acid sequence described in Sequence ID No. 3, the 8th Met is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR3, (k) An antibody containing a heavy chain variable region having a CDR3 in which the first Ser and fifth Thr in the amino acid sequence described in Sequence ID No. 3 are substituted with other amino acids. (l) An antibody containing a heavy chain variable region having a CDR3 in which the 2nd Leu, 7th Ala, and 8th Met in the amino acid sequence described in Sequence ID No. 3 are substituted with other amino acids. (m) In the amino acid sequence described in Sequence ID No. 4, the first Arg is substituted with another amino acid. An antibody containing a light chain variable region having CDR1, (n) In the amino acid sequence described in Sequence ID No. 4, the fourth Gln is substituted with another amino acid. An antibody containing a light chain variable region having CDR1, (o) In the amino acid sequence described in Sequence ID No. 4, the 9th Tyr is substituted with another amino acid. An antibody containing a light chain variable region having CDR1, (p) In the amino acid sequence described in Sequence ID No. 4, the 11th Asn is substituted with another amino acid. An antibody containing a light chain variable region having CDR1, (q) In the amino acid sequence described in Sequence ID No. 5, the second Thr is substituted with another amino acid. An antibody containing a light chain variable region having CDR2, (r) In the amino acid sequence described in Sequence ID No. 6, the first Gln is substituted with another amino acid. An antibody containing a light chain variable region having CDR3, (s) In the amino acid sequence described in Sequence ID No. 6, the third Gly is substituted with another amino acid. An antibody containing a light chain variable region having CDR3, (t) In the amino acid sequence described in Sequence ID No. 4, the 9th Tyr is substituted with another amino acid. In the amino acid sequences described in CDR1 and SEQ ID NO: 6, the third Gly is replaced by another amino acid An antibody containing a light chain variable region having a CDR3 that is substituted with, (u) An antibody containing a light chain variable region having a CDR3 in which the 5th Thr in the amino acid sequence described in Sequence ID No. 6 is substituted with another amino acid. (v) An antibody containing a light chain variable region having a CDR3 in which the first Gln and fifth Thr in the amino acid sequence described in SEQ ID NO: 6 are substituted with other amino acids. (w) In the amino acid sequence described in Sequence ID No. 2, the 9th Thr is substituted with another amino acid. In CDR2 and the amino acid sequence described in SEQ ID NO: 3, the first Ser and the fifth An antibody containing a heavy chain variable region having a CDR3 in which the Thr of the eye is 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 CDR2 in (e), [2] In the amino acid sequence described in Sequence ID No. 5, the second Thr is substituted with another amino acid. Anti-IL-6 receptor antibodies containing a light chain variable region with CDR2, [3] An anti-IL-6 receptor antibody as described in any of (a) to (y) below; (a) In the amino acid sequence described in Sequence ID No. 7, the 13th Arg is substituted with another amino acid. An antibody containing a heavy chain variable region having FR1, (b) In the amino acid sequence described in Sequence ID No. 7, the 16th Gln is replaced with another amino acid. An antibody containing a heavy chain variable region having FR1, (c) In the amino acid rupture described in Sequence ID No. 7, the 23rd Thr is substituted with another amino acid. An antibody containing a heavy chain variable region having FR1, (d) In the amino acid sequence described in Sequence ID No. 7, the 30th Thr is substituted with another amino acid. An antibody containing a heavy chain variable region having FR1, (e) The amino acid sequence described in Sequence ID No. 7 contains a heavy chain variable region having FR1 in which the 13th Arg, 16th Gln, 23rd Thr, and 30th Thr are substituted with other amino acids. Antibodies, (f) In the amino acid sequence described in Sequence ID No. 8, the 8th Arg is substituted with another amino acid. An antibody containing a heavy chain variable region having FR2, (g) In the amino acid sequence described in Sequence ID No. 9, the fourth Met is substituted with another amino acid. An antibody containing a heavy chain variable region having FR3, (h) In the amino acid sequence described in Sequence ID No. 9, the 5th Leu is substituted with another amino acid. An antibody containing a heavy chain variable region having FR3, (i) In the amino acid sequence described in Sequence ID No. 9, the 16th Arg is substituted with another amino acid. An antibody containing a heavy chain variable region having FR3, (j) In the amino acid sequence described in Sequence ID No. 9, the 27th Val is substituted with another amino acid. An antibody containing a heavy chain variable region having FR3, (k) Antibody containing a heavy chain variable region having FR3 in which the 4th Met, 5th Leu, 16th Arg, and 27th Val in the amino acid sequence described in Sequence ID No. 9 are substituted with other amino acids. body, (l) In the amino acid sequence described in Sequence ID No. 10, the third Gln is substituted with another amino acid. An antibody containing a heavy chain variable region having FR4, (m) In the amino acid sequence described in Sequence ID No. 11, the 18th Arg is substituted with another amino acid. An antibody containing a light chain variable region having FR1, (n) In the amino acid sequence described in SEQ ID NO: 12, the 11th Lys is substituted with another amino acid. An antibody containing a light chain variable region having FR2, (o) In the amino acid sequence described in Sequence ID No. 13, the 23rd Gln is substituted with another amino acid. An antibody containing a light chain variable region having FR3, (p) In the amino acid sequence described in Sequence ID No. 13, the 24th Pro is substituted with another amino acid. An antibody containing a light chain variable region having FR3, (q) In the amino acid sequence described in Sequence ID No. 13, the 27th Ile is substituted with another amino acid. An antibody containing a light chain variable region having FR3, (r) An antibody containing a light chain variable region having FR3 in which the 23rd Gln, 24th Pro, and 27th Ile in the amino acid sequence described in Sequence ID No. 13 are substituted with other amino acids. (s) In the amino acid sequence described in Sequence ID No. 14, the 10th Lys is substituted with another amino acid. An antibody containing a light chain variable region having FR4, (t) In the amino acid sequence described in Sequence ID No. 10, the 5th Ser is substituted with another amino acid. An antibody containing a heavy chain variable region having FR4, (u) An antibody containing a heavy chain variable region having FR4 in which the 3rd Gln and 5th Ser in the amino acid sequence described in Sequence ID No. 10 are substituted with other amino acids. (v) Antibody containing a heavy chain variable region having FR3 having the amino acid sequence described in SEQ ID NO: 184 body, (w) FR1 as described in (e), FR2 as described in (f), FR3 as described in (k), and FR4 as described in (l) or (u) Antibodies containing heavy chain variable regions, (x) An antibody containing a light chain variable region including FR1 as described in (m), FR2 as described in (n), FR3 as described in (r), and FR4 as described in (s), or (y) An antibody comprising the heavy chain variable region described in (w) and the light chain variable region described in (x), [4] An anti-IL-6 receptor antibody as described in any of (a) to (l) below; (a) In the amino acid sequence described in Sequence ID No. 1, the first Ser is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR1, (b) In the amino acid sequence described in Sequence ID No. 2, the 9th Thr is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR2, (c) In the amino acid sequence described in Sequence ID No. 2, the 16th Ser is replaced with another amino acid. An antibody containing a heavy chain variable region having a CDR2, (d) An antibody containing a heavy chain variable region having a CDR2 in which the 9th Thr and 16th Ser in the amino acid sequence described in Sequence ID No. 2 are substituted with other amino acids. (e) In the amino acid sequence described in Sequence ID No. 4, the first Arg is substituted with another amino acid. An antibody containing a light chain variable region having CDR1, (f) In the amino acid sequence described in Sequence ID No. 5, the second Thr is substituted with another amino acid. An antibody containing a light chain variable region having CDR2, (g) In the amino acid sequence described in Sequence ID No. 5, the fourth Arg is substituted with another amino acid. An antibody containing a light chain variable region having CDR2, (h) An antibody containing a light chain variable region having a CDR2 in which the second Thr and fourth Arg in the amino acid sequence described in Sequence ID No. 5 are substituted with other amino acids. (i) In the amino acid sequence described in Sequence ID No. 6, the 5th Thr is substituted with another amino acid. An antibody containing a light chain variable region having CDR3, (j) An antibody comprising a heavy chain variable region including CDR1 as described in (a), CDR2 as described in (d), and CDR3 having the amino acid sequence described in SEQ ID NO: 3, (k) Includes a light chain variable region comprising CDR1 as described in (e), CDR2 as described in (h), and CDR3 as described in (i). Antibodies, or (l) An antibody comprising the heavy chain variable region described in (j) and the light chain variable region described in (k), [5] An anti-IL-6 receptor antibody as described in any of (a) to (f) below; (a) In the amino acid sequence described in Sequence ID No. 1, the first Ser is replaced with another amino acid sequence. An antibody containing a heavy chain variable region including CDR1, CDR2 in which the 9th Thr and 16th Ser in the amino acid sequence 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 described in SEQ ID NO: 3 are substituted with other amino acids. (b) In the amino acid sequence described in Sequence ID No. 4, the first Arg is substituted with another amino acid. In the amino acid sequence described in CDR1, SEQ ID NO: 5, the second Thr and the fourth Arg are different. An antibody comprising a light chain variable region including CDR2 with the amino acid substituted, and CDR3 in which the 1st Gln and 5th Thr in the amino acid sequence described in SEQ ID NO: 6 are substituted with other amino acids. (c) An antibody containing a heavy chain variable region having the amino acid sequence described in SEQ ID NO: 22, (d) An antibody containing a light chain variable region having the amino acid sequence described in SEQ ID NO: 23, (e) An antibody comprising the heavy chain variable region described in (a) and the light chain variable region described in (b), or (f) An antibody comprising the heavy chain variable region described in (c) and the light chain variable region described in (d), [6] The constant region of a human antibody as described in any of (a) to (c) below; (a) In the amino acid sequence described in Sequence ID No. 19, position 329 (EU numbering 446) It is characterized by the absence of both the Gly (number 1) and the 330th (EU numbering 447) Lys. The constant region of human antibodies, (b) In the amino acid sequence described in Sequence ID No. 20, position 325 (EU numbering 446) It is characterized by the absence of both the Gly (number 1) and the 326th (EU numbering 447th) Lys. The constant region of human antibodies, (c) In the amino acid sequence described in Sequence ID No. 21, position 326 (EU numbering 446) It is characterized by the absence of both the Gly (number 1) and the 327th (EU numbering 447th) Lys. The constant region of human antibodies, [7] In the amino acid sequence described in Sequence ID No. 20, position 209 (EU numbering 330) The constant region of IgG2 in which the amino acids at positions 1, 210 (EU numbering 331) and 218 (EU numbering 339) are substituted with other amino acids. [8] In the amino acid sequence described in Sequence ID No. 20, position 276 (EU numbering 397 The IgG2 constant region in which the (th) amino acid is substituted with another amino acid, [9] In the amino acid sequence described in Sequence ID No. 20, the 14th amino acid (EU numbering 131) ), 102nd (EU numbering 219th), and / or 16th (EU numbering The constant region of IgG2 in which amino acid (position 133) is substituted with another amino acid.

[10] In the amino acid sequence described in Sequence ID No. 20, the 20th (EU numbering 137 The 1st (138th in EU numbering) and 21st (138th in EU numbering) amino acids are further placed in positions of other amino acids. The IgG2 constant region described in [9], characterized by being replaced

[11] In the amino acid sequence described in Sequence ID No. 20, the IgG2 constant region in which His at position 147 (EU numbering 268), Arg at position 234 (EU numbering 355), and / or Gln at position 298 (EU numbering 419) is substituted with other amino acids. area,

[12] In the amino acid sequence described in Sequence ID No. 20, positions 209 (EU numbering 330), 210 (EU numbering 331), and 218 (EU numbering 339) ), IgG2 constant region having an amino acid sequence in which the 276th (EU numbering 397th), 14th (EU numbering 131st), 16th (EU numbering 133rd), 102nd (EU numbering 219th), 20th (EU numbering 137th), and 21st (EU numbering 138th) amino acids are substituted with other amino acids,

[13]

[12] The IgG2 constant region having an amino acid sequence in which the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Lys are further missing,

[14] In the amino acid sequence described in Sequence ID No. 20, positions 276 (EU numbering 397), 14 (EU numbering 131), and 16 (EU numbering 133), An amino acid sequence in which the 102nd (EU numbering 219th), 20th (EU numbering 137th), and 21st (EU numbering 138th) amino acids are replaced with other amino acids. The IgG2 constant region,

[15]

[14] The IgG2 constant region having an amino acid sequence in which the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Lys are further missing,

[16] In the amino acid sequence described in Sequence ID No. 20, the 14th (EU numbering 131) Cys, the 16th (EU numbering 133) Arg, the 102nd (EU numbering 219) Cys, the 20th (EU numbering 137) Glu, the 21st (EU numbering 138) Ser, the 147th (EU numbering 268) His, the 234th (EU numbering 355) Arg and the 298th (EU numbering 419) The IgG2 constant region has an amino acid sequence in which Gln of (i) is substituted with another amino acid.

[17]

[16] The IgG2 constant region having an amino acid sequence in which the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Lys are further missing,

[18] In the amino acid sequence described in Sequence ID No. 20, the 14th (EU numbering 131st) Cys, the 16th (EU numbering 133rd) Arg, the 102nd (EU numbering 219th) Cys, the 20th (EU numbering 137th) Glu, the 21st (EU numbering 138th) Ser, the 147th (EU numbering 268th) His, the 234th (EU numbering 355th) Arg, and the 298th (EU numbering 419th) The IgG2 constant region has an amino acid sequence in which the Gln and the 313th (EU numbering 434th) Asn are substituted with other amino acids. In the IgG2 constant region described in

[19] and

[18] , the IgG2 constant region having an amino acid sequence in which the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Lys are further missing,

[20] The IgG4 constant region characterized in that the amino acid at position 289 (EU numbering position 409) in the amino acid sequence described in Sequence ID No. 21 is substituted with another amino acid.

[21] In the amino acid sequence described in Sequence ID No. 21, positions 289 (EU numbering 409), 14, 16, 20, 21, 97, 100, and 102 An IgG4 constant region having an amino acid sequence in which amino acids at positions 103, 104 and 105 (EU numbering 131, 133, 137, 138, 214, 217, 219, 220, 221, 222), 113, 114 and 115 (EU numbering 233, 234, 235) are substituted with other amino acids, and amino acid at position 116 (EU numbering 236) is missing. In the IgG4 constant region described in

[22] and

[21] , the IgG4 constant region further lacking the 326th (EU numbering 446th) Gly and the 327th (EU numbering 447th) Lys,

[23] IgG2 constant region having an amino acid sequence in which the amino acid sequence described in Sequence ID No. 20 has the amino acid sequence in which Ala at position 209 (EU numbering 330), Pro at position 210 (EU numbering 331), Thr at position 218 (EU numbering 339), Cys at position 14 (EU numbering 131), Arg at position 16 (EU numbering 133), Cys at position 102 (EU numbering 219), Glu at position 20 (EU numbering 137), and Ser at position 21 (EU numbering 138) are substituted with other amino acids.

[24]

[23] The IgG2 constant region having an amino acid sequence in which the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Lys are further missing,

[25] In the amino acid sequence described in Sequence ID No. 20, the IgG2 constant region has an amino acid sequence in which the 14th (EU numbering 131) Cys, the 16th (EU numbering 133) Arg, the 102nd (EU numbering 219) Cys, the 20th (EU numbering 137) Glu, and the 21st (EU numbering 138) Ser are replaced with other amino acids.

[26]

[25] The IgG2 constant region having an amino acid sequence in which the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Lys are further missing,

[27] A constant region having the amino acid sequence described in Sequence ID No. 24,

[28] A constant region having the amino acid sequence described in Sequence ID No. 118,

[29] A constant region having the amino acid sequence described in Sequence ID No. 25,

[30] A constant region having the amino acid sequence described in Sequence ID No. 151,

[31] A constant region having the amino acid sequence described in Sequence ID No. 152,

[32] A constant region having the amino acid sequence described in Sequence ID No. 153,

[33] A constant region having the amino acid sequence described in Sequence ID No. 164,

[34] Constant region of a human antibody having the amino acid sequence described in Sequence ID No. 194 (M40ΔGK) ,

[35] Constant region of a human antibody having the amino acid sequence described in Sequence ID No. 192 (M86ΔGK) , An antibody having a constant region as described in any of

[36] , [6], to

[35] ,

[37] The antibody according to

[36] , characterized by binding to the IL-6 receptor.

[38] Anti-human IL-6 receptor antibodies having a binding activity to the IL-6 receptor of 1 nM or less,

[39] Anti-human IL-6 receptor antibodies in which the measured isoelectric point of the full-length antibody is 7.0 or less, or the theoretical isoelectric point of the variable region is 5.0 or less.

[40] Under conditions where the antibody concentration is 100 mg / mL in a buffer solution of 20 mM Histidine-HCl, 150 mM NaCl, pH 6.5-7.0, the increase in the ratio of antibody aggregates after 1 month at 25°C is 0.3% or less. Anti-IL-6 receptor antibody characterized by, A pharmaceutical composition containing an antibody as described in any of

[41] ,

[36] , to

[40] . [Brief explanation of the drawing]

[0019] [Figure 1] This graph shows the BaF / gp130 neutralizing activity of WT and RD_6. [Figure 2]This graph shows the sensorogram of the interaction between rhIL-s6R (R&D systems) and WT. [Figure 3] This graph shows the sensorogram of the interaction between rhIL-s6R (R&D systems) and RD_6. [Figure 4-1] This figure shows a list of CDR mutations that improve affinity and neutralizing activity compared to the wild type (WT). [Figure 4-2] This figure is a continuation of Figure 4-1. [Figure 5] This figure shows a list of CDR mutations that, when combined, improve affinity and neutralizing activity. [Figure 6] This graph shows the BaF / gp130 neutralizing activity of WT and RDC23. [Figure 7] This graph shows the sensorogram of the interaction between rhIL-s6R (R&D systems) and RDC23. [Figure 8] This graph shows the sensorogram of the interaction between rhsIL-6R and WT. [Figure 9] This graph shows the sensorogram of the interaction between rhsIL-6R and RDC23. [Figure 10] This graph shows the sensorogram of the interaction between SR344 and WT. [Figure 11] This graph shows the sensorogram of the interaction between SR344 and RDC23. [Figure 12] This graph shows the BaF / gp130 neutralizing activity of WT and H53L28. [Figure 13] This graph shows the sensorogram of the interaction between SR344 and H53 / L28. [Figure 14] This graph shows the plasma concentration profile after intravenous administration of WT, H53 / L28 to mice. [Figure 15] This graph shows the plasma concentration profile after subcutaneous administration of WT and H53 / L28 to mice. [Figure 16]This graph shows the BaF / gp130 neutralizing activity of WT and PF1. [Figure 17] This graph shows the sensorogram of the interaction between SR344 and PF1. [Figure 18] This graph shows the results of high-concentration stability tests for WT and PF1. [Figure 19] This graph shows the plasma concentration profiles after intravenous administration of WT and PF1 to human IL-6 receptor transgenic mice. [Figure 20] This graph shows the concentration profile of unbound human soluble IL-6 receptor after intravenous administration of WT and PF1 to human IL-6 receptor transgenic mice. [Figure 21] This graph shows the results of gel filtration chromatography analysis of aggregate content for WT-IgG1, WT-IgG2, WT-IgG4, IgG2-M397V, and IgG4-R409K purified by hydrochloric acid elution. [Figure 22] This figure shows the results of cation exchange chromatography (IEC) analysis of WT-IgG1, WT-IgG2, and WT-IgG4. [Figure 23] This figure shows the estimated disulfide bond pattern of the hinge region of WT-IgG2. [Figure 24] This figure shows the estimated disulfide bond pattern of the hinge region of WT-IgG2-SKSC. [Figure 25] This figure shows the results of cation exchange chromatography (IEC) analysis of WT-IgG2 and IgG2-SKSC. [Figure 26] This figure shows the results of cation exchange chromatography (IEC) analysis of humanized PM1 antibody, H chain C-terminal ΔK antibody, and H chain C-terminal ΔGK antibody. [Figure 27] This figure shows a comparison of the binding amounts of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to FcγRI. [Figure 28]This graph shows a comparison of the binding amounts of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to FcγRIIa. [Figure 29] This graph shows a comparison of the binding amounts of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to FcγRIIb. [Figure 30] This graph shows a comparison of the binding amounts of WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK to FcγRIIIa (Val). [Figure 31] This graph shows the increase in aggregate size during high-concentration stability tests for WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK. [Figure 32] This graph shows the increase in Fab fragments during high-concentration stability tests for WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK. [Figure 33] This figure shows the results of cation exchange chromatography (IEC) analysis of WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK. [Figure 34] This graph shows the BaF / gp130 neutralizing activity of WT and F2H / L39-IgG1. [Figure 35] This graph shows the plasma antibody concentration profiles after subcutaneous administration of WT, PF1, and F2H / L39-IgG1 at a dose of 1.0 mg / kg to cynomolgus monkeys. [Figure 36] This graph shows the CRP concentration profiles in cynomolgus monkeys in the WT group and the F2H / L39-IgG1 administration group. [Figure 37] This graph shows the profile of unbound IL-6 receptor concentrations in cynomolgus monkeys in the WT (Worst-Two-Way) and F2H / L39-IgG1-treated groups. [Figure 38] This graph shows the plasma concentration profiles after intravenous administration of WT-IgG1 and WT-M14 to human FcRn transgenic mice. [Figure 39] This graph shows the plasma concentration profiles after intravenous administration of WT-IgG1, WT-M14, and WT-M58 to human FcRn transgenic mice. [Figure 40] This graph shows the plasma concentration profiles after intravenous administration of WT-IgG1, WT-M44, WT-M58, and WT-M73 to human FcRn transgenic mice. [Figure 41] This figure shows the effect of cation exchange chromatography on the constant region of anti-IL-6 receptor antibody WT, anti-IL-6 receptor antibody F2H / L39, anti-IL-31 receptor antibody H0L0, and anti-RANKL antibody DNS on heterogeneity. [Figure 42] This figure shows the effect of cation exchange chromatography on the heterogeneity of the CH1 domain cysteine ​​in the anti-IL-6 receptor antibody WT and the anti-IL-6 receptor antibody F2H / L39. [Figure 43] This figure shows the effect of cysteine ​​in the CH1 domain of the anti-IL-6 receptor antibody WT on the denaturation peak, as evaluated by DSC. [Figure 44] This graph shows the neutralizing activity of TOCILIZUMAB, control, and Fv5-M83 at BaF / gp130. [Figure 45] This graph shows the neutralization activity of TOCILIZUMAB, Fv3-M73, and Fv4-M73 at BaF / gp130. [Figure 46] This graph shows the plasma concentration profiles after intravenous administration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys. [Figure 47] This graph shows the changes in CRP concentration after intravenous administration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys. [Figure 48]This graph shows the changes in the neutralization rate of soluble IL-6 receptors after intravenous administration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys. [Figure 49] This chart was obtained from DSC (Differential Scanning Calorimeter) measurements of the Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody. [Figure 50] These are electrophoretic images of H0L0 antibody and Hspu2.2Lspu2.2(Hu2.2Lu2.2) antibody in high pI isoelectric focusing. [Figure 51] These are electrophoretic images of H0L0 antibody and Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody in low pI isoelectric focusing. [Figure 52] This figure shows the binding activity to the antigen glypican 3 as measured by competitive ELISA using H15L4 antibody and H0L0 antibody. [Figure 53] This figure shows the binding activity to the antigen glypican 3 as measured by competitive ELISA using Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody and H0L0 antibody. [Figure 54] This figure shows the binding activity to the antigen glypican 3 as measured by competitive ELISA using Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody and H0L0 antibody. [Figure 55] This study demonstrates the antitumor effects of H0L0 antibody, Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody, and Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody in a human liver cancer transplant mouse model. [Figure 56] This shows the plasma antibody concentrations of H0L0 antibody, Hspu2.2Lspu2.2 (Hu2.2Lu2.2) antibody, and Hspd1.8Lspd1.6 (Hd1.8Ld1.6) antibody in a human liver cancer transplant mouse model. [Figure 57] This shows the ADCC activity of each test antibody against the human hepatocellular carcinoma cell line Hep G2 cells. [Figure 58]This figure shows the IL-6 receptor neutralizing activity of 6R_b_H1L1, 6R_b_H2L2, 6R_b_H2L3, and 6R_b_H2L4 in BaF / 6R. [Figure 59] This figure shows the binding activity to the antigen glypican 3 as measured by competitive ELISA using GPC_H1L1 antibody and GPC_H2L2 antibody. [Figure 60] This figure shows the binding activity to the antigen glypican 3 as measured by competitive ELISA using GPC_H2L2 antibody and GPC_H3L3 antibody. [Figure 61] This figure shows the peak separation of A-chain and B-chain heterodimer, A-chain homodimer, and B-chain homodimer by cation exchange chromatography of 6R_a_H1H3L3, GPC3_H2H3L3, and 31R_H1aH2aL2. [Modes for carrying out the invention]

[0020] [Embodiments of the Invention] The present invention provides a method for modifying the isoelectric point of a polypeptide containing a variable region of an antibody while maintaining the binding activity of the variable region to an antigen, comprising converting the charge of at least one amino acid residue that may be exposed on the surface of the complementarity-determining region (CDR) of the polypeptide. The present invention provides a method for doing so. The present invention also provides a polypeptide comprising a variable region of an antibody with a modified isoelectric point obtained by the said method (for example, an antibody comprising a CDR selected from the group consisting of human-derived CDRs, non-human animal-derived CDRs and synthetic CDRs, 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 is It is an amino acid residue that has a different charge from the amino acid residue at the corresponding position in the wild-type CDR. This invention provides an antibody in which the isoelectric point has been modified while retaining the binding activity to the antigen compared to the original antibody.

[0021] A preferred embodiment of the present invention involves modifying the charge of at least one amino acid residue that may be exposed on the surface of an antibody. That is, by modifying the charge of an amino acid residue of an antibody and changing its isoelectric point (pI), the plasma pharmacokinetics (blood pharmacokinetics) of the antibody can be controlled. As a result, an antibody with controlled plasma pharmacokinetics can exhibit, for example, superior antitumor activity against cancer cells compared to an uncontrolled antibody.

[0022] In the method of the present invention, maintaining binding activity to the antigen means having at least 80% or more, preferably 85% or more, and particularly preferably 90% or more, of the binding activity of the peptide before modification. Furthermore, it is sufficient that the binding activity is maintained to the extent that the function of the antibody is maintained when the antibody binds to the antigen. For example, the affinity measured at 37°C under physiological conditions should be 100 nM or less, preferably 50 nM or less. More preferably, the isoelectric point is 10 nM or less, and even more preferably 1 nM or less. Whether or not the polypeptide containing the variable region of the antibody with the modified isoelectric point obtained by the method of the present invention retains binding activity to the antigen can be determined by known methods, such as BIACORE (intermolecular interaction analysis), and fine Follicle growth assay, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked immunoassay), RIA (radioactive ion assay) It can be measured by methods such as immunoassay (injection immunoassay) or fluorescence immunoassay.

[0023] Examples of the "polypeptide containing a variable region of an antibody" in the present invention include, but are not limited to, antibodies, small molecule antibodies, and scaffold proteins. In the present invention, the scaffold protein can be any structurally stable peptide that can bind to at least one antigen. Examples of such peptides include antibody variable region fragments, fibronectin, protein A domains, and LDL receptor A domains. In addition to lipocalin, other examples include 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)), and Hosse et al. (Protein Science 15:14-27 (2006)).

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

[0025] In the present invention, "antibodies" include antibodies in which the amino acid sequence has been further modified by amino acid substitution, deletion, addition and / or insertion, etc., in addition to antibodies in which the charge of amino acid residues has been modified as described above. Furthermore, antibodies in which the charge of amino acid residues has been further modified in addition to antibodies in which the amino acid sequence has been modified by amino acid substitution, deletion, addition and / or insertion, or by chimerization or humanization, etc. That is, the modification may be performed simultaneously with the process of humanizing the mouse antibody, or the humanized antibody may be further modified.

[0026] Amino acid substitution, deletion, addition and / or insertion, as well as modifications to 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 their amino acid sequences modified by amino acid substitution, deletion, addition and / or insertion, or by chimerization or humanization.

[0027] The antibody used in this invention may be derived from any animal, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, or camel antibodies. Furthermore, it may also be a modified antibody with substituted amino acid sequences, such as a chimeric antibody, or a humanized antibody. In addition, it may be any antibody, such as an antibody-modified product with various molecules attached, an antibody fragment, or a low-molecular-weight antibody.

[0028] A "chimeric antibody" is an antibody created by combining sequences from different animals. For example, an antibody consisting of the variable (V) heavy and light chain regions of a mouse antibody and the constant (C) heavy and light chain regions of a human antibody can be cited. The creation of chimeric antibodies is well known; for example, antibody V The DNA encoding the region is ligated with the DNA encoding the human antibody C region, and this is used as an expression vector. By incorporating it into a host and introducing it into the host to induce production, chimeric antibodies can be obtained.

[0029] Furthermore, the structure, manufacturing method, etc., of the low-molecular-weight antibody in this invention are not particularly limited as long as it has the ability to bind to an antigen. Some low-molecular-weight antibodies have higher activity than full-length antibodies (Orita et al., Blood (2005) 105: 562-566). In this specification, "low-molecular-weight antibody" is not particularly limited as long as it is a part of a full-length antibody (whole antibody, e.g., whole IgG). Although not required, it is preferable that the antibody fragment 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', and Fv. The amino acid sequence of VH or VL in the antibody fragment may be modified by substitution, deletion, addition and / or insertion. Furthermore, parts of VH and VL may be deleted as long as the ability to bind to the antigen is maintained. For example, among the antibody fragments mentioned above, "Fv" is the smallest antibody fragment containing a complete antigen recognition site and binding site. "Fv" is a dimer (VH-VL dimer) in which one VH and one VL are strongly bound by a non-covalent bond. Three complementary chain determining regions (CDRs) in each variable region form an antigen-binding site on the surface of the VH-VL dimer. Six CDRs confer an antigen-binding site to the antibody. However, one variable Even a region (or half of the Fv containing only the three antigen-specific CDRs) has lower affinity than the entire binding site, but it still has the ability to recognize and bind to the antigen. Therefore, molecules smaller than such an Fv are also included in the low-molecular-weight antibodies of this invention. Furthermore, the variable region of the low-molecular-weight antibody may be chimeric or humanized.

[0030] It is preferable that the low molecular weight antibody contains both VH and VL. Examples of low molecular weight antibodies include antibody fragments such as Fab, Fab', F(ab')2, and Fv, as well as scFv (single-chain Fv) which can be produced using antibody fragments (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: Examples include 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).

[0031] Antibody fragments can be obtained by treating antibodies with enzymes, such as proteases like papain and pepsin (see Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al., Science (1985) 229: 81). Also, the amino acid sequence of the antibody fragment is... It can also be manufactured using genetic engineering based on this.

[0032] Low-molecular-weight antibodies having a modified antibody fragment structure can be constructed using antibody fragments obtained by enzymatic treatment or genetic recombination. Alternatively, the gene encoding the entire low-molecular-weight antibody can be constructed, introduced into an expression vector, and then expressed in a suitable 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] Furthermore, the above "scFv" combines the two variable regions via a linker or the like as needed. It is a single-stranded polypeptide. The two variable regions contained in scFv are usually one VH and one VL, but may also be two VHs or two VLs. Generally, scFv polypeptides have VH and VL The domains contain a linker, thereby separating the VH and VL pairs necessary for antigen binding. It is formed. Typically, to form a paired portion between VH and VL within the same molecule, the linker connecting VH and VL is generally a peptide linker with a length of 10 amino acids or more. However, the linker of scFv in the present invention is not limited to such a peptide linker, as long as it does not hinder the formation of scFv. For a review of scFv, see Pluckthun, *The Pharmacology of Monoclonal Antibody*, Vol. 113 (Rosenburg and Moore ed., Springer Verlag, NY, pp. 269-315 (1994)).

[0034] Furthermore, "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 composed of two polypeptide chains. It is a dimer, and each polypeptide chain has a light chain variable region (VL) and a heavy chain variable region (VH) that are linked together by linkers that are too short to bind to each other, for example, about 5 residues. Because the linker between VL and VH, which are encoded on the same polypeptide chain, is short, they cannot form a single-chain V region fragment and instead form a dimer, resulting in a diabody having two antigen-binding sites. When VL and VH for two different epitopes (a, b) are linked by a linker of about 5 residues in the combinations VLa-VHb and VLb-VHa, and expressed simultaneously, they are secreted as bispecific Db.

[0035] Since the Diabody contains two scFv molecules, it contains four variable regions, and as a result, has two antigen-binding sites. Unlike scFv that do not form dimers, the shape of the Diabody When the goal is to achieve a specific result, the linker connecting VH and VL within each scFv molecule is usually a peptide linker of about 5 amino acids. However, the linker of scFv that forms a Diabody can be such a peptide linker as long as it does not interfere with scFv expression and does not interfere with Diabody formation. It is not limited to Chidrinker.

[0036] Among the various antibody isotypes, IgG antibodies have a sufficiently large molecular weight, therefore, the majority The essential metabolic pathway is not the one via renal excretion. IgG has an Fc region as part of its molecule. Antibodies are known to have a long in vivo half-life because they are recycled through the salvage pathway of the embryonic Fc receptor (FcRn) expressed in endothelial cells such as blood vessels. Antibodies are thought to be metabolized primarily through metabolic pathways 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). In other words, it is thought that IgG antibodies that are nonspecifically taken up by endothelial cells are recycled by binding to FcRn, while IgG antibodies that do not bind are metabolized. The plasma half-life of an IgG antibody whose Fc portion has been modified to reduce its binding activity is shortened. Conversely, to enhance the binding activity to FcRn, the amino acid residues constituting the Fc region of the IgG antibody are modified. By doing so, the plasma half-life of IgG antibodies 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, conventional methods for controlling the plasma pharmacokinetics of IgG antibodies have involved modifying the binding activity to FcRn by altering the amino acid residues constituting the Fc region. However, as shown in the examples below, it has become clear that in the present invention, the plasma half-life of the antibody is highly correlated with pI. In other words, it has been shown that it is possible to control the plasma half-life of the antibody without altering the amino acid sequence constituting Fc, which may lead to the acquisition of immunogenicity if altered.

[0037] While not intended to be bound by any particular theory, the inventors currently believe the following: The rate of nonspecific IgG antibody uptake into endothelial cells is negatively charged. This is thought to depend on the physicochemical Coulomb interaction between the cell surface and the IgG antibody. Therefore, reducing (increasing) the pI of IgG antibodies reduces (increases) the Coulomb interaction. It is thought that the reduction (increase) of nonspecific uptake into endothelial cells, resulting in a decrease (increase) in metabolism in endothelial cells, thereby controlling plasma pharmacokinetics. Since the Coulomb interaction between endothelial cells and the negative charge on the cell surface is a physicochemical interaction, this interaction is not uniquely dependent on the amino acid sequence that constitutes the antibody itself. Therefore, the method for controlling plasma pharmacokinetics discovered in this invention is not applicable only to specific antibodies, but is broadly applicable to any polypeptide containing the variable region of the antibody. Such peptides are preferably those with a molecular weight of 50,000 or more, more preferably those with a molecular weight of 100,000 or more, and even more preferably those with a molecular weight of 140,000 or more. With such peptides, the main metabolic pathway is not renal excretion, and the plasma pharmacokinetic control effect of this invention can be fully obtained. In this invention, the reduction (increase) of Coulomb interaction means an increase (decrease) in the Coulomb force, which is expressed as a repulsive force.

[0038] The polypeptide containing the FcRn binding domain in the present invention is not limited to IgG antibodies, but also Fc receptors Any protein capable of binding to (having binding activity or affinity for) the protein (FcRn) is acceptable. Preferably, the polypeptide containing the FcRn binding region in the present invention is not particularly limited, but is a protein containing the Fc region or Fc-like region of an antibody. A modified Fc region can also be used; for example, the modified Fc region of J Immunol. (1998), 160(2), 1029-35 can be used. Examples of polypeptides containing the FcRn binding region in the present invention include IgG antibodies. Modified versions of these antibodies (proteins) can also be used. However, any protein capable of binding to FcRn is included in the polypeptide containing the FcRn binding region of the present invention. In the present invention, the most preferred example of a polypeptide containing an FcRn binding region is an IgG antibody.

[0039] When using an IgG antibody as the antibody of the present invention, any subtype of IgG-type antibody molecule may be used, and it may also be a multispecific (e.g., bispecific) IgG antibody. Specific antibodies are antibodies that have specificity for two different epitopes, and include not only antibodies that recognize different antigens, but also antibodies that recognize different epitopes on the same antigen. Furthermore, even among antibody molecules, there are those whose primary metabolic pathway is renal excretion, such as scFv and Fab. As mentioned above, in the case of receptacle antibodies, pI cannot control plasma pharmacokinetics. However, the present invention is applicable to any antibody molecular form as long as it is a polypeptide containing a variable region of the antibody in which renal excretion is not the main metabolic pathway. Examples include scFv-Fc, dAb-Fc, and Fc fusion proteins. This can be achieved. Since renal excretion of these molecules is not a major metabolic pathway, it is possible to control plasma pharmacokinetics by altering pI using the method discovered in this invention. The antibody molecules to which this invention can be applied may also be antibody-like molecules. Antibody-like molecules are molecules that exert their 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, and Avimer.

[0040] Furthermore, if the antibody of the present invention is, for example, a bispecific anti-glypican 3 antibody, it can also specifically bind to epitopes of glypican 3 and antigens other than glypican 3. For example, antigens other than glypican 3 include NK cells, cytotoxic T cells, and LAK cells. Surface antigens that specifically bind to these cells can be suitably used to recruit them. For example, the antibody MUSE11 recognizes MUC1, an adenocarcinoma-associated antigen, and the antibody that recognizes LAK cell surface antigens can be suitably used. Using a bispecific antibody produced from the antibody OKT3, LAK cells were used to treat cholangiocarcinoma. It has been shown that damaging activity was exhibited (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). Recognize said MUC1 Instead of the antibody MUSE11, the glypican-3 antibody with improved plasma pharmacokinetics provided by the present invention can be suitably used. Furthermore, as the bispecific glypican-3 antibody provided by the present invention, antibodies that recognize different epitopes of the glypican-3 molecule can also be suitably used.

[0041] The above-mentioned "bispecific antibody" is, for example, one in which the heavy chain variable region and the light chain variable region are single-chain. The antibody may also be a linked structure (e.g., sc(Fv)2). Furthermore, the heavy chain variable region (VH) And the light chain variable region (VL) is linked to scFv (or sc(Fv)2), which is an Fc region (lacking the CH1 domain). It may also be an antibody-like molecule (e.g., scFv-Fc) bound to a constant region. The multispecific antibody has a first polypeptide that is VH1-linker-VL1-Fc, and a second polypeptide that is VH1-linker-VL1-Fc. The thido has a (scFv)2-Fc type structure consisting of VH2-linker-VL2-Fc. Alternatively, it may be an antibody-like molecule in which a single-domain antibody is conjugated to the Fc region (Curr Opin Drug Discov Devel. 2006, 9(2), 184-93).

[0042] In this invention, the alteration of the charge of an amino acid residue can be achieved through amino acid substitution. Amino acid substitution can be performed by the method described below. In this invention, the amino acid residues that may be exposed on the surface of the CDR region to be substituted are: From the perspective of maintaining antigen-binding activity, position 31 in the heavy chain variable region according to Kabat numbering. , the amino acid residues at positions 61, 62, 64, and 65 or the Kabatan in the light chain variable region It is preferable that the substitution is at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54, and 55 by baring. These amino acid residue substitutions are useful because they retain the function (such as antigen-binding activity) that the polypeptide containing the variable region of the antibody had before the amino acid residue substitution, and because 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 a variable region of an antibody by modifying the isoelectric point of the polypeptide. Furthermore, the present invention also includes polypeptides containing a variable region of an antibody with controlled pharmacokinetics obtained by this method.

[0044] In this invention, "controlled plasma pharmacokinetics" means that the plasma pharmacokinetics of the antibody have been modified in a desired direction by comparing the plasma pharmacokinetics of the antibody before and after modification of the amino acids constituting the antibody. That is, if it is desired to extend the half-life of the antibody (in plasma), "control of plasma pharmacokinetics" means that the plasma half-life of the antibody is extended. If it is desired to shorten the plasma half-life of the antibody, "control of plasma pharmacokinetics" means that the plasma half-life of the antibody is shortened.

[0045] In the present invention, whether or not the plasma pharmacokinetics of the antibody are modified in the desired direction, that is, whether or not the plasma pharmacokinetics are controlled as desired, can be appropriately evaluated by conducting pharmacokinetic studies using, for example, mice, rats, rabbits, dogs, monkeys, etc. Furthermore, "extension of plasma half-life" or "shortening of plasma half-life" in the present invention can be more specifically determined by any of the following parameters in addition to the plasma half-life (t1 / 2): mean plasma residence time, plasma clearance (CL), area under the concentration curve (AUC), plasma half-life, etc. This can also be understood ("Pharmacokinetics: Understanding Through Exercises" (Nanzando)). For example, following the instructions included with the in-vivo pharmacokinetic analysis software WinNonlin (Pharsight) Noncompartmental By performing the analysis, the "control of plasma kinetics" provided by the present invention can be appropriately evaluated using these parameters.

[0046] Furthermore, it is possible to sustain the function of antibodies by controlling their plasma pharmacokinetics. For example, by applying the method of the present invention to an antibody having cytotoxic activity, it is possible to sustain its function and adjust the duration of the functions of the original polypeptide, such as cytotoxic activity, antagonist activity, and agonist activity.

[0047] In this invention, "amino acid residues that can be exposed on the surface" usually refers to amino acid residues on the surface of a polypeptide constituting an antibody. "Amino acid residues on the surface of a polypeptide" refers to amino acid residues whose side chains can come into contact with solvent molecules (usually water molecules). It is not necessary for the entire side chain to come into contact with solvent molecules; if even a part of the side chain comes into contact with solvent molecules, that amino acid residue is defined as an amino acid on the surface. Those skilled in the art can create homology models of polypeptides and antibodies using commercially available software for homology modeling, etc. Based on such homology models, amino acid residues on the surface of a polypeptide constituting an appropriate antibody can be suitably selected as "amino acid residues on the surface of a polypeptide."

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

[0049] In the antibody of the present invention, the amino acid residue whose charge should be modified is preferably an amino acid residue that constitutes the heavy chain variable region or the light chain variable region of the antibody. Specifically, suitable examples of such variable regions include the complementarity-determining region (CDR) and the framework region (FR).

[0050] Those skilled in the art can appropriately select surface amino acid residues in the antibody variable region using homology models created by homology modeling or the like. That is, the H chain From among the amino acid residues 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, and H112, which are based on Kabat numbering of the variable region, 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, amino acid residues at positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 69, 72, 73, 74, 76, 77, 82, 85, 87, 89, 90, 107, 110, 112, and 114 can be exemplified as surface amino acids, but the present invention is not limited to these. Furthermore, surface amino acid residues in the H chain CDR The base can be selected by a similar homology model, namely, Kabat numbering. The underlying amino acid residue H97 is exposed on the surface of almost all antibodies. For example, the sequence The 101st serine residue in the H chain CDR of the humanized glypican-3 antibody represented by number 195 corresponds to the amino acid residue in question. Other amino acid residues in the H chain CDR of the humanized glypican-3 antibody represented by sequence number 195 include the 52nd, 54th, 62nd, 63rd, 65th, and 66th a Mino acid residues are preferred examples.

[0051] In the variable region of the light chain, the amino acid residues L1, L3, L7, based on Kabat numbering, Surface amino acid residues in the antibody variable region can be appropriately selected from 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, and L107. For example, the humanized glypican 3 antibody represented by SEQ ID NO: 195, numbers 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 43, 44, 45, 46, 48, 49 50, 54, 62, 65, 68, 70, 71, 73, 74, 75, 79, 81, 82, 84, 85, 86, 90, 105, 108, 110, 111, and 112 can be exemplified as surface amino acids, but the present invention is not limited to these. This will not happen. Furthermore, the surface amino acid residues in the L-chain CDR can be selected by a homology model similar to the homology model that determines the surface amino acid residues in the H-chain CDR. Preferred amino acid residues in the L-chain CDR of the humanized glypican-3 antibody represented by SEQ ID NO: 201 are the 24th, 27th, 33rd, 55th, and 59th amino acid residues.

[0052] In the methods provided by the present invention, "modification" of amino acid residues specifically refers to substituting one original amino acid residue with another, deleting one original amino acid residue, or creating a new amino acid residue. This refers to adding amino acid residues, but more preferably, it refers to substituting the original amino acid residue with another amino acid residue. In other words, "modification of the charge of an amino acid residue" in this invention preferably refers to amino acid substitution.

[0053] The glypican 3 antibody provided by the present invention, in order to perform the "modification of the charge of amino acid residues" described above, for example, the variable region of the H chain constituting the humanized glypican 3 antibody represented by SEQ ID NO: 195 Selected from amino acid residues at positions 19, 43, 52, 54, 62, 63, 65, 66, and 107 in the region, In both cases, the charge of one amino acid residue is suitably modified. Furthermore, for example, 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 variable region of the light chain constituting the humanized glypican-3 antibody represented by Sequence ID No. 201 is suitably modified. Of the aforementioned amino acid residues, amino acid residues other than those whose charge has been modified do not need to be modified if the desired effect of controlling plasma pharmacokinetics is achieved, but they may be appropriately modified to have the same type of charge as the modified amino acid residue, or to have no charge at all.

[0054] In the CDR of the anti-human IL-6 receptor antibody (6R_a_H1L1) provided by the present invention, in order to modify the "charge of amino acid residues" while maintaining binding activity to the antigen, for example, the charge of at least one amino acid residue selected from the 31st, 64th, and 65th amino acid residues in the H chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 221, according to Kabat numbering, is suitably modified. Also, for example, the charge of at least one amino acid residue selected from the 24th, 27th, 53rd, and 55th amino acid residues in the L chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 224, according to Kabat numbering, is suitably modified. Of the above amino acid residues, amino acid residues other than those whose charge has been modified do not need to be modified if the desired effect of controlling plasma pharmacokinetics is obtained, but they may be appropriately modified to have the same type of charge as the modified amino acid residue, or to have no charge.

[0055] In the CDR of the anti-human IL-6 receptor antibody (6R_b_H1L1) provided by the present invention, in order to modify the charge of the amino acid residue while maintaining the binding activity to the antigen, for example, the kabata in the H chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 227 is used. The charge of at least one amino acid residue selected from the 31st amino acid residue by kabat numbering is suitably modified. In addition, for example, the charge of at least one amino acid residue selected from the 24th, 53rd, 54th, and 55th amino acid residues by kabat numbering in the L chain variable region constituting the anti-human IL-6 receptor antibody represented by SEQ ID NO: 229 is suitably modified. Of the aforementioned amino acid residues, amino acid residues other than those whose charge has been modified do not need to be modified if the desired effect of controlling plasma pharmacokinetics is achieved, but they may be appropriately modified to have the same type of charge as the modified amino acid residue, or to have no charge at all.

[0056] In the CDR 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, at least one amino acid residue 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 has its charge preferably modified. Also, for example, at least one amino acid residue 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 has its charge 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 can be appropriately modified to have the same type of charge as the modified amino acid residue or no charge. In the heavy chain variable region constituting the anti-human GPC3 antibody represented by SEQ ID NO: 233, at least one amino acid residue selected from the 61st, 62nd, 64th, and 65th amino acid residues according to Kabat numbering has its charge preferably modified. Also, for example, in the light chain variable region constituting the anti-human GPC3 antibody represented by SEQ ID NO: 236, at least one amino acid residue selected from the 24th and 27th amino acid residues according to Kabat numbering has its charge 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 can be appropriately modified to have the same type of charge as the modified amino acid residue or 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, at least one amino acid residue 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 has its charge preferably modified. Also, for example, at least one amino acid residue 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 has its charge 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 can be appropriately modified to have the same type of charge as the modified amino acid residue or no charge. In the heavy chain variable region constituting the anti-human IL-31 receptor antibody represented by SEQ ID NO: 239, at least one amino acid residue selected from the 61st, 62nd, 64th, and 65th amino acid residues according to Kabat numbering has its charge preferably modified. Also, for example, at least one amino acid residue 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 has its charge 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 can be appropriately modified to have the same type of charge as the modified amino acid residue or no charge.

[0058] It is known that some amino acids carry a charge. Generally, the positively charged amino acids (cationic amino acids) include lysine (K), arginine (R), and histidine (H). The negatively charged amino acids (anionic amino acids) include aspartic acid (D), glutamic acid (E), etc. Amino acids other than these are known as uncharged amino acids.

[0059] The above-mentioned "modified amino acid residue" is preferably appropriately selected from amino acid residues included in any 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] 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, 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. 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 subjected to the modification.

[0062] The following are preferred examples of "modification of amino acid residue charge" in the glypican-3 antibody provided by the present invention. As a modification to increase the pI value, for example, at least one substitution of Q43K, D52N, and Q107R in the H chain variable region constituting the humanized glypican-3 antibody represented by SEQ ID NO: 195 can be performed, and it is particularly preferable to modify it to the amino acid sequence represented by SEQ ID NO: 198. Alternatively, for example, at least one substitution of E17Q, Q27R, and Q105R in the L chain variable region constituting the humanized glypican-3 antibody represented by SEQ ID NO: 201 can be performed, and it is particularly preferable to modify it to the amino acid sequence represented by SEQ ID NO: 204. On the other hand, as a modification to decrease the pI value, at least one substitution of K19T, Q43E, K63S, K65Q, and G66D in the H chain variable region constituting the humanized glypican-3 antibody represented by SEQ ID NO: 195 can be performed. It can be modified to the amino acid sequence represented by SEQ ID NO: 197, and is particularly preferably modified to the amino acid sequence represented by SEQ ID NO: 197. Also, for example, the L chain of the humanized glypican 3 antibody represented by SEQ ID NO: 201 is variable. At least one substitution of Q27E, K79T, and R82S in the region can be made, and is particularly preferably modified to the amino acid sequence represented by SEQ ID NO: 203.

[0063] The "amino acid residues" in the anti-human IL-6 receptor antibody (6R_a_H1L1) provided by the present invention A suitable example of "charge modification" is at least one amino acid substitution from the amino acid substitutions listed in Table 20.

[0064] The "amino acid residues" in the anti-human IL-6 receptor antibody (6R_b_H1L1) provided by the present invention A suitable example of "charge modification" is at least one amino acid substitution from the amino acid substitutions listed in Table 22.

[0065] A preferred example of "modification of amino acid residue charge" in the anti-human GPC3 antibody provided by the present invention is at least one amino acid substitution from among the amino acid substitutions listed in Table 24.

[0066] "Modification of amino acid residue charge" in the anti-human IL-31 receptor antibody provided by the present invention. A suitable example of this is at least one amino acid substitution from the amino acid substitutions listed in Table 27.

[0067] The number of amino acid residues to be modified in the present invention is not particularly limited, but for example, when modifying the variable region of an antibody, it is preferable to modify only the minimum number of amino acid residues necessary to achieve the desired controlled plasma pharmacokinetics in order to avoid reducing antigen binding activity and increasing immunogenicity. It is also preferable to appropriately combine modifications of amino acid residues that increase antigen binding activity and modifications of amino acid residues that decrease immunogenicity.

[0068] Known methods can be used to measure the antigen-binding activity of antibodies. For example, ELISA ( Methods such as enzyme-linked immunosorbent assay (EMI), EIA (enzyme immunoassay), RIA (radioimmunoassay), or fluorescence immunoassay can be used. These methods are described in the general instruction manual, "Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988".

[0069] One method for measuring the binding activity of antibodies against cells is described on pages 359-420 of Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). Specifically, BIACORE uses cells as antigens, and cells... Growth assays, such as ELISA and FACS (fluorescence-activated cell sorting), can be used for evaluation. In the ELISA format, the antibody binding activity to cells is evaluated by enzymes. The reaction is quantitatively evaluated by comparing the signal levels produced by the reaction. Specifically, the test antibody is added to an ELISA plate immobilized with each overexpression cell, and the cells that recognize the test antibody... Using enzyme-labeled antibodies, antibodies bound to cells are detected. Alternatively, in FACS, a dilution series of the test antibody is prepared, and the antibody binding titer to each overexpression cell is determined. This allows us to compare the binding activity to cells.

[0070] It is expressed on the surface of cells suspended in a buffer, etc., and is not bound to a carrier such as an ELISA plate. The binding of an antigen to an antibody against that antigen can be measured using the FACS format. Examples of flow cytometers used for such measurements include FACSCanto. TM II, FACSAria TM FACSArray TM FACSVantage TM SE, FACSCalibur TM Examples include (BD Biosciences), EPICS ALTRA HyPerSort, Cytomics FC 500, EPICS XL-MCL ADC, EPICS XL ADC, Cell Lab Quanta / Cell Lab Quanta SC (all from Beckman Coulter).

[0071] One example of a suitable method for measuring the binding activity of an antibody to an antigen is to react cells expressing the antigen with a test antibody, stain the cells with a FITC-labeled secondary antibody that recognizes the test antibody, measure the activity using FACSCalibur (BD), and analyze the fluorescence intensity using CELL QUEST Software (BD). According to this method, the antigen on the surface of the antigen-expressing cell When staining with a FITC-labeled secondary antibody that specifically recognizes the test antibody bound to it, and then measuring the fluorescence intensity using FACSCalibur, the Geometric Mean value (test Geo-Mean value) obtained by 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 formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELLQUEST Software User's Guide (BD biosciences).

[0072] Furthermore, in order to avoid increasing the immunogenicity of the antibody in the body of the person to whom it is administered, it is preferable that the modified amino acid sequence be a human sequence (a sequence found in naturally occurring antibodies of human origin), but the present invention is not limited thereto. Moreover, mutations can be suitably introduced at locations other than those modified to change the isoelectric point, so that each of the multiple FRs (FR1, FR2, FR3, FR4) becomes a human sequence after modification. A method for replacing each FR with a human sequence in this manner has been reported in 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). Furthermore, in order to alter the isoelectric point of the antibody, the charge of each FR may be changed by modifying it with another human FR (for example, FR3 may be replaced with another human FR to lower the isoelectric point of the antibody). Such humanization methods have been reported in non-patent literature (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] Furthermore, if slight modifications to the surface charge do not result in the desired controlled plasma pharmacokinetics, the desired antibody exhibiting the desired controlled plasma pharmacokinetics can be suitably obtained by repeatedly modifying the surface charge and evaluating the plasma pharmacokinetics.

[0074] Non-licensed literature (He XY, Xu Z, Melrose J, Mullowney A, Vasquez M, Queen C, Vexler V, In "Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin. J Immunol. (1998), 160(2), 1029-35)", the plasma pharmacokinetics of chimeric EP5C7.g4, a chimeric antibody (IgG4) of anti-E,P-selectin, and HuEP5C7.g4, a humanized antibody (IgG4), were compared in rhesus monkeys, and it was shown that the plasma pharmacokinetics of both were equivalent. Furthermore, a non-patent document (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) compares the plasma pharmacokinetics of ch5d8, a chimeric antibody of anti-CD154, and Hu5c8, a humanized antibody, in cynomolgus monkeys, showing that the plasma pharmacokinetics of both are equivalent. 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) shows that the plasma pharmacokinetics of the chimeric antibody cCC49 and the humanized antibody HuCC49 are equivalent in mice.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) also 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 with mouse FcRn, it is considered that the plasma pharmacokinetics and distribution of the same chimeric antibody and the same 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, using the method discovered in the present invention, when humanizing a chimeric antibody, the pI of the antibody is modified by modifying amino acid residues that can be exposed on the surface of the chimeric antibody. This allows for the production of a humanized antibody with controlled plasma pharmacokinetics (i.e., with an extended or shortened plasma half-life) compared to the chimeric antibody. The modification of amino acids that can be exposed on the surface of the humanized antibody to control plasma pharmacokinetics may be carried out simultaneously with the humanization of the antibody, or the pI of the humanized antibody may be further modified by modifying amino acid residues that can be exposed on the surface of the humanized antibody, using the humanized antibody as a starting material. The isoelectric point value in this invention can be measured by isoelectric electrophoresis, which is known to those skilled in the art. Furthermore, the theoretical isoelectric point value can be calculated using gene and amino acid sequence analysis software (such as Genetyx). In this invention, for example, sufficient plasma pharmacokinetics... This is useful when it is necessary to significantly change the isoelectric point, such as for precise control. It is particularly preferable when it is necessary to change the isoelectric point value by 1.0 or more from the theoretical isoelectric point value, and more preferably when it is necessary to change it by 3.0 or more.

[0076] Non-patent literature (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 three humanizations performed using the FR sequence of the same human antibody. It has been noted that the plasma pharmacokinetics of the humanized antibodies trastuzumab, bevacizumab, and pertuzumab are nearly equivalent. That is, when humanization is performed using the same FR sequence, the plasma pharmacokinetics are nearly equivalent. According to the method discovered in the present invention, in addition to the humanization process described above, it is possible to control the drug (in plasma) concentration by modifying the antibody's pI by modifying amino acid residues that can be exposed on the antibody surface.

[0077] Furthermore, the method of the present invention can also be applied to human antibodies. By modifying amino acid residues that can be exposed on the surface of human antibodies produced from human antibody libraries or human antibody-producing mice, the pI of the human antibody is altered, making it possible to produce human antibodies whose plasma pharmacokinetics are controlled compared to the plasma pharmacokinetics of the initially produced human antibody (i.e., their plasma half-life is extended or their plasma pharmacokinetics are shortened).

[0078] A decrease in the pI value of an antibody extends its plasma half-life. Conversely, an increase in the pI value of an antibody shortens its plasma half-life and improves its tissue distribution (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, since the immunogenicity of the antibody increases, as does its intracellular endocrinogenesis activity, the exertion of its activities such as ADCC and CDC activity is hindered by intracellular endocrinogenesis. Further improvements were needed to apply this to antibodies that exert their effects on cancer treatment through mechanisms such as cytotoxic activity inhibited by cellular activity. Specifically, improvements were needed to enhance the activity of ADCC and CDC. For antibodies that exert their effects on cancer treatment through mechanisms such as cytotoxicity, where internalization activity into cells inhibits their efficacy, it was unknown whether an increase or decrease in the pI value would enhance the tumor suppressive effect. In this invention, modified humanized antibodies with decreased pI values ​​and modified humanized antibodies with increased pI values ​​were created, and their antitumor effects were compared to verify which modification yielded a higher tumor suppressive effect. Surprisingly, the results showed that the humanized antibody with decreased pI values ​​exhibited a superior effect against liver cancer.

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

[0080] As an example of modifications aimed at improving the properties of the antibody provided by the present invention, modifications aimed at increasing the stability of the antibody (hereinafter referred to as stability modifications) are preferably mentioned. Antibodies in aqueous solution are in equilibrium between two states: the native state and an inactive, denatured state. The stability of the native state is expressed by the second law of thermodynamics (ΔG = ΔH - TΔS), and consists of the change in the Gibbs free energy of the system ΔG and its components: the change in enthalpy ΔH (due to changes in hydrophobic interactions and hydrogen bonds in the polypeptide chain) and the change in entropy ΔS (due to changes in solvation and the degree of freedom of the stereostructure). It depends on the balance (caused by the change). A positive ΔG value indicates that the protein is in its natural state. This means that it is more stable than the degraded state of quality, and if ΔG shows a larger positive value, then The stability of the protein in its native state is further increased. For a protein to denature, the forces contributing to this stabilization must be broken. For example, exposing a protein solution to high temperatures increases the degrees of freedom of its three-dimensional structure, weakening the factors that contribute to protein stabilization and causing thermal denaturation of the protein. In this case, the -TΔS term governs the denaturation. The ΔH of unfolding due to thermal denaturation of a protein is as described herein. As specifically described in the examples, it can be measured directly using DSC (differential scanning calorimetry). The DSC curve in the thermal denaturation process of a protein shows endothermic activity around a temperature specific to the test protein called the denaturation midpoint (Tm). This is the peak. By integrating this peak, the denaturation enthalpy change is obtained. Generally, the Tm value is one indicator of thermal stability. When a protein undergoes thermal denaturation by DSC... The change in heat capacity (ΔCp) can also be measured. The change in heat capacity associated with thermal denaturation is mainly due to the hydration of amino acid residues that are not exposed on the molecular surface when the protein is in its natural state, as they become exposed to solvent molecules during protein denaturation.

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

[0082] The glypican 3 antibody provided by the present invention, in order to perform the "stability modification" described above, for example, modifies 37 and 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 amino acid residues 48 and 51 is preferably modified. Also, for example, at least one amino acid residue selected from amino acid residues 2, 25, 42, 48, 50, 83, and 84 in the variable region of the light chain constituting the humanized glypican 3 antibody represented by Sequence ID No. 201 is preferably modified. Another amino acid residue is suitably modified. Of the aforementioned amino acid residues, those other than the amino acid residue whose stability has been modified do not need to be modified if the desired Tm value is obtained, but they may be appropriately modified to have a Tm value that is similar to or higher than that of the humanized glypican 3 antibody used for modification.

[0083] Stability modification can be carried out by randomly modifying each amino acid residue constituting the humanized antibody to be modified. Alternatively, it can be carried out by substituting a portion of the amino acid sequence constituting the humanized antibody to be modified with an amino acid sequence constituting an existing antibody with a high Tm value, and which corresponds to a portion of the amino acid sequence of the humanized antibody to be modified from the perspective of the conformational relationship of the antibody. The position of the substituted amino acid residue is not limited, but amino acid residues in the FR region can be preferably modified. Furthermore, amino acid residues in the CDR region... Even if the base is modified, it can be appropriately modified as long as it does not result in a reduction of antigen-binding activity. Furthermore, the number of amino acid residues to be modified is not particularly limited and can also be carried out by substituting a specific segment of the FR region with a desired segment. This can involve modifying all of the FR1, FR2, FR3, and FR4 segments of the FR region, or it can be carried out by a combination of modifications to one or more of these segments.

[0084] When modifying the FR region segment, the FR2 region of the H chain or L chain is a suitable example. For example, modifications of amino acid residues to change the FR2 of the H chain of a humanized glypican 3 antibody having the VH1b subclass represented by SEQ ID NO: 195 to the VH4 subclass, i.e., V37I by substituting valine at position 37 with isoleucine, and similarly modifications of A40P, M48I, and L51I are preferred. A concrete example is the subclass of VK2 represented by array array 201. Modification of the L-chain FR2 region of humanized glypican 3 antibodies containing VK3 to the VK3 subclass, i.e. Suitable examples include modifications to L42Q, S48A, and Q50R, as well as modifications to V2I, which corresponds to modifications to the germline sequence of FR1.

[0085] Substitution, deletion, addition and / or insertion of amino acid residues constituting antibodies, as well as modification of amino acid sequences such as humanization and chimerization, can all be suitably carried out by methods known to those skilled in the art. Similarly, when preparing the antibodies provided by the present invention as recombinant antibodies, substitution, deletion, addition and / or insertion of amino acid residues constituting the variable region and constant region of the antibody can be suitably carried out.

[0086] In this invention, antibodies derived from any animal, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, and camel antibodies, can be suitably used. Furthermore, modified antibodies in which the amino acid sequence has been substituted, such as chimeric antibodies, especially humanized antibodies, can also be suitably used. Antibody modifications to which various molecules are bound can also be suitably used.

[0087] A "chimeric antibody" is an antibody created by combining sequences from different animals. For example, an antibody composed of the variable (V) regions of the H and L chains of a mouse antibody and the constant (C) regions of the H and L chains of a human antibody is a suitable example. Methods for producing chimeric antibodies are well known. For example, 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 vector then... By culturing host cells, chimeric antibodies can be appropriately obtained or isolated from the culture medium.

[0088] Humanized antibodies, also known as reshaped human antibodies, are antibodies in which the complementarity determining region (CDR) of an antibody isolated from a non-human mammal, such as a mouse, is linked to the framework region (FR) of a human antibody. The DNA sequence encoding a humanized antibody uses multiple oligonucleotides as a template. It can be synthesized by the overlap PCR reaction used. Materials for the overlap PCR reaction, The implementation method is described in WO98 / 13388, etc. The DNA encoding the variable region of the humanized antibody of the present invention is prepared to have multiple nucleotide sequences that overlap each other. Obtained from oligonucleotides by overlap PCR, this represents the constant region of human antibodies. The coding DNA is concatenated so that the codon sequence is formed in frame. The ligated DNA is then inserted into an expression vector so that the DNA is expressed, and then introduced into the host.

[0089] Methods for identifying CDRs are publicly known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al., Nature (1989) 342, 877). General genetic engineering techniques are also publicly known (see European Patent Application Publication No. EP 125023, WO 96 / 02576). These publicly known methods... By using this method, for example, the CDR of antibodies obtained from non-human animals such as mouse antibodies can be calculated. After the determination is made, DNA encoding a recombinant antibody is constructed by linking the CDR and the FR of the human antibody. 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 CDR of the reconstituted human antibody forms an appropriate antigen-binding site. As such, the amino acid residues of FR in the variable region of the antibody can be modified as appropriate (Sato et al., Cancer Res. (1993) 53, 851-6). The amino acid residues in FR that are used for modification are those that directly bind to the antigen via non-covalent bonds (Amit et al., Science (1986) 233, 747-53). This includes residues that affect or act on the CDR structure (Chothia et al., J. Mol. Biol. (1987) 196, 901-17) and residues related to VH-VL interactions (Japanese Patent Publication No. EP239400).

[0090] The DNA in question is transformed or transduced by a commonly used expression vector into which the DNA is inserted. Humanized antibodies that encode the DNA produced by the host cells are used when the host cells are cultured. This isolates it from the culture medium.

[0091] When the antibody provided by the present invention is an antibody, a humanized antibody, or a human antibody, a C region derived from a human antibody is preferably used as the C region of the 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 may be appropriately modified to improve the stability of the antibody or its production. The chimeric antibody provided by this invention combines the V region of an antibody obtained from a non-human mammal with human antibodies. The antibody is preferably composed of the C region. On the other hand, humanized antibodies are preferably made from non-human mammals. The humanized antibody is preferably composed of the CDR of an antibody obtained from a dairy animal and the FR and C regions of a human antibody. The human antibody is preferably composed of the CDR of an antibody obtained from a human and the FR and C regions of a human antibody. The C region of a human antibody consists of a unique amino acid sequence corresponding to isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. Any C region of an antibody belonging to any isotype can be suitably used as the C region of the humanized antibody provided by the present invention. Preferably, the C region of human IgG is used, but it is not limited thereto. Furthermore, the FR of a human antibody used as the FR of a humanized antibody or human antibody is not particularly limited, and any FR of an antibody belonging to any isotype can be suitably used.

[0092] The method is similar to that described in 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) for the purpose of reducing immunogenicity. By using the method described above, it may also be possible to replace all or part of the amino acid residues constituting the FR region with germline sequences. Based on the reasonable prediction that germline sequences will have low immunogenicity, the amino acid sequences constituting the FR region of the humanized antibody are compared with the germline amino acid sequences by alignment (Abhinandan KR and Martin CR, J. Mol. Biol., (2007) 369, 852-862). Loss of binding activity to the antigen. Within the scope of the comparison, the amino acid residues constituting the FR region of different humanized antibodies. However, these can be substituted with amino acid residues in the germline sequence. Specific examples include modifications in which the 70th L is replaced with I, the 87th T with R, and the 97th T with A in the H chain variable region represented by SEQ ID NO: 195. Also, SEQ ID NO: 201 Modifications such as substituting the 25th S with A among the amino acid residues that make up the L chain variable region represented by [the symbol]. These are some examples.

[0093] The variable and constant regions of the modified chimeric antibodies, humanized antibodies, and human antibodies provided by the present invention are preferably modified by deletion, substitution, insertion, and / or addition of one or more amino acids constituting the variable and constant regions of the antibody, as long as they exhibit binding specificity to the antigen. It can be applied to.

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

[0095] In the method of the present invention, known sequences may be used as the gene sequences encoding the H chain or L chain of the antibody before mutation introduction. Alternatively, novel antibody gene sequences can be obtained by methods known to those skilled in the art. Such genes can be suitably obtained, for example, from an antibody library. Furthermore, such genes can also be obtained by cloning using known methods such as RT-PCR, which uses the mRNA of a hybridoma producing a monoclonal antibody as a template.

[0096] Many antibody libraries are already publicly known. Furthermore, methods for preparing antibody libraries are also publicly known, so those skilled in the art can obtain or prepare antibody libraries as appropriate. For example, suitable antibody libraries include the antibody phage libraries disclosed in literature such as Clackson et al., Nature (1991) 352, 624-8, Marks et al., J. Mol. Biol. (1991) 222, 581-97, Waterhouseset 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. Other known methods, such as methods for preparing libraries in eukaryotic cells (WO95 / 15393 pamphlet) and ribosome presentation methods, are preferably used. Furthermore, a technique for obtaining human antibodies by panning using a human antibody library as a starting material is known to those skilled in the art. That is, a single-chain antibody (scFv) in which the variable regions of the H and L chains of a human antibody are fused in frame is expressed on the surface of a phage by phage display. By selecting a phage bound to an antigen, the gene encoding the scFv that binds to the antigen is isolated from that phage. By identifying the sequence of this gene, the sequence of the DNA encoding the variable regions of the H and L chains of the antibody that binds to the antigen can be determined. The antibody gene having a column is appropriately inserted into an expression vector and expressed in a suitable host cell as described later, thereby obtaining a human antibody as appropriate. These methods are already well known, and examples include those disclosed in WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388.

[0097] Methods for obtaining antibody-coding genes from hybridomas that produce monoclonal antibodies can basically employ known techniques. As detailed below, in short, after an animal is immunized with a desired sensitizing antigen according to a standard immunization method, the immune cells obtained from the animal are donated to cell fusion with known parent cells using a standard cell fusion method. According to a standard screening method, monoclonal antibody-producing cells (hybridoms) are screened, and the mRNA obtained from the selected hybridomas is used as a template to synthesize the variable region (V region) cDNA of the antibody by reverse transcriptase. This cDNA is then used to determine the desired antibody... The antibody gene is preferred to fuse in-frame with the DNA encoding the normal region (C region). It will be acquired appropriately.

[0098] More specifically, the following are preferred examples, 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 an immunogenic complete antigen or an incomplete antigen containing a hapten or the like that does not exhibit immunogenicity. For example, full-length proteins, or partial polypeptides or peptides thereof, can be suitably used. The soluble GPC3 core polypeptide represented by SEQ ID NO: 207 is a suitable example. 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-mentioned forms of substances. The preparation of the antigen can be suitably carried out by methods known to those skilled in the art, for example, a method using baculovirus (e.g., WO98 / 46777) can be suitably used. If the immunogenicity of the antigen is low, the animal can be suitably immunized by the antigen bound to an immunogenic macromolecule such as albumin. Furthermore, if the sensitizing antigen is a molecule that spans the cell membrane, if necessary, a polypeptide fragment of the extracellular region of the molecule can be suitably used as the sensitizing antigen. Alternatively, cells expressing the molecule on their cell surface can be suitably used as sensitizing antigens. Furthermore, if the sensitizing antigen is an insoluble molecule, it can be solubilized by binding it with another water-soluble molecule, and the solubilized bound molecule can be suitably used as the sensitizing antigen.

[0099] Antibody-producing cells can be suitably obtained by immunizing animals with the appropriate sensitizing antigens described above. Alternatively, antibody-producing cells can be obtained by immunizing lymphocytes capable of producing antibodies in vitro. Various vertebrates and mammals can be used as animals to be immunized. In particular, rodents, lagomorphs, and primates are commonly used as animals to be immunized. Examples include rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates such as cynomolgus macaques, rhesus macaques, baboons, and chimpanzees. In addition, transgenic animals that carry a repertoire of human antibody genes in their genome are also known, and human antibodies can be suitably obtained by using such animals (see WO96 / 34096; Mendez et al., Nat. Genet. (1997) 15, 146-56). Instead of using such transgenic animals, for example, a desired human antibody having binding activity to the antigen can be suitably obtained by sensitizing human lymphocytes in vitro with the desired antigen or cells expressing the desired antigen, and then fusing them with human myeloma cells, such as U266 (see Japanese Patent Publication No. 1-59878). Furthermore, the entire repertoire of human antibody genes A desired human antibody can be suitably obtained by immunizing a transgenic animal carrying the gene in its genome with the desired antigen (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO96 / 34096, WO96 / 33735).

[0100] Immunization of animals is carried out, for example, by appropriately diluting and suspending the sensitizing antigen in phosphate-buffered saline (PBS) or physiological saline, emulsifying it by mixing it with an adjuvant if necessary, and then injecting the sensitizing antigen intraperitoneally or subcutaneously into the animal. Subsequently, preferably, the sensitizing antigen mixed with Freund's incomplete adjuvant is administered several times every 4 to 21 days. The drug is administered. Confirmation of antibody production against the sensitized antigen in immunized animals is performed by measuring the antibody titer in the animal's serum against the sensitized antigen using conventional methods, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS), or other known analytical methods. It can be done.

[0101] Hybridomas can be created by fusing antibody-producing cells obtained from animals or lymphocytes immunized with a desired sensitizing antigen with myeloma cells using a fusion agent commonly used for cell fusion (e.g., polyethylene glycol) (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 59-103). The preparation of M can be done, for example, by the method of Milstein et al. (G. Kohler and C. Milstein, Methods E This can be suitably carried out according to nzymol. (1981) 73, 3-46), etc. By culturing and proliferating hybridoma cells, 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), and flow cytometry (FACS). Subsequently, if necessary, hybridomas that produce antibodies with desired specificity, binding activity, or activity can be suitably subcloned using methods such as limiting dilution, and the monoclonal antibodies produced by these hybridomas can be isolated.

[0102] Subsequently, the gene encoding the selected antibody can be cloned from the aforementioned hybridoma or antibody-producing cells (such as sensitized lymphocytes) using a probe that can specifically bind to the gene (for example, an oligonucleotide complementary to the sequence encoding the antibody constant region). Alternatively, it can be cloned by RT-PCR using mRNA obtained from the hybridoma or antibody-producing cells (such as sensitized lymphocytes) as a template. Immunoglobulins are classified into five different classes, IgA, IgD, IgE, IgG, and IgM, based on their structural and functional differences. Furthermore, each class is classified into several isotypes (e.g., 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 of these classes and subclasses, but antibodies belonging to the IgG class are particularly preferred. This can be cited as one example.

[0103] The genes encoding the amino acid sequences that make up the heavy and light chains of antibodies can be modified as appropriate using genetic engineering techniques. For example, by modifying the nucleic acid residues that encode the amino acid sequences that make up antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies, and camel antibodies, genetically modified recombinant antibodies, such as chimeric antibodies and humanized antibodies, can be produced as appropriate with the aim of reducing heteroantigenicity against humans. Chimeric antibodies are antibodies composed of the variable regions of the heavy and light chains of antibodies derived from non-human mammals, such as mice, and the constant regions of the heavy and light chains of human antibodies. For example, they can be obtained by linking the DNA encoding the variable region of an antibody derived from a mouse with the DNA encoding the constant region of a human antibody, incorporating this into an expression vector, introducing the recombinant vector into a host, and then expressing it. Humanized antibodies, also called reshaped human antibodies, have codon sequences formed in-frame between the complementary determining region (CDR) of an antibody isolated from a non-human mammal, such as a mouse, and the framework region of a human antibody. It is an antibody that has been linked in such a way. The DNA sequence that encodes this humanized antibody is composed of multiple It can be synthesized by an overlap PCR reaction using an oligonucleotide as a template. The materials and procedures for the varwrap PCR reaction are described in WO98 / 13388, etc.

[0104] The DNA encoding the variable region of the recombinant antibody of the present invention is a DNA that overlaps with each other. Multiple oligonucleotides, each containing a creotide sequence, are obtained by overlap PCR and ligated in-frame with DNA encoding the constant region of a human antibody to form a codon sequence. The ligated DNA is then fed into an expression vector. The DNA is inserted into the host so that it is expressed, and then introduced into the host. The antibody encoded by the DNA is expressed by culturing the host. The expressed antibody is obtained by purifying it as appropriate from the host culture medium, etc. (see EP239400; WO96 / 02576). Linked via CDR The FR of the humanized antibody to be conjugated is selected to have a complementarity-determining region that forms a good antigen-binding site for the antigen. If necessary, the amino acid residues constituting the FR of the variable region of the antibody selected so that the complementarity-determining region of the reconstituted human antibody forms an appropriate antigen-binding site for the antigen can be modified by appropriate substitution (K. Sato et al., Cancer Res. (1993) 53, 851-856).

[0105] In addition to the humanization modifications described above, modifications may be made to improve the biological properties of the antibody, such as its binding activity to the antigen it recognizes. Modifications in this invention can be suitably carried out by methods such as site-directed mutation (see, for example, Kunkel, Proc. Natl. Acad. Sci. USA (1985) 82, 488), PCR mutation, and cassette mutation. Generally, the amino acid sequences constituting the modified antibody with improved biological properties have 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 97%, 98%, 99%, etc.) identity and / or similarity. The amino acid sequence of the antibody to be modified (i.e., the antibody on which the modified antibody is based) has a similarity to the amino acid sequence of the antibody to be modified. In this specification, sequence identity and / or similarity means sequence identity After the sequence has been aligned and gaps introduced as necessary to maximize uniformity, this refers to the percentage of amino acid residues that are identical (the same residue) or similar (amino acid residues classified into the same group based on the general side-chain properties of amino acids) to the amino acid residues that make up the antibody on which the modified antibody was based. Typically, natural amino acid residues are (1) hydrophobic based on the properties of their side chains. (2) Neutral hydrophilic: A Sparagine, glutamine, cysteine, threonine and serine; (3) Acidic: Asparagus (4) Basic: arginine, histidine, and lysine; (5) Residues that affect chain orientation: glycine and proline; and (6) Aromatic: tyrosine, triglycerides. It can be classified into the groups of putophan and phenylalanine.

[0106] Furthermore, as a modification aimed at enhancing antibody function, for example, improving the cytotoxic activity exhibited by antibodies, including humanized antibodies, is a suitable specific embodiment. Suitable examples of cytotoxic activity include antibody-dependent cell-mediated cytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC) activity. In the present invention, CDC activity refers to cytotoxic activity mediated by the complement system. On the other hand, ADCC activity refers to the activity in which, when a specific antibody attaches to the cell surface antigen of a target cell, Fcγ receptor-possessing cells (immune cells, etc.) bind to the Fc portion via the Fcγ receptor, thereby damaging the target cell. Whether or not the test antibody has ADCC activity, or whether or not it has CDC activity Whether or not this occurs can be measured by known methods (for example, Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Editor, John E, Coligan et al., John Wiley & Sons, Inc., (1993), etc.).

[0107] Specifically, the first step is to prepare effector cells, complement solution, and target cells. (1) Preparation of effector cells Splenectomy cells were isolated from CBA / N mice and other mice using RPMI1640 medium (Invitrogen). The cells are then washed with the same medium containing 10% fetal bovine serum (FBS, HyClone), and the cell concentration is reduced to 5x10. 6 Effector cells can be prepared by adjusting the cell / ml concentration. (2) Preparation of complement solution Baby Rabbit Complement (CEDARLANE) diluted 10-fold in 10% FBS-containing medium (Invitrogen). By doing so, a complement solution can be prepared. (3) Preparation of target cells Target cells can be radiolabeled by culturing cells expressing the antigen protein to which the test antibody binds with 0.2 mCi of 51Cr-sodium chromate (GE Healthcare Biosciences) in 10% FBS-containing DMEM medium at 37°C for 1 hour. Suitable cells expressing the antigen protein to which the test antibody binds include cells transformed with the gene encoding the antigen protein, as well as cells from ovarian cancer, prostate cancer, breast cancer, uterine cancer, liver cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, and colorectal cancer. After radiolabeling, the cells are washed three times with 10% FBS-containing RPMI1640 medium to reduce the cell concentration to 2x10⁻⁶. 5 By preparing the cells / ml, the target Cells can be prepared.

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

[0109] On the other hand, when measuring CDC activity, a 96-well flat-bottom plate (Becton Dickinson) is used, target Cells and 50 μl each of the test antibody are added, and the reaction is carried out on ice for 15 minutes. Afterward, 100 μl of complement solution is added to the reaction mixture, which is then incubated in a carbon dioxide incubator for 4 hours. The antibody is incubated. The final concentration of the test antibody can be used as appropriate within the range of 0 to 3 μg / ml. After culturing, 100 μl of supernatant was collected, and the radioactivity of the supernatant was measured using a gamma counter. It is determined that cytotoxic activity can be calculated in a similar manner to the measurement of ADCC activity.

[0110] On the other hand, when measuring cytotoxic activity using antibody conjugates, 50 μl each of target cells and the test antibody conjugate are added to a 96-well flat-bottom plate (Becton Dickinson), and the reaction is carried out on ice for 15 minutes. The plate is then incubated in a carbon dioxide incubator for 1 to 4 hours. The final antibody concentration is used as appropriate, within the range of 0 to 3 μg / ml. It is possible. After culturing, 100 μl of supernatant is collected and the radiation present in the supernatant is measured with a gamma counter. Activity is measured. Cytotoxic activity can be calculated in a similar manner to the measurement of ADCC activity.

[0111] The variable regions of the antibody's H and L chains are typically defined by three CDRs and four FRs, as described above. It is composed of such amino acids. In a preferred embodiment of the present invention, the amino acid residue to be "modified" can be appropriately selected from, for example, the amino acid residues that constitute CDR or FR.

[0112] Furthermore, a person skilled in the art could use public databases such as Kabat to find the amino acid sequence that constitutes the variable region (FR) of an antibody, which is actually present in living organisms such as humans or mice. This allows for the acquisition of suitable results.

[0113] In a preferred embodiment of the present invention, a humanized antibody is provided whose plasma pharmacokinetics are controlled by the method of the present invention. The humanized antibody is, for example, a humanized antibody comprising a complementation-determining region (CDR) derived from a non-human animal, a framework region (FR) derived from a 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 is originally The amino acid residues at the corresponding positions of the CDR or FR of the antibody have a different charge from the amino acid residues. Compared to chimeric antibodies that share the same C region, humanized antibodies exhibit controlled plasma pharmacokinetics. It is the body.

[0114] Furthermore, in a preferred embodiment of the present invention, a human antibody whose plasma pharmacokinetics are controlled by the method of the present invention is provided. This human antibody, for example, has a human-derived complementarity-determining region (CDR). ), a human antibody comprising a human-derived framework region (FR) 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 different charge from the amino acid residue at the corresponding position in the CDR or FR of the original antibody, This is a human antibody with controlled plasma pharmacokinetics compared to chimeric antibodies that share the same C region.

[0115] The above-mentioned human constant region preferably refers to a region including the wild-type human Fc region, but modified Fc can also be suitably used. The above-mentioned "modified Fc" may include Fc in which the amino acid residues constituting the Fc have been modified, or Fc in which the modifications applied to the Fc portion have been modified. This may also include such modifications. Modifying the type of glycosylation added to the Fc portion is a suitable example of such modification. In this specification, an antibody with reduced fucose content bound to the Fc region is specifically disclosed as a reference example.

[0116] An antibody with reduced fucose content bound to the antibody's Fc region refers to an antibody that, compared to a control antibody, has significantly less bound fucose and is preferably undetectable. Typically, this refers to the two glycan-binding sites located in the Fc region of the two H chains that make up one antibody molecule. Fucose is added to the N-glycosidic linked sugar chain. In the present invention, "antibody with reduced fucose content bound to the antibody Fc region" refers to an antibody having 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 to such a normal antibody as a control. The fucose content can be measured using the analytical methods specifically shown below as reference examples. Methods for producing antibodies with reduced fucose content are described as reference examples of the present invention, but also suitably exemplified by methods using animal cells lacking fucosyltransferase (Biotechnol Bioeng. (2004), 87(5), 614-22), methods using animal cells with modified complex branched glycosylation (Biotechnol Bioeng. (2006) 93(5), 851-61), etc. These can be cited as examples. Furthermore, preferred methods for producing cells other than animal cells as host cells include methods using plant cells (Nature Biotechnology (2006) 24, 1591-7) or yeast cells (Nature Biotechnology (2006) 24, 210-5). It is possible.

[0117] A preferred embodiment of the manufacturing method of the present invention is a method for producing a polypeptide containing an antibody variable region with a modified isoelectric point, (a) at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide Modify the nucleic acid encoding the polypeptide containing the amino acid residue so that the charge is converted, (b) Culture host cells so that the nucleic acid is expressed, (c) A method comprising recovering a polypeptide containing an antibody variable region from a host cell culture. Furthermore, a preferred embodiment of the manufacturing method of the present invention is a method for producing a polypeptide containing an antibody variable region in which plasma pharmacokinetics are controlled, (a) at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide Modify the nucleic acid encoding the polypeptide containing the amino acid residue so that the charge is converted, (b) Culture host cells so that the nucleic acid is expressed, (c) A method comprising recovering a polypeptide containing an antibody variable region from a host cell culture. Furthermore, the present invention also includes polypeptides containing a variable region of an antibody with controlled plasma pharmacokinetics produced by the said method.

[0118] The present invention also provides a method for producing a multispecific polypeptide comprising a first polypeptide and a second polypeptide having a variable region of the antibody. Furthermore, the present invention provides a multispecific polypeptide produced by said method. A preferred embodiment of the production method of the present invention is a method that includes modifying both or either 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 as to increase the difference in isoelectric points between the first polypeptide and the second polypeptide. That is, by changing the charge of the amino acid residues of the first polypeptide and the second polypeptide, the difference in isoelectric points (pI) of the polypeptides can be increased, and a multispecific antibody can be produced by utilizing this difference in isoelectric points. In detail, the production method includes the following steps (a) to (c). (a) At least the surface of the CDR region of the first polypeptide and the second polypeptide that may be exposed This includes modifying a nucleic acid encoding a polypeptide containing an amino acid residue such that the charge of another amino acid residue is altered, wherein the modification of the nucleic acid modifies both or either 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 such that the difference in isoelectric points between the first polypeptide and the second polypeptide increases compared to before the modification. (b) Culture host cells so that the nucleic acid is expressed, (c) Recovering multispecific antibodies from host cell cultures.

[0119] In this invention, polypeptides typically refer to peptides and proteins having a length of about 10 amino acids or more. While they are usually polypeptides of biological origin, they are not particularly limited and may include, for example, polypeptides consisting of artificially designed sequences. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Furthermore, fragments of the above-mentioned polypeptides are also included in the polypeptides of this invention. In the present invention, "multispecific polypeptide comprising a first polypeptide and a second polypeptide having a variable region of an antibody" means a polypeptide comprising a variable region of an antibody that binds to at least two different antigens or different epitopes within the same antigen, and the polypeptide comprising the variable region of an antibody includes, as described above, antibodies, small molecule antibodies, scaffold proteins, etc.

[0120] In the present invention, "increasing the difference in isoelectric points of polypeptides" means that, in two or more polypeptides, the isoelectric points of each polypeptide are not equal, or the difference in isoelectric points between the two or more polypeptides is increased by modifying the charge of the surface amino acids. The difference in isoelectric points can be observed, for example, by using a method such as isoelectric focusing electrophoresis. Furthermore, 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 a variable region of an antibody, (a) Modify both or either 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 the difference in isoelectric points between the first polypeptide and the second polypeptide is 1.0 or more, preferably 1.2 or more, and more preferably 1.5 or more, the charge of at least one amino acid residue that can be exposed on the surface of the CDR region is altered. (b) Culture host cells so that the nucleic acid is expressed, (c) A method for producing multispecific antibodies is provided, which includes recovering multispecific antibodies from host cell cultures.

[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 a variable region of the antibody. A preferred embodiment of the purification method of the present invention includes modifying both or either 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 as to increase the difference in isoelectric points between the first polypeptide and the second polypeptide. 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) can be introduced into the polypeptide, and a multispecific antibody can be purified by utilizing this difference in isoelectric point. The purification method includes the following steps (a) to (c). (a) At least the surface of the CDR region of the first polypeptide and the second polypeptide that may be exposed This method involves modifying a nucleic acid encoding a polypeptide containing an amino acid residue such that the charge of another amino acid residue is altered, wherein the modification of the nucleic acid results in a first positive charge compared to the original state. Modify either or both of the nucleic acids encoding amino acid residues of the first polypeptide and the nucleic acids encoding amino acid residues of the second polypeptide so that the difference in isoelectric points between the lipeptide and the second polypeptide increases. (b) Culture host cells so that the nucleic acid is expressed, (c) Purify the multispecific antibody from the host cell culture by standard chromatography.

[0123] Furthermore, the present invention also includes a method for producing multispecific antibodies that includes a step of purification by the purification method described above.

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

[0125] Furthermore, the nucleic acid in this invention is typically held (inserted) into a suitable vector and introduced into a host cell. The vector is not particularly limited as long as it stably holds the inserted nucleic acid. For example, if E. coli is used as the host, the pBluescript vector (Stratagene) is preferred as the cloning vector, but various commercially available vectors can be used. Therefore, 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 is a vector that expresses the polypeptide in vitro, in E. coli, in cultured cells, or in living organisms. For example, the pBEST vector (Promega) is preferred for in vitro expression, the pET vector (Invitrogen) for E. coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. (1988) 8, 466-472) for living organisms. The insertion of the DNA of the present invention into the vector is performed by conventional methods, for example, using restriction enzyme sites. This can be preferably carried out by a ligase reaction (Current protocols in Molecular Biology edit). Ausubel et al. (1987) Publish. John Wiley & Sons.Section 11.4-11.11).

[0126] There are no particular restrictions on the host cells mentioned above, and various host cells can be used depending on the purpose. Examples of cells used to express polypeptides 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), and animal cells (e.g., Examples include CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells, and plant cells. Vector introduction into host cells can be performed by known methods such as calcium phosphate precipitation, electro-pulsed poroscopy (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection, and microinjection.

[0127] To secrete polypeptides (antibodies) expressed in host cells into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, appropriate secretion signals can be suitably incorporated into the target antibody. These signals can preferably be endogenous signals specific to the target polypeptide (antibody) or heterologous signals.

[0128] In the above manufacturing method, if the polypeptide (antibody) of the present invention is secreted into the culture medium, the recovery of the polypeptide (antibody) is carried out by recovering the culture medium. If the antibody of the present invention is produced inside a cell, the cell is first lysed, and then the antibody is recovered.

[0129] For the purification of the antibodies 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, hydroxyl apatite chromatography, and lectin chromatography can be suitably used.

[0130] In the present invention, the polypeptides that modify nucleic acids are preferably homopolymers of the first polypeptide, homopolymers of the second polypeptide, and heteropolymers of the first polypeptide and the second polypeptide. Examples of homopolymers of the first polypeptide, homopolymers of the second polypeptide, and heteropolymers of the first polypeptide and the second polypeptide are those described in the examples, but are not limited thereto.

[0131] Standard chromatography methods used in this invention include, but are not limited to, cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, hydrophobic charge interaction chromatography, and chromatographic focusing.

[0132] In the above method of the present invention, the first polypeptide and the second polypeptide preferably include 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 includes 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 include a heavy chain constant region. More preferably, the heavy chain constant region is one which results in a pI difference between the first polypeptide and the second polypeptide. Examples of such heavy chain constant regions include the heavy chain constant regions of antibodies that have a pI difference. It is also possible to introduce a pI difference between the first and second polypeptides using the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, which originally have a difference in pI. Alternatively, a non-wild-type human constant region can be created by modifying only the amino acids in the heavy chain constant regions of the first and second polypeptides that are affected by the difference in isoelectric points between these subclasses, or by simultaneously modifying adjacent amino acids that do not affect their isoelectric points, thereby introducing a pI difference between the two constant regions. Examples of modification sites for introducing a pI difference into the constant region include the H chain. In the EU numbering of the constant region, H chains 137, 196, 203, 214, 217, and 233 The following are examples: the 1st, 268th, 274th, 276th, 297th, 355th, 392nd, 419th, and 435th. Furthermore, since removing the glycans in the heavy chain constant region creates a difference in pI, the 297th glycan addition site can also be considered a modification site for introducing pI chains. The present invention also includes a method that combines, with the method in which the first polypeptide and the second polypeptide described above include a heavy chain constant region, a method in which the first polypeptide and the second polypeptide described above 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 polymer with the third polypeptide.

[0135] Furthermore, the multispecific polypeptide produced by the above method is also included in the present invention.

[0136] Furthermore, if 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, the Kabat numbering of positions 31, 61, 62, 64 and 65 in the heavy chain variable region One possible embodiment is to ensure that at least one amino acid residue selected from the amino acid residues has an electric charge. Furthermore, if a light chain variable region is included, in order to "increase the difference in isoelectric points", for example, Kabat numbering can be used in the light chain variable region. One possible embodiment is one in which at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54, and 55 has an electric charge. Of the amino acid residues of the first polypeptide indicated by the above numbering, the amino acid residues other than the amino acid residue having the electric charge may have the same type of charge as the amino acid residue having the electric charge, or they may have no charge or the opposite charge, as long as there is a difference in the isoelectric points of the first polypeptide and the second polypeptide.

[0137] The multispecific antibody of the present invention is preferably characterized in that the second polypeptide has a charge opposite to or no charge to the charged amino acid residue of the first polypeptide. Specifically, the second polypeptide includes a heavy chain variable region, and amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region are selected according to Kabat numbering. In other words, it is a multispecific antibody in which at least one amino acid residue has the opposite charge to or is uncharged to the amino acid residue selected to have a charge in the variable region contained in the first polypeptide. Furthermore, if a light chain variable region is included, the amino acids at positions 24, 27, 53, 54 and 55 in the light chain variable region according to Kabat numbering. The antibody is multispecific in which at least one amino acid residue selected from the acid residues has a charge opposite to or no charge to the amino acid residue selected in the variable region of the first polypeptide that has a charge. Of the amino acid residues of the second polypeptide indicated by the above numbering, the amino acid residues other than the amino acid residue having the charge may have the same type of charge as the amino acid residue having the charge, may be chargeless, or may have the opposite charge, as long as there is a difference in the isoelectric points of the first polypeptide and the second polypeptide.

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

[0139] In the above antibody, "having the same type of charge" means, for example, amino acid residues in the heavy chain variable region according to the Kabat numbering, or EU numbering in the heavy chain constant region. This means that each amino acid residue in the result of the rang contains an amino acid residue that belongs to either group (a) or (b) below. (a) Glutamic acid (E), aspartic acid (D) (b) Lysine (K), Arginine (R), Histidine (H)

[0140] Furthermore, "having opposite charges" means, for example, the Kabat numbering or EU numbering in a second polypeptide having a heavy chain variable region and / or a heavy chain constant region. If at least one amino acid residue among the amino acid residues obtained by the process is an amino acid residue at a corresponding position in the heavy chain variable region and / or heavy chain constant region of the first polypeptide, and is an amino acid residue belonging to either group (a) or (b) above, then the remaining amino acid residues are amino acid residues belonging to a different group.

[0141] In other words, the present invention provides a multispecific antibody in which the amino acid residue having the same type of charge is selected from amino acid residues included in either group (a) or (b) above.

[0142] Furthermore, if the original (pre-modification) amino acid residue already has an electric charge, modifying it to become an amino acid residue without an electric charge is also one of the preferred embodiments of the present invention.

[0143] In the present invention, it is preferable that amino acid residues are modified so as to increase the difference in isoelectric points (pI) between the first polypeptide and the second polypeptide. Furthermore, if there are multiple amino acid residues introduced by the modification, a small number of these amino acid residues may be uncharged.

[0144] The present invention also relates to a composition (pharmaceutical) comprising a polypeptide (e.g., an antibody such as an IgG antibody) whose plasma pharmacokinetics are controlled by the method of the present invention, and a pharmaceutically acceptable carrier.

[0145] In this invention, a pharmaceutical composition generally refers to a drug used for the treatment or prevention of a disease, or for examination and diagnosis.

[0146] The pharmaceutical compositions of the present invention can be suitably formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of an injectable sterile solution or suspension with water or other pharmaceutically acceptable liquid. For example, they can be suitably formulated by mixing them in appropriate combination with a pharmacokinetically acceptable carrier or medium, specifically sterile water or saline solution, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., in a unit dose form generally accepted for pharmaceutical practice. The amount of active ingredient in these formulations is set so as to yield an appropriate dose within the indicated range.

[0147] Sterile compositions for injection can be suitably formulated in accordance with standard formulation procedures using a vehicle such as distilled water for injection.

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

[0149] Examples of oily liquids include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol can be suitably used in combination as solubilizers. Furthermore, buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants can be suitably added. The injection solution prepared as described above is usually filled into appropriate ampoules.

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

[0151] The method of administration may be appropriately selected depending on the patient's age and symptoms. The dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody may be appropriately set in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, the dosage may be set or prepared to 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can set an appropriate dosage and method of administration considering these conditions.

[0152] Furthermore, the present invention provides nucleic acids encoding antibodies (e.g., humanized glypican-3 antibodies) whose plasma pharmacokinetics are controlled by the method of the present invention. A vector carrying such nucleic acid is also included in the present invention.

[0153] Furthermore, the present invention provides a host cell containing the nucleic acid. The type of host cell is not particularly limited and includes, for example, bacterial cells such as E. 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. In vitro and in vivo production systems can be suitably used as the production system. Eukaryotic cells and prokaryotic cells are preferably used as host cells in this process.

[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 African clawed frog oocytes (Valle et al., Nature (1981) 291, 338-340), and insect cells, such as Sf9, Sf21, and Tn5, are preferably exemplified. For the expression of the antibodies of the present invention, CHO-DG44, CHO-DX11B, COS7 cells, HEK293 cells, and BHK cells are preferably used. CHO cells are particularly preferred as host cells when aiming for high-volume expression using animal cells. For example, recombinant vectors can be suitably introduced into host cells by methods such as the calcium phosphate method, the DEAE dextran method, the cationic ribosome DOTAP (Boehringer Mannheim) method, electroporation, and lipofection.

[0155] Examples of plant cells include those derived from Nicotiana tabacum. And duckweed (Lemna minor) is known as a protein production system, and this cell is The antibodies of the present invention can be produced by a method of culturing fungal cells. As fungal cells, protein expression systems using cells of the genus Saccharomyces (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe) and filamentous fungi (e.g., Aspergillus niger) are known and can be used as host cells to produce the antibodies of the present invention.

[0156] When prokaryotic cells are used, a production system using bacterial cells is preferably used. In addition to the aforementioned Escherichia coli (E. coli), a production system using Bacillus subtilis (B. subtilis) is also known. These bacterial cells can all be suitably used for the production of antibodies according to the present invention.

[0157] In order to produce antibodies using the host cells of the present invention, the host cells transformed with an expression vector containing a polynucleotide encoding the antibody of the present invention are cultured, and in the culture In this state, polynucleotides encoding antibodies are expressed. Culturing can be suitably carried out according to known methods. For example, when animal cells are used as the host, DMEM, MEM, RPMI1640, and IMDM can be suitably used as the culture medium. In this case, FBS and fetal bovine serum (FCS) Serum replacement solutions such as those listed above are preferably used in combination. Cells can also be cultured by serum-free culture. Although it depends on the host cell, the cells can be preferably cultured under conditions of pH approximately 6 to 8. Culture is usually carried out at approximately 30 to 40°C for approximately 15 to 200 hours, with the culture medium being changed, aerated, and stirred as needed.

[0158] On the other hand, a system for producing the antibody of the present invention in vivo is, for example, a system using animals. Production systems using animals or plants can be suitably used. That is, polynucleotides encoding the antibody of the present invention are introduced into these animals or plants, and the glypican 3 antibody is produced and recovered within the body of the animal or plant. The term "host" in the present invention includes these animals and plants.

[0159] When animals are used as hosts, production systems using mammals or insects are available. Suitable mammals include goats, pigs, sheep, mice, and cattle (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Transgenic animals are also used when mammals are employed.

[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. A polynucleotide fragment containing this fusion gene is then injected into a goat embryo, and the embryo is transplanted into a female goat. The target antibody is obtained from the milk produced by the transgenic goat born from the embryo-receiving goat or its offspring. To increase the amount of antibody-containing milk produced by the transgenic goat, hormones are appropriately administered to the transgenic goat (Ebert et al., Bio / Technology (1994) 12, 699-702).

[0161] Furthermore, silkworms can be used as insects to produce the antibodies of the present invention. When silkworms are used, a baculovirus in which a polynucleotide encoding the target antibody has been inserted into its viral genome is used to infect the 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 plants are used to produce the antibodies of the present invention, for example, tobacco may be used as the plant. When tobacco is used, the recombinant vector obtained by inserting the polynucleotide encoding the target antibody into a plant expression vector, for example pMON 530, is a bacterium such as Agrobacterium tumefaciens. It can be introduced into A. This bacterium is used to infect tobacco, for example, Nicotiana tabacum (Ma et al., Eur. J. Immunol. (1994) 24, 131-8). ), the desired glypican 3 antibody can be obtained from infected tobacco leaves. Furthermore, similar bacteria have been used to infect duckweed (Lemna minor), and cloned infected duckweed... The desired glypican-3 antibody can be obtained from the cells of (Cox KM et al. Nat. Biotechnol. (2006) 24(12), 1591-7).

[0163] The antibodies of the present invention obtained in this manner can be isolated from host cells or extracellularly (e.g., culture medium, milk) and purified as substantially pure and homogeneous antibodies. While the separation and purification methods commonly used for the purification of polypeptides may be suitably used for antibody separation and purification, they are not limited to these methods. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc., can be appropriately selected and combined to suitably separate and purify the antibodies.

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

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

[0166] This invention relates to the variable region and constant region of TOCILIZUMAB, a humanized anti-IL-6 receptor IgG1 antibody. This invention provides a pharmaceutical composition consisting of a second-generation molecule superior to TOCILIZUMAB, which enhances pharmacological efficacy, improves plasma retention to reduce administration frequency and sustain therapeutic effects, and improves immunogenicity, safety, and physical properties by modifying the amino acid sequence of the region, as well as a method for producing such pharmaceutical compositions. Furthermore, this invention provides an antibody constant region suitable for use as a pharmaceutical.

[0167] The present invention relates to an anti-IL-6 receptor antibody that has excellent antigen-binding activity, neutralizing activity, plasma retention, stability, and / or homogeneity, and reduces the risk of immunogenicity. Preferably, the anti-IL-6 receptor antibody is a humanized PM-1 antibody (TOCILIZUMAB). Specifically, the present invention provides humanized PM-1 antibodies with enhanced antigen-binding activity through amino acid substitution, humanized PM-1 antibodies with enhanced neutralizing activity, humanized PM-1 antibodies with improved plasma retention, humanized PM-1 antibodies with reduced immunogenicity risk, humanized PM-1 antibodies with improved stability, and humanized PM-1 antibodies with improved uniformity.

[0168] Humanized PM-1 antibodies bind to human IL-6 receptors and inhibit the binding of human IL-6 to human IL-6 receptors. In this specification, the correspondence between the amino acid sequence of the humanized PM-1 antibody and the sequence number 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 heavy chain CDR1 (HCDR1) Sequence ID: 1 Amino acid sequence of heavy chain CDR2 (HCDR2) Sequence ID: 2 Amino acid sequence of heavy chain CDR3 (HCDR3) Sequence ID: 3 Amino acid sequence of heavy chain FR1 (HFR1) Sequence ID: 7 Amino acid sequence of heavy chain FR2 (HFR2) Sequence ID: 8 Amino acid sequence of heavy chain FR3 (HFR3) Sequence ID: 9 Amino acid sequence of heavy chain FR4 (HFR4) Sequence ID: 10 The amino acid sequence of the light chain CDR1 (LCDR1) is sequence number 4. The amino acid sequence of the light chain CDR2 (LCDR2) is shown in SEQ ID NO: 5 The amino acid sequence of the light chain CDR3 (LCDR3) is shown in Sequence ID: 6 The amino acid sequence of the light chain FR1 (LFR1) is shown in SEQ ID NO: 11 The amino acid sequence of the light chain FR2 (LFR2) is shown in sequence number 12. The amino acid sequence of the light chain FR3 (LFR3) is shown in SEQ ID NO: 13 The amino acid sequence of the light chain FR4 (LFR4) is shown in SEQ ID NO: 14

[0169] <Affinity-neutralizing activity-enhancing antibody> The present invention provides an anti-human IL-6 receptor antibody with high binding and / or neutralizing activity to the human IL-6 receptor. More specifically, the present invention provides the antibodies described in (a) to (y) below, and a method for producing the antibody. (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 Sequence ID No. 1 is substituted with another amino acid. The substituted amino acids are not particularly limited, but examples include Trp (RD_68), Thr (RD_37), and Asp (RD_8). Substitution with 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. Sequence ID: 26 shows the amino acid sequence in which the first Ser is replaced with Trp in the amino acid sequence described in Sequence ID: 1. Sequence ID: 27 shows the amino acid sequence in which the first Ser is replaced with Thr in the amino acid sequence described in Sequence ID: 1. Sequence ID: 28 shows the amino acid sequence in which the first Ser is replaced with Asp in the amino acid sequence described in Sequence ID: 1. Sequence ID: 29 shows the amino acid sequence in which the first Ser is replaced with Asn in the amino acid sequence described in Sequence ID: 1. Sequence ID: 30 shows the amino acid sequence in which the first Ser is replaced with Arg in the amino acid sequence described in Sequence ID: 1. Sequence ID: 31 shows the amino acid sequence in which the first Ser is replaced with Val in the sequence described in Sequence ID: 1. Sequence ID: 32 shows the amino acid sequence in which the first Ser is replaced with Phe in the amino acid sequence described in Sequence ID: 1. Sequence ID: 33 shows the amino acid sequence in which the first Ser is replaced with Ala in the amino acid sequence described in Sequence ID: 1. Sequence ID: 34 shows the amino acid sequence in which the first Ser is replaced with Gln in the sequence described in Sequence ID: 1. Sequence ID: 35 shows the amino acid sequence in which the first Ser is replaced with Tyr in the sequence described in Sequence ID: 1. Sequence ID: 36 shows the amino acid sequence in which the first Ser is replaced with Leu in the amino acid sequence described in Sequence ID: 1. Sequence ID: 37 shows the amino acid sequence in which the first Ser is replaced with His in the sequence described in Sequence ID: 1. Sequence ID: 38 shows the amino acid sequence in which the first Ser is replaced with Glu in the amino acid sequence described in Sequence ID: 1. Sequence ID: 39 shows the amino acid sequence in which the first Ser is replaced with Cys in the amino acid sequence described in Sequence ID: 1.

[0170] (b) An anti-human IL-6 receptor antibody having a heavy chain CDR1 in which the 5th Trp in the amino acid sequence (HCDR1) described in Sequence ID No. 1 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution to Ile(RD_9) or Val(RD_30) is possible. preferable. Sequence ID: 40 shows the amino acid sequence in which the 5th Trp is replaced with Ile in the amino acid sequence described in Sequence ID: 1. Sequence ID: 41 shows the amino acid sequence in which the 5th Trp is replaced with Val in the sequence described in Sequence ID: 1.

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

[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) described in Sequence ID No. 2 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution to Arg(RD_79) is preferred. Sequence ID: 43 shows the amino acid sequence in which the 8th Thr is replaced with Arg in the amino acid sequence described in Sequence ID: 2.

[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) described in Sequence ID No. 2 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution to Ser(RD_12) or Asn(RD_61) is preferred. Sequence ID: 44 shows the amino acid sequence in which the 9th Thr is replaced with Ser in the sequence described in Sequence ID: 2. Sequence ID: 45 shows the amino acid sequence in which the 9th Thr is replaced with Asn in the amino acid sequence described in Sequence ID: 2.

[0174] (f) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the first Ser in the amino acid sequence (HCDR3) described in Sequence ID No. 3 is substituted with another amino acid. The substituted amino acid is not particularly limited, but could be Ile(RD_2), Val(RD_4), Thr(RD_80), or Leu Substitution with (RD_5) is preferable. Sequence ID: 46 shows the amino acid sequence in which the first Ser is replaced with Ile in the amino acid sequence described in Sequence ID: 3. Sequence ID: 47 shows the amino acid sequence in which the first Ser is replaced with Val in the sequence described in Sequence ID: 3. Sequence ID: 48 shows the amino acid sequence in which the first Ser is replaced with Thr in the sequence described in Sequence ID: 3. Sequence ID: 49 shows the amino acid sequence in which the first Ser is replaced with Leu in the amino acid sequence described in Sequence ID: 3.

[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) described in Sequence ID No. 3 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution to Thr(RD_84) is preferred. Sequence ID: 50 shows the amino acid sequence in which the second Leu is replaced with Thr in the sequence described in Sequence ID: 3.

[0176] (h) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the 5th Thr in the amino acid sequence (HCDR3) described in Sequence 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. Another preferred substitution is the substitution of the fifth Thr with Ser(RDC_14H). Sequence ID: 51 shows the amino acid sequence in which the 5th Thr is replaced with Ala in the sequence described in Sequence ID: 3. Sequence ID: 52 shows the amino acid sequence in which the 5th Thr is replaced with Ile in the amino acid sequence described in Sequence ID: 3. Sequence ID: 53 shows the amino acid sequence in which the 5th Thr is replaced with Ser in the sequence described in Sequence ID: 3.

[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) described in Sequence ID No. 3 is substituted with another amino acid. The substituted amino acid sequence is not particularly limited, but substitution to Ser(RD_81) or Val(PF_3H) is preferred. Sequence ID: 54 shows the amino acid sequence in which the 7th Ala is replaced with Ser in the sequence described in Sequence ID: 3. The sequence in which the 7th Ala is replaced with Val in the amino acid sequence described in Sequence ID No. 3. Number 55 is shown.

[0178] (j) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the 8th Met is substituted with another amino acid in the amino acid sequence (HCDR3) described in SEQ ID NO: 3. The substituted amino acid sequence is not particularly limited, but substitution to Leu(PF_4H) is preferred. Sequence ID: 56 shows the amino acid sequence in which the 8th Met is replaced with Leu in the amino acid sequence described in Sequence ID: 3.

[0179] (k) An anti-human IL-6 receptor antibody having a heavy chain CDR3 in which the first Ser and fifth Thr in the amino acid sequence (HCDR3) described in Sequence ID No. 3 are substituted with other amino acids. The substituted amino acids are not particularly limited, but the first Ser is replaced with Leu and the fifth Thr is replaced with Ala. It is preferable to substitute (RD_6). Other preferred substitutions include substituting the first Ser with Val and the fifth Thr with Ala (RDC_2H), substituting the first Ser with Ile and the fifth Thr with Ala (RDC_3H), substituting the first Ser with Thr and the fifth Thr with Ala (RDC_4H), Substitution of the 1st Ser to Val and substitution of the 5th Thr to Ile (RDC_5H), the 1st Ser to Ile Substitution of and substitution of the 5th Thr to Ile (RDC_6H), substitution of the 1st Ser to Thr and substitution of the 5th Thr to Ile (RDC_7H), or substitution of the 1st Ser to Leu and substitution of the 5th Thr to Ile One possible example is the substitution (RDC_8H). In the amino acid sequence described in Sequence ID No. 3, the first Ser is changed to Leu and the fifth Thr is changed to Ala. The substituted sequence is shown at sequence number 57. In the amino acid sequence described in Sequence ID No. 3, the first Ser becomes Val and the fifth Thr becomes Ala. The substituted sequence is shown at sequence number 58. In the amino acid sequence described in Sequence ID No. 3, the first Ser is changed to Ile and the fifth Thr is changed to Ala. The substituted sequence is shown at sequence number 59. In the amino acid sequence described in Sequence ID No. 3, the first Ser becomes Thr, and the fifth Thr becomes Ala. The substituted sequence is shown at sequence number 60. In the amino acid sequence described in Sequence ID No. 3, the first Ser becomes Val and the fifth Thr becomes Ile. The substituted sequence is shown at sequence number 61. In the amino acid sequence described in Sequence ID No. 3, the first Ser becomes Ile and the fifth Thr becomes Ile. The substituted sequence is shown at sequence number 62. In the amino acid sequence described in Sequence ID:3, the first Ser becomes Thr, and the fifth Thr becomes Ile. The substituted sequence is shown at sequence number 63. In the amino acid sequence described in Sequence ID No. 3, the first Ser is changed to Leu and the fifth Thr is changed to Ile. The substituted sequence is shown at sequence number 64.

[0180] (l) In the amino acid sequence (HCDR3) described in Sequence ID No. 3, the second Leu and the seventh Ala Anti-human IL-6 receptor antibodies that have a heavy chain CDR3 in which the 8th Met is substituted with another amino acid. body. The substituted amino acids are not particularly limited, but for example, the second Leu is replaced with Thr, the seventh Ala with Val, and 8 It is preferable to replace the i-th Met with Leu (RD_78). Sequence ID: 65 shows the amino acid sequence in Sequence ID: 3 in which the second Leu is replaced with Thr, the seventh Ala with Val, and the eighth Met with Leu.

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

[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) described in Sequence ID No. 4 is substituted with another amino acid. The substituted amino acid sequence is not particularly limited, but substitution to Arg(RD_26) or Thr(RD_20) is preferred. Sequence ID: 67 shows the amino acid sequence in which the fourth Gln is replaced with Arg in the amino acid sequence described in Sequence ID: 4. Sequence ID: 68 shows the amino acid sequence in which the fourth Gln is replaced with Thr in the sequence described in Sequence ID: 4.

[0183] (o) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the 9th Tyr in the amino acid sequence (LCDR1) described in Sequence ID No. 4 is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution to Phe(RD_73) is preferred. Sequence ID: 69 shows the amino acid sequence in which the 9th Tyr is replaced with Phe in the amino acid sequence described in Sequence ID: 4.

[0184] (p) An anti-human IL-6 receptor antibody having a light chain CDR1 in which the 11th Asn is substituted with another amino acid in the amino acid sequence (LCDR1) described in Sequence ID No. 4. The substituted amino acid sequence is not particularly limited, but substitution to Ser(RD_27) is preferred. Sequence ID: 70 shows the amino acid sequence in which the 11th Asn is replaced with Ser in the sequence described in Sequence ID: 4.

[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 Sequence ID No. 5 is substituted with another amino acid. The substituted amino acid sequence is not particularly limited, but substitution to Gly is preferred. Sequence ID: 71 shows the amino acid sequence in which the second Thr is replaced with Gly in the amino acid sequence described in Sequence ID: 5.

[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 Sequence ID No. 6 is substituted with another amino acid. The substituted amino acid sequence is not particularly limited, but substitution to Gly(RD_28), Asn(RD_29), or Ser(RDC_15L) is preferred. Sequence ID: 72 shows the amino acid sequence in which the first Gln is replaced with Gly in the amino acid sequence described in Sequence ID: 6. Sequence ID: 73 shows the amino acid sequence in which the first Gln is replaced with Asn in the amino acid sequence described in Sequence ID: 6. Sequence ID: 74 shows the amino acid sequence in which the first Gln is replaced with Ser in the sequence described in Sequence ID: 6.

[0187] (s) In the amino acid sequence described in Sequence ID No. 6, the third Gly is substituted with another amino acid. An anti-human IL-6 receptor antibody possessing a light chain CDR3. The substituted amino acid sequence is not particularly limited, but substitution to Ser is preferred. Sequence ID: 75 shows the amino acid sequence in which the third Gly is replaced with Ser in the sequence described in Sequence ID: 6.

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

[0189] (u) In the amino acid sequence (LCDR3) described in Sequence ID No. 6, the 5th Thr is in the same position as another amino acid. Anti-human IL-6 receptor antibody with substituted light chain CDR3. The substituted amino acid is not particularly limited, but substitution to Arg(RD_23) or Ser is preferred. The sequence in which the 5th Thr in the amino acid sequence described in Sequence ID:6 is replaced with Arg is the sequence Number 76 is shown. Sequence ID: 77 shows the amino acid sequence in which the 5th Thr is replaced with Ser in the sequence described in Sequence ID: 6.

[0190] (v) An anti-IL-6 receptor antibody having a light chain CDR3 in which the first Gln and fifth Thr in the amino acid sequence (LCDR3) described in SEQ ID NO: 6 are substituted with other amino acids. The substituted amino acids are not particularly limited, but it is preferable to substitute the first Gln with Gly and the fifth Thr with Ser (RD_22). Other preferred substitutions include the substitution of the first Gln with Gly and the substitution of the fifth Thr with Arg (RDC_11L). Sequence ID: 78 shows the amino acid sequence in Sequence ID: 6, in which the first Gln is replaced with Gly and the fifth Thr is replaced with Ser. Sequence ID: 79 shows the amino acid sequence in Sequence ID: 6, in which the first Gln is replaced with Gly and the fifth Thr is replaced with Arg.

[0191] (w) An anti-IL-6 receptor antibody comprising a heavy chain CDR2 in which the 9th Thr 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 the 1st Ser and 5th Thr in the amino acid sequence (HCDR3) described in SEQ ID NO: 3 are substituted with other amino acids. In the amino acid sequence (HCDR2) described in SEQ ID NO: 2, the 9th Thr is preferably substituted with Asn. Furthermore, preferred amino acid combinations after substitution of the 1st Ser and 5th Thr in the amino acid sequence (HCDR3) described in SEQ ID NO: 3 include 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), and Leu and Ile (RDC_8H). One could list these:

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

[0193] The present invention provides an antibody containing at least one of the amino acid substitutions described in (a) to (y) above, and a method for producing the antibody. Therefore, the antibody of the present invention also includes antibodies containing amino acid substitutions other than those described in (a) to (y) above, in addition to the amino acid substitutions described in (a) to (y) above. Furthermore, the antibody of the present invention also includes antibodies in which multiple amino acid substitutions described in (a) to (y) above are combined. Examples of amino acid substitutions described in (a) to (y) above include substitutions of the CDR amino acid sequence with other amino acids. Other types of amino acid substitutions include, for example, substitutions of amino acid sequences in other CDR regions. Examples include deletions, additions, and / or insertions. Furthermore, examples include substitutions, deletions, additions, and / or insertions of the amino acid sequence of the FR region. Also, examples include substitutions, deletions, additions, and / or insertions of the amino acid sequence of the constant region.

[0194] Furthermore, the antibody of the present invention incorporates the high affinity CDR discovered in the present invention into the humanized PM-1 antibody. This also includes antibodies transplanted into any other framework. Furthermore, the antibodies of the present invention include antibodies in which the high affinity CDR discovered in the present invention has been transplanted into a framework other than the humanized PM-1 antibody. As a result, in antibodies with reduced affinity, mutations were introduced into the framework portion to obtain the antibody with the original affinity (see, for example, Curr Opin Biotechnol. 1994 Aug;5(4):428-33), and mutations were introduced into the CDR portion to obtain the antibody with the original affinity. It contains antibodies that have been introduced (see, for example, US2006 / 0122377).

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

[0196] Furthermore, antibodies containing any of the amino acid substitutions described in (a) to (y) above can also be expressed as shown in (1) or (2) below, for example. Here, we will use the antibody in (a) as an example, but antibodies in (b) to (y) can be expressed in the same way. (1) In the amino acid sequence described in Sequence ID No. 1, the first Ser is substituted with another amino acid. An antibody containing a heavy chain variable region having CDR1 with a specified amino acid sequence. (2) As CDR1, an antibody comprising a heavy chain having an amino acid sequence in which the first Ser in the amino acid sequence set forth in SEQ ID NO: 1 is replaced with another amino acid Antibody containing a heavy chain having an amino acid sequence in which the first Ser in the amino acid sequence set forth in SEQ ID NO: 1 is replaced 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, and 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 etc.

[0198] <Antibody with reduced immunogenic risk of CDR sequence> Further, the present invention provides an anti-IL-6 receptor antibody with reduced immunogenicity, particularly a humanized PM-1 antibody. It is considered that when a T-cell epitope that binds to HLA exists in the antibody sequence, the immunogenicity of the antibody is high Therefore, by replacing 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 replaces the amino acids in the amino acid sequence of the antibody, particularly in the CDR sequence, with other amino acids The present invention provides a humanized anti-human IL-6 receptor antibody, particularly a humanized PM-1 light chain variable region, in which the T-cell epitope is removed and immunogenicity is reduced. The present invention also provides an antibody containing the light chain variable region. More specifically, the present invention relates to the amino acid sequence (LCDR2) described in Sequence ID No. 5, where the second The present invention provides a light chain CDR2 in which the Thr group is substituted with another amino acid. Furthermore, the present invention includes the light chain CDR2. The present invention provides a light chain variable region. Furthermore, the present invention provides an anti-IL-6 receptor antibody containing the light chain variable region. The substituted amino acid sequence is not particularly limited, but substitution to Gly is preferred. Sequence ID: 71 shows the amino acid sequence in which the second Thr is replaced with Gly in the amino acid sequence described in column number: 5. This amino acid substitution is preferably carried out in the light chain variable region of the humanized PM-1 antibody.

[0200] <H53 / L28のFRおよびCDR> The present invention also provides anti-human IL-6 receptor antibodies 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 pI with a high correlation coefficient. Therefore, two antibodies against different antigens... When we attempted to modify the pI of the variable region in various antibodies, we succeeded in controlling the plasma half-life without modifying the Fc region, regardless of the antigen type. The rate of nonspecific uptake of antibodies into endothelial cells is related to the physicochemical Coulomb phase between the negatively charged cell surface and IgG. It is thought to depend on the interaction. By reducing the pI of IgG, the Coulomb interaction is reduced. This reduces nonspecific uptake into endothelial cells, which in turn reduces metabolism in endothelial cells and increases plasma retention.

[0201] In other words, the present invention provides an anti-human IL-6 receptor antibody, particularly a humanized PM-1 antibody, in which the isoelectric point is lowered and plasma retention is increased by substituting the amino acid sequence of the anti-IL-6 receptor 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), and H105 (SEQ ID NO: 10) of humanized PM-1 are substituted. (the third amino acid of), 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 third amino acid of), L18 (the 18th amino acid of SEQ ID NO: 11), L45 (the 11th amino acid of SEQ ID NO: 12), L79 ( (10th amino acid of sequence number 14), H31 (1st amino acid of sequence number 1), L24 (1st amino acid of sequence number 4) and / or L53 (4th amino acid of sequence number 5) The amino acid is replaced with another amino acid that lowers the isoelectric point. This makes it possible to lower the isoelectric point without affecting the binding activity or stability of humanized PM-1. In addition, in humanized PM-1 antibodies, when the mouse sequence is humanized, several amino acid residues remain in the mouse sequence to maintain binding activity. Specifically, in the Kabat numbering mentioned above, In the PM-1 antibody, 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 are directly derived from the mouse sequence. Regarding HFR1, it is possible to convert it to a human sequence as HFR1 by substituting H13, H16, H23, and H30. It is thought that it is possible to produce antibodies with an even lower immunogenicity risk than the t-transferred antibody PM-1. Furthermore, since humanized PM-1 is an antibody that has been humanized by CDR grafting, Regarding stability, there is room for improvement. For example, it is thought that the antibody can be stabilized by substituting amino acid residues exposed on the surface in the variable region of the antibody with hydrophilic amino acids. Furthermore, modifying the CDR sequence into a consensus sequence... Antibodies can also be stabilized by the following: In the humanized PM-1 antibody, the above-mentioned Kabat numbering involves the substitution of Met to Ile at H69 (the 4th amino acid of SEQ ID NO: 9) (stabilization of the hydrophobic core structure), and the substitution of Leu to Ser at H70 (the 5th amino acid of SEQ ID NO: 9). Substitution (hydrophilization of surface-exposed residues), Thr to Asn of H58 (the 9th amino acid in SEQ ID NO: 2) Substitution to (modification of the consensus sequence of heavy chain CDR2), H65 (sequence number: 2, 16th A Substitution of Ser to Gly in amino acids (substitution of Gly in the β-turn portion, consensus of heavy chain CDR2) Modification of the sequence, or replacement of Thr to Ser in L93 (the 5th amino acid of sequence number 6) Antibodies can be stabilized by substitution (hydrophilization of surface-exposed residues). Furthermore, by substituting Thr (L51), the second residue of LCDR2 (SEQ ID NO: 5), with Gly, binding activity and stability can be improved. By removing T-cell epitopes predicted in silico without affecting the system. It is possible to reduce the risk of immunogenicity. By combining these amino acid substitutions, it is possible to obtain anti-IL-6 receptor antibodies with improved plasma pharmacokinetics, immunogenicity, and stability. This is possible.

[0202] Examples of such antibodies include those listed in any of (1) to (37) below. (1) In the amino acid sequence described in Sequence ID No. 7, the 13th Arg is substituted with another amino acid. An antibody containing a heavy chain variable region having FR1. The substituted amino acid sequence is not particularly limited, but substitution to Lys is preferred. Sequence ID: 80 shows the amino acid sequence in which the 13th Arg is replaced with Lys in the sequence ID: 7. (2) In the amino acid sequence described in Sequence ID No. 7, the 16th Gln is substituted with another amino acid. An antibody containing a heavy chain variable region having FR1. The substituted amino acid sequence is not particularly limited, but substitution with Glu is preferred. Sequence ID: 81 shows the amino acid sequence in which the 16th Gln is replaced with Glu in the sequence described in Sequence ID: 7. (3) In the amino acid sequence described in Sequence ID No. 7, the 23rd Thr is substituted with another amino acid. An antibody containing a heavy chain variable region having FR1. The substituted amino acid sequence is not particularly limited, but substitution with Ala is preferred. Sequence ID: 82 shows the amino acid sequence in which the 23rd Thr is replaced with Ala in the sequence described in Sequence ID: 7. (4) In the amino acid sequence described in Sequence ID No. 7, the 30th Thr is substituted with another amino acid. An antibody containing a heavy chain variable region having FR1. The substituted amino acid sequence is not particularly limited, but substitution to Ser is preferred. Sequence ID: 83 shows the amino acid sequence in which the 30th Thr is replaced with Ser in the sequence described in Sequence ID: 7. (5) A heavy chain variable region having FR1 in which the 13th Arg, 16th Gln, 23rd Thr, and 30th Thr in the amino acid sequence described in Sequence ID No. 7 is replaced with other amino acids. Contains antibodies. The substituted amino acids are not particularly limited, but substitutions of the 13th Arg with Lys, the 16th Gln with Glu, the 23rd Thr with Ala, and the 30th Thr with Ser are preferred. Sequence ID: 84 shows the amino acid sequence described in Sequence ID: 7, in which the 13th Arg is replaced with Lys, the 16th Gln with Glu, the 23rd Thr with Ala, and the 30th Thr with Ser. (6) The amino acid sequence described in Sequence ID: 8, in which the 8th Arg is replaced with another amino acid. An antibody containing a heavy chain variable region with a defined FR2. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. Sequence ID: 85 shows the amino acid sequence in which the 8th Arg is replaced with Glu in the sequence described in Sequence ID: 8. (7) In the amino acid sequence described in Sequence ID No. 9, the fourth Met is substituted with another amino acid. An antibody containing a heavy chain variable region with a defined FR3. The substituted amino acid is not particularly limited, but substitution to Ile is preferred. Sequence ID: 86 shows the amino acid sequence in which the fourth Met is replaced with Ile in the amino acid sequence described in Sequence ID: 9. (8) In the amino acid sequence described in Sequence ID No. 9, the fifth Leu is substituted with another amino acid. An antibody containing a heavy chain variable region with a defined FR3. The substituted amino acid sequence is not particularly limited, but substitution to Ser is preferred. Sequence ID: 87 shows the amino acid sequence in which the 5th Leu is replaced with Ser in the sequence described in Sequence ID: 9. (9) In the amino acid sequence described in Sequence ID No. 9, the 16th Arg is substituted with another amino acid. An antibody containing a heavy chain variable region with FR3. The substituted amino acid is not particularly limited, but substitution to Lys is preferred. The amino acid sequence described in Sequence ID No. 9 has been modified by substituting Arg at position 16 with Lys. Shown in column number 88. (10) In the amino acid sequence described in Sequence ID No. 9, the 27th Val is replaced by another amino acid. An antibody containing a heavy chain variable region with a replaced FR3. The substituted amino acid sequence is not particularly limited, but substitution with Ala is preferred. Sequence ID: 89 shows the amino acid sequence in which the 27th Val is replaced with Ala in the sequence described in Sequence ID: 9. (11) Heavy chain variable FR3 having the amino acid sequence described in Sequence ID No. 9 (HFR3) in which the 4th Met, 5th Leu, 16th Arg, and 27th Val are substituted with other amino acids. Antibodies containing a specific region. The substituted amino acids are not particularly limited, but substitutions of the 4th Met with Ile, the 5th Leu with Ser, the 16th Arg with Lys, and the 27th Val with Ala are preferred. Sequence ID: 90 shows the amino acid sequence in Sequence ID: 9 in which the 4th Met is replaced with Ile, the 5th Leu with Ser, the 16th Arg with Lys, and the 27th Val with Ala. (12) In the amino acid sequence described in Sequence ID No. 10 (HFR4), the third Gln is different from other amino acids. An antibody containing a heavy chain variable region with FR4 substituted with an acid. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. Sequence ID: 91 shows the amino acid sequence in which the third Gln is replaced with Glu in the amino acid sequence described in Sequence ID: 10. (13) In the amino acid sequence described in Sequence ID No. 11 (LFR1), the 18th Arg is different from other A An antibody containing a light chain variable region with FR1 substituted with a methyl amino acid. The substituted amino acid is not particularly limited, but substitution to Ser is preferred. Sequence ID: 92 shows the amino acid sequence in which the 18th Arg is replaced with Ser in the sequence described in Sequence ID: 11. (14) In the amino acid sequence described in Sequence ID No. 12 (LFR2), the 11th Lys is different from the other A An antibody containing a light chain variable region with FR2 substituted with a methyl amino acid. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. Sequence ID: 93 shows the amino acid sequence in which the 11th Lys is replaced with Glu in the sequence described in Sequence ID: 12. (15) In the amino acid sequence described in Sequence ID No. 13, the 23rd Gln is replaced by another amino acid An antibody containing a light chain variable region with a substituted FR3. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. Sequence ID: 94 shows the amino acid sequence in which the 23rd Gln is replaced with Glu in the amino acid sequence described in Sequence ID: 13. (16) In the amino acid sequence described in Sequence ID No. 13, the 24th Pro is replaced by another amino acid. An antibody containing a light chain variable region with a substituted FR3. The substituted amino acid sequence is not particularly limited, but substitution with Ala is preferred. Sequence ID: 95 shows the amino acid sequence in which the 24th Pro is replaced with Ala in the amino acid sequence described in Sequence ID: 13. (17) In the amino acid sequence described in Sequence ID No. 13, the 27th Ile is replaced by another amino acid An antibody containing a light chain variable region with a substituted FR3. The substituted amino acid sequence is not particularly limited, but substitution with Ala is preferred. Sequence ID: 96 shows the amino acid sequence in which the 27th Ile is replaced with Ala in the sequence described in Sequence ID: 13. (18) In the amino acid sequence described in Sequence ID No. 13 (LFR3), the 23rd Gln and 24th It contains a light chain variable region with FR3 in which the Pro and 27th Ile are substituted with other amino acids. Antibodies. The substituted amino acids are not particularly limited, but substitution of the 23rd Gln with Glu, the 24th Pro with Ala, and the 27th Ile with Ala is preferred. Sequence ID: 97 shows the amino acid sequence described in Sequence ID: 13, in which the 23rd Gln is replaced with Glu, the 24th Pro is replaced with Ala, and the 27th Ile is replaced with Ala. (19) In the amino acid sequence described in Sequence ID No. 14 (LFR4), the 10th Lys is different from the other A An antibody containing a light chain variable region with FR4 substituted with a methyl amino acid. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. Sequence ID: 98 shows the amino acid sequence in which the 10th Lys molecule is replaced with Glu in the sequence described in Sequence ID: 14. (20) In the amino acid sequence described in Sequence ID No. 10 (HFR4), the 5th Ser is different from other amino acids. An antibody containing a heavy chain variable region with FR4 substituted with an acid. The substituted amino acid is not particularly limited, but substitution to Thr is preferred. Sequence ID: 132 shows the amino acid sequence in which the 5th Ser is replaced with Thr in the sequence described in Sequence ID: 10. (21) In the amino acid sequence described in Sequence ID No. 10 (HFR4), the 3rd Gln and the 5th An antibody containing a heavy chain variable region having FR4 in which Ser is substituted with another amino acid. The substituted amino acids are not particularly limited, but substitution of the third Gln with Glu and the fifth Ser with Thr is preferred. Sequence ID: 133 shows the amino acid sequence in Sequence ID: 10, in which the third Gln is replaced with Glu and the fifth Ser is replaced with Thr. An antibody comprising a humanized PM-1 heavy chain variable region with the amino acid substitutions described in (22)(5), (6), (11), and (21). An antibody containing a humanized PM-1 light chain variable region with the amino acid substitutions described in (23), (13), (14), (18), and (19). An antibody comprising the heavy chain variable region described in (24)(22) and the light chain variable region described in (23). (25) An antibody containing a heavy chain variable region having a CDR1 in which the first Ser in the amino acid sequence described in Sequence ID No. 1 (HCDR1) is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution to Asp is preferred. Sequence ID: 28 shows the amino acid sequence in which the first Ser is replaced with Asp in the amino acid sequence described in Sequence ID: 1. (26) In the amino acid sequence described in Sequence ID No. 2, the 16th Ser is placed in place of another amino acid. An antibody containing a heavy chain variable region with a replaced CDR2. The substituted amino acid is not particularly limited, but substitution with Gly is preferred. Sequence ID: 99 shows the amino acid sequence in which the 16th Ser is replaced with Gly in the amino acid sequence described in Sequence ID: 2. (27) An antibody containing a heavy chain variable region having a CDR2 in which the 9th Thr and 16th Ser in the amino acid sequence described in Sequence ID No. 2 (HCDR2) are substituted with other amino acids. The substituted amino acids are not particularly limited, but substitution of the 9th Thr with Asn and the 16th Ser with Gly is preferred. Sequence ID: 100 shows the amino acid sequence described in Sequence ID: 2, in which the 9th Thr is replaced with Asn and the 16th Ser is replaced with Gly. (28) An antibody containing a light chain variable region having a CDR1 in which the first Arg is substituted with another amino acid in the amino acid sequence described in Sequence ID No. 4 (LCDR1). The substituted amino acid is not particularly limited, but substitution with Gln is preferred. Sequence ID: 101 shows the amino acid sequence in which the first Arg is replaced with Gln in the sequence described in Sequence ID: 4. (29) In the amino acid sequence described in Sequence ID No. 5, the fourth Arg is substituted with another amino acid. An antibody containing a light chain variable region with a modified CDR2. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. Sequence ID: 102 shows the amino acid sequence in which the fourth Arg is replaced with Glu in the amino acid sequence described in Sequence ID: 5. (30) An antibody containing a light chain variable region having a CDR2 in which the second Thr and fourth Arg in the amino acid sequence described in Sequence ID No. 5 (LCDR2) are substituted with other amino acids. The substituted amino acids are not particularly limited, but substitution of the second Thr with Gly and the fourth Arg with Glu is preferred. Sequence ID: 103 shows the amino acid sequence described in Sequence ID: 5, in which the second Thr is replaced with Gly and the fourth Arg is replaced with Glu. (31) An antibody containing a light chain variable region having a CDR3 in which the 5th Thr in the amino acid sequence described in Sequence ID No. 6 (LCDR3) is substituted with another amino acid. The substituted amino acid is not particularly limited, but substitution to Ser is preferred. Sequence ID: 77 shows the amino acid sequence in which the 5th Thr is replaced with Ser in the sequence described in Sequence ID: 6. (32)(25) and (27) Antibody containing a heavy chain variable region with amino acid substitutions as described above body. An antibody comprising a light chain variable region with the amino acid substitutions described in (33), (28), (30), and (31). An antibody comprising the heavy chain variable region described in (34)(32) and the light chain variable region described in (33). (35) Heavy chain variable region having the amino acid sequence (VH of H53 / L28) described in Sequence ID No. 104 Antibodies containing this substance. (36) Light chain variable region having the amino acid sequence described in Sequence ID No. 105 (VL of H53 / L28) Antibodies containing this substance. An antibody having the heavy chain variable region described in (37)(35) and the light chain variable region described in (36).

[0203] The amino acid substitutions described in any of (1) to (37) above are preferably performed on the humanized PM-1 antibody. The present invention provides an antibody containing at least one of the amino acid substitutions described in any of (1) to (37) above, and a method for producing the antibody. Therefore, the antibody of the present invention also includes antibodies containing amino acid substitutions other than those described in (1) to (37) above, in addition to the amino acid substitutions described in any of (1) to (37) above. Furthermore, the antibody of the present invention also includes antibodies in which multiple amino acid substitutions described in any of (1) to (37) above are combined. Examples of amino acid substitutions described in (1) to (37) above include the amino acid sequence substitutions of FR and CDR described above. Other amino acid substitutions include substitutions, deletions, and additions of FR and CDR sequences other than those mentioned above. Examples include substitution and / or insertion. Other examples include substitution, deletion, addition, and / or insertion of amino acid sequences in the constant region. Furthermore, in addition to the amino acid modifications described above, other modifications that lower the isoelectric point without reducing the activity of the anti-IL-6 receptor antibody include, for example, the substitution of the 15th Lys and / or 16th Ser in the amino acid sequence described in SEQ ID NO: 2 with other amino acids. The substituted amino acids are not particularly limited, but it is preferable to substitute the 15th Lys with Gln and the 16th Ser with Asp. SEQ ID NO: 121 shows the sequence in which the 15th Lys is substituted with Gln and the 16th Ser with Asp in the amino acid sequence of SEQ ID NO: 2. Such amino acid substitutions may also be performed on the amino acid sequence described in SEQ ID NO: 100. SEQ ID NO: 122 shows the sequence in which the 15th Lys is substituted with Gln and the 16th Gly is substituted with Asp in the amino acid sequence of SEQ ID NO: 100. Accordingly, the present invention provides an antibody comprising a heavy chain variable region having a CDR2 in which the 15th Lys and / or the 16th Ser in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 100 is substituted with another amino acid. Furthermore, another modification that lowers the isoelectric point is to replace the fourth Gln in the amino acid sequence described in SEQ ID NO: 4 with another amino acid. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. In the amino acid sequence of SEQ ID NO: 4, the fourth The amino acid sequence in which Gln is replaced with Glu is shown in SEQ ID NO: 123. This amino acid substitution may also be performed on the amino acid sequence of SEQ ID NO: 101. The amino acid sequence in which the fourth Gln in the amino acid sequence of SEQ ID NO: 101 is replaced with Glu is shown in SEQ ID NO: 124. Accordingly, the present invention provides an antibody containing a light chain variable region having CDR1 in which the fourth Gln in the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 101 is replaced with another amino acid. Furthermore, another modification that lowers the isoelectric point is to replace the 6th His amino acid in the amino acid sequence described in Sequence ID No. 5 with another amino acid. The substituted amino acid is not particularly limited, but substitution with Glu is preferred. The amino acid sequence in which the sixth His is replaced with Glu is shown in SEQ ID NO: 125. This amino acid substitution may also be performed on the amino acid sequence of SEQ ID NO: 103. The amino acid sequence in which the sixth His is replaced with Glu in the amino acid sequence of SEQ ID NO: 103 is shown in SEQ ID NO: 126. Accordingly, the present invention provides an antibody containing a light chain variable region having a CDR2 in which the sixth His is replaced with another amino acid in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 103. Furthermore, the amino acid sequence of the heavy chain FR3 described in SEQ ID NO: 90 has an immunogenicity risk. One modification to reduce the load is to replace the 27th Ala (Kabat numbering H89) with Val. The amino acid sequence in which the 27th Ala is replaced with Val in the amino acid sequence described in SEQ ID NO: 90 is shown in SEQ ID NO: 127. Therefore, the present invention includes a heavy chain variable region having FR3 in which the 27th Ala is replaced with Val in the amino acid sequence of SEQ ID NO: 90. To provide antibodies. Furthermore, in the amino acid sequence of heavy chain FR3 described in SEQ ID NO: 9 or SEQ ID NO: 90, only Regarding the 6th Arg (Kabat numbering H71), which is the remaining mouse sequence, H71 is Arg By using the human VH1 subclass (sequence number: 128) or human VH3 subclass (sequence number: 129) human sequence stored in the FR3 sequence, the framework can be used as Therefore, it is considered possible to produce anti-human IL-6 receptor antibodies that are completely human sequences. Accordingly, the present invention relates to the heavy chain variable having FR3 as described in SEQ ID NO: 128 or SEQ ID NO: 129. The present invention provides antibodies containing a specific region. Furthermore, the amino acid sequence of the heavy chain FR4 described in SEQ ID NO: 10 has been improved to enhance stability. One possible modification is to replace the fifth Ser (Kabat numbering H107) with Ile. This can be done. The amino acid sequence in which the 5th Ser is replaced with Ile in the amino acid sequence described in SEQ ID NO: 10 is shown in SEQ ID NO: 130. This amino acid sequence may also be performed on the amino acid sequence of SEQ ID NO: 91. The amino acid sequence in which the 5th Ser is replaced with Ile in the amino acid sequence described in SEQ ID NO: 91 is shown in SEQ ID NO: 131. Accordingly, the present invention provides an antibody containing a heavy chain variable region having FR4 in which the 5th Ser is replaced with Ile in the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 91. Such amino acid substitutions are found in the humanized PM-1 antibody, H53 / L28 (the heavy chain variable region of SEQ ID NO: 104). (Antibody containing the light chain variable region of SEQ ID NO: 105), or PF1 antibody (SEQ ID NO: 22 It is preferable that the substitution is performed on an antibody containing 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 containing at least such amino acid substitutions and a method for producing the antibody. Accordingly, the antibodies of the present invention also include antibodies containing 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 mentioned above. This can be achieved. Other examples include substitution, deletion, addition, and / or insertion of amino acid sequences in the constant region.

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

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

[0206] The present invention also provides a humanized PM-1 antibody in which the amino acid substitutions described in any of (1) to (37) above have been further modified by amino acid substitutions described in any of (a) to (y) above to improve the binding activity and / or neutralizing activity. One embodiment of such an antibody is a heavy chain variable region having the amino acid sequence (PF1_H) described in SEQ ID NO: 22, and a sequence Examples include, but are not limited to, antibodies (PF1) having a light chain variable region with the amino acid sequence (PF1_L) described in item 23.

[0207] Furthermore, the present invention provides an anti-IL-6 receptor antibody described in any of (A) to (I) below. (A) CDR1 having the amino acid sequence (VH5-M83 CDR1) described in SEQ ID NO: 165, SEQ ID NO: A heavy chain variable region having a CDR2 having the amino acid sequence described in :166 (CDR2 of VH5-M83), and a 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 A light chain variable region having CDR2 having the amino acid sequence described in 68 (CDR2 of VL5), and CDR3 having the amino acid sequence described in SEQ ID NO: 79 (CDR3 of VL5), (C) An antibody containing the heavy chain variable region of (A) and the light chain variable region of (B), (D) CDR1 having the amino acid sequence (VH3-M73 CDR1) described in SEQ ID NO: 169, SEQ ID NO: A heavy chain variable region having a CDR2 having the amino acid sequence described in :170 (VH3-M73 CDR2), and a CDR3 having the amino acid sequence described in SEQ ID NO: 171 (VH3-M73 CDR3), (E) CDR1 having the amino acid sequence described in SEQ ID NO: 172 (CDR1 of VL3), SEQ ID NO: 1 A light chain variable region having CDR2 having the amino acid sequence described in 73 (CDR2 of VL3), and CDR3 having the amino acid sequence described in SEQ ID NO: 79 (CDR3 of VL3), (F) An antibody containing a heavy chain variable region (D) and a light chain variable region (E), (G) CDR1 having the amino acid sequence (VH4-M73 CDR1) described in SEQ ID NO: 169, SEQ ID NO: A heavy chain variable region having a CDR2 having the amino acid sequence described in :174 (VH4-M73 CDR2), and a CDR3 having the amino acid sequence described in SEQ ID NO: 171 (VH4-M73 CDR3), (H) CDR1 having the amino acid sequence (VL1 CDR1) described in SEQ ID NO: 175, SEQ ID NO: 1 CDR2 having the amino acid sequence described in 73 (CDR2 of VL1), amino acid described in SEQ ID NO: 79 A light chain variable region having a CDR3 having an acid sequence (CDR3 of VL1), An antibody containing the heavy chain variable region of (I) (G) and the light chain variable region of (H).

[0208] Furthermore, the present invention provides an anti-IL-6 receptor antibody as described in any of (a) to (q) below. (a) An antibody containing 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, the 35th Trp , 51st Tyr, 63rd Ser, 65th Lys, 66th Gly, 99th Val, 103rd Ile an antibody containing a heavy chain variable region having an amino acid sequence in which at least one of the following amino acids is substituted with another amino acid: the 108th Tyr, the 111th Glu, and the 113th Thr. (c) In the amino acid sequence (H96-IgG1 variable region) described in SEQ ID NO: 159, the 65th Lys An antibody containing a heavy chain variable region having an amino acid sequence in which the 66th Gly, 99th Val, 103rd Ile, 111th Glu, and 113th Thr are substituted with other amino acids. (d) In the amino acid sequence (H96-IgG1 variable region) described in SEQ ID NO: 159, the 35th Trp , 51st Tyr, 63rd Ser, 65th Lys, 66th Gly, 99th Val, 103rd Ile an antibody containing a heavy chain variable region having an amino acid sequence in which the 108th Tyr is substituted with another amino acid, (e) An antibody containing a heavy chain variable region having the amino acid sequence (F2H-IgG1 variable region) described in SEQ ID NO: 160, (f) A heavy chain variable region having the amino acid sequence (VH5-M83 variable region) described in Sequence ID No. 161 Antibodies containing (g) The 27th Gln and / or 55th amino acid in the amino acid sequence (PF1L) described in Sequence ID No. 23 An antibody having a light chain variable region having an amino acid sequence in which His is substituted with another amino acid, (h) Includes a light chain variable region having the amino acid sequence (L39 variable region) described in SEQ ID NO: 162. antibody, (i) Light chain variable region having the amino acid sequence (VL5-kappa variable region) described in SEQ ID NO: 163 Antibodies containing, (j) Heavy chain variable region having the amino acid sequence (VH3-M73 variable region) described in SEQ ID NO: 176 Antibodies containing, (k) Heavy chain variable region having the amino acid sequence (VH4-M73 variable region) described in SEQ ID NO: 178 Antibodies containing, (l) Light chain variable region having the amino acid sequence (VL3-kappa variable region) described in Sequence ID No. 177 Antibodies containing the region, (m) Light chain variable region having the amino acid sequence (VL1-kappa variable region) described in Sequence ID No. 179 Antibodies containing the region, (n) An antibody containing the heavy chain variable region of (e) and the light chain variable region of (h), (o) An antibody containing the heavy chain variable region of (f) and the light chain variable region of (i) (a combination of variable regions of FV5-M83) , (p) (j) heavy chain variable region and (l) light chain variable region antibody (FV4-M73 variable region combination) ), (q) An antibody containing the heavy chain variable region of (k) and the light chain variable region of (m) (a combination of variable regions of FV3-M73) .

[0209] In the amino acid substitutions in the heavy chain variable region described in (a) to (d) above, the substituted amino acids are not particularly limited, but it is preferable that the 35th Trp is substituted with Val, the 51st Tyr with Phe, the 63rd Ser with Thr, the 65th Lys with Gln, the 66th Gly with Asp, the 99th Val with Leu, the 103rd Ile with Ala, the 108th Tyr with Val, the 111th Glu with Gln, and the 113th Thr with Ile. Also, the light (g) described above In amino acid substitutions in the chain variable region, the substituted amino acids are not particularly limited, but it is preferable that the 27th Gln is substituted with Glu and the 55th His is substituted with Glu. In addition, substitutions, deletions, insertions, and / or additions of amino acids other than those described above may also be performed.

[0210] The constant region of the antibody of the present invention is not particularly limited, and any constant region may be used. For example, constant regions having natural sequences such as IgG1, IgG2, and IgG4, or modified constant regions created by substitution, deletion, addition, and / or insertion of amino acids in a constant region having a natural sequence can be used. Examples of modified constant regions include the constant regions described later. Furthermore, the following antibodies can be cited as examples of antibodies using the variable region of the present invention described above. (1) An antibody containing a heavy chain having the amino acid sequence (H96-IgG1) described in SEQ ID NO: 134, (2) An antibody containing a heavy chain having the amino acid sequence (F2H-IgG1) described in Sequence ID No. 135, (3) An antibody containing a heavy chain having the amino acid sequence (VH5-IgG1) described in SEQ ID NO: 137, (4) An antibody containing a heavy chain having the amino acid sequence (VH5-M83) described in SEQ ID NO: 139, (5) An antibody containing a light chain having the amino acid sequence (L39) described in SEQ ID NO: 136, (6) An antibody containing a light chain having the amino acid sequence (VL5-kappa) described in SEQ ID NO: 138, (7) An antibody containing a heavy chain having the amino acid sequence (VH3-M73) described in SEQ ID NO: 180, (8) An antibody containing a heavy chain having the amino acid sequence (VH4-M73) described in Sequence ID No. 182, (9) An antibody containing a light chain having the amino acid sequence (VL3-kappa) described in Sequence ID No. 181, (10) An antibody containing a light chain having the amino acid sequence (VL1-kappa) described in Sequence ID No. 183, (11) An antibody containing the heavy chain of (2) and the light chain of (5), (12) An antibody containing the heavy chain of (3) and the light chain of (6), (13) An antibody (FV5-M83) containing the heavy chain of (4) and the light chain of (6), (14) An antibody (FV4-M73) containing the heavy chain of (7) and the light chain of (9), (15) An antibody (FV3-M73) containing the heavy chain of (8) and the light chain of (10), (16) An antibody having activity equivalent to that of any of the antibodies described in (1) to (15). Here, "having equivalent activity" means that the binding activity to the antigen and / or neutralizing activity are equivalent. In the present invention, equivalent activity does not necessarily mean identical activity; for example, it means having 50% or more activity, preferably 70% or more activity, and more preferably 90% or more activity. It means that something is happening.

[0211] Furthermore, the present invention provides a CDR or FR as described in any of (i) to (xxii) below. (i) Heavy chain FR1 having the amino acid sequence described in SEQ ID NO: 84 (heavy chain FR1 of VH5) (ii) Heavy chain FR1 having the amino acid sequence described in Sequence ID No. 186 (heavy chain FR1 of VH3 and VH4) (iii) Heavy chain FR2 having the amino acid sequence described in Sequence ID No. 85 (heavy chain FR2 of VH3, VH4, VH5) ) (iv) Heavy chain FR3 having the amino acid sequence described in SEQ ID NO: 184 (heavy chain FR3 of VH3, VH4, and VH5) (v) Heavy chain FR4 having the amino acid sequence described in SEQ ID NO: 133 (heavy chain FR4 of VH3, VH4, VH5) ) (vi) Light chain FR1 having the amino acid sequence described in SEQ ID NO: 92 (light chain FR1 of VL1, VL3, and VL5) (vii) Light chain FR2 having the amino acid sequence described in SEQ ID NO: 93 (light chain FR2 of VL1, VL3, and VL5) ) (viii) Light chain FR3 having the amino acid sequence described in SEQ ID NO: 97 (light chain FR3 of VL1, VL3, and VL5) (ix) Light chain FR4 having the amino acid sequence described in SEQ ID NO: 98 (light chain FR4 of VL1, VL3, and VL5) (x) Heavy chain CDR1 having the amino acid sequence described in SEQ ID NO: 169 (heavy chain CDR1 of VH3 and VH4) (xi) Heavy chain CDR1 having the amino acid sequence described in SEQ ID NO: 165 (heavy chain CDR1 of VH5), (xii) Heavy chain CDR2 having the amino acid sequence described in Sequence ID No. 170 (heavy chain CDR2 of VH3), (xiii) Heavy chain CDR2 having the amino acid sequence described in Sequence ID No. 174 (heavy chain CDR2 of VH4), (xiv) Heavy chain CDR2 having the amino acid sequence described in Sequence ID No. 166 (heavy chain CDR2 of VH5), (xv) Heavy chain CDR3 having the amino acid sequence described in SEQ ID NO: 171 (heavy chain CDR3 of VH3 and VH4) , (xvi) Heavy chain CDR3 having the amino acid sequence described in SEQ ID NO: 167 (heavy chain CDR3 of VH5), (xvii) Light chain CDR1 having the amino acid sequence described in Sequence ID No. 175 (VL1 light chain CDR1), (xviii) Light chain CDR1 having the amino acid sequence described in Sequence ID No. 172 (VL3 light chain CDR1), (xix) Light chain CDR1 having the amino acid sequence described in Sequence ID No. 101 (VL5 light chain CDR1), (xx) Light chain CDR2 having the amino acid sequence described in SEQ ID NO: 173 (VL1, VL3 light chain CDR2) , (xxi) Light chain CDR2 having the amino acid sequence described in Sequence ID No. 168 (VL5 light chain CDR2), (xxii) Light chain CDR3 having the amino acid sequence described in SEQ ID NO: 79 (light chain CDR3 of VL1, VL3, and VL5)

[0212] The antibodies of the present invention also include antibody fragments and modifications thereof that contain the amino acid substitutions described above. Examples of antibody fragments include Fab, F(ab')2, Fv, or single-chain Fv (scFv) obtained by linking the Fv of the H chain and L chain with an appropriate linker, single H chain domains or single L chain domains (e.g., Nat Biotechnol. 2005 Sep;23(9):1126-36.), Unibody (WO2007059782 A1), and SMIP (WO2007014278 A2). The origin of the antibody is not particularly limited, but examples include human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies. Furthermore, the antibodies of the present invention may be chimeric antibodies, humanized antibodies, fully humanized antibodies, etc. Specifically, antibodies are treated with enzymes, such as papain or pepsin, to generate antibody fragments, or genes encoding these antibody fragments are constructed, introduced into an expression vector, and then expressed in suitable host cells (e.g., Co, MS et al., J. Immunol. (1994) 152). 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Pluckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 497-515, Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods. in Enzymology (1989) 121, 663-66, Bird, RE et al., TIBTECH (1991) 9, 132-137).

[0213] scFv is obtained by linking the V region of the heavy chain and the V region of the light chain of an antibody. In this scFv, the V region of the heavy chain and the V region of the light chain are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). The V region of the heavy chain and the V region of the light chain in the scFv may be derived from any of the antibodies described above. As the peptide linker that links the V regions, for example, any single-chain peptide consisting of 12-19 amino acid residues can be used.

[0214] <Antibody permanent region> The present invention also provides an improved antibody constant region in which any of the amino acids described in (i) to (xxi) below are substituted. The term "constant region" refers to the constant regions of the IgG1, IgG2, and IgG4 types. The amino acid sequences of the human IgG1, human IgG2, and human IgG4 constant regions are known (human IgG1 constant region: SEQ ID NO: 19, human IgG2 constant region: SEQ ID NO: 20, human IgG4 constant region: SEQ ID NO: 21). The human IgG4 constant region is a sequence that has been modified to improve the stability of the hinge portion (Mol Immunol. 1993 Jan;30(1):105-8.). Furthermore, the present invention provides an antibody containing the antibody constant region in which the amino acids are substituted. The antibody constant region is preferably a human antibody constant region. Furthermore, the antibody constant region in which the amino acids of the present invention are substituted may include other amino acid substitutions or modifications, as long as it includes the amino acid substitutions described in any of (i) to (xxi) below. Therefore, in the present invention, when the amino acid substitution of the present invention is performed on an IgG2 constant region in which one or more amino acids have already been substituted and / or modified from the amino acid sequence described in SEQ ID NO: 20, or when one or more amino acids are substituted and / or modified after the amino acid substitution of the present invention has been performed, it also corresponds to an IgG2 constant region in which the amino acid substitution of the present invention has been performed on an IgG2 constant region having the amino acid sequence described in SEQ ID NO: 20. The same applies to the IgG1 constant region containing the mino acid sequence and the IgG4 constant region described in SEQ ID NO: 21. Also, EU numbering (Sequences of proteins of immunological interest, NIH) (See Publication No. 91-3242) The 297th glycan can be any glycan structure. Furthermore, the presence of glycans is not required (for example, a constant region produced in host cells that do not have glycans attached, such as E. coli).

[0215] (i) Improved stability of IgG2 in the acidic region in the constant state One embodiment of the IgG2 constant region in which the amino acids of the present invention are substituted is an IgG2 constant region having the amino acid sequence described in SEQ ID NO: 20, wherein the 276th amino acid (397th in EU numbering) An example is the IgG2 constant region in which Met is replaced with another amino acid. The substituted amino acid is particularly The amino acid described in SEQ ID NO: 20 is preferred, although it is not limited to this. By substituting the 276th Met (397th in EU numbering) in the acid sequence with another amino acid, it is possible to improve the stability of antibodies under acidic conditions.

[0216] (ii) Improvement of heterogeneity in the constant region of IgG2 Furthermore, one embodiment of the IgG2 constant region in which the amino acids of the present invention are substituted is an IgG2 constant region having the amino acid sequence described in SEQ ID NO: 20, wherein the 14th (EU numbering 131st) Examples include the IgG2 constant region in which the Cys (Cys), the 16th (133rd in EU numbering) Arg, and the 102nd (219th in EU numbering) Cys are substituted with other amino acids. The substituted amino acids are not particularly limited, but the 14th (131st in EU numbering) Cys is substituted with Ser. It is preferable that the 16th (133rd in EU numbering) Arg be replaced with Lys. It is preferable that the 102nd (219th in EU numbering) Cys be replaced with Ser. Preferred (IgG2-SKSC). By performing these substitutions, it is possible to reduce heterogeneity originating from the hinge region of IgG2. The constant region of IgG2 with the amino acids substituted according to the present invention has the three types described above. The IgG2 constant region contains at least one of the amino acid substitutions. However, it is preferable that the 14th Cys and the 102nd Cys are substituted with other amino acids, or that all three of the above amino acids are substituted.

[0217] (iii) Reduction of binding of the constant region of IgG2 to FcγR Furthermore, as one embodiment of the IgG2 constant region in which the amino acids of the present invention are substituted, in an IgG2 constant region having the amino acid sequence described in SEQ ID NO: 20, the 209th (EU330) Ala is replaced with Ser. This provides an IgG2 constant region in which the 210th (EU331) Pro is replaced with Ser, and / or the 218th (EU339) Thr is replaced with Ala. It has already been reported that substitution of the 209th (EU330) Ala and the 210th (EU331) Pro can reduce binding to the Fcγ receptor (Eur J Immunol. 1999 Aug;29(8):2613-24.), but this modification is undesirable from the standpoint of immunogenicity risk because it introduces a non-human peptide that can become a T-cell epitope. Therefore, by simultaneously substituting the 218th (EU339) Thr with Ala, only human peptides are used as the 9-12 amino acids that can become T-cell epitopes. It is possible to reduce the binding of IgG2 to the Fcγ receptor. The IgG2 constant region in which the amino acids of the present invention are substituted is one of the three amino acid substitutions described above. While substitution of at least one amino acid is sufficient, it is preferable that all three amino acids described above be substituted. Therefore, a preferred embodiment of the amino acid-substituted IgG2 constant region of the present invention is an IgG2 constant region having the amino acid sequence described in Sequence ID No. 20, in which the 209th (EU330) Ala is substituted with Ser, the 210th (EU331) Pro is substituted with Ser, and the 218th (EU339) Thr is substituted with Ala. It is possible.

[0218] (iv) Improvement of C-terminal heterogeneity of the constant region of IgG2 In the present invention, in the constant region of IgG2 having the amino acid sequence described in Sequence ID No. 20, 32 The 5th (446th in EU numbering) Gly and the 326th (44th in EU numbering) Provides a constant region of IgG2 lacking the 7th Lys. Both of these amino acids are deleted. This makes it possible to reduce heterogeneity originating from the C-terminus of the antibody's heavy chain for the first time.

[0219] (v) Improvement of plasma retention by modifying the constant region of IgG2 Furthermore, as one embodiment of the amino acid-substituted IgG2 constant region of the present invention, an IgG2 constant region having the amino acid sequence described in Sequence ID No. 20 is provided in which the 147th (268th in EU numbering) His, the 234th (355th in EU numbering) Arg, and the 298th (419th in EU numbering) Gln are replaced with other amino acids. These amino acid substitutions can improve the retention of antibodies in plasma. The substituted amino acids are not particularly limited, but it is preferable that the 147th (EU numbering 268th) His is substituted with Gln, the 234th (EU numbering 355th) Arg is substituted with Gln, and the 298th (EU numbering 419th) Gln is substituted with Glu. The constant region of IgG2 with the amino acids of the present invention substituted contains the three types of amino acids mentioned above. The IgG2 constant region contains at least one amino acid substitution among amino acid substitutions, It is preferable that all three of the above amino acids are substituted.

[0220] (vi) Improved stability of IgG4 in the acidic region in the constant state The present invention provides an IgG4 constant region having the amino acid sequence described in SEQ ID NO: 21, in which the 289th Arg (EU numbering 409th) is substituted with another amino acid. The substituted amino acid is not particularly limited, but a substitution with Lys is preferred. By substituting the 289th Arg (409th in EU numbering) in the amino acid sequence described in column number 21 with another amino acid, it is possible to improve the stability of the antibody under acidic conditions.

[0221] (vii) Improvement of C-terminal heterogeneity of the constant region of IgG4 The present invention relates to the constant region of IgG4 having the amino acid sequence described in SEQ ID NO: 21, wherein the 326th (446th in EU numbering) Gly and the 327th (44th in EU numbering) Provides an IgG4 constant region lacking the 7th Lys. Both of these amino acids are deleted. This makes it possible to reduce heterogeneity originating from the C-terminus of the antibody's heavy chain for the first time.

[0222] (viii) Improvement of C-terminal heterogeneity of the constant region of IgG1 The present invention relates to the constant region of IgG1 having the amino acid sequence described in SEQ ID NO: 19, wherein the 329th (446th in EU numbering) Gly and the 330th (44th in EU numbering) Provides a constant region of IgG1 lacking the 7th Lys. Both of these amino acids are deleted. This makes it possible to reduce heterogeneity originating from the C-terminus of the antibody's heavy chain for the first time.

[0223] (ix) The present invention relates to an amino acid sequence in which the 317th Asn (434th in EU numbering) in the constant region of IgG1 having the amino acid sequence described in Sequence ID No. 19 is replaced with another amino acid. This provides a constant IgG1 region having the following characteristics. The substituted amino acid is not particularly limited, but substitution with Ala is preferred.

[0224] (x) The present invention relates to the amino acid sequence described in Sequence ID No. 20, wherein the 209th (EU numbering 330th) Ala, the 210th (EU numbering 331st) Pro, the 218th (EU numbering 339th) Thr, the 276th (EU numbering 397th) Met, and the 14th (EU numbering This provides an IgG2 constant region having an amino acid sequence in which Cys (EU numbering 131), Arg (EU numbering 133), Cys (EU numbering 219), Glu (EU numbering 137), and Ser (EU numbering 138) are substituted with other amino acids. The substituted amino acids are not particularly limited, but the 209th Ala is replaced with Ser, and the 210th Pro is to Ser, the 218th Thr is to Ala, the 276th Met is to Val, the 14th Cys is to Ser, It is preferable to substitute the 16th Arg with Lys, the 102nd Cys with Ser, the 20th Glu with Gly, and the 21st Ser with Gly.

[0225] (xi) The present invention provides an IgG2 constant region having an amino acid sequence in which, in the amino acid sequence described in Sequence ID No. 20, Ala at position 209 (EU numbering 330), Pro at position 210 (EU numbering 331), Thr at position 218 (EU numbering 339), Met at position 276 (EU numbering 397), Cys at position 14 (EU numbering 131), Arg at position 16 (EU numbering 133), Cys at position 102 (EU numbering 219), Glu at position 20 (EU numbering 137), and Ser at position 21 (EU numbering 138) are substituted with other amino acids, and Gly at position 325 (EU numbering 446) and Lys at position 326 (EU numbering 447) are missing. The substituted amino acids are not particularly limited, but the 209th Ala is replaced with Ser, and the 210th Pro is to Ser, the 218th Thr is to Ala, the 276th Met is to Val, the 14th Cys is to Ser, It is preferable to substitute the 16th Arg with Lys, the 102nd Cys with Ser, the 20th Glu with Gly, and the 21st Ser with Gly.

[0226] (xii) The present invention provides an IgG2 constant region having an amino acid sequence in which the amino acid sequence described in Sequence ID No. 20 is replaced by other amino acids: Met at position 276 (EU numbering 397), Cys at position 14 (EU numbering 131), Arg at position 16 (EU numbering 133), Cys at position 102 (EU numbering 219), Glu at position 20 (EU numbering 137), and Ser at position 21 (EU numbering 138). The substituted amino acids are not particularly limited, but it is preferable that Met at position 276 be substituted with Val, Cys at position 14 with Ser, Arg at position 16 with Lys, Cys at position 102 with Ser, Glu at position 20 with Gly, and Ser at position 21 with Gly.

[0227] (xiii) The present invention provides an IgG2 constant region having an amino acid sequence in which, in the amino acid sequence described in Sequence ID No. 20, the 276th (EU numbering 397th) Met, the 14th (EU numbering 131st) Cys, the 16th (EU numbering 133rd) Arg, the 102nd (EU numbering 219th) Cys, the 20th (EU numbering 137th) Glu, and the 21st (EU numbering 138th) Ser are substituted with other amino acids, and the 325th (EU numbering 446th) Gly and the 326th (EU numbering 447th) Lys are missing. The substituted amino acids are not particularly limited, but it is preferable that Met at position 276 be substituted with Val, Cys at position 14 with Ser, Arg at position 16 with Lys, Cys at position 102 with Ser, Glu at position 20 with Gly, and Ser at position 21 with Gly.

[0228] (xiv) The present invention relates to the amino acid sequence described in Sequence ID No. 20, in which the 14th (EU numbering 131st) Cys, the 16th (EU numbering 133rd) Arg, the 102nd (EU numbering 219th) Cys, the 20th (EU numbering 137th) Glu, the 21st (EU numbering 138th) Ser, the 147th (EU numbering 268th) His, and the 234th (EU numbering Arg (number 355 in EU numbering) and Gln (number 298 in EU numbering, number 419 in EU numbering) are among the others. The present invention provides an IgG2 constant region having an amino acid sequence in which the amino acids are substituted and the 325th (EU numbering 446th) Gly and 326th (EU numbering 447th) Lys are missing. The substituted amino acids are not particularly limited, but it is preferable that the 14th Cys is substituted with Ser, the 16th Arg with Lys, the 102nd Cys with Ser, the 20th Glu with Gly, the 21st Ser with Gly, the 147th His with Gln, the 234th Arg with Gln, and the 298th Gln with Glu.

[0229] (xv) The present invention relates to the amino acid sequence described in Sequence ID No. 20, in which the 14th (EU numbering 131st) Cys, the 16th (EU numbering 133rd) Arg, the 102nd (EU numbering 219th) Cys, the 20th (EU numbering 137th) Glu, the 21st (EU numbering 138th) Ser, the 147th (EU numbering 268th) His, and the 234th (EU numbering Arg (355th in EU numbering), Gln (298th in EU numbering, 419th in EU numbering), and Asn (313th in EU numbering, 434th in EU numbering) are substituted with other amino acids, and 325 This provides an IgG2 constant region having an amino acid sequence lacking the 446th (EU numbering) Gly and the 326th (EU numbering) Lys. The substituted amino acids are not particularly limited, but for example, Cys at position 14 is replaced by Ser, Arg at position 16 by Lys, Cys at position 102 by Ser, Glu at position 20 by Gly, Ser at position 21 by Gly, His at position 147 by Gln, Arg at position 234 by Gln, Gln at position 298 by Glu, and Asn at position 313. It is preferable that this be replaced with Ala.

[0230] (xvi) The present invention relates to the amino acid sequence described in Sequence ID No. 21, specifically the 289th (EU numbering 409th) Arg, the 14th Cys, the 16th Arg, the 20th Glu, the 21st Ser, the 97th Arg, the 100th Ser, the 102nd Tyr, the 103rd Gly, and the 104th Pro and the 105th Pro (EU numbering 131, 133, 137, 138, 214, 217, 219, 220, 221, 222), the 113th Glu, the 114th Phe and the 115th (EU numbering 233, 234, 235 The present invention provides an IgG4 constant region having an amino acid sequence in which the Leu at position 116 (EU numbering 236) is substituted with another amino acid and the Gly at position 116 (EU numbering 236) is missing. The substituted amino acids are not particularly limited, but examples include: Cys at position 14 (EU numbering 131) being replaced by Ser, Arg at position 16 (EU numbering 133) being replaced by Lys, Glu at position 20 (EU numbering 137) being replaced by Gly, Ser at position 21 (EU numbering 138) being replaced by Gly, Arg at position 97 (EU numbering 214) being replaced by Thr, Ser at position 100 (EU numbering 217) being replaced by Arg, and Tyr at position 102 (EU numbering 219) being replaced by Se It is preferable to substitute r with Cys for the 103rd (EU numbering 220) Gly, Val for the 104th (EU numbering 221) Pro, Glu for the 105th (EU numbering 222) Pro, Pro for the 113th (EU numbering 233) Glu for the 113th (EU numbering 233) Pro, Val for the 114th (EU numbering 234) Phe, Ala for the 115th (EU numbering 235) Leu, and Lys for the 289th (EU numbering 409) Arg.

[0231] (xvii) The present invention relates to the amino acid sequence described in Sequence ID No. 21, specifically the 289th (EU numbering 409th) Arg, the 14th Cys, the 16th Arg, the 20th Glu, the 21st Ser, the 97th Arg, the 100th Ser, the 102nd Tyr, the 103rd Gly, and the 104th Pro And the 105th Pro (EU numbering 131, 133, 137, 138, 214, 217, 219, 220, 221, 222), the 113th Glu, the 114th Phe and the 115th Leu (EU numbering 233, 234, 235) are substituted with other amino acids, and the 116th (EU numbering 236) Gly, Gly number 326 (EU number 446) and Gly number 327 (EU number 447) This provides an IgG4 constant region having an amino acid sequence in which the Lys group of ) is missing. The substituted amino acid is not particularly limited, but the 14th (EU numbering 131) Cys is S To er, the 16th (EU numbering 133) Arg becomes Lys, the 20th (EU numbering 137) Glu becomes Gly, the 21st (EU numbering 138) Ser becomes Gly, the 97th (EU numbering 214) Arg becomes Thr, the 100th (EU numbering 217) Ser becomes Arg, the 102nd (EU numbering 219) Tyr becomes Ser, and the 103rd (EU numbering 220) It is preferable to substitute Gly with Cys, the 104th (EU numbering 221) Pro with Val, the 105th (EU numbering 222) Pro with Glu, the 113th (EU numbering 233) Glu with Pro, the 114th (EU numbering 234) Phe with Val, the 115th (EU numbering 235) Leu with Ala, and the 289th (EU numbering 409) Arg with Lys.

[0232] (xviii) The present invention relates to an IgG1 constant region having the amino acid sequence described in Sequence ID No. 19, wherein the 317th Asn (434th in EU numbering) is substituted with another amino acid, and the 329th Gly (446th in EU numbering) and 330th (447th in EU numbering) This provides an IgG1 constant region having an amino acid sequence lacking Lys (of the eye). The amino acid after substitution of Asn position 317 (434th in EU numbering) is particularly limited. However, substitution with Ala is preferable.

[0233] (xix) Furthermore, the present invention can list the following preferred embodiments of IgG2 in which the heterogeneity of the hinge region is improved and / or the binding activity to the Fcγ receptor is reduced. In IgG2 having a constant region consisting of the amino acid sequence described in Sequence ID No. 20, the 209th Ala, 210th Pro, 218th Thr, 14th Cys, 16th Arg, and 10 An antibody in which the second Cys, 20th Glu, and 21st Ser are substituted with other amino acids. The substituted amino acid is not particularly limited, but is Ala at position 209 (EU numbering 330). Ser, the 210th (EU numbering 331) Pro, Ser, the 218th (EU numbering It is preferable to replace Thr in 339) with Ala, Cys at the 14th position (EU numbering 131) with Ser, Arg at the 16th position (EU numbering 133) with Lys, Cys at the 102nd position (EU numbering 219) with Ser, Glu at the 20th position (EU numbering 137) with Gly, and Ser at the 21st position (EU numbering 138) with Gly. An example of such a constant IgG2 region is the amino acid sequence of SEQ ID NO: 191(M86). The constant region of IgG2 can be cited as an example. Furthermore, another preferred embodiment of the IgG2 constant region of the present invention is an IgG2 constant region in which the 325th Gly and 326th Lys are further deleted in order to reduce C-terminal heterogeneity. An example of such an antibody is a sequence IgG2 having a constant region consisting of amino acid sequence number: 192 (M86ΔGK) is an example. can.

[0234] (xx) Furthermore, the present invention can list the following preferred embodiments of an IgG2 steady-state region in which the heterogeneity of the hinge region is improved. In the constant region of IgG2 having the amino acid sequence described in Sequence ID No. 20, the 14th Cys The IgG2 constant region in which the 16th Arg, 102nd Cys, 20th Glu, and 21st Ser are substituted with other amino acids. The substituted amino acids are not particularly limited, but it is preferable to substitute Cys at position 14 (EU numbering 131) with Ser, Arg at position 16 (EU numbering 133) with Lys, Cys at position 102 (EU numbering 219) with Ser, Glu at position 20 (EU numbering 137) with Gly, and Ser at position 21 (EU numbering 138) with Gly. An example of such a constant IgG2 region is the amino acid sequence of SEQ ID NO: 193(M40). The constant region of IgG2 can be cited as an example. Furthermore, another preferred embodiment of the IgG2 constant region of the present invention is an IgG2 constant region in which the 325th Gly and 326th Lys are further deficient in the above-mentioned IgG2 constant region. An example of such an antibody is the amino acid sequence of SEQ ID NO: 194 (M40ΔGK). The constant region of IgG2 can be cited as an example.

[0235] (xxi)M14ΔGK, M17ΔGK, M11ΔGK, M31ΔGK, M58, M73, M83, M86ΔGK, M40ΔGK The present invention also relates to an antibody constant region (M14ΔGK) having the amino acid sequence described in SEQ ID NO: 24. The present invention provides an antibody constant region (M17ΔGK) having the amino acid sequence described in SEQ ID NO: 116. Furthermore, the present invention provides an antibody constant region (M17ΔGK) having the amino acid sequence described in SEQ ID NO: 25. The present invention provides an antibody constant region (M11ΔGK). Furthermore, the present invention provides the A described in Sequence ID No. 118. The present invention provides a constant region (M31ΔGK) having a mino acid sequence. Furthermore, the present invention relates to Sequence ID: 151 The present invention provides a constant region (M58) having the amino acid sequence described above. Furthermore, the present invention provides Sequence ID: The present invention provides a constant region having the amino acid sequence described in 153 (M73). Furthermore, the present invention provides a sequence The present invention provides a constant region having the amino acid sequence described in No. 164 (M83). This provides a constant region having the amino acid sequence described in Sequence ID No. 192 (M86ΔGK). Furthermore, the present invention provides a constant region having the amino acid sequence described in SEQ ID NO: 194 (M40 ΔGK). These antibody constant regions are optimized antibody constant regions that have properties such as reduced binding activity to the Fcγ receptor, reduced immunogenicity risk, improved stability under acidic conditions, reduced heterogeneity, improved plasma retention, and / or higher stability in the formulation compared to the IgG1 constant region.

[0236] The present invention provides an antibody comprising an antibody constant region as described in any of (i) to (xxi) above. As long as it has the above-described antibody constant region, the type of antigen, the origin of the antibody, etc., are not limited, and any antibody may be used. An example of a preferred antibody is an antibody that binds to the IL-6 receptor. Another example of a preferred antibody is a humanized antibody. An example of such an antibody is an antibody having a variable region of a humanized PM1 antibody....

Claims

1. A method for modifying the isoelectric point of a polypeptide containing an antibody variable region while maintaining the binding activity of the variable region to an antigen, wherein the complementarity-determining region (CDR) of the polypeptide A method comprising modifying the charge of at least one amino acid residue that may be exposed on the surface.

2. The method according to claim 1, wherein the polypeptide comprising the variable region of the antibody further comprises an FcRn binding region.

3. The method according to claim 1, wherein the polypeptide containing the variable region of the antibody is an IgG antibody.

4. The method according to claim 1, wherein the polypeptide containing the variable region of the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

5. The method according to claim 1, wherein the polypeptide containing the variable region of the antibody is a multispecific polypeptide that binds to at least two types of antigens.

6. The method according to claim 1, wherein the modification of the charge of the amino acid residue is an amino acid substitution.

7. Claim 1, wherein the modification of the charge of the amino acid residue is such that the theoretical isoelectric point changes by 1.0 or more. Methods used.

8. The amino acid residues that can be exposed on the surface of the CDR region are selected from the amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region according to Kabat numbering, or from the amino acid residues at positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat numbering. The method according to claim 1, wherein at least one amino acid residue.

9. A polypeptide comprising a variable region of an antibody with a modified isoelectric point, obtained by the method according to any one of claims 1 to 8.

10. A method for controlling the plasma pharmacokinetics of a polypeptide, comprising modifying the isoelectric point of the polypeptide containing an antibody variable region by the method according to any one of claims 1 to 8.

11. The control of the aforementioned pharmacokinetics affects plasma clearance (CL), area under the concentration curve (AUC), and mean plasma retention. The method according to claim 10, wherein the parameter is elongated or decreased, either the retention time or the plasma half-life (t1 / 2).

12. A polypeptide comprising a variable region of an antibody whose plasma pharmacokinetics are controlled, obtained by the method of claim 10.

13. A method for producing a polypeptide containing an antibody variable region with an altered isoelectric point, (a) At least one amino acid residue that may be exposed on the surface of the CDR region of the polypeptide (b) Modify the nucleic acid encoding a polypeptide containing the amino acid residue so that the load is altered, and (b) culture host cells so that the nucleic acid is expressed. (c) Recovering polypeptides containing antibody variable regions from host cell cultures. A method that includes this.

14. The method according to claim 13, wherein the polypeptide containing the variable region of the antibody further contains an FcRn binding region.

15. The method according to claim 13, wherein the polypeptide containing the variable region of the antibody is an IgG antibody.

16. The method according to claim 13, wherein the polypeptide containing the variable region of the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

17. The method according to claim 13, wherein the polypeptide containing the variable region of the antibody is a multispecific polypeptide that binds to at least two antigens.

18. The method according to claim 13, wherein the modification of the charge of the amino acid residue is an amino acid substitution.

19. Claim 1, wherein the modification of the charge of the amino acid residue is such that the theoretical isoelectric point changes by 1.0 or more. The method described in 3.

20. The amino acid residues that can be exposed on the surface of the CDR region are selected from the amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region according to Kabat numbering, or from the amino acid residues at positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat numbering. The method according to claim 13, wherein at least one amino acid residue.

21. A polypeptide comprising a variable region of an antibody with an altered isoelectric point, obtained by the method according to any one of claims 13 to 20.

22. A method for producing a polypeptide containing an antibody variable region with controlled plasma pharmacokinetics, comprising modifying the isoelectric point of the polypeptide containing the antibody variable region by the method according to any one of claims 13 to 20.

23. The control of the aforementioned pharmacokinetics affects plasma clearance (CL), area under the concentration curve (AUC), and mean plasma retention. The method according to claim 22, wherein the parameter is elongated or decreased, either the retention time or the plasma half-life (t1 / 2).

24. A polypeptide comprising a variable region of an antibody whose plasma pharmacokinetics are controlled, manufactured by the method of claim 22.

25. A method for producing a multispecific polypeptide comprising a first polypeptide and a second polypeptide having a variable region of an antibody, (a) at least one amino acid residue that can be exposed on the surface of the CDR region of the polypeptide The method involves modifying a nucleic acid encoding a polypeptide containing the amino acid residue such that its charge is altered, wherein the modification of the nucleic acid involves modifying both or either 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 such that the difference in isoelectric points between the first polypeptide and the second polypeptide increases compared to before the modification. (b) Culture host cells so that the nucleic acid is expressed, (c) Recovering multispecific antibodies from host cell cultures. A method that includes this.

26. The method according to claim 25, wherein the step of recovering a multispecific polypeptide comprising a first polypeptide and a second polypeptide from a host cell culture is performed by standard chromatography.

27. In the modification of the nucleic acid, a standard cross-polymer of a homopolymer of the first polypeptide, a homopolymer of the second polypeptide, and a heteropolymer of the first polypeptide and the second polypeptide is used. The method according to claim 25, wherein the nucleic acid is modified such that the peaks obtained by analysis using matrix imaging are more separated compared to the peaks before modification.

28. The method according to claim 25, wherein the multispecific polypeptide is a multispecific antibody.

29. A multispecific antibody produced by the method described in claim 27.

30. The multispecific antibody according to claim 29, wherein the multispecific antibody is a bispecific antibody.

31. An antibody comprising a CDR selected from the group consisting of human-derived CDRs, non-human animal-derived CDRs, and synthetic CDRs, a human-derived framework region (FR), and a human constant region, wherein the CDR At least one amino acid residue that can be exposed on the surface is the amino acid at the corresponding position in the wild-type CDR. This antibody features an amino acid residue with a different charge than the original antibody, resulting in an antibody with a modified isoelectric point while maintaining its antigen-binding activity.

32. The antibody according to claim 31, wherein the human constant region includes a human Fc region.

33. The antibody according to claim 31, wherein the plasma pharmacokinetics are controlled by altering the isoelectric point.

34. The 31st, 61st, 62nd, 64th and 65th positions in the heavy chain variable region according to Kabat numbering. Mino acid residues, or positions 24, 27, and 53 according to Kabat numbering in the light chain variable region. An IgG antibody in which the charge of at least one amino acid residue selected from the amino acid residues at positions 1, 54, and 55 is modified, and the isoelectric point is modified compared to before the modification of the amino acid residue.

35. The antibody according to claim 34, wherein the modified amino acid residue is selected from amino acid residues belonging to either group (a) or (b) below; (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 the Kabat numbering of the heavy chain variable region of the first polypeptide at positions 31, 61, 62, and 64 and the amino acid residue at position 65, or 24 Kabat numbering in the light chain variable region A multispecific antibody in which at least one amino acid residue selected from the amino acid residues at positions 1, 27, 53, 54, and 55 is charged, and the isoelectric points of the first polypeptide and the second polypeptide are different from each other.

37. Kabat numbering positions 31, 61, and 62 in the heavy chain variable region of the second polypeptide , amino acid residues at positions 64 and 65, or Kabat numbering in the light chain variable region The antibody according to claim 36, wherein the charge of at least one amino acid residue selected from the amino acid residues at positions 24, 27, 53, 54, and 55 is opposite to the charge of the amino acid residue selected in the first polypeptide, or has no charge.

38. The antibody according to claim 36, wherein the combination of an amino acid residue having the aforementioned charge and an amino acid residue having the opposite charge is selected from amino acid residues belonging to either group (a) or (b) below; (a) Glutamic acid (E), aspartic acid (D); (b) Lysine (K), Arginine (R), Histidine (H).

39. A multispecific antibody comprising the first polypeptide and the second polypeptide, wherein the homopolymer of the first polypeptide and the homopolymer of the second polypeptide are chromatographically determined. The antibody according to claim 36, which is a peak separated by analysis using CG.

40. A composition comprising the antibody described in claims 31 to 39 and a pharmaceutically acceptable carrier.

41. A nucleic acid encoding a polypeptide constituting the antibody according to claims 31 to 39.

42. A host cell having nucleic acid as described in claim 41.

43. A method for producing an antibody according to claims 31 to 39, comprising the steps of culturing the host cells described in claim 42 and recovering polypeptides from the cell culture.

44. In a polypeptide containing the variable region of an antibody, amino acids that can be exposed on the surface of the complementarity-determining region (CDR) of the polypeptide while maintaining its binding activity to the antigen. A method for substituting residues, which involves Kabat numbering in at least the heavy chain variable region. The amino acid residues selected are those at positions 31, 61, 62, 64, and 65, or those at positions 24, 27, 53, 54, and 55 in the light chain variable region, determined by Kabat numbering. A method of substituting at least one amino acid residue.

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