HK1129420A - Antibody modification method for purifying bispecific antibody - Google Patents

Description

Antibody modification process for purification of bispecific antibodies

Technical Field

The present invention relates to an antibody modification method for purifying a bispecific antibody, a method for isolating the bispecific antibody, a pharmaceutical composition containing the bispecific antibody as an active ingredient, and the like.

Background

Antibodies have drawn attention as drugs because of their high stability in blood and low side effects. Among them, there is a bispecific antibody that can recognize two antigens (antigen a and antigen B) at the same time (non-patent document 1). MDX-210, which is currently undergoing clinical trials, is an IgG-type bispecific antibody obtained by reconstituting HER-2/neu-expressing cancer cells with monocytes expressing Fc γ RI (non-patent document 2). In general, genetic recombination techniques are often used to produce antibodies. Specifically, the present invention relates to a technique for producing a protein by cloning a DNA encoding an antibody protein from an antibody-producing cell such as a hybridoma or an antibody-producing lymphocyte or a phage library presenting an antibody gene, integrating the DNA into an appropriate vector, and introducing the vector into a host cell. An IgG-type bispecific antibody using gene recombination technology is produced by introducing two genes constituting the H chain and L chain of IgG, which are targets, and four genes in total into a cell, and then secreting the antibody by co-expression. In the above expression, when the wild-type H chain and L chain constituting genes are expressed, two kinds of H chain associations or H chain and L chain associations occur at random, and therefore the ratio of the target bispecific antibody is extremely small. Specifically, the target bispecific antibody is only one of ten, and the production efficiency is reduced. The low production efficiency of the target antibody not only hinders the purification of the target antibody but also increases the variation in lot-to-lot variation and the like, resulting in an increase in production cost.

As an effective method for producing a bispecific antibody for developing a bispecific antibody, a common L chain acquisition technique for acquiring a common L chain of two H chains and a Knobs-into-holes technique for heterozygously associating H chains have been reported. Specifically, a common L chain capable of retaining two types of antigen binding activity to each H chain that recognizes antigen a and antigen B is found from a Phage library (Phage library), and the like, and the amino acid side chain present in the CH3 region of one H chain is replaced with a larger side chain (knob), and the amino acid side chain present in the CH3 region of the other H chain is replaced with a smaller side chain (hole, void), whereby the knob is disposed in the void, and formation of an H chain heterodimer is promoted, and a bispecific antibody of interest can be efficiently obtained (patent document 1, non-patent document 3, non-patent document 4).

However, when the Knobs-into-holes technique is used to obtain the H chain heterodimer, as shown in non-patent documents 3 and 4, the content of the desired A chain and B chain heterodimer can be increased to about 95% at the maximum by the Knobs-into-holes technique, and the remaining 5% are an A chain homodimer and a B chain homodimer and become impurities. In order to develop bispecific antibodies as drugs, it is necessary to purify the a chain and B chain heterodimers as high as possible from the three molecular species (a chain homodimers, B chain homodimers, a chain and B chain heterodimers) generated when a common L chain (non-patent document 3 and non-patent document 4) is used. Therefore, it is necessary to remove 5% of the remaining impurities, i.e., a chain homodimer and B chain homodimer, and purify the a chain B chain hybrid dimer to a high purity that can be developed as a drug. When using the common L chain without using the Knobs-into-holes technique, theoretically, the A chain homodimer, the A chain B chain heterodimer, and the B chain homodimer are generated at 1:2:1, and 50% of impurities-the A chain homodimer and the B chain homodimer must be removed.

In chromatographic separation at the pharmaceutical manufacturing level, there are several methods for separating a-chain B-chain hybrid dimers and a-chain homodimers, B-chain homodimers. As a method for selectively purifying a-chain B-chain hybrid dimers, the following method is reported in non-patent document 5: mouse IgG2a was used for the A chain, rat IgG2B was used for the B chain, and the A chain-B chain heterodimer was purified by controlling the elution pH of protein A based on the difference in affinity between protein A and each H chain of mouse IgG2a and rat IgG2B, but since the constant regions of mouse and rat were used, it was difficult to apply this method to human drugs from the viewpoint of antigenicity. This method cannot isolate a-chain B-chain hybrid dimers containing H-chains of the same subclass, and thus its utilization is limited.

Non-patent document 6 reports a method for purifying a chain B chain hybrid dimers by hydrophobic interaction chromatography, but it is difficult to sufficiently separate peaks of a target a chain B chain hybrid dimer containing anti-CD 3 mouse IgG2a and anti-CD 19 mouse IgG1, and it is considered that separation is performed by using H chains of different subclasses and utilizing differences in hydrophobicity, and therefore, it is not always possible to separate a chain B chain hybrid dimer containing H chains of the same subclasses.

Non-patent document 7 reports a method for purifying a chain B chain hybrid dimer by thiophilic affinity chromatography, which is difficult to use as a method for separating a chain B chain hybrid dimer containing H chains of the same subclass because mouse IgG1 and rat IgG2a are used and free cysteines (thiol groups) in the hinge region are used, and free cysteines are involved in aggregation during storage, and thus are not suitable for the development of stable pharmaceutical preparations.

Affinity chromatography using an antigen is reported in non-patent document 8. However, affinity chromatography using protein or peptide antigens has problems in terms of cost and stability of the column, and thus the preparation of drugs using affinity chromatography is not a conventional method. In addition, in order to purify the a chain B chain hybrid dimer bound to two antigens, two affinity chromatographies must be performed, with a predictable increase in cost. It has also been reported that an antibody recognizing only the steric structure of an antigen or an antibody having a low affinity and a target function is difficult to adopt affinity chromatography using an antigen. Therefore, the purification of bispecific antibodies using affinity chromatography is not universal.

As described above, the purification of A chain B chain hybrid dimers of bispecific antibodies can be carried out only in a limited range, and no method has been reported for purifying A chain B chain hybrids of bispecific antibodies having the same H chain subclass and constant region sequence to a high purity acceptable as a pharmaceutical. When two antibodies constituting a bispecific antibody have the same constant region sequence, it is necessary to isolate a chain B chain hybrid dimer only based on the difference in the variable region sequence, but the amino acid sequence of the variable region of the antibody has very high homology between the antibodies (non-patent document 9), and it is difficult to purify the a chain B chain hybrid dimer to a pharmaceutically acceptable high purity only based on the difference in the variable region sequence.

Patent document 1: international publication No. 96/27011.

Non-patent document 1: marvin JS and Zhu Z, "Recombinant aproaces toIgG-like bispecific antibiotics", acta. Pharmacol. sin., June 2005, Vol.26(6), p.649-58.

Non-patent document 2: segal D.M. et al, Current Opinion in Immunology, 1999, Vol.11, p.558-562.

Non-patent document 3: merchant AM et al 7., "An effective route to humanbispecific IgG.", nat. Biotechnol., Jul 1998, Vol.16(7), p.677-81.

Non-patent document 4: carter P, "Bispecific human IgG by design", J.Immunol.methods, Feb 2001, Vol.248(1-2), p.7-15.

Non-patent document 5: lindhofer H et al 4, "sensitive species-corrected species/light chain pairing in rat/mouse quadra. improvements for implementation-step purification of bispecific analytes", J.Immunol., Jul 1, 1995, Vol.155(1), p.219-25.

Non-patent document 6: manzke O et al 4., "Single-step purification of biochemical monoclonal antibodies for immunological use by hydrolytic interaction chromatography.", J.immunological methods., Oct 13, 1997, Vol.208(1), p.65-73.

Non-patent document 7: kreutz FT et al 3., "Efficient bispecific monoclonal antibody purification using a genetic affinity chromatography", J.chromatography.B.biomed.Sci.appl.Appl., Sep 4, 1998, Vol.714(2), p.161-70.

Non-patent document 8: gupta S and Suresh M, "Affinity chromatography and" chromatography of biochemical monoclonal antibodies ", J.biochem.Biophys.methods., May 31, 2002, Vol.51(3), p.203-16.Review.

Non-patent document 9: carl Branden, Introduction to Protein Structure 2 division, Newton Press.

Disclosure of Invention

The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for efficiently modifying an amino acid in an antibody variable region of a bispecific antibody, a pharmaceutical composition containing a modified bispecific antibody, and a method for producing a pharmaceutical composition of a bispecific antibody. The invention also provides bispecific antibodies with modified heavy chain constant regions, pharmaceutical compositions containing the modified bispecific antibodies, and methods for making the bispecific antibody pharmaceutical compositions.

The present inventors have conducted intensive studies on a method for substituting an amino acid in a variable region of an antibody as a method for efficiently purifying a bispecific antibody, which has been difficult to purify as a target substance by using a commonly used column.

As a result, they have found a method for purifying a bispecific antibody efficiently by a column using the difference in isoelectric point between two antibodies constituting the bispecific antibody by modifying amino acids present on the surface of the antibody variable region, introducing the difference in isoelectric point between the H chains of the two antibodies, and utilizing the difference in isoelectric point. Specifically, a modification site capable of controlling only the isoelectric point without reducing the function (activity) of the antibody is found in the H chain of the antibody. The inventors also confirmed that the bispecific antibody obtained by the method of the present invention actually remains functional.

As described above, the present inventors have succeeded in developing a method for purifying an arbitrary bispecific antibody with high efficiency using a commonly used column by substituting amino acids in the variable region of the antibody, and have completed the present invention.

The present inventors have also found a method for purifying a bispecific antibody efficiently by a column using different subclasses of constant regions having different isoelectric points in the respective H chains for the constant regions of the two H chains constituting the bispecific antibody, utilizing the difference in isoelectric points. The inventors have confirmed that the bispecific antibody obtained by the method of the present invention actually retains the function.

The present invention relates to a method for substituting an amino acid in an antibody variable region for efficient purification using a chromatography column, a pharmaceutical composition containing a modified bispecific antibody, and a method for producing a pharmaceutical composition containing a bispecific antibody, and further relates to a bispecific antibody in which a heavy chain constant region is modified, a pharmaceutical composition containing a modified bispecific antibody, and a method for producing a pharmaceutical composition containing a bispecific antibody, and more specifically to the following:

[1] a method for producing a multispecific antibody, the multispecific antibody comprising a1 st polypeptide and a 2nd polypeptide, the method comprising the steps of:

(a) modifying one or both of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide to produce a difference in isoelectric point between the 1 st polypeptide and the 2nd polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) recovering the multispecific antibody from the host cell culture.

[2] [1] the method, wherein, the step (a) of modifying nucleic acid, so that the 1 st polypeptide homopolymer, the 2nd polypeptide homopolymer, and the 1 st and 2nd polypeptide hybrid polymer through the use of standard chromatography analysis and formation of separation of peaks.

[3] [1] the method according to, wherein the 1 st polypeptide and the 2nd polypeptide each contain a heavy chain variable region.

[4] [3] the method according to [3], wherein the multispecific antibody comprises a3 rd polypeptide containing a light chain variable region, and the 1 st polypeptide and the 2nd polypeptide form multimers with the 3 rd polypeptide, respectively.

[5] [1] to [4], wherein the 1 st polypeptide and the 2nd polypeptide each contain a heavy chain constant region.

[6] [5] the method according to [5], wherein the heavy chain constant regions contained in the 1 st polypeptide and the 2nd polypeptide are heavy chain constant regions having isoelectric points different from each other.

[7] [6] the method according to any one of the above aspects, wherein the heavy chain constant regions having different isoelectric points are IgG1 and IgG4, or IgG1 and IgG 2.

[8] [1] the method according to, wherein the multispecific antibody is a bispecific antibody.

[9] A multispecific antibody produced by the method of [1 ].

[10] A method for purifying a multispecific antibody, which multispecific antibody comprises a1 st polypeptide and a 2nd polypeptide, the method comprising:

(a) modifying one or both of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide to produce a difference in isoelectric point between the 1 st polypeptide and the 2nd polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) the multispecific antibodies are purified from the host cell culture by standard chromatography.

[11] [10] the method, wherein, the step (a) of modifying nucleic acid, so that 1 st polypeptide homopolymer, 2nd polypeptide homopolymer, and 1 st polypeptide and 2nd polypeptide hybrid polymer through the use of standard chromatography analysis to form separation of peaks.

[12] [10] the method according to any one of the above 1 and 2, wherein the polypeptide comprises a heavy chain variable region.

[13] [12] the method according to [12], wherein the multispecific antibody comprises a3 rd polypeptide containing a light chain variable region, and the 1 st polypeptide and the 2nd polypeptide form multimers with the 3 rd polypeptide, respectively.

[14] [10] to [13], wherein the 1 st polypeptide and the 2nd polypeptide each contain a heavy chain constant region.

[15] [14] the method according to any one of the above 1 st and 2nd polypeptides, wherein the heavy chain constant regions are heavy chain constant regions having isoelectric points different from each other.

[16] [15] the method according to any one of the above aspects, wherein the heavy chain constant regions having different isoelectric points are IgG1 and IgG4, or IgG1 and IgG 2.

[17] [10] the method according to, wherein the multispecific antibody is a bispecific antibody.

[18] A method for producing a multispecific antibody, which comprises a step of purification by the method of [10 ].

[19] A multispecific antibody produced by the method of [18 ].

[20] A multispecific antibody, comprising a polypeptide 1 and a polypeptide 2, wherein the polypeptide 1 comprises a heavy chain variable region and/or a heavy chain constant region, and at least one amino acid residue selected from amino acid residues 10, 12, 23, 39, 43 and 105 of the heavy chain variable region according to Kabat numbering, or amino acid residues 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, 435 of the heavy chain constant region according to EU numbering, has an electric charge, and the isoelectric points of the polypeptide 1 and polypeptide 2 are different from each other.

[21] [20] the multispecific antibody according to [20], wherein the 2nd polypeptide comprises a heavy chain variable region and/or a heavy chain constant region, and at least one amino acid residue selected from the group consisting of amino acid residues No. 10, No.12, No.23, No. 39, No. 43 and No. 105 of the heavy chain variable region according to Kabat numbering, and amino acid residues No. 137, No. 196, No. 203, No. 214, No. 217, No. 233, No. 268, No. 274, No. 276, No. 297, No. 355, No. 392, No. 419 and No. 435 of the heavy chain constant region according to EU numbering, has a charge opposite to or no charge from an amino acid residue having a charge selected from the group consisting of the heavy chain variable region and/or the heavy chain variable region contained in the 1 st polypeptide.

[22] [20] the multispecific antibody according to, wherein the combination of the amino acid residue having a charge and the amino acid residue having a charge opposite to that of the amino acid residue is selected from the amino acid residues contained in any group of (a) or (b):

(a) glutamic acid (E), aspartic acid (D);

(b) lysine (K), arginine (R), histidine (H)

[23] A multispecific antibody in which the isoelectric points of the 1 st and 2nd polypeptides differ, wherein the homomultimer of the 1 st polypeptide, the homomultimer of the 2nd polypeptide, and the hetero-multimer of the 1 st and 2nd polypeptides form separate peaks by analysis using standard chromatography.

[24] [23] the multispecific antibody according to, wherein the 1 st polypeptide and the 2nd polypeptide comprise a heavy chain variable region.

[25] [24] the multispecific antibody, wherein the multispecific antibody comprises a3 rd polypeptide containing a light chain variable region, and the 1 st polypeptide and the 2nd polypeptide form multimers with the 3 rd polypeptide, respectively.

[26] [23] the multispecific antibody of any one of [25] to [23], wherein the 1 st polypeptide and the 2nd polypeptide each comprise a heavy chain constant region.

[27] [26] the multispecific antibody according to any one of the above 1 st polypeptide and 2nd polypeptide, wherein the heavy chain constant regions contained in the polypeptides are heavy chain constant regions that differ from each other in isoelectric point.

[28] [27] the multispecific antibody according to, wherein the heavy chain constant regions differing in isoelectric point are IgG1 and IgG4, or IgG1 and IgG 2.

[29] The multispecific antibody of [23], wherein the multispecific antibody is a bispecific antibody.

[30] A composition comprising the multispecific antibody of any one of [23] to [29] and a pharmaceutically acceptable carrier.

[31] A nucleic acid encoding a polypeptide constituting the multispecific antibody of any one of [23] to [29 ].

[32] A host cell having the nucleic acid as described in [31 ].

[33] A method for producing a multispecific antibody according to any one of [23] to [29], which comprises culturing the host cell according to [32 ]; a step of recovering the polypeptide from the cell culture.

[34] [25] the multispecific antibody according to any one of [ a 1] to [ a 7] in which the variable region of the 1 st polypeptide comprises an amino acid sequence as described in any one of [ b 1] to [ b 3] below, the variable region of the 2nd polypeptide comprises an amino acid sequence as described in any one of [ b 1] to [ b 3] below, and the variable region of the 3 rd polypeptide comprises an amino acid sequence as described in (c1) or (c2) below:

(a1)SEQ ID NO.7

(a2)SEQ ID NO.8

(a3)SEQ ID NO.9

(a4)SEQ ID NO.10

(a5)SEQ ID NO.11

(a6)SEQ ID NO.12

(a7)SEQ ID NO：13

(b1)SEQ ID NO.14

(b2)SEQ ID NO.15

(b3)SEQ ID NO.16

(c1)SEQ ID NO.17

(c2)SEQ ID NO.18。

[35] [34] the multispecific antibody, wherein the variable region of the 1 st polypeptide comprises the amino acid sequence of SEQ ID NO.11, the variable region of the 2nd polypeptide comprises the amino acid sequence of SEQ ID NO.16, and the variable region of the 3 rd polypeptide comprises the amino acid sequence of SEQ ID NO. 17.

[36] [34] the multispecific antibody, wherein the variable region of the 1 st polypeptide comprises the amino acid sequence of SEQ ID NO.12, the variable region of the 2nd polypeptide comprises the amino acid sequence of SEQ ID NO.16, and the variable region of the 3 rd polypeptide comprises the amino acid sequence of SEQ ID NO. 18.

[37] [34] the multispecific antibody of any one of [36], wherein the 1 st polypeptide and the 2nd polypeptide comprise a human IgG4 constant region, and the 3 rd polypeptide comprises a human kappa constant region.

Drawings

FIG. 1 shows the results of evaluation of the clotting activity of the humanized bispecific antibody (humanized A69(hA69 a)/humanized B26(hB26-F123e 4)/humanized BBA (hAL-F123j 4)). The results of the evaluation showed that the coagulation activity was equal to or higher than that of the chimeric bispecific antibody.

FIG. 2 is a graph showing the results of antibody modeling using the humanized A69-H chain variable region (hA69a) and humanized BBA (hAL-F123j4), and the humanized hB26-H chain variable region (hB26-F123e4) and humanized BBA (hAL-F123j 4). The side chain is emphasized for amino acids that can change the surface charge. Numbering is carried out using the sequence numbering of the Kabat database (Kabat EA et al, 1991.Sequences of Proteins of Immunological interest. NIH).

FIG. 3 is a photograph showing the results of isoelectric focusing analysis using a homodimer of a humanized A69 antibody which was unmodified and modified in the variable region, and a homodimer of a humanized B26 antibody which was unmodified and modified in the variable region. And (3) confirming an analysis result: by modification, the isoelectric point changes.

FIG. 4 is a graph showing the results of cation exchange chromatography using a homodimer of humanized A69 antibody with variable region modifications. And (3) confirming an analysis result: the peaks are shifted compared to the unmodified antibody.

FIG. 5 is a graph showing the results of cation exchange chromatography using a homodimer of a humanized B26 antibody modified in the variable region. And (3) confirming an analysis result: the peaks are shifted compared to the unmodified antibody.

FIG. 6 is a graph showing the results of evaluating clotting activity using a humanized bispecific antibody (H chain constant region using the nanobs-into-holes technique) in which the variable region was modified. The evaluation results showed coagulation activity equivalent to that of the unmodified antibody.

FIG. 7 is a photograph showing the results of isoelectric focusing electrophoretic analysis using homodimers of humanized A69 antibody with variable region (CDR) modifications. And (3) confirming an analysis result: the bands were shifted compared to the unmodified antibody.

Fig. 8 is a graph showing the results of evaluating the binding activity to factor IXa as an antigen using a homodimer of the humanized a69 antibody in which variable regions (CDRs) were modified. The results of the evaluation showed that the modified antibody retained the same binding activity as the unmodified antibody.

FIG. 9 is a diagram showing the results of cation exchange chromatography analysis of an unmodified humanized bispecific antibody prepared using humanized A69-H chain-hA 69a, humanized B26-H chain-hB 26-F123e4 and humanized BBA-L chain-hAL-F123 j4 as an unmodified antibody. In the analysis results, both homodimers were not separated from the bispecific antibody and eluted as one peak.

FIG. 10 is a graph showing the results of cation exchange chromatography using a humanized bispecific PF antibody prepared using hA69-PF, which is a humanized A69-H chain modifier, hA26-PF, which is a humanized B26-H chain modifier, and humanized BBA-L chain-hAL-s 8. In the analysis results, the two homodimers and the bispecific antibody were separated, and eluted as three peaks in the order of hA69-PF homodimer, humanized bispecific PF antibody, and hB26-PF homodimer.

FIG. 11 is a photograph showing the results of isoelectric focusing electrophoresis analysis using purified humanized A69 antibody-PF homodimer and humanized B26-PF antibody homodimer, humanized bispecific PF antibody. And (3) confirming an analysis result: the bispecific antibody of interest can be purified.

FIG. 12 is a graph showing the results of evaluating clotting activity using a purified humanized bispecific PF antibody (H chain constant region is wild type). The evaluation results showed coagulation activity equivalent to that of bispecific antibody (KiH) obtained by the konds-into-holes technique in the H chain constant region.

FIG. 13 shows chromatograms obtained when bispecific antibodies were purified from culture supernatants containing humanized A69 antibody homodimer, humanized B26 antibody homodimer, and humanized bispecific antibody using a column commonly used for preparation.

FIG. 14 is a graph showing the results of evaluating clotting activity using a humanized bispecific antibody (H chain constant region is wild type) purified using a column commonly used in the preparation. The evaluation results showed coagulation activity equivalent to that of the humanized bispecific PF antibody.

FIG. 15 is a photograph showing the results of isoelectric focusing electrophoresis analysis using unmodified, IgG 2-and IgG 4-humanized PM-1 antibodies. The analysis results confirmed that: the isoelectric point is changed by modification. A represents an unmodified humanized PM-1 antibody, B represents an IgG2 humanized PM-1 antibody, and C represents an IgG4 humanized PM-1 antibody.

FIG. 16 is a photograph showing the results of isoelectric focusing electrophoresis analysis using each of the co-expressed antibodies of the unmodified, IgG 2-and IgG 4-humanized PM-1 antibodies. The analysis results showed that each subclass antibody and subclass hybrid antibody could be separated by pI difference. A represents an unmodified humanized PM-1 antibody/IgG 2 humanized PM-1 antibody co-expressed antibody, B represents an unmodified humanized PM-1 antibody/IgG 4 humanized PM-1 antibody co-expressed antibody, and C represents a humanized PM-1 antibody purified product (batch).

FIG. 17 is a graph showing the results of cation exchange chromatography using an unmodified, IgG 2-or IgG 4-humanized PM-1 antibody expressed alone. And (3) confirming an analysis result: the peaks are shifted compared to the unmodified antibody.

FIG. 18 shows the results of cation exchange chromatography analysis of unmodified, IgG 2-and IgG 4-humanized PM-1 antibodies, respectively. In the analysis results, three main peaks of homodimers and hybrid dimers of each subclass were mainly observed in the combination of unmodified humanized PM-1 antibody/IgG 2-humanized PM-1 antibody and the combination of unmodified humanized PM-1 antibody/IgG 4-humanized PM-1 antibody. A represents an unmodified humanized PM-1 antibody/IgG 2 humanized PM-1 antibody co-expressed antibody, and B represents an unmodified humanized PM-1 antibody/IgG 4 humanized PM-1 antibody co-expressed antibody.

FIG. 19 shows the results of purification of homodimers and heterodimers from antibodies co-expressing unmodified humanized PM-1 antibody/IgG 4 humanized PM-1 antibody by cation exchange chromatography. As a result, the IgG4 humanized PM-1 antibody homodimer, the unmodified humanized PM-1/IgG4 humanized PM-1 hybrid antibody, and the unmodified humanized PM-1 antibody homodimer were eluted in this order as three peaks, and thus they could be separated. Arrows indicate approximate compositional ranges.

FIG. 20 is a graph showing the results of secondary chromatography using unmodified humanized PM-1 antibody homodimers, unmodified humanized PM-1/IgG4 humanized PM-1 hybrid antibodies, and IgG4 humanized PM-1 antibody homodimers purified by cation exchange chromatography. The results confirmed that: the desired subclass hybrid antibody can be purified.

FIG. 21 is a photograph showing the results of isoelectric focusing electrophoretic analysis using unmodified humanized PM-1 antibody homodimers, unmodified/humanized PM-1 hybrid antibodies humanized with IgG4, and humanized PM-1 antibody homodimers humanized with IgG4 purified by cation exchange chromatography. And (3) confirming an analysis result: the desired subclass hybrid antibody can be purified. A represents an unmodified humanized PM-1 antibody/IgG 4 humanized PM-1 antibody co-expression antibody, B represents an unmodified humanized PM-1 antibody separation component, C represents an unmodified humanized PM-1/IgG4 humanized PM-1 hybrid antibody separation component, and D represents an IgG4 humanized PM-1 antibody separation component.

FIG. 22 is a graph showing the results of evaluating the neutralizing activity of human IL-6 using unmodified humanized PM-1 antibody homodimer, unmodified humanized PM-1/IgG4 humanized PM-1 hybrid antibody, IgG4 humanized PM-1 antibody homodimer purified by cation exchange chromatography. The results of the evaluation showed that any antibody had the same neutralizing activity as the purified humanized PM-1 antibody. A and B represent BaF3 cell lines expressing human gp130, and C and D represent BaF3 cell lines co-expressing human gp 130/human IL-6 receptor. Black circles (●) indicate humanized PM-1 antibody purified product (bulk), white boxes (□) indicate unmodified humanized PM-1 antibody, white triangles (Δ) indicate IgG4 humanized PM-1 antibody, and Xs indicate unmodified humanized PM-1/IgG4 humanized PM-1 hybrid antibody.

Detailed Description

First, the present invention provides an antibody modification method for preparing a multispecific antibody. A preferred embodiment of the production method of the present invention is a method comprising modifying either or both of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide to differentiate the isoelectric points of the 1 st polypeptide and the 2nd polypeptide. That is, a difference in isoelectric point (pI) can be introduced into the polypeptide by changing the charge of the amino acid residues of the 1 st polypeptide and the 2nd polypeptide, and a multispecific antibody can be produced using the difference in isoelectric point. Specifically, the method comprises the following steps (a) to (c).

(a) Modifying one or both of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide to produce a difference in isoelectric point between the 1 st polypeptide and the 2nd polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) recovering the multispecific antibody from the host cell culture.

The polypeptide of the present invention generally refers to a polypeptide or protein having a length of about 10 amino acids or more. The polypeptide is usually derived from a living organism, but is not particularly limited, and may be, for example, a polypeptide having an artificially designed sequence. It may be in any form of natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. Also, fragments of the above polypeptides are included in the polypeptide of the present invention.

In the present invention, the phrase "the isoelectric points of the polypeptides differ" means that the isoelectric points of two or more polypeptides are different from each other by changing the surface amino acid charges. The difference in isoelectric point can be observed by using, for example, isoelectric focusing electrophoresis. In the present invention, it is preferable to control the isoelectric point without changing the structure or function (activity) of the polypeptide.

That is, the present invention provides a method for producing a multispecific antibody, the multispecific antibody comprising a1 st polypeptide and a 2nd polypeptide, the method comprising the steps of:

(a) modifying one or both of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide so that the difference in isoelectric points between the 1 st polypeptide and the 2nd polypeptide is 0.5 or more, preferably 0.7 or more, and more preferably 0.9 or more;

(b) culturing the host cell to express the nucleic acid;

(c) recovering the multispecific antibody from the host cell culture.

The invention also provides antibody modification methods for purifying multispecific antibodies. A preferred embodiment of the purification method of the present invention is a method comprising modifying both or either one of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide to differentiate the isoelectric points of the 1 st polypeptide and the 2nd polypeptide. That is, by changing the charge of the amino acid residues of the 1 st polypeptide and the 2nd polypeptide, a difference in isoelectric point (pI) is introduced into the polypeptides, and the multispecific antibody can be purified by utilizing the difference in isoelectric point. Specifically, the method comprises the following steps (a) to (c).

(a) Modifying one or both of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide to produce a difference in isoelectric point between the 1 st polypeptide and the 2nd polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) the multispecific antibodies are purified from the host cell culture by standard chromatography.

The method for producing a multispecific antibody comprising the step of purifying by the above-mentioned purification method is also encompassed in the present invention.

The nucleic acid of the present invention is generally cloned (inserted) into an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it can stably hold the inserted nucleic acid, and for example, when Escherichia coli is used as the host, pBluescript vector (manufactured by Stratagene) is preferable as the vector for cloning, and various commercially available vectors can be used. For the production of the multispecific antibody (polypeptide) of the present invention, the expression vector is particularly effective when used. The expression vector is not particularly limited as long as it is a vector for expressing a polypeptide in vitro, in Escherichia coli, in cultured cells, or in an individual organism, and examples thereof include pBEST vector (プロメガ) for in vitro expression, pEP vector (Invitrogen) for in Escherichia coli expression, pME18S-SL3 vector (GenBank accession No. AB009864) for in vivo expression, and pME18S vector (Mol Cell biol.8: 466-472(1988)) for in vivo expression. The insertion of the DNA of the present invention into a vector can be carried out by a conventional method, for example, by a ligase reaction using a restriction enzyme cleavage site (Current protocols in Molecular biology. Ausubel et al (1987) publishing. John Wiley & sons. section. 11.4-11.11).

The host cell is not particularly limited, and various host cells can be used according to the purpose. Examples of cells for expressing a polypeptide are bacterial cells (e.g.Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g.Yeast, Aspergillus), insect cells (e.g.Drosophila melanogaster S2, Spodoptera frugiperda SF9), animal cells (e.g.CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. The vector can be introduced into the host cell by a known method such as calcium phosphate precipitation, electric pulse electroporation (Current protocols in molecular biology apparatus. Ausubel et al (1987) Publish.John Wiley & sons. section9.1-9.9), lipofection, and microinjection.

For secretion of the polypeptide expressed in the host cell in the lumen of the endoplasmic reticulum, in the periplasm, or in the extracellular environment, an appropriate secretion signal may be incorporated into the polypeptide of interest. These signals may be endogenous to the polypeptide of interest or may be signals of different species.

In the above production method, for the recovery of the multispecific antibody (polypeptide), the medium is recovered when the polypeptide of the present invention is secreted into the medium. When the polypeptide of the present invention is produced in a cell, the cell is first lysed, and then the polypeptide is recovered.

When the polypeptide of the present invention is recovered from a recombinant cell culture and purified, a known method including ammonium phosphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, or the like can be used.

The invention also relates to compositions (medicaments) comprising the multispecific antibodies of the invention and a pharmaceutically acceptable carrier.

In the present invention, a pharmaceutical composition generally refers to a drug used for the treatment or prevention, or examination or diagnosis of a disease.

The pharmaceutical composition of the present invention can be formulated according to methods known in the art. For example, it can be used parenterally in the form of an injection of a sterile solution or suspension with water or a pharmaceutically acceptable liquid other than water. For example, the pharmaceutical composition can be mixed with a pharmaceutically acceptable carrier or vehicle, specifically, sterile water or physiological saline, a vegetable oil, an emulsifier, a suspending agent, a surfactant, a stabilizer, a flavoring agent, an excipient, a carrier, a preservative, a binder, etc., in an appropriate combination, in a unit dosage form required for generally accepted pharmaceutical practice, to prepare a preparation. The amount of the active ingredient in these preparations is set to an appropriate capacity to obtain the indicated range.

The sterile composition for injection can be formulated using a carrier such as distilled water for injection in accordance with the usual formulation practice.

Examples of the aqueous solution for injection include: isotonic solution containing physiological saline, glucose or other auxiliary agent (such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride). Suitable solubilizing aids such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), nonionic surfactants (polysorbate 80(TM), HCO-50, etc.) may also be used in combination.

The oily liquid may be oleum Sesami or soybean oil, and the dissolution auxiliary agent may be benzyl benzoate and/or benzyl alcohol. Buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may also be added. The prepared injection is usually filled in a suitable ampoule bottle.

The pharmaceutical compositions of the present invention are preferably administered non-orally. For example, the composition can be prepared into injection, nasal administration, pulmonary administration, or transdermal administration. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

The administration method may be appropriately selected depending on the age and symptoms of the patient. The amount of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody to be administered may be set, for example, in the range of 0.0001mg to 1000mg per 1 kg of body weight. Or, for example, each patient may be administered in an amount of 0.001 to 100000mg, to which the present invention is not limited. The administration amount and the administration method vary depending on the body weight, age, symptoms and the like of the patient, and the administration amount and the administration method can be appropriately set by those skilled in the art in consideration of these conditions.

The multispecific antibody of the present invention may also be combined with other pharmaceutical ingredients as needed to make a formulation.

The invention also provides nucleic acids encoding polypeptides that comprise the multispecific antibodies of the invention. Also, a vector carrying the nucleic acid is included in the present invention.

The invention further provides host cells having the above-described nucleic acids. The host cell is not particularly limited, and examples thereof include Escherichia coli and various animal cells. The host cell may be used, for example, in the form of a production system for the production or expression of the antibody or polypeptide of the present invention. Production systems for preparing polypeptides include in vitro and in vivo production systems. In vitro production systems include those using eukaryotic cells and those using prokaryotic cells.

Examples of eukaryotic cells that can be 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 cells), HeLa, Vero, etc., amphibian cells such as Xenopus laevis ovum (Valle, et al, Nature (1981) 291: 338-340), and insect cells such as Sf9, Sf21, Tn 5. For expression of the antibody of the present invention, CHO-DD44, CHO-DX11B, COS7 cells, HEK293 cells, and BHK cells are preferably used. Among animal cells, CHO cells are particularly preferable for the purpose of mass expression. The vector can be introduced into the host cell by, for example, the calcium phosphate method, the DEAE dextran method, the method using cationic liposome DOTAP (manufactured by Boehringer Mannheim), the electroporation method, lipofection, or the like.

Plant cells such as tobacco-derived (Nicotiana tabacum) cells and duckweed (Lemma minor) are known as protein production systems, and the antibodies of the present invention can be produced by callus culture of the cells. It is known to use yeasts such as cells of the genus Saccharomyces (Saccharomyces) (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Saccharomyces pombe), and the like); and filamentous fungi such as cells of the genus Aspergillus (Aspergillus niger) and the like can be used as hosts for the production of the antibody of the present invention.

When prokaryotic cells are used, there are production systems using bacterial cells. Bacterial cells in addition to the above E.coli (E.coli), production systems using Bacillus subtilis are also known and can be used for antibody production according to the present invention.

When the host cell of the present invention is used for producing an antibody, the host cell transformed with an expression vector containing a polynucleotide encoding the antibody of the present invention may be cultured to express the polynucleotide. For example, when animal cells are used as hosts, DMEM, MEM, RPMI1640, or IMDM can be used as the culture medium. In this case, the cells can be cultured in serum-free culture by using a serum replacement solution such as FBS or Fetal Calf Serum (FCS). The pH during the culture is preferably about 6 to 8. The culture is usually carried out at about 30 to 40 ℃ for about 15 to 200 hours, and the medium may be replaced, aerated, or stirred as necessary.

Examples of systems for producing polypeptides in vivo include production systems using animals and production systems using plants. The target polynucleotide is introduced into these animals or plants, and the polypeptide is produced in vivo in the animals or plants and recovered. The "host" of the present invention includes such animals and plants.

When animals are used, there are production systems using mammals and insects. As the mammal, goat, pig, sheep, mouse, cow, etc. can be used (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). When mammals are used, transgenic animals can be used.

For example, a polynucleotide encoding an antibody of the present invention and a gene encoding a polypeptide specifically produced in milk such as goat beta casein are prepared as a fusion gene. Then, the polynucleotide fragment containing the fusion gene is injected into a goat embryo, and the embryo is transferred into a female goat. The antibody of interest can be obtained from the milk produced by the transgenic goat produced by the goat having received the embryo, or by the offspring thereof. In order to increase the amount of milk containing the antibody produced by the transgenic goat, the transgenic goat may be appropriately administered with a hormone (Ebert et al, Bio/Technology (1994) 12: 699-702).

As the insect producing the antibody of the present invention, silkworm can be used, for example. When silkworms are used, the target antibody can be obtained from the body fluid of silkworms by infecting silkworms with a baculovirus into which a polynucleotide encoding the target antibody has been inserted (Susumu et al, Nature (1985) 315: 592-594).

When plants are used for the production of the antibody of the present invention, tobacco can be used, for example. When tobacco is used, a polynucleotide encoding an antibody of interest is inserted into a plant expression vector, for example, pMON 530, and the vector is introduced into a bacterium such as Agrobacterium tumefaciens (Agrobacterium tumefaciens). The bacteria are infected into tobacco, such as tobacco (Nicotiana tabacum), from which the desired antibodies can be obtained (Ma et al, Eur.J. Immunol. (1994) 24: 131-. The same bacteria were infected with duckweed and cloned to obtain the desired antibody from cells of duckweed (Cox K.M. et al, nat. Biotechnol.2006 Dec; 24 (12): 1591-.

The antibody obtained as described above can be isolated from the inside or outside of the host cell (medium, milk, etc.) and purified as a substantially pure and homogeneous antibody. The antibody may be isolated or purified by any isolation or purification method usually used for polypeptide purification, without any limitation. For example, the antibody can be isolated and purified by appropriately selecting and combining a chromatography column, a filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, dialysis, recrystallization, and the like.

Examples of the chromatography include: affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (stratgies for Protein Purification and chromatography: A Laboratory Course Manual. Ed Daniel R. Marshak et al, (1996) Cold Spring Harbor Laboratory Press). These chromatographies can be carried out using liquid chromatography such as HPLC, FPLC and the like. The column used in affinity chromatography includes protein A column and protein G column. For example, columns using protein a are: hyper D, POROS, sepharose f.f. (Pharmacia), and the like.

If necessary, an appropriate protein-modifying enzyme may be allowed to act before or after purification of the antibody, whereby any modification may be applied, or the peptide may be partially removed. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.

As described above, the method for producing a multispecific antibody of the present invention, which comprises the steps of culturing a host cell of the present invention and recovering the polypeptide from the culture, is also one of the preferred embodiments of the present invention.

The "multispecific antibody" of the present invention is an antibody that can specifically bind to at least two different antigens. Preferred multispecific antibodies obtained by the preparation method or purification method of the invention are: bispecific antibodies (BsAb) that specifically bind to two antigens (also referred to as two classes of specific antibodies).

In the present invention, the "different antigens" are not necessarily different from each other, and may be included in the "different antigens" of the present invention, for example, when the epitopes are different from each other. Thus, for example, different epitopes within a single molecule are included in different antigens of the present invention, and two antibodies that recognize different epitopes within the single molecule can be used as antibodies that recognize different antigens in the present invention.

The multispecific antibodies of the present invention are molecules comprising antibodies or antibody fragments that are specific for two or more different antigens.

In the above method of the present invention, "modification of nucleic acid" includes modifying nucleic acid to obtain a peak formed by separating the 1 st polypeptide and the 2nd polypeptide by analysis using standard chromatography.

In the method of the present invention, the "modified nucleic acid" refers to a modified nucleic acid corresponding to an amino acid residue introduced by the "modification" of the present invention. More specifically, a nucleic acid encoding an original (before modification) amino acid residue is modified to a nucleic acid encoding an amino acid residue introduced by the modification.

In general, the term "encoding" refers to a codon encoding an amino acid residue of interest obtained by subjecting an original nucleic acid to genetic manipulation such as insertion, deletion or substitution of at least one nucleotide or mutation. That is, the codon encoding the original amino acid residue is replaced with a codon encoding an amino acid residue introduced by modification. The modification of the nucleic acid can be carried out appropriately by using a technique known in the art, for example, a site-specific mutagenesis method, a PCR mutagenesis method, or the like.

Examples of the modification position in the present invention include: (1) amino acid residues located in the surface of the polypeptide, (2) amino acid residues located in the variable region, preferably the FR region, and (3) amino acid residues located in the constant region.

An "amino acid located on the surface of a polypeptide" is an amino acid whose side chain can be contacted with a solvent molecule (usually a water molecule), and it is not necessary that the entire side chain is contacted with the solvent molecule, and when a part of the side chain is contacted with the solvent molecule, the amino acid is an amino acid located on the surface. Those skilled in the art can prepare a homology model of a polypeptide or antibody by homology modeling using commercially available software, and the like, and thereby can select amino acids having appropriate residues on the surface.

The surface amino acids in the antibody variable region can be appropriately selected by those skilled in the art through a homology model prepared by homology modeling or the like, for example, in the H chain FR region, H1, H3, H5, H8, H10, H12, H13, H15, H16, H19, H23, H25, H26, H39, H42, H43, H46, H68, H71, H72, H73, H75, H76, H81, H82b, H83, H85, H86, H105, H108, H110, H112 are surface amino acids, but the present invention is not limited thereto. The CDR regions of the H chain can likewise be surface amino acids selected by homology modeling, for example H97 with almost the entire antibody exposed on the surface. In the FR region of the L chain, L1, L3, L7, L8, L9, L11, L12, L16, L17, L18, L20, L22, L38, L39, L41, L42, L43, L45, L46, L49, L57, L60, L63, L65, L66, L68, L69, L70, L74, L76, L77, L79, L80, L81, L85, L100, L103, L105, L106, L107, L108 are exemplified as surface amino acids, but the present invention is not limited thereto. The CDR regions of the L chain can likewise be selected for surface amino acids by homology modeling.

In the present invention, the amino acid residues located in the variable region include amino acid residues located in the heavy chain variable region (VH) or the light chain variable region (VL), and preferably amino acid residues located in the scaffold region (FR).

In the present invention, in the FR region other than the CDR, the amino acids exposed on the surface include, for example: h10, H12, H23, H39, H43, H105, but is not limited thereto.

In the present invention, the polypeptide obtained by modifying a nucleic acid is preferably a homo-multimer of the 1 st polypeptide, a homo-multimer of the 2nd polypeptide, and a hetero-multimer of the 1 st polypeptide and the 2nd polypeptide. For example, as described in the following examples, homomultimers of the 1 st polypeptide have: homodimers of the humanized A69-H chain and the humanized BBA-L chain, homomultimers of the 2nd polypeptide having: homodimers of a humanized B26-H chain and a humanized BBA-L chain, hybrid multimers of the 1 st and 2nd polypeptides have: humanized A69-H chain and a hybrid dimer of humanized B26-H chain and humanized BBA-L chain, but is not limited thereto.

The standard chromatography methods of the present invention are: cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, hydroxyapatite chromatography, hydrophobic charge interaction chromatography, chromatofocusing, and the like.

In the above method of the present invention, the 1 st polypeptide and the 2nd polypeptide preferably contain a heavy chain variable region (VH). The variable region may comprise, for example, a Complementarity Determining Region (CDR), a scaffold region (FR).

In the method of the present invention, the number of amino acid residues to be modified is not particularly limited, and for example, when the variable region of an antibody is modified, it is preferable to modify the minimum number of amino acid residues necessary for isolating the target polypeptide so as not to decrease the binding activity to an antigen or to increase the antigenicity.

It is also preferable that the modified amino acid sequence is a human sequence so as not to increase antigenicity, but the present invention is not limited thereto. Furthermore, mutations may be introduced into modified FRs (FR1, FR2, FR3, FR4) at positions other than the positions where the respective FRs are human sequences and modifications are introduced by changing the isoelectric point. The above-mentioned method of replacing each FR with a human sequence is reported in the non-patent literature (Ono K et al, mol. Immunol.1999 Apr; 36 (6): 387-395). In order to change the isoelectric point of each FR, it is also possible to modify it to be FR of a person other than one having a changed isoelectric point (for example, FR3 is exchanged with a person other than one having a low isoelectric point). The above humanization methods are reported in the non-patent literature (Dall' Acqua WF., methods.2005 May; 36 (1): 43-60).

If separation of the target polypeptide cannot be achieved with a small change in surface charge, a desired multispecific antibody can be obtained by repeating evaluation of the change in surface charge and separation of the polypeptide.

In the above method of the present invention, the multispecific antibody preferably comprises a3 rd polypeptide comprising a light chain variable region. Preferably, the 1 st and 2nd polypeptides form multimers with the 3 rd polypeptide, respectively.

In the above method of the present invention, it is preferable that the 1 st polypeptide and the 2nd polypeptide contain a heavy chain constant region. More preferably, in the heavy chain constant region, the 1 st polypeptide and the 2nd polypeptide produce a difference in pI. The heavy chain constant region has: the heavy chain constant region of the antibody having a difference in pI can be introduced into the 1 st and 2nd polypeptides using the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4 which originally have a difference in pI; it is also possible to prepare a non-wild-type human constant region by simultaneously modifying only the amino acids causing the difference in isoelectric point between these subclasses in the heavy chain constant regions of the 1 st and 2nd polypeptides, or the adjacent amino acids having no influence on these isoelectric points, and introduce a difference in pI into both constant regions. According to the EU numbering of the H chain constant region, the modification positions used to introduce a difference in pI into the constant region are, for example: 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, 435 of the H chain.

The pI difference can be produced by removing the sugar chain of the heavy chain constant region. Therefore, the 297 th position of the sugar chain-applying site also serves as a modification site for introducing the pI chain.

In the present invention, the method of the present invention wherein the 1 st polypeptide and the 2nd polypeptide contain a heavy chain constant region also includes a method wherein the 1 st polypeptide and the 2nd polypeptide contain a heavy chain variable region, and/or a method wherein the multispecific antibody contains a3 rd polypeptide containing a light chain variable region, and the 1 st polypeptide and the 2nd polypeptide are each combined with a method wherein the 3 rd polypeptide forms a multimer.

Multispecific antibodies prepared by the above-described methods are also encompassed by the present invention.

In the multispecific antibody provided by the present invention, in order to achieve the above-mentioned "isoelectric point difference" when the polypeptide 1 comprises a heavy chain variable region and/or a heavy chain constant region, at least one of amino acid residues No. 10, No.12, No.23, No. 39, No. 43 and No. 105 of the Kabat numbering of the heavy chain variable region, or amino acid residues No. 137, No. 196, No. 203, No. 214, No. 217, No. 233, No. 268, No. 274, No. 276, No. 297, No. 355, No. 392, No. 419 and No. 435 of the EU numbering of the heavy chain constant region may be charged. In the amino acid residue of the 1 st polypeptide represented by the above numbering, amino acid residues other than the amino acid residue having the charge may have the same charge as the amino acid residue having the charge, may have no charge, or may have opposite charges, as long as the isoelectric points of the 1 st polypeptide and the 2nd polypeptide differ.

The aforementioned multispecific antibody of the present invention is preferably characterized in that: the 2nd polypeptide has a charge opposite to or no charge of the amino acid residue having a charge of the 1 st polypeptide. Specific examples are the following multispecific antibodies: the polypeptide of claim 2 comprises a heavy chain variable region and/or a heavy chain constant region, and at least one amino acid residue selected from the group consisting of amino acid residues nos. 10, 12, 23, 39, 43 and 105 by Kabat numbering of the heavy chain variable region, and amino acid residues nos. 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419 and 435 by EU numbering of the heavy chain constant region has a charge opposite to or not at the charged amino acid residue in the heavy chain variable region and/or the heavy chain constant region contained in the polypeptide of claim 1. In the amino acid residues of the 2nd polypeptide represented by the above numbering, amino acid residues other than the amino acid residue having the charge may have the same charge as the amino acid residue having the charge, may have no charge, or may have opposite charges, as long as the 1 st polypeptide and the 2nd polypeptide have a difference in isoelectric point.

For the purpose of lowering the isoelectric point, for example, a sequence of IgG2 or IgG4 for No. 137, a sequence of IgG1 or IgG2 or IgG4 for No. 196, a sequence of IgG2 or IgG4 for No. 203, a sequence of IgG2 for No. 214, a sequence of IgG1 or IgG3 or IgG4 for No. 217, a sequence of IgG1 or IgG3 or IgG4 for No. 233, a sequence of IgG4 for No. 268, a sequence of IgG2 or IgG3 or IgG4 for No. 274, a sequence of IgG1 or IgG2 or IgG4 for No. 276, a sequence of IgG4 for No. 355, a sequence of IgG3 for No. 392, a sequence of IgG4 for No. 419, a sequence of IgG1 or IgG2 or IgG4 for No. 435 are preferable. For example, for increasing the isoelectric point, a sequence using IgG1 or IgG3 for No. 137, a sequence using IgG3 for No. 196, a sequence using IgG1 or IgG3 for No. 203, a sequence using IgG1 or IgG3 or IgG4 for No. 214, a sequence using IgG2 for No. 217, a sequence using IgG2 for No. 233, a sequence using IgG1 or IgG2 or IgG3 for No. 268, a sequence using IgG1 for No. 274, a sequence using IgG3 for No. 276, a sequence using IgG1 or IgG2 or IgG3 for No. 355, a sequence using IgG1 or IgG2 or IgG4 for No. 392, a sequence using IgG1 or IgG2 or IgG3 for No. 419, and a sequence using IgG3 for No. 435 are preferable.

These sequences may be used as long as they can generate a sufficient difference in isoelectric point between two H chains, and not all of them are necessarily used.

Among amino acids, those having a charge are known. As the amino acid having a positive charge (positively charged amino acid), lysine (K), arginine (R) and histidine (H) are usually mentioned. As the negatively charged amino acid (negatively charged amino acid), aspartic acid (D), glutamic acid (E), and the like are known.

The "amino acid residue having a charge" is preferably selected from amino acid residues contained in any one of the following groups (a) and (b), and is not particularly limited.

(a) Glutamic acid (E), aspartic acid (D)

(b) Lysine (K), arginine (R), histidine (H)

In the above antibody, "having homogeneous charges" means that, for example, any one of the Kabat-numbered amino acid residues in the heavy chain variable region or the EU-numbered amino acid residues in the heavy chain constant region has an amino acid residue contained in any one of the groups (a) or (b).

The phrase "having opposite charges" means that, for example, in the 2nd polypeptide having a heavy chain variable region and/or a heavy chain constant region, at least one of the amino acid residues in the Kabat numbering or the EU numbering is an amino acid residue at a position corresponding to the heavy chain variable region and/or the heavy chain constant region contained in the 1 st polypeptide, and when the amino acid residues contained in any one of the groups (a) or (b) are contained, the remaining amino acid residues have amino acid residues contained in different groups.

That is, the present invention provides a multispecific antibody, wherein the amino acid residues having the same charge are selected from the amino acid residues contained in any one of the above (a) or (b).

When the original (unmodified) amino acid residue has a charge, it is also one of the preferred embodiments of the present invention to modify the amino acid residue to an amino acid residue having no charge.

In the present invention, it is preferable that the amino acid residues are modified so that the isoelectric points (pI) of the 1 st polypeptide and the 2nd polypeptide are different from each other, and when a plurality of amino acid residues are introduced by the modification, a small number of the amino acid residues may contain an amino acid residue having no charge.

The present invention also provides a multispecific antibody, wherein the variable region of the 1 st polypeptide comprises an amino acid sequence described in any one of (a1) to (a7) below, the variable region of the 2nd polypeptide comprises an amino acid sequence described in any one of (b1) to (b3) below, and the variable region of the 3 rd polypeptide comprises an amino acid sequence described in (c1) or (c2) below.

(a1)SEQ ID NO.7

(a2)SEQ ID NO.8

(a3)SEQ ID NO.9

(a4)SEQ ID NO.10

(a5)SEQ ID NO.11

(a6)SEQ ID NO.12

(a7)SEQ ID NO.13

(b1)SEQ ID NO.14

(b2)SEQ ID NO.15

(b3)SEQ ID NO.16

(c1)SEQ ID NO.17

(c2)SEQ ID NO.18

The above amino acid sequence is intended to more specifically exemplify the amino acids which can be used for modification in the present invention, and is not limited to the case where the variable region is composed of these amino acids.

One of the preferred embodiments of the above-mentioned multispecific antibody is the following multispecific antibody: the variable region of the 1 st polypeptide has the amino acid sequence of SEQ ID NO.11, the variable region of the 2nd polypeptide has the amino acid sequence of SEQ ID NO.16, and the variable region of the 3 rd polypeptide has the amino acid sequence of SEQ ID NO. 17.

Another preferred embodiment is exemplified by the following multispecific antibodies: the variable region of the 1 st polypeptide has the amino acid sequence of SEQ ID NO.12, the variable region of the 2nd polypeptide has the amino acid sequence of SEQ ID NO.16, and the variable region of the 3 rd polypeptide has the amino acid sequence of SEQ ID NO. 18.

One of the further preferred embodiments of the above multispecific antibody is the following multispecific antibody: the 1 st and 2nd polypeptides contain the human IgG4 constant region and the 3 rd polypeptide contains the human kappa constant region.

The term "antibody" in the present invention is used in the broadest sense as long as it shows a desired biological activity, and includes monoclonal antibodies, polyclonal antibodies, antibody mutants (chimeric antibodies, humanized antibodies, low molecular weight antibodies (also including antibody fragments), multispecific antibodies, and the like). In the present invention, the antibody modification method of the present invention is preferably used for obtaining these antibodies.

As described above, the term "antibody" in the present invention includes an antibody in which the charge of an amino acid residue is changed, and the amino acid sequence of the antibody is further changed by amino acid substitution, deletion, addition, and/or insertion. Also included are antibodies in which the charge of amino acid residues is further altered in antibodies whose amino acid sequences have been altered by substitution, deletion, addition and/or insertion of amino acids, or by chimerization or humanization. That is, the modification may be performed simultaneously with the step of humanizing the mouse antibody, or the humanized antibody may be further modified.

Amino acid substitutions, deletions, additions and/or insertions, and changes in amino acid sequences such as humanization and chimerization can be performed according to methods known in the art. Similarly, the variable and constant regions of the antibody used in the production of the antibody of the present invention in the form of a recombinant antibody may be modified in amino acid sequence by amino acid substitution, deletion, addition and/or insertion, or by chimerization or humanization.

The antibody of the present invention may be an antibody derived from any animal, such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. Further, for example, a modified antibody obtained by replacing an amino acid sequence of a chimeric antibody or a humanized antibody thereof may be used. The antibody may be any antibody such as a modified antibody, an antibody fragment, or a low molecular weight antibody to which various molecules are bound.

"chimeric antibody" refers to an antibody prepared by combining sequences from different animals. For example, the antibody may contain the variable (V) regions of the heavy and light chains of a mouse antibody and the constant (C) regions of the heavy and light chains of a human antibody. Chimeric antibodies are well known and can be obtained, for example, by ligating a DNA encoding a V region of an antibody with a DNA encoding a C region of a human antibody, integrating the resulting DNA into an expression vector, introducing the vector into a host, and producing the antibody.

"humanized antibody", also called a reshaped (reshaped) human antibody, is obtained by grafting Complementarity Determining Regions (CDRs) of an antibody derived from a mammal other than a human, for example, a mouse antibody, to CDRs of a human antibody. Methods for identifying CDRs are well known (Kabat et al, Sequence of proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md.; Chothia et al, Nature (1989) 342: 877). In addition, conventional gene recombination methods are also known (European patent application laid-open Nos. EP125023 and WO 96/02576). Thus, for example, the CDR of a mouse antibody can be identified by a known method, an antibody in which the CDR is linked to a scaffold region (FR) of a human antibody can be obtained, and a humanized antibody can be produced by using a system obtained by a conventional expression vector. The DNA can be synthesized by PCR using a plurality of prepared oligonucleotides having overlapping portions in the terminal regions of both CDR and FR as primers (see the method described in WO 98/13388). The FRs of human antibodies that are joined via the CDRs can be selected so that the CDRs form a good antigen-binding portion. Amino acid modifications of FRs in the antibody variable region may also be made as necessary to allow the CDRs of the reshaped human antibody to form an appropriate antigen binding site (Sato, K. et al, Cancer Res. (1993) 53: 851. 856). Amino acid residues in modifiable FRs include moieties that bind to the antigen directly, or by non-covalent bonds (Amit et al, Science (1986) 233: 747-53), moieties that have an effect or effect on the structure of the CDRs (Chothia et al, J.mol.biol. (1987) 196: 901-17), and moieties that are associated with VH-VL interactions (EP 239400).

When the antibody of the present invention is a chimeric antibody or a humanized antibody, a region derived from a human antibody is preferably used for the C region of the antibody. For example, C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4 can be used for the H chain, and C.kappa.and C.lambda.can be used for the L chain. In order to improve the stability of the antibody or its production, the human antibody C region may be modified as necessary. The chimeric antibody of the present invention preferably contains a variable region derived from an antibody derived from a mammal other than human and a constant region derived from a human antibody. The humanized antibody preferably contains CDRs from an antibody derived from a mammal other than human, and FRs and C regions from a human antibody. The constant region derived from a human antibody has an amino acid sequence unique to each of IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE and the like isotypes. The constant region used in the humanized antibody of the present invention may be a constant region of an antibody belonging to any isotype. The constant region of human IgG is preferably used, but not limited thereto. Furthermore, the FR derived from a human antibody to be used in a humanized antibody is not particularly limited, and may be an antibody of any isotype.

The variable and constant regions of the chimeric and humanized antibodies of the present invention may be modified by deletion, substitution, insertion, addition or the like within a range that can exhibit the binding specificity of the original antibody.

Chimeric antibodies and humanized antibodies obtained using human-derived sequences have reduced antigenicity in humans and can therefore be administered to humans for therapeutic purposes and the like.

The low molecular weight antibody can be used as an antibody because of its dynamic properties in vivo and because it can be produced at low cost using Escherichia coli, plant cells, etc.

The antibody fragment is a kind of low molecular weight antibody. The low molecular weight antibody includes an antibody having an antibody fragment as a part of its structure. The low molecular weight antibody of the present invention is not particularly limited in its structure, production method, and the like, as long as it has an ability to bind to an antigen. Among the low molecular weight antibodies, there are antibodies with higher activity than the full-length antibodies (Orita et al, Blood (2005) 105: 562-566). In the present specification, the "antibody fragment" is not particularly limited as long as it is a part of a full-length antibody (for example, full-length IgG), and preferably contains a heavy chain variable region (VH) or a light chain variable region (VL). Examples of preferred antibody fragments are, for example: fab, F (ab ') 2, Fab', Fv, etc. The amino acid sequence of VH or VL in an antibody fragment may be modified by substitution, deletion, addition and/or insertion. Furthermore, VH and VL may be partially deleted as long as they retain the ability to bind to an antigen. For example, "Fv" in the above antibody fragments is the smallest antibody fragment containing a complete antigen recognition site and binding site. "Fv" is a dimer in which one VH and one VL are strongly bound by a non-covalent bond (VH-VL dimer). An antigen-binding site can be formed on the surface of the VH-VL dimer based on three complementary Chain Determining Regions (CDRs) of each variable region. The six CDRs confer an antigen binding site on the antibody. However, one variable region (or half of an Fv with only three CDRs specific for an antigen) has a lower affinity than the entire binding site, but still has the ability to recognize and bind antigen. Accordingly, molecules smaller than Fv as described above are also included in the antibody fragments of the present invention. The variable regions of the antibody fragments may be chimeric or humanized.

The low molecular weight antibody preferably contains both VH and VL. Examples of low molecular weight antibodies are: utilizing Fab, Fab ', F (ab')₂And antibody fragments such as Fv, and scFv (signal chain Fv) and The like which can be prepared using The antibody fragments (Huston et al, Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; Pluckthun "The pharmaceutical of Monoclonal Antibodies" Vol.113, Resenburg and Moore (eds.), Springer Verlag, New York, pp.269-315, (1994)); diabodies (Holliger et al, Proc. Natl. Acad. Sci. USA (1993) 90: 6444-8; EP 404097; WO 93/11161; Johnson et al, Method in enzymology (1991) 203: 88-98; Holliger et al, Protein Engineering (1996) 9: 299-305; Perisic et al, Structure (1994) 2: 1217-26; John et al, Protein Engineering (1999)12 (7): 597-604; Atwell et al, mol. Immunol. 1996) 33: 1301-12); sc (fv)2(Hudson et al, J immunol. methods (1999) 231: 177-89; Orita et al, Blood (2005) 105: 562-566); triabodies (Journal of immunological Methods (1999) 231: 177-89); tandem diabodies (cancer research (2000) 60: 4336-41).

Antibody fragments can be obtained by treating the antibody with an enzyme such as papain, trypsin, etc. (see Morimoto et al, J. biochem. Biophys. methods (1992) 24: 107-17; Brennan et al, Science (1985) 229: 81). Can be prepared by gene recombination based on the amino acid sequence of the antibody fragment.

A low molecular weight antibody having a structure in which an antibody fragment is modified can be constructed using an antibody fragment obtained by enzyme treatment or gene recombination. Alternatively, genes encoding the totality of the low molecular weight antibodies can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (see, e.g., 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).

The "scFv" is a single-chain polypeptide in which two variable regions are bound via a linker or the like as necessary. The two variable regions contained in the scFv are usually a VH and a VL, or two VH or two VL. Typically, scFv polypeptides contain a linker between the VH and VL domains, whereby paired portions of VH and VL necessary for binding to antigen can be formed. In general, in order to form a pair portion between VH and VL in the same molecule, a linker connecting VH and VL is generally made into a peptide linker of 10 amino acids or more in length. The linker of the scFv of the present invention is not limited to the above polypeptide linker as long as it does not interfere with the formation of the scFv. For general discussion of scFv, reference may be made to Pluckthun "The Pharmacology of Monoclonal antibodies", Vol.113(Rosenburg and Moore ed., Springer Verlag, NY, pp.269-315 (1994)).

In addition, "diabody (Db)" refers to a bivalent antibody fragment constructed by gene fusion (P. Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404, 097; WO93/11161, etc.). The diabody is a dimer composed of two polypeptide chains, in which a light chain variable region (VL) and a heavy chain variable region (VH) are bound to each other at positions where they cannot bind to each other via a short linker of, for example, about 5 residues, in the same chain. VL and VH encoded on the same polypeptide chain cannot form a single-chain V-region fragment due to the short linker between them, but form a dimer, so the bivalent miniantibody has two antigen-binding sites. In this case, if VL and Vh corresponding to two different epitopes (a, b) are simultaneously expressed and ligated as a combination of VLa-VHb and VLb-VHa, using a linker of about 5 residues, they are secreted as bispecific Db.

The diabody contains two molecules of scFv and therefore four variable regions. The result has two antigen binding sites. In order to form a bivalent small antibody, a linker connecting VH and VL within each scFv molecule may be made to be about 5 amino acids, as is the case with an scFv that does not form a dimer. However, the linker of the scFv that forms the diabody is not limited to the peptide linker as long as it does not inhibit the expression of scFv and does not inhibit the formation of diabody.

Further preferred in the present invention are bispecific antibodies as multispecific antibodies.

The "bispecific antibody" refers to an antibody having a structure in which a heavy chain variable region and a light chain variable region are linked to each other to form a single chain (for example, sc (fv) 2). The antibody-like molecule may be an antibody-like molecule (e.g., scFv-Fc) in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked to form scFv (or sc (fv)2) and which is bound to an Fc region (a constant region lacking the CH1 domain). Multispecific scFv-Fc-containing antibodies have a structure of the (scFv)2-Fc type, which is formed by: the 1 st polypeptide is VH 1-linker-VL 1-Fc, and the 2nd polypeptide is VH 2-linker-VL 2-Fc. Also, the antibody may be an antibody-like molecule comprising a single domain antibody bound to an Fc region (curr. Opin. drug Discov. Devel.2006, (9), (2), 184-93).

In the method of the present invention, a gene encoding an H chain or an L chain of an antibody before mutation introduction (which may be simply referred to as "the antibody of the present invention" in the present specification) can be obtained using a known sequence or according to a method known in the art. For example, it can be obtained by cloning a gene encoding an antibody from a library of antibodies or a hybridoma producing a monoclonal antibody.

Many antibody libraries are known, and methods for preparing antibody libraries are also known, and therefore, those skilled in the art can obtain appropriate antibody libraries. For example, for antibody phage libraries reference can be made to Clackson et al, Nature 1991, 352: 624-8; marks et al, j.mol.biol.1991, 222: 581-97; waterhouses et al, Nucleic Acids Res.1993, 21: 2265-6; griffiths et al, EMBO j.1994, 13: 3245-60; vaughan et al, nature biotechnology 1996, 14: 309-14; and Japanese patent application laid-open No. Hei 20-504970. Other methods may be known, such as a method of preparing a library of eukaryotic cells (WO 95/15393) or a ribosome display method. Also known is a technique for obtaining a human antibody by panning using a human antibody library. For example, the variable region of a human antibody may be made into a single chain antibody (scFv), expressed on the surface of a phage by phage display, and a phage that binds to an antigen may be selected. Analysis of the genes of the selected phage allows the determination of the DNA sequences encoding the variable regions of the human antibodies that bind the antigen. If the DNA sequence of scFv that binds to an antigen is known, an appropriate expression vector can be prepared based on the sequence to obtain a human antibody. Such methods are known and reference may be made to WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO 95/15388.

The method for obtaining a gene encoding an antibody from a hybridoma can be basically obtained by using a known technique as follows: a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen, the immunization is carried out according to a conventional immunization method, the obtained immune cell is fused with a known mother cell by a conventional cell fusion, a monoclonal antibody-producing cell (hybridoma) is selected by a conventional screening method, cDNA of a variable region (V region) of an antibody is synthesized from mRNA of the obtained hybridoma using a reverse transcriptase, and the cDNA is linked to DNA encoding a constant region (C region) of the antibody.

More specifically, the sensitizing antigen used for obtaining the antibody genes encoding the above-mentioned H chain and L chain includes both a complete antigen having immunogenicity and an incomplete antigen containing a hapten or the like which does not exhibit immunogenicity, but is not limited to this example. For example, a full-length protein or a partial peptide of the target protein can be used. In addition, it is known that a substance composed of a polysaccharide, a nucleic acid, a lipid, or the like can be used as an antigen, and the antigen of the antibody of the present invention is not particularly limited. The antigen can be prepared by a method known in the art, for example, a method using baculovirus (for example, WO 98/46777). Hybridomas can be prepared, for example, according to the method of Milstein et al (G.Kohler and C.Milstein, Methods enzymol.1981, 73: 3-46). When the immunogenicity of the antigen is low, the antigen can be conjugated to a large immunogenic molecule such as albumin, and the antigen can be immunized. If necessary, the antigen may be combined with other molecules to prepare a soluble antigen. When a transmembrane molecule of a receptor or the like is used as an antigen, a part of the extracellular region of the receptor may be used as a fragment, or a cell expressing the transmembrane molecule on the cell surface may be used as an immunogen.

Antibody-producing cells can be obtained by immunizing an animal with the above-mentioned appropriate sensitizing antigen. Or immunizing in vitro lymphocyte capable of producing antibody to obtain antibody producing cell. The animals to be immunized may be mammals of various kinds, and animals of the order rodentia, rabbits, primates are generally used. Examples of the plant include rodents such as mice, rats and hamsters. Rabbit, etc. Primate animals such as cynomolgus monkey, macaque, baboon, chimpanzee, etc. In addition, transgenic animals having all the components of human antibody genes are known, and human antibodies can be obtained using the above animals (see WO 96/34096; Mendez et al, nat. Genet.1997, 15: 146-56). For example, a desired human antibody having a binding activity to an antigen can be obtained by sensitizing human lymphocytes to the desired antigen or cells expressing the desired antigen in vitro and fusing the sensitized lymphocytes with human myeloma cells such as U266, instead of using the above-mentioned transgenic animal (see Japanese examined patent publication (Kokoku) No. 1-59878). In addition, a desired human antibody can be obtained by immunizing a transgenic animal having all the components of human antibody genes with a desired antigen (see WO93/1227, WO92/03918, WO94/02602, WO96/34096, and WO 96/33735).

Immunization of animals can be carried out, for example, as follows: the sensitizing antigen is diluted and suspended with Phosphate Buffered Saline (PBS) or physiological saline, and the like, emulsified with an adjuvant if necessary, and then injected into the abdominal cavity or subcutaneous tissue of an animal. The sensitizing antigen mixed in incomplete freon's adjuvant is then preferably administered several times every 4 to 21 days. Confirmation of antibody production can be carried out by measuring the titer of the target antibody in the serum of an animal by a commonly used method.

Hybridomas can be prepared by fusing antibody-producing cells obtained from animals immunized with a desired antigen or from lymphocytes with myeloma cells using commonly used fusing agents (e.g., polyethylene glycol) (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, 59-103). The binding specificity of the antibody produced by the hybridoma can also be measured by a known analytical method such as immunoprecipitation, Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Then, the hybridomas producing the antibodies whose target specificity, affinity, or activity have been measured are subcloned by limiting dilution or the like, if necessary.

Then, a gene encoding a selected antibody is cloned from a hybridoma or an antibody-producing cell (e.g., a sensitized lymphocyte) using a probe capable of specifically binding to the antibody (e.g., an oligonucleotide complementary to a sequence encoding a constant region of the antibody). It can also be cloned from mRNA by RT-PCR. Immunoglobulins are classified into five distinct classes, IgA, IgD, IgE, IgG and IgM. And these classes are subdivided into subclasses (isotypes) (e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1, IgA-2, etc.). In the present invention, the H chain and L chain used for the production of the antibody may be derived from an antibody belonging to any of these classes and subclasses, and are not particularly limited, with IgG being particularly preferred.

Here, genes encoding the H chain and the L chain can be modified by genetic engineering methods. For example, an artificially modified recombinant antibody, for example, a chimeric antibody or a humanized antibody can be appropriately prepared for an antibody such as a mouse antibody, a rat antibody, a rabbit antibody, a hamster antibody, a sheep antibody or a camel antibody in order to reduce antigenicity against a human. The chimeric antibody is an antibody comprising the variable regions of the H chain and L chain of a non-human mammal such as a mouse antibody and the constant regions of the H chain and L chain of a human antibody, and can be obtained by: the DNA encoding the variable region of the mouse antibody is ligated with the DNA encoding the constant region of the human antibody, integrated into an expression vector, introduced into a host and produced. Humanized antibodies, also known as reshaped human antibodies, were synthesized as follows: a DNA sequence designed to link Complementarity Determining Regions (CDRs) of a non-human mammal such as a mouse antibody is synthesized from a plurality of oligonucleotides having overlapping portions at their prepared terminal portions by a PCR method. The resulting DNA is ligated with a DNA encoding a human antibody constant region, and then integrated into an expression vector, which is introduced into a host to produce the antibody (see EP239400, WO 96/02576). The FRs of the human antibody connected via the CDRs are selected from those whose complementarity determining regions form a good antigen-binding site. Amino acids in the scaffold region of the variable region of an antibody may be substituted as necessary to form an appropriate antigen-binding site in the complementarity determining region of the reshaped human antibody (K.Sato et al, Cancer Res.1993, 53: 851. 856).

In addition to the above-mentioned humanization, modification may be made to improve the biological properties of an antibody such as binding to an antigen. The modification of the present invention can be carried out by site-specific mutagenesis (for example, refer to Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis, cassette mutagenesis, and the like. Generally, an antibody mutant having improved biological properties has 70% or more, more preferably 80% or more, and still more preferably 90% or more (e.g., 95% or more, 97%, 98%, 99%, etc.) amino acid sequence homology and/or similarity with the amino acid sequence of the variable region of the original antibody. In the present specification, the homology and/or similarity of the sequences are defined as the ratio of amino acid residues which are identical (identical residues) or similar (generally classified into amino acid residues of the same group according to the characteristics of the amino acid side chains) to the original antibody residues after the sequence homology is maximized by reforming the sequences and introducing gaps as necessary. In general, natural amino acid residues are classified according to the nature of their side chains as (1) hydrophobic: alanine, isoleucine, valine, methionine and leucine; (2) neutral hydrophilicity: asparagine, glutamine, cysteine, threonine, and serine; (3) acidity: aspartic acid and glutamic acid; (4) alkalinity: arginine, histidine and lysine; (5) residues that affect the orientation of the chain: glycine and proline; and (6) aromatic: tyrosine, tryptophan and phenylalanine.

In general, six Complementarity Determining Regions (CDRs), which are present in all of the H chain and L chain variable regions, interact to form the antigen binding site of an antibody. One of the variable domains is known to have a lower affinity than the one containing the entire binding site, but still have the ability to recognize and bind to the antigen. Therefore, the H chain and L chain-encoding antibody gene of the present invention may encode a fragment portion containing each antigen-binding site of the H chain and L chain, as long as the polypeptide encoded by the gene can maintain the binding property to a desired antigen.

According to the method of the present invention, as described above, for example, the actual activity can be maintained, and a bispecific antibody can be efficiently obtained.

The heavy chain variable region is generally composed of 3 CDR regions and 4 FR regions, as described above. In a preferred embodiment of the present invention, the amino acid residues to be "modified" may be appropriately selected from amino acid residues located in a CDR region or an FR region. In general, amino acid residues in CDR regions can be modified to reduce their ability to bind to antigen. Therefore, in the present invention, the amino acid residues to be "modified" are not particularly limited, and are preferably appropriately selected from amino acid residues located in the FR region.

In organisms such as humans or mice, the sequences of FRs that can be used as variable regions of antibodies can be obtained appropriately by those skilled in the art using public databases and the like. More specifically, the amino acid sequence information of the FR region can be obtained by the method of the example described later.

All prior art documents cited in this specification are incorporated herein by reference.

Examples

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

EXAMPLE 1 humanization of bispecific antibody with hybrid L chain

In Japanese patent application No. 2005-112514, bispecific antibodies comprising a combination of anti-FactorIXa antibody A69-VH, anti-FactorX antibody B26-VH, and hybrid L chain (BBA) having the highest effect of shortening the clotting time were humanized as follows.

Homology search of 1-1 human antibody

Human antibody amino acid sequence data were obtained from the generally disclosed Kabat database (ftp:// ftp. ebi. ac. uk/pub/databases/Kabat /) and IMGT database (http:// IMGT. cines. fr /), and homology searches were performed using the constructed databases, divided into a mouse A69-H variable region (amino acid sequence: SEQ ID NO.19), a mouse B26-H variable region (amino acid sequence: SEQ ID NO.20), and a mouse BBA-L variable region (amino acid sequence: SEQ ID NO. 21). As a result, it was confirmed that: the humanized antibody has high homology with the human antibody sequence shown below, and thus is used in the scaffold region (hereinafter referred to as FR) of the humanized antibody.

(1) A69-H variable region: KABATID-000064(Kabat database)

(Kipps et al, J.Clin.invest.1991; 87: 2087-

(2) B26-H chain variable region: EMBL accession number AB063872(IMGT database)

(unpublished data)

(3) BBA-L variable region: KABATID-024300(Kabat database)

(Welschof et al, J.Immunol.method.1995; 179: 203-

A humanized antibody was prepared by grafting complementary epitope regions (hereinafter referred to as CDRs) of each mouse antibody to FRs of the human antibodies of (1) to (3).

The human antibody secretion signal sequence having high homology to the human antibody of (4) to (6) was searched using the homology search website (http:// www.ncbi.nlm.nih.gov/BLAST /) generally disclosed in NCBI. The secretion signal sequence obtained by the search and shown below was used.

(4) A69-H variable region: genbank accession No. AF 062257.

(5) B69-H chain variable region: genbank accession number AAC 18248.

(6) BBA-L variable region: genbank accession number AAA 59100.

Construction of 1-2 humanized antibody Gene expression vector

In the nucleotide sequence encoding the amino acid sequence from the secretion signal sequence to the antibody variable region, 12 oligo DNAs of about 50 nucleotides were prepared alternately so that about 20 nucleotides were hybridized on the 3' end side. The synthetic oligo DNA is designed to encode a human sequence on the 5 'terminal side and a mouse sequence on the 3' terminal side, or all bases encode a human sequence. Then, the 5 ' -end of the antibody variable region gene was annealed, and the 3 ' -end of the antibody variable region gene was annealed together with a primer having an XhoI cleavage sequence, thereby preparing a primer having a SfiI cleavage sequence and encoding the 5 ' -end sequence of the intron sequence.

mu.L of each synthetic oligo DNA prepared to 2.5. mu.M was mixed, and 1 XTaKaRa ExTaq buffer, 0.4mM dNTPs, and 0.5 unit TaKaRa Ex Taq (all prepared by Takara Shuzo Co., Ltd.) were added to prepare 48. mu.L of a reaction solution. After incubation at 94 ℃ for 5 minutes, the reactions containing 94 ℃ for 2 minutes, 55 ℃ for 2 minutes, and 72 ℃ for 2 minutes were subjected to 2 cycles to carry out the assembly and chain extension reactions of each synthetic oligo DNA. Next, 1. mu.L of primers (10. mu.M each) annealing to the 5 '-end and 3' -end of the antibody gene was added, and a reaction containing 94 ℃ for 30 seconds, 50 ℃ for 30 seconds, and 72 ℃ for 1 minute was performed for 35 cycles, followed by a reaction at 72 ℃ for 5 minutes to amplify the antibody variable region gene. After PCR, the total amount of the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the target size (about 400dp) was purified using QIA quick Gel Extraction kit (QIAGEN) according to the method described in the attached instruction, and eluted with 30. mu.L of sterile water. This fragment was cloned using pGEM-T Easy vector system (Promega) according to the method of the attached instructions. The nucleotide sequence of each DNA fragment was determined by means of the ABI PRISM3730XL DNA sequencer or ABI PRISM 3700 DNA sequencer (Applied Biosystems) using the BigDye terminator cycle sequencing kit (Applied Biosystems) according to the method described in the attached instructions.

The plasmid into which the H chain variable region fragment of the humanized antibody variable region gene sequence confirmed to be correct was digested with XhoI and SfiI, and the plasmid into which the L chain variable region fragment was inserted was digested with EcoRI, and then the reaction mixture was subjected to 1% agarose gel electrophoresis. The DNA fragment of the desired size (about 400bp) was purified by the method according to the attached instructions using QIAquick gel purification kit (QIAGEN), and eluted with 30. mu.L of sterilized water. Then, an expression vector for animal cells was prepared as follows. In order to preferentially express IgG4 in which the H chain is a heterozygous combination, an amino acid substitution for the CH3 portion of IgG4 was used with reference to the knobs-into-hole technique of IgG1 (Merchant AM et al, Nature Biotechnology, 1998, Vol.16, p.677-681). And to facilitate dimer formation of the H chain, amino acid substitutions (-ppcPSCp- → -pppPcp-) are introduced into the hinge. A humanized A69H chain expression vector was prepared by integrating the constant region genes substituted with Y39C and T366W into pCAGGS (Niwa et al, Gene, 1991, Vol.108, 193-199) having a chicken β actin promoter, and inserting a humanized A69H chain variable region antibody Gene fragment into the resulting expression vector. Furthermore, a humanized B26H chain expression vector was prepared by integrating the constant region genes substituted with E356C, T366S, L368A and Y407V into pCAGGS and inserting a humanized B26H chain variable region antibody gene fragment into the resulting expression vector. In addition, a plasmid (pCAG-g. kappa.DNA) obtained by inserting a wild-type antibody L chain constant region into pCAGGS was digested with EcoRI to prepare an expression vector into which a humanized BBA L chain variable region antibody gene fragment was inserted. The ligation reaction was carried out by transforming E.coli strain DH5 alpha (manufactured by Toyo Seiki Co., Ltd.) using Rapid DNA ligation kit (Roche Diagnostics).

Expression of 1-3 humanized bispecific antibodies

Expression of humanized bispecific antibodies was performed using the following method.Expression of humanized bispecific antibody from human fetal kidney cancer cells was performed using the method described in examples 1-2 or the following method. HEK293H strain (Invitrogen) from human fetal kidney cancer cells was suspended in DMEM medium (Invitrogen) containing 10% fetal bovine serum at 5-6X 10⁵Cell density per mL, 10mL in each of adherent cell culture dishes (10 cm diameter, CORNING) in CO₂Incubator (37 ℃, 5% CO)₂) After the culture was performed overnight, the medium was removed by suction, and 6.9mL of CHO-S-SFM-II (Invitrogen) medium containing 1% fetal bovine serum (Invitrogen) was added. The plasmid DNA mixture (13.8. mu.g in total) prepared in 1-2 was mixed with 20.7. mu.L of 1. mu.g/mL polyethyleneimine (Polysciences Inc.) and 690. mu.L of CHO-S-SFM-II medium, allowed to stand at room temperature for 10 minutes, added to the cells in each dish, and cultured in CO₂Incubator (37 ℃, 5% CO)₂) Internal culture for 4-5 hr. Then 6.9mL of CHO-S-SFM-II (Invitrogen) medium containing 1% fetal bovine serum (Invitrogen) was added thereto in CO₂Culturing in an incubator for 3 days. The culture supernatant was recovered, centrifuged (about 2000g, 5 min, room temperature) to remove the cells, and the cells were further filtered through a 0.22 μm filter MILLEX^(R)GV (Millipore) and the samples were stored at 4 ℃ before use.

Purification of 1-4 humanized bispecific antibodies

To the culture supernatant obtained in example 1-2, 100. mu.L of rProtein A Sepharose was added^TMFast Flow (Amersham Biosciences) was mixed at 4 ℃ for 4 hours or more by inversion. The solution was transferred to a 0.22 μm filter cup Ultrafree^(R)-MC (Millipore) with 500. mu.L containing 0.01% Tween^(R)20 TBS was washed three times and 100. mu.L of TBN containing 0.01% Tween^(R)20 of a 50mM sodium acetate solution was suspended in rrProtein agarose^TMThe resin was allowed to stand at pH 3.3 for 2 minutes, and then the antibody was eluted. 6.7. mu.L of 1.5M Tris-HCl pH7.8 was immediately added for neutralization.

Concentration quantification of 1-5 humanized bispecific antibodies

The measurement was carried out according to two methods as shown below.

Goat coat anti-human IgG (Biosource International) was prepared to 1. mu.g/mL using coating buffer and immobilized on Nunc-Immuno plates (Nunc). Blocking was performed with dilution buffer (D.B.) and then a sample of culture supernatant diluted appropriately with d.b. was added. Human IgG4 (humanized anti-TF antibody, see WO99/51743) diluted in a 3-fold series with D.B. to 11 gradients from 2000ng/mL was similarly added as a standard for antibody concentration calculation. After washing 3 times, goat anti-human IgG and alkaline phosphatase were reacted (Biosource International). After 5 washes, Sigma 104^(R)Phosphatase substrate (Sigma-Aldrich) was developed as a substrate, and absorbance at 405nm was measured by an absorbance reader model 3550(Bio-Rad Laboratories)) at 665nm as a reference wavelength. The concentration of human IgG in the culture supernatant was calculated from the calibration curve of the standard using microplate management III (Bio-Rad Laboratories) software.

Quantification was also performed using Biacore 1000(Biacore) using protein a immobilized sensor chip CM5 (Biacore). Specifically, a protein A (SIGMA) solution diluted to 50. mu.g/mL with 10mM sodium acetate aqueous solution (pH4.0, BIACORE) was reacted at 5. mu.L/min for 30 minutes in an activated sensor chip, and then a blocking operation was performed according to the manufacturer's instructions to prepare a sensor chip immobilized with protein A. The concentrations of the culture supernatant and the purified product were measured using this sensor chip and using Biacore 1000 (BIACORE). The fixation and concentration determination of the sensor chip were performed using HBS-EP Buffer (BIACORE). As a standard for concentration measurement, a humanized IgG4 antibody (humanized anti-TF antibody, see WO99/51743) was diluted in HBS-EP buffer in a 2-fold series from 4000ng/mL to 6 gradients.

Evaluation of blood coagulation Activity of 1-6 humanized bispecific antibody

To determine whether the bispecific antibody has altered clotting ability in hemophilia a blood, the effect of the antibody on Activated Partial Thrombin Time (APTT) using factor VIII-poor plasma was investigated. 50 μ L of antibody solutions at various concentrations, 50 μ L of factor VIII-poor plasma (B)iomerieux) and 50. mu.L of APTT reagent (Dade Behring) were warmed at 37 ℃ for 3 minutes. The coagulation reaction was performed by adding 50. mu.L of 20mM CaCl₂(Dade Behring) was added to the mixture to initiate. The time required to clot was determined by KC10A (Amelung) with CR-A (Amelung) attached.

The factor VIII-like activity (%) of the bispecific antibody was calculated from the clotting time of the case where the bispecific antibody was added using a calibration curve prepared with the clotting time of factor VIII-poor plasma being 0% and the clotting time of normal plasma being 100%.

1-7 acquisition of humanized bispecific antibody maintaining blood coagulation Activity

In the evaluation of the blood coagulation activity, the humanized bispecific antibody having low blood coagulation ability was subjected to attention on the increase in the activity thereof, and the amino acid of the humanized antibody FR was modified. Specifically, a mutation was introduced into the variable region of a humanized antibody by the method described in the attached specification using QuikChange site-specific mutagenesis kit (Stratagene). The plasmid into which the H chain variable region fragment confirmed as the variable region gene sequence of the humanized antibody of interest was inserted was digested with XhoI and SfiI, and the plasmid into which the L chain variable region fragment was inserted was digested with EcoRI, and then the reaction mixture was subjected to 1% agarose gel electrophoresis. The DNA fragment of the desired size (about 400bp) was purified by the method described in the attached instruction using QIAquick gel purification kit (QIAGEN), and eluted with 30. mu.L of sterile water. Then, expression plasmids for animal cells were prepared as described in example 1-2. Humanized bispecific antibodies were prepared according to the methods described in examples 1-3, 1-4, and 1-5, and the blood coagulation activity was evaluated according to the methods described in examples 1-6.

Amino acid modifications of the FR sequence and evaluation of blood clotting ability were repeated to obtain a humanized bispecific antibody (humanized A69(hA69 a)/humanized B26(hB26-F123e 4)/humanized BBA (hAL-F123j4)) having equivalent activity to the chimeric bispecific antibody (A69/B26/BBA) (FIG. 1). The variable region sequence of each antibody is represented as SEQ id no below.

(1) Humanized A69 antibody VH (hA69a) SEQ ID NO.1 (base sequence), SEQ ID NO.2 (amino acid sequence)

(2) Humanized B26 antibody VH (hB26-F123e4) SEQ ID NO.3 (base sequence), SEQ ID NO.4 (amino acid sequence)

(3) Humanized BBA antibody VHL (hAL26-F123j4) SEQ ID NO.5 (base sequence), SEQ ID NO.6 (amino acid sequence)

Example 2 determination of the position of amino acid modification in the variable region for the isolation of bispecific antibody in the expression in the preparation of bispecific antibody, two H chains and one L chain were used, and then three antibodies, homodimer of humanized A69-H chain and humanized BBA-L chain, homodimer of humanized B26-H chain and humanized BBA-L chain, and heterodimer of humanized A69-H chain and humanized B26-H chain and humanized BBA-L chain, were expressed. These three antibodies were isolated, and in order to purify only bispecific antibodies, the isoelectric point of the humanized a69H chain variable region was lowered, and the isoelectric point of the humanized B26H chain variable region was raised, to thereby modify the amino acids.

First, in order to confirm the amino acid residues exposed on the surface of the variable regions of the humanized a69 antibody and the humanized B26 antibody, antibody Fv region models of the humanized a69 antibody and the humanized B26 antibody were prepared by homology modeling using MOE software (Chemical Computing Group Inc.). As shown in fig. 2, according to detailed analysis of this model, among the exposed amino acids in the FR Sequences other than CDRs, H10, H12, H23, H39, H43, and H105(Kabat numbering, Kabat EA et al, 1991.Sequences of Proteins of immunologicalcalest. nih) are candidate amino acids that can change the isoelectric point without decreasing the activity.

[ example 3] modification of amino acids in the variable region of humanized bispecific antibody

At the positions selected in example 2, amino acid modifications were made in order to prepare modified antibodies. Specifically, the H chain variable region of humanized A69 antibody (hA69a, SEQ ID NO.1) and the H chain variable region of humanized B26 antibody (hB26-F123e4, SEQ ID NO.3) were prepared by the method described in the attached specification using QuikChange site-specific mutagenesis kit (Stratagene), and mutations were introduced into them. The plasmid into which the fragment of the H chain variable region confirmed as the target humanized antibody variable region gene sequence was inserted was digested with XhoI and SfiI, and then the reaction mixture was subjected to 1% agarose gel electrophoresis. The DNA fragment of the desired size (about 400bp) was purified by the method described in the attached instruction using QIAquick gel purification kit (QIAGEN), and eluted with 30. mu.L of sterile water. The prepared DNA fragment was inserted into an expression plasmid in which amino acids in the constant region were substituted and an expression plasmid having a wild-type constant region according to the method shown in example 1-2 with reference to the konbs-into-hole technique, to prepare an H chain expression vector. Then, humanized bispecific antibodies were prepared according to the methods described in examples 1-3, 1-4, and 1-5. The variable region sequences of the modified humanized antibodies are shown in SEQ ID NOs as described in table 1 below.

TABLE 1

EXAMPLE 4 isoelectric focusing electrophoresis analysis of modified humanized antibody

In order to evaluate the change in surface charge due to amino acid modification of the variable region, preparation of modified antibody and analysis by isoelectric focusing electrophoresis were performed

Antibodies comprising five homodimers of hA69, hA69a-p18, hA69-p8, hA69-p17 and hA69-p16 were prepared by simultaneously expressing a humanized BBA-L chain (hAL-F123j4) expression vector in combination with each of the H chain expression vectors of hA69-p18, hA69-p8, hA69-p17, hA69-p16 and unmodified hA69a modified by modifying the humanized A69-H chain. Similarly, antibodies composed of homodimers of hB26-F123e4, hB26-p19 and hB26-p15 were prepared by simultaneously expressing a humanized BBA-L chain expression vector in combination with each of the H chain expression vectors of hB26-p19, hB26-p15 and unmodified hB26-F123e4, which were obtained by modifying a humanized B26-H chain. Isoelectric focusing electrophoresis was performed as follows. Phastsystem Cassette (produced by Amercham Bioscience) was used, and Phast-Gel Dry IEF (produced by Amercham Bioscience) was Gel-swollen with the following swelling solution for about 30 minutes.

20% Glycerol 0.95mL

MilliQ Water 0.95mL

Bio-Lite 7/9 (manufactured by BioRad) 10. mu.L

Bio-Lite 3/10 (manufactured by BioRad) 10. mu.L

Pharmalyte 8-10.5 for IEF (produced by Amercham Bioscience) 80. mu.L

The swollen gel was used, and electrophoresis was performed by Phastsystem (produced by Amercham Bioscience) according to the following procedure. The sample was added to the gel at step 2. pI marker pI calibration curve kit (Amersham Biosciences) was used.

Step 1: 2000V 2.5mA 3.5W 15 ℃ 75Vh

Step 2: 200V 2.5mA 3.5W 15 ℃ 15Vh

And step 3: 2000V 2.5mA 3.5W 15 ℃ 410Vh

The gel after electrophoresis was fixed with 20% TCA, and Silver stained with Silver staining kit, protein (Amersham Biosciences), according to the instructions attached to the kit. After staining, the isoelectric point of the sample was calculated from the known isoelectric point of the pI marker.

The results of analysis of homodimers of the unmodified and modified humanized a69 antibody and the humanized B26 antibody are shown in fig. 3. The movement of the bands is observed in isoelectric focusing electrophoresis by the change of surface charge. The isoelectric point of each antibody, which is presumed by reference to the pI marker, is about 8.4 for modified hA69-p18, about 8.2 for hA69-p17, and about 8.1 for hA69-p8, relative to 8.8 for the unmodified hA69a homodimer, and the difference in isoelectric point of about 0.7 at the maximum can be produced by modification. Similarly, for humanized B26 homodimer, the isoelectric point difference was about 0.3 maximum by modification, with modified hB26-p19 being about 9.3 and hB26-p15 being about 9.4 relative to 9.1 for unmodified hB26-F123e 4. This study showed that the isoelectric point can be changed by charge-changing the surface amino acids of the selected variable regions H12, H23, H39, H43, and H105.

EXAMPLE 5 cation exchange chromatography analysis of modified humanized antibody

Using the modified antibody prepared in example 4, cation exchange chromatography analysis was performed according to the following method to evaluate the effect of modification on the separation of the two antibodies. The retention time of homodimers of the humanized a69 antibody and the humanized B26 antibody was calculated under the following cation exchange chromatography analysis conditions.

Column: ProPac WCX-10, 4 x 250mm, (Dionex)

Mobile phase: a: 10mmol/L NaH₂PO₄/Na₂HPO₄，pH6.25

B：10mmol/L NaH₂PO₄/Na₂HPO₄，500mmol/L NaCl，pH6.25

Flow rate: 1.0mL/min

Gradient: 10% B (5 minutes) → (40 minutes) → 60% B → (5 minutes) → 100% B (5 minutes)

And (3) detection: 220nm

The results of analysis of homodimers of the unmodified and modified five humanized a69 antibodies are shown in fig. 4, and the results of analysis of homodimers of the unmodified and modified three humanized B26 antibodies are shown in fig. 5. The retention time of the homodimer of the unmodified humanized a69 antibody and the homodimer of the humanized B26 antibody were both about 25 minutes, and separation of both homodimers, and thus separation of the target bispecific antibody, could not be performed. The humanized a69 antibody, which was altered to reduce the isoelectric point of the unmodified antibody, exhibited a shift in peak compared to the unmodified antibody, and the retention time was shortened to about 22.4 minutes, about 21.2 minutes, and about 20.2 minutes with the number of alterations. The humanized B26 antibody with the change in isoelectric point of the variable region was altered to increase the retention time to about 28.4 minutes to about 29.4 minutes with a different number of changes observed to shift the peaks compared to the unaltered antibody. The retention time was changed by changing the surface amino acid charges of the variable regions H12, H23, H39, H43, and H105 selected in this study.

According to the isoelectric points determined in example 4, the pIs of the unmodified hA69a homodimer and the unmodified hB26-F123e4 homodimer were 0.3 different, but the retention times of both were about 25 minutes and could not be separated (FIG. 9), while the pIs of the unmodified hA69 homodimer and hB26-p19 were 0.5 different, resulting in a difference of about 2.6 minutes, the pIs of the hA69-p18 and hB26 homodimer were 0.7 different, resulting in a difference of about 3.4 minutes, the largest difference being 1.3 between the pIs of hA69-p16 and hB26-p15, resulting in a difference of about 9.2 minutes. Thus, the separation of the two homodimers was made possible for the first time by modification.

EXAMPLE 6 evaluation of clotting Activity of modified humanized bispecific antibody

Through the analysis of example 4 and example 5, we can observe the change of surface charge, and make two kinds of humanized antibody H chain (hA69-p8, hB26-p15) and humanized L chain (hAL-F123j4) expression, prepare humanized bispecific antibody. To efficiently promote the hybrid dimer, the H chain vector used was an expression vector incorporating the IgG4 constant region using the konbs-into-holes technique. Using the prepared humanized bispecific antibody, clotting activity was evaluated according to the following method.

To determine whether a bispecific antibody can alter the clotting ability of hemophilia a blood, the effect of the antibody on Activated Partial Thrombin Time (APTT) using factor VII-depleted plasma was investigated. 50 mu is addedL mixtures of various concentrations of antibody solution, 50. mu.L of factor VIII-poor plasma (Biomerieux) and 50. mu.L of LAPTT reagent (Dade Behring) were warmed at 37 ℃ for 3 minutes. The coagulation reaction was performed by adding 50. mu.L of 20mM CaCl₂(Dade Behring) was added to the mixture to initiate. The time required to clot was determined by KC10A (Amelung) with CR-A (Amelung) attached.

The factor VIII-like activity (%) of the bispecific antibody was calculated from the clotting time of the case where the bispecific antibody was added using a calibration curve prepared with the clotting time of factor VIII-poor plasma being 0% and the clotting time of normal plasma being 100%.

The results of the activity evaluation are shown in FIG. 6. The humanized bispecific antibody with the modified variable region showed the same clotting activity as the unmodified humanized bispecific antibody, and thus it was revealed that the modification of the variable region in this example had no effect on the activity of the antibody.

EXAMPLE 7 preparation and evaluation of humanized antibody having CDR modified

Analysis of the model of the humanized anti-a 69 antibody prepared in example 2 confirmed that H97 is a surface-exposed amino acid. In the antibodies shown in Table 1, hA69-N97R of the humanized A69-H chain had a sequence in which asparagine No. 97 present in CDR3 was modified to arginine. An expression vector having hA69-N97R was prepared according to the method of example 1-2, and expressed together with humanized BBA-L chain-hAL-F123 j4 to prepare a modified antibody. The change in surface charge of the antibody was evaluated, and isoelectric focusing electrophoresis was performed according to the method of example 4. As shown in FIG. 7, the isoelectric point of the unmodified antibody (hA69a/hAL-F123j4) was 8.9, while that of the modified antibody (hA69a-N97R/hAL-F123j4) was 9.1, and a change in surface charge was observed in the amino acid substitution of the CDR.

To evaluate the function of the modified antibody, the binding activity to the antigen, factor IXa, was evaluated as follows. Factor Ixa. beta. (Enzyme Research laboratories) diluted to 1. mu.g/mL with coating buffer (100mM sodium bicarbonate, pH 9.6, 0.02% sodium azide) was dispensed at 100. mu.L/well intoNunc-Immuno plate (Nunc-Immuno)^TM96MicroWell^TM MaxiSorp^TM(Nalge Nunc International)), incubated at 4 ℃ overnight. By containing tween^(R)20 of PBS (-) was washed 3 times, and then diluted with a buffer (50mM Tris-HCl, pH8.1, 1% bovine serum albumin, 1mM MgCl)₂0.15M NaCl, 0.05% Tween^(R)20, 0.02% sodium azide) the plates were blocked at room temperature for 2 hours. The buffer was removed, then 100. mu.L/well of purified antibody diluted with dilution buffer was added and incubated at room temperature for 1 hour. The plates were washed 3 times, then 100. mu.L/well of alkaline phosphatase-labeled goat anti-mouse IgG (BIOSOURCE) diluted to 1/4000 with dilution buffer was added and incubated at room temperature for 1 hour. Plates were washed 5 times, 100. mu.L/well chromogenic substrate (Sigma) was added, and incubated at room temperature for 30 minutes. Absorbance at 405nm (control 655nm) was measured using a microplate reader model 3550(Bio-Rad Laboratories). As a result, as shown in fig. 8, the CDR-modified antibody and the unmodified antibody showed the same binding activity due to the change in charge. Also shown is: when the surface charge is changed as described above, the modified site is not only FR as shown in example 5 but also CDR.

EXAMPLE 8 preparation and evaluation of humanized Dual-specific PF antibody

Among the antibodies shown in Table 1, humanized A69-H chain hA69a, humanized B26-H chain hB26-F123e4 and humanized BBA-L chain hAL-F123j4(SEQ ID NO.5) were used as unmodified antibodies to prepare unmodified humanized bispecific antibodies. Among the antibodies shown in Table 1, humanized bispecific PF antibodies were prepared using a humanized A69-H chain modifier hA69a-PFL, a humanized B26-H chain modifier hB26-PF, and a humanized BBA-L chain hAL-s8(SEQ ID NO.17) as modified antibodies. H chain Using an expression vector having a wild-type constant region, an expression vector was constructed as described in example 1-2, and antibodies were prepared in accordance with the methods of examples 1-3, 1-4, and 1-5. Using a mixed solution containing the two homodimers and the bispecific antibody, cation exchange chromatography was performed according to the method shown in example 5.

The results of the analysis of the unmodified humanized bispecific antibody and the humanized bispecific PF antibody are shown in fig. 9 and 10. In this result, in the unmodified humanized bispecific antibody, the two homodimers were not separated from the bispecific antibody and eluted as one peak, whereas in the humanized bispecific PF antibody, the two homodimers and the target bispecific antibody were separated, and three peaks were eluted sequentially in the order of hA69-PF homodimer, humanized bispecific PF antibody, hB26-PF homodimer. Three peaks were obtained upon cation exchange chromatography, from which two homodimers and the humanized bispecific PF antibody could be purified. The fraction was concentrated by Amicon Ultra, MWCO 10000(Millipore), dialyzed overnight at low temperature against 20mM sodium acetate, 150mM NaCl, ph6.0, and the concentration was measured.

After each antibody was purified, isoelectric focusing electrophoresis was performed according to the method shown in example 4. As shown in fig. 1, three bands of the antibody before the cation exchange chromatography analysis were present, and it was confirmed that each antibody could be purified by the cation exchange chromatography. The isoelectric points of the homodimer of the humanized a69-PF antibody, the humanized bispecific PF antibody and the homodimer of the humanized B26-PF antibody were about 7.9, about 8.6 and about 9.2, the difference between the isoelectric point of the homodimer of the humanized a69-PF antibody and the isoelectric point of the humanized dual specific PF antibody was about 0.7, and the difference between the isoelectric point of the homodimer of the humanized B26-PF antibody and the isoelectric point of the humanized dual specific PF antibody was about 0.6.

Next, the purified bispecific PF antibody was evaluated for clotting activity according to the method shown in example 6, and compared with the following three antibodies: bispecific antibodies expressed using the IgG4 constant region using the above-described knobs-into-holes technique; bispecific antibodies comprising hA69a (SEQ ID NO.2), hB26-F123e4(SEQ ID NO.4), hAL-F123j4(SEQ ID NO.6) without variable region modification; bispecific antibodies with variable regions using IgG4 constant regions using the knobs-into-holes technique were identical to the purified bispecific PF antibody. The evaluation results are shown in fig. 12. The clotting activity of the bispecific PF antibody having an IgG4 constant region using the nanobs-into-holes technique was equivalent to that of the bispecific PF antibody having a wild-type constant region and purified by ion exchange chromatography, indicating that the variable region modification of H10, H12, H23, H39, H43, and H105 of this example had no effect on the activity, and the bispecific antibody could be purified at high purity.

EXAMPLE 8 construction of humanized bispecific antibody-expressing cell line

To prepare the modified humanized bispecific antibody, an antibody-expressing cell line was constructed as follows.

Using a wild-type H chain constant region gene of human IgG4 as a template, a primer on the 5 'end side, which had been designed to encode a NheI recognition sequence (GCTAGC) consisting of the nucleotide sequence of two amino acids (Ala-Ser) on the N-terminal side of the H chain constant region, and a primer having a NotI recognition site and being designed to anneal to the 3' end side, the H chain constant region was PCR-amplified and ligated to a vector prepared by digesting a pBluescriptKS + vector (Toyobo) with NheI and NotI (both prepared by Takara Shuzo) to prepare pBCH4 (containing an IgG4 constant region gene). PCR was carried out using the primers complementary to the base sequence on the 5 '-end side of the H chain variable region of the humanized A69-H chain antibody (hA69-KQ) and the humanized B26-H chain antibody (hB26-PF) shown in Table 1 and having a Kozak sequence (CCACC) and an EcoRI recognition sequence, and a primer having a base sequence on the 3' -end side of the NheI recognition sequence, and the resulting PCR product was digested with EcoRI and NheI (both prepared by Takara Shuzo), inserted into pBCH4 similarly digested with EcoRI and NheI, and the variable region and the constant region were ligated. The humanized A69-H chain antibody vector thus prepared was digested with EcoRI and NotI (both prepared by Takara Shuzo), and cloned into an expression vector pCXND3 for animal cells similarly digested with EcoRI and NotI.

The construction scheme of this vector pCXND3 is shown below. In order to cleave the H chain gene of the antibody of DHFR-. DELTA.E-. gamma.VH-PM 1-f (see WO92/19759) from the vector, digestion was carried out using restriction enzymes EcoRI and SmaI, only one side of the vector was recovered, and then EcoRI-NotI-BamHI aptamer (prepared by Takara Shuzo) was cloned. This vector was named pCHOI. The DHFR Gene expression site of pCHOI was cloned into the HindIII site of the restriction enzyme of pCXN (Niwa et al, Gene 1991, 108: 193-200), and the resulting vector was named pCXND 3. The humanized B26-H chain antibody vector thus prepared was digested with EcoRI and NotI (both prepared by Takara Shuzo), and cloned into an expression vector pCXZD1 for animal cells similarly digested with EcoRI and NotI. The pCXZD1 vector is an expression vector obtained by replacing the neomycin resistance gene of the pCXND3 vector with a bleomycin resistance gene. PCR was performed using a synthetic oligonucleotide complementary to the base sequence on the 5 '-terminal side of the L chain variable region of a humanized BBA-L chain antibody (hAL-AQ, SEQ ID NO.18) and having a Kozak sequence, and a synthetic oligonucleotide complementary to the base sequence on the 3' -terminal side of the BsiWI site, and the resulting PCR product was cloned into a pBCL vector obtained by inserting the human kappa constant region into a pBluescriptKS + vector. The human L variable region was connected to the constant region by a BsiWI site. The prepared L chain gene fragment was cloned into an expression vector pUCAG. The vector pUCAG was obtained by ligating a 2.6kbp fragment obtained by digesting pCXN (Niwa et al, Gene 1991, 108: 193-. The L chain was cloned into pUCAG, and the resulting vector was digested with the restriction enzyme BamHI and cloned into the expression vector pHycDHFR-4b containing the hygromycin resistance gene. The three prepared expression vectors are made into linear chain connection by restriction enzymes, and then genes are introduced into CHO-DG44 cells to establish antibody expression cell strains.

The stably expressing cell line was prepared as follows. The gene was introduced by electroporation using GenePulseII (Bio-Rad). Each antibody expression vector was mixed with 0.75mL of CHO cells (1X 10) suspended in PBS⁷cells/mL) were mixed, the mixture was cooled on ice for 10 minutes, transferred to a sample cup, and then pulsed at 1.5kV, 25 μ FD capacity. After 10 minutes of recovery at room temperature, the electroporated cells were suspended in 40mL of CHO-S-SFMII medium (Invitrogen) containing HTsupplement (Invitrogen) at a 1-fold concentration. A10-fold dilution was prepared using the same medium and dispensed into 96-well culture plates at 100. mu.L/well. In CO₂Incubator (5% CO)₂) After 24 hours of medium culture, 0.5mg/mL geneticin (Invitrogen), 0.6mg/mL bleomycin (Invitrogen), and 0.4mg/mL hygromycin B (Invitrogen) were added thereto and cultured for 2 weeks. Will show drug resistanceColonies of the sexual transformed cells were sequentially cultured in an enlarged scale, and a large amount of the established high-producing cell line was used to obtain a culture supernatant.

EXAMPLE 9 isolation and purification of humanized bispecific antibody by commonly used column for preparation

The bispecific antibody was purified from the culture supernatant obtained in example 8 according to the following procedure. The culture supernatant was added to an rProtein A Sepharose Fast Flow column (Amersham Biosciences, 50mm I.D.. times.9.9 cm H. times.194.3 mL-resin) equilibrated with an equilibration buffer (20mmol/L sodium phosphate buffer, 1mol/L NaCl, pH7.0), washed with a washing buffer 1 (50mmol/L sodium acetate buffer, pH6.0), and then eluted with 100mmol/L acetic acid. Immediately after elution, the column was diluted 3-fold with 20mmol/L sodium acetate buffer, pH 6.0.

The resulting purified solution was added to a preparative conventional column SP TOYOPERL 650M (imperial ソ —, 26mm i.d. × 22.3cm h. ═ 118.3 mL-resin) equilibrated with Solvent a (20mmol/L sodium acetate buffer, ph 6.0). The separation is performed by antibody surface charge differences in the solutions and gradients shown below.

Solvent A: 20mmol/L sodium acetate buffer, pH6.0

Solvent B: 20mmol/L sodium acetate buffer, 1mol/L NaCl, pH6.0

Flow rate: 10 mL/min (113 cm/hr) was 5.3 mL/min (60 cm/hr) only at the time of elution

Gradient: 0 → 15% B step gradient 3 Column Volume (CV) flushing liquid

15 → 22% B gradient 2.5CV

22 → 30% B gradient 6CV

30 → 100% B step gradient 3CV Wash liquid

The results of elution show that the three peaks shown in figure 13 were detected and that bispecific antibodies could also be isolated and purified using a preparative conventional column.

EXAMPLE 10 evaluation of Activity of modified humanized bispecific antibody

For the humanized bispecific antibody prepared in example 9, the clotting activity was evaluated according to the method shown in example 6. The evaluation results are shown in fig. 14. Compared with the humanized bispecific PF antibody prepared in example 8, the clotting activity was equivalent to that of the humanized bispecific antibody purified in example 9. For example, the amino acid sequences of hA69-PFL and hA69-KQ, the variable regions were slightly different, and the activity was not affected by the use of an antibody purified by a column commonly used for preparation.

As shown above, in the preparation of bispecific antibody, the H chain variable region can be modified without changing the structure or the function (activity) of the antibody, and only the surface charge can be changed, so that the target humanized bispecific antibody and the antibody forming two homodimers can be separated and purified. By using this method, it was shown that the bispecific antibody can be isolated and purified by a conventional column for preparation, and thus can be used as a method for preparing a drug containing the bispecific antibody.

[ example 11] preparation of subclass hybrid antibody

11-1 cloning of human IgG2 antibody H chain constant region Gene

In order to clone the gene of the H chain constant region of human IgG2 antibody, the following procedure was performed.

For amplification of cDNA fragments, 50. mu.L of each reaction solution (1. mu.L each of 20. mu. M K62 primer (5 'cac cgt ctc ctc agc ctc cac caa 3'/SEQ ID NO.22), K63 primer (5 'gtggca ctc att tac ccg gag aca 3'/SEQ ID NO.23), 5. mu.L of MTC multi-tissue cDNA set (peripheral blood leukocytes) (manufactured by Clontech), 4. mu.L of 5 XPrime STAR buffer, 4. mu.L of 2.5mM dNTPs, and 1. mu.L of Prime STAR HS DNA polymerase (manufactured by TaKaRa, supra)) was prepared, and PCR was performed. PCR was performed using a thermal cycling GeneAmp PCR system 9700(Parkin Elmer) by heating at 98 ℃ for 2 minutes, then performing 30 cycles of reactions containing 98 ℃ for 10 seconds, 60 ℃ for 5 seconds, and 72 ℃ for 2 minutes, and finally heating at 72 ℃ for 10 minutes. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. Amplified fragments of the target size (about 1000bp) were purified according to the method of the attached instructions according to the QIAqucick gel purification kit (QIAGEN), eluted with 50. mu.L of sterile water, and then subjected to γ -Taq treatment for adding A (adenosine) to the ends of the amplified fragments. The γ -Taq treatment was carried out by incubating 10. mu.L of γ Taq reaction solution (1. mu.L of 10 Xγ Taq reaction solution, 1. mu.L of 2.5mM dNTPs, 1. mu.L of rTaq, and 7. mu.L of the amplified fragment) at 72 ℃ for 30 minutes. The gamma-Taq-treated fragment was cloned into pCR2.1-TOPO vector (Invitrogen) and the base sequence was determined. The nucleotide sequence of each DNA fragment was determined by a DNA sequencer ABI PRISM3730xL Genetic analyzer (Applied Biosystems) using BigDye terminal 3.1 cycle sequencing kit (Applied Biosystems) according to the method described in the attached specification.

When the determined nucleotide sequence was compared with access.no. BX640623, the nucleotide sequence of the translated amino acid sequence was different from that of the nucleotide sequence of BX640623, and the nucleotide sequence was modified to be the same as the amino acid sequence of BX640623 by performing amino acid substitution using QuickChange site-specific mutation kit (Stratagene), which is considered to be a mutation caused by insertion during PCR amplification. The QuickChange site-specific mutagenesis kit (Stratagene) was carried out according to the method described in the attached instructions. Furthermore, in order to link the human IgG2-H chain constant region gene and the target variable region gene, the first two amino acids (Ala-Ser) of the human IgG2-H chain constant region were mutated to form a restriction enzyme NheI recognition sequence (GCTAGC). The base sequence and amino acid sequence of the human IgG2-H chain constant region used in this experiment are shown in SEQ ID NO.24 and SEQ ID NO.25, respectively.

11-2 construction of expression vector for subclass-substituted antibody

An antibody expression vector in which the H chain constant regions of human IgG1, human IgG2, and human IgG4 were linked to the H chain variable region of the humanized PM-1 antibody was prepared as follows.

PCR was carried out using a synthetic oligonucleotide having a Kozak sequence complementary to the base sequence on the 5 '-end side of the H chain variable region of a humanized anti-human interleukin 6 receptor antibody (humanized PM-1 antibody) shown in non-patent literature (Sato K et al, Cancer Research 1993, 53: 851-856) and a synthetic oligonucleotide having a recognition sequence for the restriction enzyme NheI and complementary to the base sequence on the 3' -end side, and the resulting PCR product was cloned into a pB-CH vector obtained by inserting a human IgG1-H chain constant region (see SatoK et al, Cancer Research 1993, 53: 851-856) into a pBluescript KS + vector (TOYOBO). The H chain Gene fragment in which the H chain variable region and the constant region were ligated was inserted into the pCAGGS vector (Niwa et al 1991 Gene, 108: 193-199) whose expression was controlled by the chicken beta actin promoter. The H chain variable region gene of the humanized PM-1 antibody amplified by PCR was ligated to the human IgG4 constant region gene (see WO99/51743) and NheI at the 5' end of the human IgG2-H chain gene prepared in example 11-1, and inserted into the pCAGGS vector. Various H chain expression vectors express H chains by linking the H chain variable region of humanized PM-1 antibody to the human H chain constant region via NheI sequence.

Similarly, PCR was carried out using a synthetic oligonucleotide complementary to the base sequence on the 5 '-terminal side of the L chain variable region of the humanized PM-1 antibody and having a Kozak sequence, and a synthetic oligonucleotide complementary to the base sequence on the 3' -terminal side of the restriction enzyme BsiWI recognition sequence, and the resulting PCR product was cloned into a pB-CL vector obtained by inserting the human kappa constant region into a pBluescript KS + vector (TOYOBO). The L chain gene fragment formed by connecting the L chain variable region and the constant region is inserted into a pCAGGS vector whose expression is controlled by a chicken beta actin promoter. The L variable region of the humanized PM-1 antibody was linked to the human kappa chain constant region by BsiWI sequence to express the L chain.

11-3 expression of subclass hybrid antibodies

Subclass hybrid antibody 2 humanized PM-1 antibody H chain expression vectors having various constant regions of human IgG1, human IgG2, and human IgG4, respectively, can be combined and co-expressed together with a humanized PM-1 antibody L chain expression vector in an expression cell. The expression of each antibody can be carried out by the method of example 4-2 or the following method. HEK293H strain derived from human fetal kidney cancer cells (Invitrogen) was suspended in a DMEM medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) at 10mL each, 5-6X 10⁵Cell density per mL was seeded on each of adherent cell culture dishes (10 cm diameter, CORNING) in CO₂Incubator (37 ℃, 5% CO)₂) After internal culture for one day and night, the medium was removed by suction, and 6.9mL of CHO-S-SFM-II (Invitrogen) medium was added. Using the plasmid DNA prepared in 11-2, a mixture for expression of each subclass antibody and a mixture for expression of a hybrid antibody (total 13.8. mu.g) were prepared as follows.

(1) 6.9. mu. g L chain expression vector, 6.9. mu.g IgG1-H chain expression vector

(2) 6.9. mu. g L chain expression vector, 6.9. mu.g IgG2-H chain expression vector

(3) 6.9. mu. g L chain expression vector, 6.9. mu.g IgG4-H chain expression vector

(4) 6.9. mu. g L chain expression vector, 3.45. mu.g IgG1-H chain expression vector, 3.45. mu.g IgG2-H chain expression vector

(5) 6.9. mu. g L chain expression vector, 3.45. mu.g IgG2-H chain expression vector, 3.45. mu.g IgG4-H chain expression vector

(6) 6.9. mu.g of the chain expression vector, 3.45. mu.g of the IgG1-H chain expression vector, and 3.45. mu.g of the IgG4-H chain expression vector

Each mixture was mixed with 20.7. mu.L of 1. mu.g/mL polyethyleneimine (Polysciences Inc.) and 690. mu.L of CHO-S-SFMII medium, allowed to stand at room temperature for 10 minutes, added to the cells in each dish, and cultured in CO₂Incubator (37 ℃, 5% CO)₂) Internal culture for 4-5 hr. Then 6.9mL CHO-S-SFM-II (Invitrogen) medium was added under CO₂Culturing in an incubator for 3 days. The culture supernatant was recovered, centrifuged (about 2000g, 5 min, room temperature) to remove the cells, and sterilized by passing through a 0.22 μm filter MILLEX (R) -GV (Millipore). The samples were stored at 4 ℃ prior to use.

Purification of 11-4 subunit hybrid antibodies

In examples 11 to 3The culture supernatant obtained by the above method was supplemented with 100. mu.L of protein A Sepharose^TMFast Flow (Amersham Biosciences) was mixed at 4 ℃ for 4 hours or more by inversion. The solution was transferred to a 0.22 μm filter bowl Ultrafree^(R)-MC (Millipore), washed 3 times with 500. mu.L TBS and then suspended in rProtein A Sepharose^TMThe resin was allowed to stand in 100. mu.L of 50mM aqueous sodium acetate solution, pH3.0, for 2 minutes, and then the antibody was eluted. 6.7. mu.L of 1.5M Tris-HCl, 150mM NaCl, pH8.0 was immediately added for neutralization. The resulting antibody solution was dialyzed against PBS for activity measurement and against 20mM acetate buffer pH6.0 containing 150mM NaCl for DSC measurement, to replace the buffer. Hereinafter, an antibody having a purified H chain constant region of human IgG1 will be referred to as "unmodified humanized anti-PM-1 antibody", an antibody having a H chain constant region of human IgG2 will be referred to as "IgG 2-humanized anti-PM-1 antibody", and an antibody having a H chain constant region of human IgG4 will be referred to as "IgG 4-humanized anti-PM-1 antibody".

11-5 concentration quantification of subclass hybrid antibodies

mu.L of the solution containing the antibody obtained in example 11-4 was supplied to an ND-1000 spectrometer (NanoDrop), or 50. mu.L of the solution was supplied to a spectrophotometer DU-600(BECKMAN), and the absorbance at 280nm was measured. The antibody concentration was calculated from the obtained values by the following equation. The blank was PBS or 20mM acetate buffer containing 150mM NaCl, pH 6.0.

Antibody concentration (mg/mL) ═ absorbance × dilution factor ÷ 14.6 × 10

EXAMPLE 12 analysis of subclass hybrid antibody

12-1, isoelectric focusing electrophoresis analysis of subclass hybrid antibodies

To evaluate the change in surface charge due to constant region displacement, analysis was performed by isoelectric focusing electrophoresis.

Isoelectric focusing electrophoresis was performed as follows. Phastsystem Cassette (produced by Amercham bioscience) was used, and Phast-Gel Dry IEF (produced by Amercham bioscience) was Gel-swollen with the following swelling solution for about 30 minutes.

20% Glycerol 1.5mL

Pharmalyte 8-10.5 for IEF (produced by Amercham Bioscience) 100. mu.L

The swollen gel was used, and electrophoresis was performed by Phastsystem (produced by Amercham Bioscience) according to the following procedure. The sample was added to the gel at step 2. pI marker pI calibration curve kit (Amersham Biosciences) was used.

Step 1: 2000V 2.5mA 3.5W 15 ℃ 75Vh

Step 2: 200V 2.5mA 3.5W 15 ℃ 15Vh

And step 3: 2000V 2.5mA 3.5W 15 ℃ 410Vh

The gel after electrophoresis was fixed with 20% TCA, and Silver stained with Silver staining kit, protein (Amersham Biosciences), according to the instructions attached to the kit. After staining, the isoelectric point of the sample was calculated from the known isoelectric point of the pI marker.

The results of the analysis of unmodified, IgG2 and IgG4 humanized PM-1 antibodies are shown in FIG. 15. By substitution of the subclasses, movement of bands was observed in isoelectric focusing electrophoresis. The isoelectric point of each antibody estimated by reference to the pI marker was about 8.9 for the IgG2 humanized PM-1 antibody and about 8.7 for the IgG4 humanized PM-1 antibody, relative to 9.3 for the unmodified humanized PM-1 antibody, and the difference in isoelectric point was about 0.7 at the most by substitution. This study showed that the isoelectric point can be changed by replacing the constant region of the antibody subclass.

The results of the co-expression antibody analysis of the unmodified, IgG2 humanized PM-1 antibody and the unmodified, IgG4 humanized PM-1 antibody are shown in FIG. 16. Thus, in any combination, homodimers and hybrid dimers of each subclass were observed as 3 main bands, and the isoelectric point of each antibody estimated by reference to the pI marker was 9.2 for the unmodified humanized PM-1/IgG2 human PM-1 antibody and 9.0 for the unmodified humanized PM-1/IgG2 human PM-1 hybrid antibody. In this study, it was shown that by co-expressing the expression vectors of each subclass of antibody in combination, subclass hybrid antibodies can be prepared, which are separated by isoelectric points.

12-2 cation exchange chromatography of subclass hybrid antibodies

Using the subclass hybrid antibody prepared in example 11, analysis by cation exchange chromatography was performed according to the following method to evaluate the effect of subclass substitution on the separation. The cation exchange chromatography analysis conditions were as follows to calculate retention times of the unmodified humanized PM-1 antibody, the IgG2 humanized PM-1 antibody, the IgG4 humanized PM-1 antibody, and the hybrid antibody of the unmodified humanized PM-1 antibody/IgG 2 humanized PM-1 antibody, the hybrid antibody of the unmodified humanized PM-1 antibody/IgG 4 humanized PM-1 antibody.

Column: ProPac WCX-10, 4 x 250mm, (Dionex)

Mobile phase: a: 25mmol/L MES/NaOH, pH6.1

B：25mmol/L MES/NaOH，250mmol/L NaCl，pH6.1

Flow rate: 0.5mL/min

Gradient: 10% B (5 minutes) → (105 minutes) → 67% B → (1 minute) → 100% B (5 minutes)

And (3) detection: 280nm

The results of analysis of unmodified, IgG 2-and IgG 4-expressed humanized PM-1 antibodies expressed alone are shown in FIG. 17. The retention times of the unmodified humanized PM-1 antibody, the IgG2 humanized PM-1 antibody and the IgG4 humanized PM-1 antibody were about 60.2 minutes, 30.5 minutes and 30.3 minutes, respectively, and the retention times were slightly less than 30 minutes by subclass substitution. On the other hand, in isoelectric focusing electrophoresis, the retention times of the IgG2 humanized PM-1 antibody and IgG4 humanized PM-1 antibody, which showed differences in pI, were approximately the same. Results of antibody co-expression analysis of unmodified, IgG2, and unmodified, IgG4 humanized PM-1 antibodies are shown in FIG. 18. In the combination of unmodified humanized PM-1 antibody/IgG 2 humanized PM-1 antibody, and unmodified humanized PM-1 antibody/IgG 4 humanized PM-1 antibody combination, three main peaks of homodimer and heterodimer of each subclass were observed. Regarding retention time, the hybrid antibody of unmodified humanized PM-1/IgG2 humanized PM-1 was about 43.8 minutes, and the hybrid antibody of unmodified humanized PM-1/IgG4 humanized PM-1 was about 45.1 minutes, separated from each homodimer with a retention time difference of 10 minutes or more. This study showed that by co-expressing the expression vectors of each subclass of antibody in combination, subclass hybrid antibodies could be prepared, which could be separated by ion exchange chromatography.

EXAMPLE 13 separation and purification of subclass hybrid antibody by cation exchange chromatography

The antibody solution obtained in example 1 was concentrated with Amicon-Ultra4(Amicon), packaged in easy Sep (トミ -Seiko), dialyzed against 5mM citric acid buffer (pH6.5), and the buffer was replaced to purify the subclass hybrid antibody under the following conditions.

Column: poly GAT A, 4.6X 100mm, particle size 3 μm, pore size 150nm (Poly LC)

Mobile phase: a: 25mmol/L MES/NaOH, pH6.1

B: 25mmol/L MES/NaOH, 250mmol/L sodium acetate, pH6.1

Flow rate: 1.0mL/min

Gradient: 35% B (5 minutes) → (54 minutes) -65% B → (1 minute) → 100% B (5 minutes)

And (3) detection: 280nm

Approximately 100. mu.g of each injection was used to prepare peaks for unmodified humanized PM-1 antibody, unmodified humanized PM-1 antibody/IgG 4 humanized PM-1 subclass hybrid antibody, IgG4 humanized PM-1 antibody. The chromatogram obtained during the preparation is shown in FIG. 19. The fractions for peak preparation were mixed, concentrated with Amicon-Ultra4(Amicon), and then packed into easy Sep (トミ -Seiko), and the buffer was replaced by PBS for activity measurement and by 20mM acetate buffer containing 150mM NaCl at pH6.0 for DSC measurement. The prepared peaks were re-analyzed under the same conditions as described above, and the results are shown in FIG. 20. It was thus shown that the subclass hybrid antibody can be prepared and purified by ion exchange chromatography.

Since the present technology can isolate antibodies having a common H chain variable region using constant regions of subclasses having different pI values, even if different H chain variable regions having no difference in pI are linked to H chain constant regions of subclasses having different pI values, bispecific antibodies can be isolated by ion exchange chromatography. In addition, when different H chain variable regions are present, the difference in pI between molecules can be further increased by combining the technique of introducing mutations into the variable regions as described in example 9, and separation and purification can be facilitated. When it is difficult to introduce mutations into the H chain variable region, they can be converted into naturally occurring IgG subclass sequences, and the bispecific antibody can be isolated and purified by ion exchange chromatography without considering antigenicity.

EXAMPLE 14 isoelectric focusing electrophoresis for preparation of purified product of subclass hybrid antibody

To evaluate the purity of the preparations, analysis was carried out by isoelectric focusing electrophoresis.

Isoelectric focusing electrophoresis was performed as follows. Phastsystem Cassette (produced by Amercham bioscience) was used, and Phast-Gel Dry IEF (produced by Amercham bioscience) was Gel-swollen with the following swelling solution for about 30 minutes.

MilliQ Water 1.5mL

Pharmalyte 5-8 for IEF (produced by Amercham Bioscience) 50. mu.L

Pharmalyte 8-10.5 for IEF (produced by Amercham Bioscience) 50. mu.L

The swollen gel was used, and electrophoresis was performed by Phastsystem (produced by Amercham Bioscience) according to the following procedure. The sample was added to the gel at step 2. pI marker pI calibration curve kit (Amersham Biosciences) was used.

Step 1: 2000V 2.5mA 3.5W 15 ℃ 75Vh

Step 2: 200V 2.5mA 3.5W 15 ℃ 15Vh

And step 3: 2000V 2.5mA 3.5W 15 ℃ 410Vh

The gel after electrophoresis was fixed with 20% TCA, and Silver stained with Silver staining kit, protein (Amersham Biosciences), according to the instructions attached to the kit.

FIG. 21 shows the results of analysis of purified products of the subclass hybrid antibodies. By ion exchange chromatography, homodimers of each subclass can be purified to almost no.

EXAMPLE 15 evaluation of Activity of purified product of subclass hybrid antibody preparation

15-1 establishment of BaF3 cell line expressing human gp130 and BaF3 cell line co-expressing human gp 130/human IL-6 receptor

In order to obtain a cell line showing IL-6 dependent proliferation, a BaF3 cell line expressing human gp130 was established as follows.

The full-length human gp130 cDNA was amplified by PCR (Hibi et al, Cell 1990, 63: 1149-1157(GenBank # NM-002184)), the DHFR gene expression site of pCHOI was removed, and cloned into the expression vector pCOS2Zeo into which the expression site of the bleomycin-resistant gene was inserted, to construct pCOS2Zeo/gp 130.

Mu.g of pCOS2Zeo/gp130 and BaF3 cells (0.8X 10) suspended in PBS⁷Individual cells) were mixed, and pulsed at 0.33kV and 950. mu. FD using GenePulser (Bio-Rad). The gene-introduced BaF3 cells were treated by electroporation and cultured overnight in RPMI16540 medium (Invitrogen) containing 0.2ng/mL mouse interleukin-3 (Peprotech) and 10% fetal bovine serum (hereinafter referred to as FBS, HyClone), to which 10ng/mL human interleukin-6 (R) was added&D) 100ng/mL human interleukin-6 soluble receptor (R)&D system) and contains 10%RPMI1640 medium of FBS was screened to establish a BaF3 cell line expressing human gp130 (hereinafter referred to as BaF3/gp 130).

15-2 evaluation of neutralizing Activity of purified product of subclass hybrid antibody on human IL-6

IL-6 neutralizing activity was evaluated using BaF3/gp130, which showed IL-6-dependent proliferation, as shown below. The purified unmodified humanized PM-1 antibody, unmodified/IgG 4-humanized PM-1 subclass hybrid antibody, and IgG 4-humanized PM-1 antibody were diluted to 10 μ g/mL with RPMI1640 containing 10% FBS, and using this solution, 7 serial dilutions having a dilution ratio of 3 were prepared, and 50 μ L of each dilution was dispensed into each well of a 96-well plate (coriig). Next, BaF3/gp130 was washed 3 times with 10% FBS-containing RPMI1640 medium, and suspended in 60ng/mL human interleukin-6 (TORAY), 60ng/mL soluble human IL-6 receptor (manufactured by this company), and 10% FBS-containing RPMI1640 medium at a concentration of 5X 10⁴Each 50. mu.L of each cell/mL was mixed in each well and then dispensed with an antibody sample. The human soluble IL-6 receptor was prepared as follows. The gene encoding amino acids No.1 to 344 of the human soluble receptor IL-6 receptor was introduced into CHO cells, and then purified and prepared from the culture supernatant. At 37 deg.C, 5% CO₂After culturing for 72 hours under the above conditions, WST-8 reagent (Cell Counting Kit-8, Kyowa Kagaku Co., Ltd.) diluted 2-fold with PBS was added thereto at 20. mu.L/well, and absorbance at 450nm (see wavelength: 620nm) was immediately measured using SUNRISECLASSIC (TECAN). After 2 hours of incubation, the absorbance at 450nm (see wavelength 620nm) was measured again, and the neutralization activity of IL-6 was evaluated using the change in absorbance for 2 hours as an index.

As a result, as shown in FIG. 22, the neutralizing activity of the purified unmodified humanized PM-1 antibody, unmodified/IgG 4-humanized PM-1 subclass hybrid antibody, and IgG 4-humanized PM-1 antibody were equivalent to that of the purified product (bulk) of the humanized PM-1 antibody. As shown above, the subclass hybrid antibody does not lose the original antigen-binding ability, and has a neutralizing antibody function.

Industrial applicability

In the method of the present invention, the isoelectric point can be controlled without changing the structure or function (activity) of the amino acid substitution number by a small number, and thus, by using a commonly used column, the bispecific antibody can be purified efficiently and with high purity for drug development, and is very useful for drug development.

By using the method of the present invention, a bispecific antibody that actually retains the activity can be obtained with high efficiency.

Sequence listing

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Claims (37)

1. A method for producing a multispecific antibody, the multispecific antibody comprising a1 st polypeptide and a 2nd polypeptide, the method comprising the steps of:

(a) modifying one or both of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide to produce a difference in isoelectric point between the 1 st polypeptide and the 2nd polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) recovering the multispecific antibody from the host cell culture.

2. The method of claim 1, wherein the modification of step (a) is a modification of the nucleic acid such that the homo-multimer of the 1 st polypeptide, the homo-multimer of the 2nd polypeptide, and the hybrid-multimer of the 1 st polypeptide and the 2nd polypeptide form separate peaks by analysis using standard chromatography.

3. The method of claim 1, wherein said 1 st polypeptide and said 2nd polypeptide comprise a heavy chain variable region.

4. The method of claim 3, wherein said multispecific antibody comprises a3 rd polypeptide comprising a light chain variable region, and said 1 st polypeptide and said 2nd polypeptide each form multimers with said 3 rd polypeptide.

5. The method of any one of claims 1-4, wherein said 1 st polypeptide and said 2nd polypeptide comprise a heavy chain constant region.

6. The method according to claim 5, wherein the heavy chain constant regions contained in the 1 st polypeptide and the 2nd polypeptide are heavy chain constant regions having isoelectric points different from each other.

7. The method of claim 6, wherein the heavy chain constant regions having different isoelectric points are IgG1 and IgG4, or IgG1 and IgG 2.

8. The method of claim 1, wherein said multispecific antibody is a bispecific antibody.

9. A multispecific antibody produced by the method of claim 1.

10. A method for purifying a multispecific antibody, which multispecific antibody comprises a1 st polypeptide and a 2nd polypeptide, the method comprising:

(a) modifying one or both of a nucleic acid encoding an amino acid residue of the 1 st polypeptide and a nucleic acid encoding an amino acid residue of the 2nd polypeptide to produce a difference in isoelectric point between the 1 st polypeptide and the 2nd polypeptide;

(b) culturing the host cell to express the nucleic acid;

(c) the multispecific antibodies are purified from the host cell culture by standard chromatography.

11. The method of claim 10, wherein the modification of step (a) is a modification of the nucleic acid such that the homo-multimer of the 1 st polypeptide, the homo-multimer of the 2nd polypeptide, and the hybrid-multimer of the 1 st polypeptide and the 2nd polypeptide form separate peaks by analysis using standard chromatography.

12. The method of claim 10, wherein said 1 st polypeptide and said 2nd polypeptide comprise a heavy chain variable region.

13. The method of claim 12, wherein said multispecific antibody comprises a3 rd polypeptide comprising a light chain variable region, and said 1 st polypeptide and said 2nd polypeptide each form multimers with said 3 rd polypeptide.

14. The method of any one of claims 10-13, wherein said 1 st polypeptide and said 2nd polypeptide comprise a heavy chain constant region.

15. The method according to claim 14, wherein the heavy chain constant regions contained in the 1 st polypeptide and the 2nd polypeptide are heavy chain constant regions having isoelectric points different from each other.

16. The method of claim 15, wherein the heavy chain constant regions having different isoelectric points are IgG1 and IgG4, or IgG1 and IgG 2.

17. The method of claim 10, wherein said multispecific antibody is a bispecific antibody.

18. A method of making a multispecific antibody, the method comprising the step of purifying by the method of claim 10.

19. A multispecific antibody produced by the method of claim 18.

20. A multispecific antibody, comprising a polypeptide 1 and a polypeptide 2, wherein the polypeptide 1 comprises a heavy chain variable region and/or a heavy chain constant region, and at least one amino acid residue selected from amino acid residues 10, 12, 23, 39, 43 and 105 of the heavy chain variable region according to Kabat numbering, or amino acid residues 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, 435 of the heavy chain constant region according to EU numbering, has an electric charge, and the isoelectric points of the polypeptide 1 and polypeptide 2 are different from each other.

21. The multispecific antibody according to claim 20, wherein the 2nd polypeptide comprises a heavy chain variable region and/or a heavy chain constant region, and at least one amino acid residue selected from amino acid residues 10, 12, 23, 39, 43 and 105 according to Kabat numbering of the heavy chain variable region, or amino acid residues 137, 196, 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, 435 of the heavy chain constant region according to EU numbering has a charge opposite to or no charge of an amino acid residue having a charge selected from the heavy chain variable region and/or the heavy chain variable region contained in the 1 st polypeptide.

22. The multispecific antibody of claim 20, wherein the combination of an amino acid residue having the above charge and an amino acid residue having a charge opposite to that of the amino acid residue is selected from the amino acid residues contained in any group of (a) or (b) below:

(a) glutamic acid (E), aspartic acid (D);

(b) lysine (K), arginine (R), histidine (H).

23. A multispecific antibody in which the isoelectric points of the 1 st and 2nd polypeptides differ, wherein the homomultimer of the 1 st polypeptide, the homomultimer of the 2nd polypeptide, and the hetero-multimer of the 1 st and 2nd polypeptides form separate peaks by analysis using standard chromatography.

24. The multispecific antibody of claim 23, wherein the 1 st polypeptide and the 2nd polypeptide comprise a heavy chain variable region.

25. The multispecific antibody of claim 24, wherein the multispecific antibody comprises a3 rd polypeptide comprising a light chain variable region, and the 1 st polypeptide and the 2nd polypeptide each form multimers with the 3 rd polypeptide.

26. The multispecific antibody of any one of claims 23-25, wherein the 1 st polypeptide and the 2nd polypeptide comprise heavy chain constant regions.

27. The multispecific antibody according to claim 26, wherein the heavy chain constant regions contained in the 1 st polypeptide and the 2nd polypeptide are heavy chain constant regions that differ from each other in isoelectric point.

28. The multispecific antibody of claim 27, wherein the heavy chain constant regions which differ in isoelectric point are IgG1 and IgG4, or IgG1 and IgG 2.

29. The multispecific antibody of claim 23, wherein the multispecific antibody is a bispecific antibody.

30. A composition comprising the multispecific antibody of any one of claims 23-29 and a pharmaceutically acceptable carrier.

31. A nucleic acid encoding a polypeptide constituting a multispecific antibody according to any one of claims 23 to 29.

32. A host cell having the nucleic acid of claim 31.

33. A method of producing a multispecific antibody according to any one of claims 23 to 29, comprising the step of culturing a host cell according to claim 32; a step of recovering the polypeptide from the cell culture.

34. The multispecific antibody of claim 25, wherein the variable region of polypeptide 1 comprises the amino acid sequence of any one of (a1) to (a7) below, the variable region of polypeptide 2 comprises the amino acid sequence of any one of (b1) to (b3) below, and the variable region of polypeptide 3 comprises the amino acid sequence of (c1) or (c2) below:

(a1)SEQ ID NO.7

(a2)SEQ ID NO.8

(a3)SEQ ID NO.9

(a4)SEQ ID NO.10

(a5)SEQ ID NO.11

(a6)SEQ ID NO.12

(a7)SEQ ID NO：13

(b1)SEQ ID NO.14

(b2)SEQ ID NO.15

(b3)SEQ ID NO.16

(c1)SEQ ID NO.17

(c2)SEQ ID NO.18。

35. the multispecific antibody of claim 34 wherein the variable region of polypeptide 1 comprises the amino acid sequence of SEQ ID No.11, the variable region of polypeptide 2 comprises the amino acid sequence of SEQ ID No.16 and the variable region of polypeptide 3 comprises the amino acid sequence of SEQ ID No. 17.

36. The multispecific antibody of claim 34 wherein the variable region of polypeptide 1 comprises the amino acid sequence of SEQ ID No.12, the variable region of polypeptide 2 comprises the amino acid sequence of SEQ ID No.16 and the variable region of polypeptide 3 comprises the amino acid sequence of SEQ ID No. 18.

37. The multispecific antibody of any one of claims 34-36, wherein polypeptides 1 and 2 comprise a human IgG4 constant region and polypeptide 3 comprises a human kappa constant region.