Antibody production method and antibody

Abstract

To provide means for increasing the yield of an antibody in which at least three amino acid residues of a framework region 3 are substituted with arginine residues or lysine residues, in the production of the antibody by a protein expression system using an animal cell.SOLUTION: The foregoing problem is solved by culturing an animal cell into which a gene encoding an antibody has been introduced in the presence of a polyanionic compound, and causing the animal cell to produce an antibody.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] The present invention relates to a method for producing an antibody and the antibody. [Background technology]

[0002] A technique is known for controlling the affinity of an antibody for an antigen by modifying the amino acid sequence of the framework region (FR) while maintaining the amino acid sequence of the antibody's complementarity-determining region (CDR). FRs are regions present in the variable regions of the antibody's light and heavy chains, other than the CDRs. For example, Patent Document 1 describes a method for controlling the affinity for an antigen by replacing at least three amino acid residues in the FR3 of an antibody with charged amino acid residues. Antibodies obtained by the method described in Patent Document 1 are expected to be used in pharmaceuticals, in vitro diagnostic agents, reagents, etc. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Application Publication No. 2018 / 0179298 Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have cultured animal cells transfected with genes encoding antibodies in which at least three amino acid residues in FR3 have been substituted with arginine or lysine residues by conventional methods to obtain such antibodies. However, the amount of antibody obtained was not fully satisfactory. The present inventors aimed to provide a means for increasing the yield of such antibodies. [Means for solving the problem]

[0005] Generally, in antibody production using a protein expression system using animal cells, antibody yield increases as the scale of cell culture is expanded. However, this increases production costs. The present inventors investigated means for increasing antibody yield without expanding the scale of cell culture. As a result, the present inventors found that the antibody yield increases when animal cells into which genes encoding the above-mentioned antibodies have been introduced are cultured in the presence of a polyanionic compound, thereby completing the present invention.

[0006] The present invention provides a method for producing an antibody, which comprises culturing animal cells introduced with a gene encoding the antibody in the presence of a polyanionic compound to cause the animal cells to produce the antibody, wherein the polyanionic compound is at least one selected from the group consisting of anionic polysaccharides and anionic polyamino acids, and the antibody has at least three amino acid residues in FR3 substituted with arginine residues or lysine residues. The present invention also provides antibodies produced by this production method. [Effects of the Invention]

[0007] According to the present invention, the yield of the antibody can be increased in the production of the antibody using a protein expression system that uses animal cells. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a graph showing the viability (%) of a CHO-S cell line stably expressing rituximab when cultured in a medium supplemented with glucose, sucrose, or trehalose. [Figure 2] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R3 mutant of rituximab when cultured in a medium supplemented with glucose, sucrose, or trehalose. [Figure 3] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R5 mutant of rituximab when cultured in a medium supplemented with glucose, sucrose, or trehalose. [Figure 4]1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing rituximasib when cultured in a medium supplemented with dextran sulfate sodium, chondroitin sulfate sodium, or heparin sodium. [Figure 5] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R3 mutant of rituximab when cultured in a medium supplemented with dextran sulfate sodium, chondroitin sulfate sodium, or heparin sodium. [Figure 6] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R5 mutant of rituximab when cultured in a medium supplemented with dextran sulfate sodium, chondroitin sulfate sodium, or heparin sodium. [Figure 7] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing rituximasib was cultured in a medium supplemented with glucose, sucrose, or trehalose. [Figure 8] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R3 mutant of rituximasib was cultured in a medium supplemented with glucose, sucrose, or trehalose. [Figure 9] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R5 mutant of rituximasib was cultured in a medium supplemented with glucose, sucrose, or trehalose. [Figure 10] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing rituximasib was cultured in a medium supplemented with dextran sulfate sodium, chondroitin sulfate sodium, or heparin sodium. [Figure 11] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R3 mutant of rituximasib was cultured in a medium supplemented with dextran sulfate sodium, chondroitin sulfate sodium, or heparin sodium. [Figure 12]1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R5 mutant of rituximasib was cultured in a medium supplemented with dextran sulfate sodium, chondroitin sulfate sodium, or heparin sodium. [Figure 13] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing rituximasib when cultured in a medium containing dextran sulfate sodium at a concentration of 0.1, 1, or 10 mg / mL. [Figure 14] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R5 mutant of rituximasib when cultured in a medium containing dextran sulfate sodium at a concentration of 0.1, 1, or 10 mg / mL. [Figure 15] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing rituximasib was cultured in a medium containing dextran sulfate sodium at a concentration of 0.1, 1, or 10 mg / mL. [Figure 16] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R5 mutant of rituximasib was cultured in a medium containing dextran sulfate sodium at a concentration of 0.1, 1, or 10 mg / mL. [Figure 17] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing rituximasib when cultured in a medium containing dextran sulfate sodium with a molecular weight of 5,000 or 50,000. [Figure 18] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R5 mutant of rituximasib when cultured in a medium containing dextran sulfate sodium with a molecular weight of 5,000 or 50,000. [Figure 19] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing rituximasib was cultured in a medium containing dextran sulfate sodium with a molecular weight of 5,000 or 50,000. [Figure 20]1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R5 mutant of rituximasib was cultured in a medium containing dextran sulfate sodium with a molecular weight of 5,000 or 50,000. [Figure 21] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R3 mutant of rituximasib when cultured in a medium containing dextran sulfate sodium with a molecular weight of 5,000, 50,000, or 500,000. [Figure 22] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R3 mutant of rituximasib was cultured in a medium containing dextran sulfate sodium with a molecular weight of 5,000, 50,000, or 500,000. [Figure 23] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing rituximasib when cultured in a medium containing heparin sodium at a concentration of 0.1, 1, or 10 mg / mL. [Figure 24] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R5 mutant of rituximasib when cultured in a medium containing heparin sodium at a concentration of 0.1, 1, or 10 mg / mL. [Figure 25] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing rituximasib was cultured in a medium containing heparin sodium at a concentration of 0.1, 1, or 10 mg / mL. [Figure 26] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R5 mutant of rituximasib was cultured in a medium containing heparin sodium at a concentration of 0.1, 1, or 10 mg / mL. [Figure 27] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing rituximasib when cultured in a medium containing sodium polyaspartate or sodium polyglutamate. [Figure 28]1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R3 mutant of rituximasib when cultured in a medium containing sodium polyaspartate or sodium polyglutamate. [Figure 29] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R5 mutant of rituximasib when cultured in a medium containing sodium polyaspartate or sodium polyglutamate. [Figure 30] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing rituximasib was cultured in a medium containing sodium polyaspartate or sodium polyglutamate. [Figure 31] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R3 mutant of rituximasib was cultured in a medium containing sodium polyaspartate or sodium polyglutamate. [Figure 32] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R5 mutant of rituximasib was cultured in a medium containing sodium polyaspartate or sodium polyglutamate. [Figure 33] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing rituximasib when cultured in a medium containing dextran or dextran sulfate sodium. [Figure 34] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R3 mutant of rituximasib when cultured in a medium containing dextran or dextran sulfate sodium. [Figure 35] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R5 mutant of rituximasib when cultured in a medium containing dextran or dextran sulfate sodium. [Figure 36] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing rituximasib was cultured in a medium containing dextran or dextran sulfate sodium. [Figure 37]1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R3 mutant of rituximasib was cultured in a medium containing dextran or dextran sulfate sodium. [Figure 38] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R5 mutant of rituximasib was cultured in a medium containing dextran or dextran sulfate sodium. [Figure 39] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing rituximasib when cultured in a medium containing polyproline or sodium alginate. [Figure 40] 1 is a graph showing the survival rate (%) of a CHO-S cell line stably expressing the R3 mutant of rituximasib when cultured in a medium containing polyproline or sodium alginate. [Figure 41] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing rituximasib was cultured in a medium containing polyproline or sodium alginate. [Figure 42] 1 is a graph showing the antibody concentration (mg / L) in the supernatant when a CHO-S cell line stably expressing the R3 mutant of rituximasib was cultured in a medium containing polyproline or sodium alginate. [Figure 43] 1 is a graph showing the affinity of trastuzumab and its variants for antigens. [Figure 44] 1 is a graph showing the affinity of an anti-lysozyme antibody and its mutants for antigens. [Figure 45] 1 is a graph showing the affinity of anti-interleukin (IL)-6 antibodies and their mutants for antigens. [Figure 46] 1 is a graph showing the affinity of anti-IL-8 antibodies and their mutants for antigens. [Figure 47] 1 is a graph showing the affinity of rituximab and its variants for antigens. DETAILED DESCRIPTION OF THE INVENTION

[0009] In the antibody production method of this embodiment, animal cells into which an antibody-encoding gene has been introduced are cultured ex vivo in the presence of a polyanionic compound to cause the animal cells to produce the antibody. Here, the antibody encoded by the gene introduced into the animal cells and the antibody produced by the animal cells are antibodies in which at least three amino acid residues in FR3 have been substituted with arginine residues or lysine residues (hereinafter also referred to as "mutants").

[0010] As used herein, the term "antibody" may be any of IgG, IgA, IgM, IgD, and IgE, but is preferably IgG. When producing IgG, it is preferable to use an animal cell expression vector incorporating a gene encoding a full-length light chain and a gene encoding a full-length heavy chain as antibody-encoding genes. The "antibody" may also be an antibody fragment. Preferably, the antibody fragment comprises the variable region of the light chain. Examples of such antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, domain antibodies (dAbs), reduced IgG (rIgG), diabodies, triabodies, etc.

[0011] Animal cells introduced with a gene encoding an antibody (hereinafter also referred to as "transgenic cells") are animal cells into which the gene has been introduced so as to be able to express the antibody as a protein. Transgenic cells can be, for example, animal cells into which the gene has been integrated into the intracellular genome, or animal cells that harbor the gene as an episomal vector. Animal cells into which an antibody-encoding gene has been introduced in this manner are called stable expression strains, and are capable of constitutively expressing the antibody as a protein. Alternatively, transgenic cells may be animal cells that transiently express the antibody as a protein upon transfection with the gene. The antibody expressed by the transgenic cells is secreted from the cells into the culture supernatant.

[0012] Animal cells are host cells that express proteins through gene transfer. Examples of animal cells include mammalian cells and insect cells. Among these, mammalian cells are particularly preferred. Mammalian cells may be primary cultured cells prepared from tissues or established cell lines. Mammalian cell lines are preferably used. Examples of cell lines include CHO cells (including CHO-K1 and CHO-S), HEK293 cells (including HEK293T and HEK293E), BHK cells, COS cells, HeLa cells, VERO cells, and 3T3 cells. Cell lines are available from ATCC (American Type Culture Collection), ECACC (European Collection of Cell Cultures), etc. Commercially available cell lines such as Expi293™ cells and ExpiCHO™ cells may also be used.

[0013] The gene encoding the antibody may be incorporated into an animal cell expression vector. The type of animal cell expression vector is not particularly limited and may be, for example, a plasmid vector or a viral vector. The gene encoding the antibody includes a gene encoding the antibody light chain and a gene encoding the antibody heavy chain. When the gene encoding the antibody is incorporated into an animal cell expression vector, the gene encoding the antibody light chain and the gene encoding the antibody heavy chain may be incorporated into a single vector, or may be incorporated into two vectors, respectively.

[0014] Introduction of an antibody-encoding gene into animal cells can be carried out by known transfection methods, including, but not limited to, lipofection, calcium phosphate co-precipitation, viral vector methods, and electroporation.

[0015] Culturing in the presence of a polyanionic compound can be carried out, for example, by culturing transgenic cells in a medium supplemented with a polyanionic compound. Culturing transgenic cells in the presence of a polyanionic compound increases the yield of mutants produced by the cells compared to culturing in the absence of a polyanionic compound. While the details of the mechanism by which the yield increases are unclear, it is believed that electrostatic interactions between mutants secreted into the culture supernatant from transgenic cells and the polyanionic compound in the medium contribute to this. However, the present invention is not bound by any particular theory.

[0016] "Culturing in the absence of polyanionic compounds" refers to culturing transfected cells in a medium that is substantially free of polyanionic compounds. "Medium that is substantially free of polyanionic compounds" refers to a medium that contains only trace amounts of polyanionic compounds, even if any, and to which polyanionic compounds are not actively added. An example of a medium containing a trace amount of polyanionic compounds is when a polyanionic compound is included in the additives described below.

[0017] The polyanionic compound refers to a polymer or salt thereof whose monomer unit is an anionic compound having an anionic functional group. Examples of the anionic functional group include sulfate groups and carboxyl groups. The salt of the polyanionic compound is preferably a pharmaceutically acceptable salt, such as a sodium salt or a potassium salt. In this embodiment, an anionic polysaccharide and / or an anionic polyamino acid is used as the polyanionic compound. Examples of the anionic polysaccharide include sulfated polysaccharides and alginates. Examples of the anionic polyamino acid include polyglutamic acid, polyaspartic acid, and salts thereof. The polyanionic compound may be one type or two or more types.

[0018] Examples of sulfated polysaccharides include dextran sulfate and its salts, glycosaminoglycans, and sulfate-containing proteoglycans. Pharmaceutically acceptable salts of dextran sulfate are preferred, including sodium and potassium salts. Sodium dextran sulfate is particularly preferred.

[0019] The alginate is preferably a pharmaceutically acceptable salt of alginic acid, such as sodium alginate, potassium alginate, and ammonium alginate, with sodium alginate being particularly preferred.

[0020] Glycosaminoglycans refer to acidic polysaccharides containing amino sugars. Examples of glycosaminoglycans include heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, pentosan sulfate, and salts thereof. Chondroitin sulfate has a variety of structures depending on the type of sugar component and the position of the sulfate group. Examples of types of chondroitin sulfate include chondroitin sulfate A (chondroitin 4-sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D (chondroitin 6-sulfate having a sulfate group at the 2-position of glucuronic acid), and chondroitin sulfate E (chondroitin sulfate having sulfate groups at the 4- and 6-positions of N-acetylgalactosamine). Chondroitin sulfate B is the same as dermatan sulfate. Any chondroitin sulfate may be used in this embodiment.

[0021] The glycosaminoglycan salt is preferably a pharmaceutically acceptable salt, such as a sodium salt or a potassium salt. Sodium salts are particularly preferred. In this embodiment, sodium heparin, sodium heparan sulfate, sodium chondroitin sulfate, sodium dermatan sulfate, sodium keratan sulfate, or sodium pentosan sulfate is preferably used. Among these, sodium heparin and sodium chondroitin sulfate are particularly preferred.

[0022] Proteoglycans are compounds in which glycosaminoglycans are covalently bonded to proteins. Examples of proteoglycans containing sulfate groups include proteins covalently bonded to heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and salts thereof. Such proteins are known and include, for example, aggrecan, versican, neurocan, brevican, decorin, biglycan, syndecan, and glypican.

[0023] The anionic polyamino acid preferably contains multiple acidic amino acids. The acidic amino acids contained in the anionic polyamino acid are either one or both of glutamic acid and aspartic acid. The anionic polyamino acid may contain neutral amino acids and / or basic amino acids, but if it contains basic amino acids, the number of basic amino acid residues is less than the number of acidic amino acid residues. The anionic polyamino acid preferably does not contain basic amino acids, and more preferably consists only of acidic amino acids. The anionic polyamino acid may be a polymer of L-amino acids, a polymer of D-amino acids, or a polymer containing both L- and D-amino acids. The anionic polyamino acid salt is preferably a pharmaceutically acceptable salt, such as a sodium salt or a potassium salt. Sodium polyglutamate and sodium polyaspartate are particularly preferred.

[0024] The average molecular weight of dextran sulfate sodium can be from about 5,000 to about 500,000. As used herein, "average molecular weight" refers to the weight average molecular weight measured by gel permeation chromatography (GPC). When a commercially available polyanionic compound is used, the molecular weight of the compound may be the value disclosed by the manufacturer or supplier.

[0025] The lower limit of the polyanionic compound concentration in the medium is preferably higher than 0.01 mg / mL. The lower limit may be, for example, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, or 0.1 mg / mL. The upper limit of the polyanionic compound concentration in the medium is not particularly limited, but may be, for example, 20 mg / mL or less. The upper limit may be, for example, 19 mg / mL, 18 mg / mL, 17 mg / mL, 16 mg / mL, 15 mg / mL, 14 mg / mL, 13 mg / mL, 12 mg / mL, 11 mg / mL, or 10 mg / mL. For example, transfected cells can be cultured in a medium containing a polyanionic compound at a concentration of 0.05 mg / mL to 20 mg / mL, more preferably 0.1 mg / mL to 10 mg / mL.

[0026] The polyanionic compound may be added to the medium in advance, or may be added to the medium during cell culture. When the transfected cells are stable expression strains, they are preferably cultured in a medium to which a polyanionic compound has been added in advance. When the transfected cells are cells that transiently express an antibody by transfection, the polyanionic compound is preferably added to the medium, for example, 3 to 4 hours after the completion of the gene transfer procedure. Alternatively, the medium may be replaced with a medium to which a polyanionic compound has been added in advance.

[0027] The culture period is not particularly limited and can be appropriately determined depending on, for example, the type of animal cell, the type of protein expression system, the culture method, etc. When the animal cell is a stable expression strain, the culture period can be, for example, 5 to 14 days. When the animal cell transiently expresses a mutant by transfection of a gene encoding the mutant, the culture period can be, for example, 24 hours to 7 days from the completion of the gene introduction procedure.

[0028] The medium is not particularly limited, and examples include media commonly used for in vitro animal cell culture. Such media are known, and examples include MEM, DMEM, and RPMI-1640. Commercially available media such as CD FortiCHO™ medium and Expi293™ Expression medium may also be used. If necessary, additives such as fetal bovine serum (FBS), L-glutamine, antibiotics, antimycotics, and antiaggregating agents may be mixed into the medium. When the transfected cells are stably expressing strains into which a gene expressing a selection marker such as a drug resistance gene has been introduced, it is preferable to add a selection agent to the medium. Selection markers and their corresponding selection agents are known.

[0029] Culture conditions for animal cells may be selected from known conditions depending on the cell type, etc. For example, general mammalian cells can be cultured at 37°C in a 5% CO2 atmosphere. The culture method may be selected depending on the scale of culture. Culture methods include, for example, batch culture, fed-batch culture, and continuous culture. Continuous culture includes chemostat culture and perfusion culture. Batch culture is suitable for small-scale culture at the laboratory level, for example. Fed-batch culture and continuous culture are suitable for large-scale culture at the industrial level, for example.

[0030] When the antibody production method of this embodiment is performed by batch culture, a medium containing transgenic cells and a polyanionic compound is added to a culture vessel or culture device and cultured. Hereinafter, a culture vessel or culture device containing transgenic cells cultured in a medium containing a polyanionic compound is also referred to as a "culture system." In batch culture, the medium is not supplied to the culture system and the cell culture (cells and medium) is not removed from the culture system during the culture period. When the antibody production method of this embodiment is performed by fed-batch culture, a medium containing transgenic cells and a polyanionic compound is added to a culture vessel or culture device and cultured. During the culture period, predetermined components (medium components, nutrient sources, the above-mentioned additives, polyanionic compound, etc.) are continuously or intermittently supplied to the culture system. When the antibody production method of this embodiment is performed by chemostat culture, the medium containing transgenic cells and a polyanionic compound is continuously supplied to a culture vessel or culture device to which the medium is added. At this time, the cell culture is continuously removed from the culture system in an amount equal to the supplied medium to maintain the culture system in a steady state. When the antibody production method of this embodiment is carried out by perfusion culture, a culture medium containing transfected cells and a polyanionic compound is continuously supplied to a culture vessel or culture device to which the medium has been added. At this time, a culture supernatant in an amount equal to the supplied medium is continuously removed from the culture system to maintain the culture system in a steady state. In perfusion culture, cells are not removed.

[0031] The culture vessel or culture device can be appropriately selected depending on the type of cell, culture method, etc. Examples of the culture vessel include dishes, microplates, culture flasks (T-flasks), spinner flasks, roller bottles, etc. Examples of the culture device include stirred bioreactors, fluidized bed bioreactors, fixed bed culture vessels, etc.

[0032] After culturing transgenic cells in vitro in the presence of a polyanionic compound, the produced mutant is recovered from the culture supernatant or the cells. Mutants expressed by transgenic cells are usually secreted extracellularly, so they are present in the culture supernatant. Therefore, a solution of the mutant can be obtained by removing the cells from the cell culture and recovering only the culture supernatant. If a portion of the mutant expressed by transgenic cells accumulates intracellularly, the cells can be lysed in a solution containing an appropriate solubilizing agent to release the mutant, thereby obtaining a solution of the mutant. If necessary, the cell lysate can be centrifuged to recover the supernatant containing the mutant. The mutant released into the liquid can be purified by known methods such as gel filtration chromatography or affinity chromatography.

[0033] The antibody production method of this embodiment increases the yield of mutants compared to the yield of mutants obtained by culturing in the absence of polyanionic compounds. Meanwhile, the antibody function of the mutants produced by this embodiment is equivalent to that of mutants obtained by culturing in the absence of polyanionic compounds. For example, the affinity of the mutants obtained by the antibody production method of this embodiment for an antigen is the same as that of the mutants obtained by culturing in the absence of polyanionic compounds. The affinity for an antigen can be evaluated, for example, by immunological measurement such as ELISA, or by kinetic parameters in antigen-antibody reactions. An example of an indicator of affinity by immunological measurement is the 50% effective concentration (EC50). An example of a kinetic parameter is the dissociation constant (K D ), binding rate constant (k on ) and dissociation rate constant (k off The kinetic parameters of the antigen-antibody reaction can be obtained by surface plasmon resonance (SPR) technology. The following describes the mutants.

[0034] The mutant obtained by the antibody production method of this embodiment is an antibody in which at least three amino acid residues in FR3 are substituted with arginine or lysine residues, and there are no particular limitations on the antigen recognized by the mutant or the animal species from which the mutant is derived. Here, FR refers to the region present in each of the light and heavy chain variable regions of an antibody, other than the CDRs. The FR serves as a scaffold connecting the three CDRs and contributes to the structural stability of the CDRs. Therefore, the amino acid sequences of FRs are highly conserved among antibodies of the same species. The variable regions of the heavy and light chains each contain three CDRs, CDR1, CDR2, and CDR3, and four FRs, FR1, FR2, FR3, and FR4. These are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus of the variable region.

[0035] The variant may be, for example, an antibody of human, mouse, rat, hamster, rabbit, goat, horse, or chicken origin. The variant may be a chimeric antibody, a humanized antibody, a bispecific antibody, etc.

[0036] In the mutant, at least three amino acid residues in FR3 are substituted with arginine or lysine residues, thereby improving the affinity of the mutant for the antigen compared to the antibody before the substitution. Hereinafter, the antibody before the substitution of at least three amino acid residues in FR3 with arginine or lysine residues is also referred to as the "original antibody." Hereinafter, the substitution of at least three amino acid residues in FR3 of the original antibody with arginine or lysine residues is also referred to as "modifying." The mutant may be an antibody described in U.S. Patent Application Publication No. 2018-0179298. U.S. Patent Application Publication No. 2018-0179298 is incorporated herein by reference.

[0037] In this embodiment, the mutant is preferably an antibody in which at least three amino acid residues in the light chain FR3 have been substituted with arginine or lysine residues. In one embodiment, the at least three amino acid residues in FR3 of the mutant that have been substituted with arginine or lysine residues include at least three residues selected from the group consisting of positions 63, 65, 67, 70, and 72 of the light chain as defined by the Chothia method. For example, the positions of arginine or lysine residues in the light chain FR3 of the mutant may be any of the following positions (1) to (5):

[0038] (1) positions 63, 65, and 67 of the light chain as defined by the Chothia method; (2) positions 65, 67, and 70 of the light chain as defined by the Chothia method; (3) positions 63, 65, 67, and 70 of the light chain as defined by the Chothia method; (4) positions 65, 67, 70, and 72 of the light chain as defined by the Chothia method; and (5) Positions 63, 65, 67, 70, and 72 of the light chain as defined by the Chothia method.

[0039] The Chothia method is known as one of the methods for numbering amino acid residues in CDRs (hereinafter also referred to as the "numbering method") to define the boundaries and lengths of CDRs. When the amino acid residues in a CDR are numbered according to the numbering method, the amino acid residues in FRs are also numbered. The numbers assigned to amino acid residues according to the numbering method indicate the position of the corresponding amino acid residue in the amino acid sequence of the light chain or heavy chain. Herein, the boundaries and lengths of CDRs and FR3s are defined according to the Chothia method (see Chothia C. and Lesk AM., Canonical Structures for the Hypervariable Regions of Immunoglobulins., J. Mol. Biol., vol. 196, pp. 901-917, 1987). According to the Chothia method, light chain FR3 is defined as the region consisting of amino acid residues 57 to 88 of the light chain. Hereinafter, when the position of an amino acid residue in the light chain of an antibody is described, unless otherwise specified, the position of the amino acid residue refers to the position defined by the Chothia method.

[0040] In the mutant, amino acid residues other than positions 63, 65, 67, 70, and 72 in the light chain, for example, amino acid residues selected from positions 57 to 62, 74, 76, 77, and 79 to 81 in the light chain, may be arginine or lysine residues. Here, positions 57 to 62, 74, 76, 77, and 79 to 81 in the light chain refer to the amino acid residue positions excluding Vernier zone residues and non-exposed residues from the amino acid sequence of light chain FR3. "Vernier zone residues" are amino acid residues in the FR amino acid sequence that contribute to the structural stability of the CDR. "Non-exposed residues" are amino acid residues that are folded inside the molecule and not exposed on the surface.

[0041] In the mutant, the amino acid residues that are modified from the amino acid residues of the original antibody may all be arginine residues or all lysine residues, or some of the amino acid residues that are modified from the amino acid residues of the original antibody may be arginine residues and the rest may be lysine residues.

[0042] In this embodiment, the variant preferably has a CDR with a neutral or negative electrical characteristic. As used herein, the "CDR electrical characteristic" is determined by the following formula (I):

[0043] X = [the number of basic amino acid residues in the amino acid sequence of the CDR contained in one antigen-binding site] - [the number of acidic amino acid residues in the amino acid sequence of the CDR contained in one antigen-binding site] Formula (I) (Wherein, when X is −2 or less, the electrical characteristic of the CDR is a negative charge; When X is −1, 0 or 1, the electrical properties of the CDRs are neutral; When X is 2 or more, the electrical characteristic of the CDR is a positive charge.

[0044] "One antigen-binding site" is a site consisting of one heavy chain variable region and one light chain variable region, and is the site in an antibody that binds to an antigen. The number of antigen-binding sites an antibody has varies depending on the class and form of the antibody. For example, when an antibody is an IgG or F(ab')2, the antibody has two antigen-binding sites. When an antibody is a Fab, the antibody has one antigen-binding site. "CDRs contained in one antigen-binding site" refer to all CDRs present in one heavy chain variable region and one light chain variable region that constitute the one antigen-binding site. In other words, the CDRs contained in one antigen-binding site are a total of six CDRs: CDR1, CDR2, and CDR3 in one heavy chain variable region, and CDR1, CDR2, and CDR3 in one light chain variable region.

[0045] As can be seen from formula (I) above, the electrical properties of a CDR are determined based on the number of acidic amino acid residues and basic amino acid residues in the amino acid sequence of the CDR contained in one antigen-binding site. When X calculated by formula (I) above is -2 or less, i.e., when the number of acidic amino acid residues is 2 or more greater than the number of basic amino acid residues in the amino acid sequence of the CDR contained in one antigen-binding site, the electrical properties of the CDR are determined to be negatively charged. When X calculated by formula (I) above is -1, 0, or 1, i.e., when the difference between the number of acidic amino acid residues and the number of basic amino acid residues in the amino acid sequence of the CDR contained in one antigen-binding site is 0 or 1, the electrical properties of the CDR are determined to be neutral.

[0046] In other words, when the total number of acidic amino acid residues contained in the six CDRs (light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3) is two or more times greater than the total number of basic amino acid residues, the electrical property of the CDR is defined as negatively charged. Furthermore, when the difference between the total number of acidic amino acid residues and the total number of basic amino acid residues contained in the six CDRs (light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3) is 0 or 1, the electrical property of the CDR is defined as neutral. Herein, acidic amino acid residues refer to aspartic acid residues and glutamic acid residues, and basic amino acid residues refer to arginine residues and lysine residues. Herein, histidine residues are not included in the basic amino acid residues.

[0047] Genes encoding the mutants can be obtained using known DNA recombinant technology and other molecular biology techniques, for example, see U.S. Patent Application Publication No. 2018 / 0179298. First, a polynucleotide encoding the amino acid sequence of the original antibody is obtained. For example, if a hybridoma producing the original antibody is available, polynucleotides encoding the light chain and heavy chain of the original antibody are synthesized using RNA extracted from the hybridoma via reverse transcription and rapid amplification of cDNA ends (RACE). Next, polynucleotides encoding amino acid sequences with modified light chain FR3 and / or heavy chain FR3 are prepared from these polynucleotides. For example, a polynucleotide encoding a light chain with modified FR3 can be obtained by amplifying the polynucleotide encoding the light chain of the original antibody as a template using PCR with primers for modifying at least three amino acid residues in the light chain FR3. The obtained polynucleotide and a polynucleotide encoding the heavy chain of the original antibody can be used as genes encoding the mutants. If necessary, the genes encoding the mutants may be incorporated into an animal cell expression vector.

[0048] If a hybridoma producing the original antibody is not available, an antibody-producing hybridoma can be prepared by known methods, such as the method described in Kohler and Milstein, Nature, vol. 256, pp. 495-497, 1975. Alternatively, polynucleotides encoding the light chain and heavy chain of the antibody can be synthesized using RNA obtained from the spleen of an animal such as a mouse immunized with an antigen of interest. Alternatively, the amino acid sequence or nucleotide sequence of the antibody gene of an antibody that specifically binds to the antigen of interest can be obtained from a known database, and gene synthesis can be performed based on these sequences to obtain the antibody gene. Examples of databases include GeneBank, abYsis, and IMGT.

[0049] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0050] Example 1: Examination of the effects of sugars and sulfated polysaccharides Animal cells transfected with genes encoding rituximab and its variants were cultured in a medium containing sugars or sulfated polysaccharides to examine whether the antibody yield increases.

[0051] (1) Identification of the rituximab mutant gene Polynucleotides encoding the light chains of rituximab and its R3 and R5 mutants were synthesized by PCR using a plasmid DNA containing a gene encoding rituximab (a mouse / human chimeric antibody) as a template, similar to the method described in U.S. Patent Application Publication No. 2018 / 0179298. Polynucleotides encoding the heavy chain of rituximab were also synthesized. The R3 mutant was an antibody in which the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Chothia method were substituted with arginine residues. The R5 mutant was an antibody in which the amino acid residues at positions 63, 65, 67, 70, and 72 of the light chain defined by the Chothia method were substituted with arginine residues. Polynucleotides encoding the light chains of each mutant and a polynucleotide encoding the heavy chain of rituximab were inserted into the Freedom™ pCHO 1.0 Expression Vector (Thermo Fisher Scientific). As a result, we obtained plasmid DNAs for animal cell expression containing genes encoding each of the rituximab mutants, as well as plasmid DNAs for animal cell expression containing a gene encoding rituximab without substitution of amino acid residues in the light chain FR3.

[0052] Based on the nucleotide sequence of the gene encoding rituximab, the amino acid sequences of the light and heavy chains of the antibody (IgG) were determined. These amino acid sequences are shown below. The amino acid sequences of the light chains of each of the mutants (IgG) prepared are also shown below. The underlined parts indicate the positions substituted with arginine residues.

[0053] Rituximab heavy chain QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWG AGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

[0054] Rituximab light chain QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2)

[0055] - Rituximab R3 mutant light chain QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF R G R G R GTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3)

[0056] -Rituximasib R5 mutant light chain QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRF R G R G R GT R Y R LTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4)

[0057] (2) Introduction of antibody-encoding genes into animal cells [reagent] CHO-S cells (Thremo Fisher Sientific) CD FortiCHO™ Medium (Thremo Fisher Scientific) Anticoagulant (Thremo Fisher Scientific) OptiPRO(TM) SFM (Thremo Fisher Sientific) FreeStyle™ MAX Reagent (Thremo Fisher Scientific) L-glutamine (Thremo Fisher Scientific) Puromycin dihydrochloride (Thremo Fisher Scientific) Methotrexate hydrate (Sigma Aldrich)

[0058] CHO-S cells were grown in CD FortiCHO™ medium containing 8 mM L-glutamine at 37°C and 85% humidity in an 8% CO atmosphere with shaking (130 rpm). Depending on the number of samples, 30 mL of cell culture (1.0 x 10) was added. 6 Plasmid DNA encoding rituximab, the R3 mutant, and the R5 mutant was digested with the restriction enzyme PvuI and purified by ethanol precipitation to obtain linearized plasmid DNA. A DNA solution with the following composition was prepared using the linearized plasmid DNA. A transfection reagent with the following composition was also prepared and allowed to stand for 5 minutes.

[0059] [DNA solution] Amount (μL) equivalent to 50μg of plasmid DNA solution OptiPRO(TM) SFM appropriate amount (mL) Total 1.5 mL

[0060] [Transfection reagent] FreeStyle™ MAX Reagent 80 μL OptiPRO(TM) SFM 1420μL Total 1.5 mL

[0061] The prepared DNA solution and transfection reagent were mixed and allowed to stand for 10 minutes. The resulting mixture (3 mL) was added to a cell culture (30 mL) and cultured with shaking (150 rpm) at 37°C in a 5% CO2 atmosphere. After 48 hours, puromycin and methotrexate were added to each culture to select for cells into which the gene had been introduced. Medium replacement and subculture were continued until viability and cell proliferation ability were restored, yielding CHO-S cells stably expressing rituximab and each mutant (hereinafter referred to as "stable expression strains"). Cultures of these stable expression strains were frozen and stored using standard methods.

[0062] (3) Cultivation of stable expression strains Each frozen stable cell line was thawed and cultured in CD FortiCHO™ medium containing 1 / 100 of the volume of an anticoagulant and 8 mM L-glutamine, and passaged multiple times. Test media were prepared by adding sugars or sulfated polysaccharides to CD FortiCHO™ medium containing 8 mM L-glutamine. Sugars included D(+) glucose (Fujifilm Wako Pure Chemical Corporation), sucrose (Kishida Chemical Co., Ltd.), or trehalose dihydrate (Fujifilm Wako Pure Chemical Corporation) at a concentration of 50 mM. Sulfated polysaccharides included chondroitin sulfate C sodium (Fujifilm Wako Pure Chemical Corporation), dextran sulfate sodium (molecular weight 5000) (Merck), or heparin sodium (Fujifilm Wako Pure Chemical Corporation) at a concentration of 1 mg / mL. The passaged stable cell lines were centrifuged at 800 rpm for 5 minutes to completely remove the medium. Each recovered stable expression clone was diluted to 3.0 x 10 5 The cells were seeded at a concentration of 1000 cells / mL on each test medium and cultured for 14 days. For comparison, each stable expression strain was cultured for 14 days in medium without added sugars or sulfated polysaccharides.

[0063] (4) Evaluation of survival and antibody production of stable expression cells A portion of the cell culture was collected as a sample at the start of culturing in each test medium (day 0) and on days 3, 7, 10, and 14 after the start. A portion of the sample was used to count live and dead cells. The sample was also centrifuged, and the antibody concentration in the supernatant was measured. Cells were counted using a hemocytometer after staining with trypan blue. The survival rate of each stable expression strain was calculated from the number of live and dead cells. The antibody concentration in the supernatant was measured by sandwich ELISA. The specific procedures were as follows.

[0064] A solution of anti-human IgG Fc antibody (Bethyl Laboratories) diluted to 2 μg / mL in KPL coating solution (SeraCare Life Sciences) was added to each well of a 96-well plate (Corning) and incubated overnight at 4°C. After removing the solution from each well, 1% BSA / PBS was added and incubated at room temperature for 3 hours. After removing the solution from each well, the plate was washed three times with PBS containing 0.1% Tween® 20 (PBST). Culture supernatant was added to each well and incubated at room temperature for 1 hour. After removing the solution from each well, the plate was washed three times with PBST. After removing the solution from each well, the plate was washed three times with PBST. After adding a solution of HRP-labeled anti-human IgG Fc antibody (Bethyl Laboratories) diluted to 0.3 μg / mL in 1% BSA / PBS, the plate was incubated at room temperature for 1 hour. After removing the solution from each well, the plate was washed three times with PBST. The color reaction was developed using the KPL ABTS® Peroxidase Substrate System (SeraCare Life Sciences) and stopped using KPL ABTS® Peroxidase Stop Solution (SeraCare Life Sciences). The absorbance at 405 nm was measured using a SpectraMax® 190 microplate reader (Molecular Devices). The antibody concentration in each supernatant was obtained using a standard curve of a reference antibody.

[0065] (5) Results The survival rates (%) of each stable expression strain cultured in a medium supplemented with saccharides or sulfated polysaccharides are shown in Figures 1 to 6. The antibody yields of each stable expression strain cultured in a medium supplemented with saccharides or sulfated polysaccharides are shown in Figures 7 to 12 as antibody concentrations (mg / L) in the supernatant. In the figures, "WT" refers to rituximab in which no amino acid residues in the light chain FR3 have been substituted (the same applies to Figure 13 and subsequent figures).

[0066] As shown in Figures 1 to 3, the addition of any of the sugars did not significantly affect the survival rates of the stable expression strains of rituximab and each mutant. As shown in Figures 4 to 6, when dextran sulfate sodium and heparin sodium were added, the survival rates of each stable expression strain on days 10 and 14 tended to be slightly higher than when chondroitin sulfate was added or when nothing was added.

[0067] As shown in Figure 7, the addition of any sugar resulted in a lower yield of rituximab than when no sugar was added. As shown in Figure 8, the yield of the R3 mutant on day 14 was slightly higher when sucrose was added than when no sugar was added. As shown in Figure 9, the yield of the R5 mutant on days 7, 10, and 14 was slightly higher when sucrose or glucose was added than when no sugar was added. These results suggest that the addition of sugars may slightly improve the yield of rituximab and each mutant. However, the increase in yield of the mutants due to the addition of sugars was not fully satisfactory.

[0068] As shown in Figure 10, the yield of rituximab on day 14 was slightly higher when dextran sulfate sodium or heparin sodium was added than when chondroitin sulfate sodium was added or when nothing was added. On the other hand, as shown in Figures 11 and 12, the yields of the R3 and R5 mutants on days 7, 10, and 14 were significantly increased with the addition of any sulfated polysaccharide compared to when nothing was added. Specifically, the yield of the R3 mutant on day 14 was approximately twice as high with the addition of sulfated polysaccharides as with no addition. Furthermore, the yield of the R5 mutant on day 14 was 3.7-fold higher with the addition of chondroitin sulfate sodium than with no addition, and approximately 10-fold higher with the addition of dextran sulfate sodium and heparin sodium than with no addition. These results suggest that the addition of sulfated polysaccharides significantly improves the yields of the R3 and R5 mutants of rituximab.

[0069] Example 2: Examination of the concentration and molecular weight of dextran sulfate sodium The stably expressing strains of rituximab and the R5 mutant prepared in Example 1 were cultured in media containing dextran sodium sulfate of various concentrations or molecular weights to examine whether the antibody yield increases.

[0070] (1) Cultivation of stable expression strains Frozen stocks of stable strains expressing rituximab and the R5 mutant were thawed, cultured, and passaged as described in Example 1. To examine molecular weight, test media were prepared by adding 5,000 molecular weight dextran sulfate sodium (Merck) or 50,000 molecular weight dextran sulfate sodium (Fujifilm Wako Pure Chemical Industries, Ltd.) to CD FortiCHO™ medium containing 8 mM L-glutamine at a concentration of 1 mg / mL. To examine concentration, test media were prepared by adding 5,000 molecular weight dextran sulfate sodium (Merck) to 8 mM L-glutamine at concentrations of 0.1, 1, or 10 mg / mL. As described in Example 1, each stable strain was inoculated into each test medium and cultured for 11 or 12 days. For comparison, each stable strain was cultured for 11 or 12 days in medium without dextran sulfate sodium.

[0071] (2) Evaluation of survival and antibody production of stable expression cells For experiments using test media containing different concentrations of dextran sulfate sodium, aliquots of the cell culture were collected as samples at the start of culture (day 0) and on days 4, 7, 9, and 11. For experiments using test media containing dextran sulfate sodium of different molecular weights, aliquots of the cell culture were collected as samples at the start of culture (day 0) and on days 5, 7, 9, and 12. Cell viability was calculated and antibody concentrations in the supernatants were measured in the same manner as in Example 1.

[0072] (3) Results The survival rates (%) of the stably expressing strains of rituximab and the R5 mutant in test media containing different concentrations of dextran sulfate sodium are shown in Figures 13 and 14, and the antibody concentrations (mg / L) in the supernatants are shown in Figures 15 and 16. The survival rates (%) of the stably expressing strains of rituximab and the R5 mutant in test media containing dextran sulfate sodium of different molecular weights are shown in Figures 17 and 18, and the antibody concentrations (mg / L) in the supernatants are shown in Figures 19 and 20.

[0073] As shown in Figures 13 and 14, the viability of the stable cells expressing rituximab and the R5 mutant on day 11 tended to be higher with increasing dextran sulfate sodium concentration compared to cells without dextran sulfate sodium. As shown in Figure 15, the yield of rituximab on day 11 increased approximately 1.4-fold when the dextran sulfate sodium concentration in the medium was 0.1 or 1 mg / mL compared to cells without dextran sulfate sodium. At a concentration of 10 mg / mL, the yield of rituximab decreased compared to cells without dextran sulfate sodium. As shown in Figure 16, the yield of the R5 mutant on day 11 improved with increasing dextran sulfate sodium concentration, increasing up to approximately 5.4-fold compared to cells without dextran sulfate sodium. Even at the lowest concentration of 0.1 mg / mL, the yield of the R5 mutant increased approximately 3.3-fold compared to cells without dextran sulfate sodium. This improvement in yield of the R5 mutant was significant even when taking into account the improvement in viability due to the addition of dextran sulfate sodium.

[0074] As shown in Figures 17 and 18, when dextran sulfate sodium with a molecular weight of 5,000 and dextran sulfate sodium with a molecular weight of 50,000 were added, the survival rates of the stable cells expressing rituximab and the R5 mutant on day 12 tended to be higher than when dextran sulfate sodium was not added. As shown in Figure 19, the yield of rituximab was slightly increased by the addition of dextran sulfate sodium compared to when dextran sulfate sodium was not added. As shown in Figure 20, the yield of the R5 mutant on day 12 when dextran sulfate sodium was added increased by up to approximately 3.7-fold compared to when dextran sulfate sodium was not added.

[0075] Example 3: Examination of the molecular weight of dextran sulfate sodium (2) The strain stably expressing the R3 mutant of rituximasib prepared in Example 1 was cultured in media containing dextran sulfate sodium of various molecular weights to examine whether the antibody yield increases.

[0076] (1) Cultivation of stable expression strains A frozen stock of a stable strain expressing the R3 mutant was thawed, cultured, and passaged as in Example 1. Test media were prepared as in Example 2 by adding 5,000 molecular weight dextran sulfate sodium (Merck), 50,000 molecular weight dextran sulfate sodium (Fujifilm Wako Pure Chemical Industries, Ltd.), or 500,000 molecular weight dextran sulfate sodium (Fujifilm Wako Pure Chemical Industries, Ltd.) to CD FortiCHO™ medium containing 8 mM L-glutamine at a concentration of 1 mg / mL. Each stable strain was inoculated into each test medium as in Example 1 and cultured for 12 days. For comparison, each stable strain was cultured for 12 days in medium without dextran sulfate sodium.

[0077] (2) Evaluation of survival and antibody production of stable expression cells Aliquots of the cell culture were collected as samples at the start of the culture (day 0) and on days 5, 7, 9, and 12 after the start. The cell viability was calculated and the antibody concentration in the supernatant was measured in the same manner as in Example 1.

[0078] (3) Results The viability (%) of the strain stably expressing the R3 mutant is shown in Figure 21, and the antibody concentration (mg / L) in the supernatant is shown in Figure 22. As shown in Figure 21, the viability of the strain stably expressing the R3 mutant on day 12 tended to be higher with the addition of dextran sulfate sodium than with no dextran sulfate sodium addition. As shown in Figure 22, the yield of the R3 mutant on day 12 was significantly increased by the addition of dextran sulfate sodium, increasing by a maximum of approximately 4.6-fold compared to the case without dextran sulfate sodium addition. Differences in the molecular weight of dextran sulfate sodium had little effect on the yield improvement effect of the R3 mutant.

[0079] Example 4: Examination of heparin sodium concentration The stably expressing strains of rituximab and the R5 mutant prepared in Example 1 were cultured in media containing various concentrations of sodium heparin to examine whether the antibody yield increased.

[0080] (1) Cultivation of stable expression strains Frozen stocks of each stable expression strain of rituximab and the R5 mutant were thawed, cultured, and passaged in the same manner as in Example 1. Test media were prepared in the same manner as in Example 2 by adding heparin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.) to CD FortiCHO™ medium containing 8 mM L-glutamine at a concentration of 0.1, 1, or 10 mg / mL. Each stable expression strain was inoculated into each test medium and cultured for 12 days in the same manner as in Example 1. For comparison, each stable expression strain was cultured for 12 days in medium without heparin sodium.

[0081] (2) Evaluation of survival and antibody production of stable expression cells Aliquots of the cell culture were collected as samples at the start of the culture (day 0) and on days 5, 7, 9, and 12 after the start. The cell viability was calculated and the antibody concentration in the supernatant was measured in the same manner as in Example 1.

[0082] (3) Results The survival rates (%) of the stable expression cells of rituximab and the R5 mutant are shown in Figures 23 and 24, and the antibody concentrations (mg / L) in the supernatants are shown in Figures 25 and 26. As shown in Figures 23 and 24, the survival rates of the stable expression cells on day 12 were higher with the addition of heparin sodium than without it. In particular, when 10 mg / mL of heparin sodium was added, the survival rates of the stable expression cells on day 12 were significantly higher than when it was not added.

[0083] As shown in Figure 25, the yield of rituximab was almost unchanged regardless of the concentration of heparin sodium added. As shown in Figure 26, the yield of the R5 mutant on day 12 improved with increasing heparin sodium concentration, increasing by approximately 4.2-fold compared to the case without heparin sodium. Even at the lowest concentration of 0.1 mg / mL, the yield of the R5 mutant increased by approximately 2.2-fold compared to the case without heparin sodium. As mentioned above, the viability of the stably expressing rituximab strain was significantly increased by the addition of 10 mg / mL heparin sodium, but the yield of rituximab remained unchanged. This suggests that the improvement in yield of the R5 mutant is still significant, even when taking into account the improvement in viability due to the addition of heparin sodium.

[0084] Example 5: Examination of the effect of anionic polyamino acids Animal cells transfected with genes encoding rituximab and its variants were cultured in a medium containing sodium polyaspartate or sodium polyglutamate as anionic polyamino acids to examine whether the antibody yield increases.

[0085] (1) Cultivation of stable expression strains Frozen stocks of stable expression strains of rituximab, the R3 mutant, and the R5 mutant were thawed, cultured, and passaged as in Example 1. Test media were prepared by adding poly-(α,β)-DL-sodium aspartate (Merck) or poly-L-γ-sodium glutamate (Merck) to a concentration of 1 mg / mL in CD FortiCHO™ medium containing 8 mM L-glutamine. Each stable expression strain was inoculated into each test medium as in Example 1 and cultured for 12 days. For comparison, each stable expression strain was cultured for 12 days in medium without the addition of anionic polyamino acids.

[0086] (2) Evaluation of survival and antibody production of stable expression cells Aliquots of the cell culture were collected as samples at the start of the culture (day 0) and on days 5, 7, 9, and 12 after the start. The cell viability was calculated and the antibody concentration in the supernatant was measured in the same manner as in Example 1.

[0087] (3) Results The viability (%) of the stably expressing strains of rituximab, the R3 mutant, and the R5 mutant is shown in Figures 27 to 29, and the antibody concentration (mg / L) in the supernatant is shown in Figures 30 to 32. In the figures, "PASP" refers to sodium polyaspartate, and "PGA" refers to sodium polyglutamate. As shown in Figures 27 to 29, the viability of each stably expressing strain on day 12 tended to be higher with the addition of anionic polyamino acids than without. As shown in Figure 30, the yield of rituximab was almost the same with or without the addition of anionic polyamino acids. As shown in Figure 31, the yield of the R3 mutant was significantly increased with the addition of anionic polyamino acids compared to without their addition. As shown in Figure 32, the yield of the R5 mutant was significantly increased with the addition of anionic polyamino acids compared to without their addition. In particular, the yield of the R5 mutant on day 12 when sodium polyaspartate was added was approximately 4.5-fold higher than when anionic polyamino acids were not added. These results demonstrate that the yields of the R3 and R5 mutants of rituximab can be improved not only by sulfated polysaccharides but also by anionic polyamino acids.

[0088] Example 6: Examination of the effect of neutral polysaccharides We cultured animal cells transfected with genes encoding rituximab and its variants in a medium containing dextran as a neutral polysaccharide to examine whether antibody yields increased. For comparison, we also performed experiments using a medium containing dextran sulfate sodium, an anionic polysaccharide.

[0089] (1) Cultivation of stable expression strains Frozen stocks of stable expression strains of rituximab, the R3 mutant, and the R5 mutant were thawed, cultured, and passaged as in Example 1. Test media were prepared by adding dextran (molecular weight 5,000) (Sigma) or dextran sulfate sodium (molecular weight 5,000) (Sigma) to a concentration of 1 mg / mL in CD FortiCHO™ medium containing 8 mM L-glutamine. Each stable expression strain was inoculated into each test medium as in Example 1 and cultured for 12 days. For comparison, each stable expression strain was cultured for 12 days in medium without any polysaccharides.

[0090] (2) Evaluation of survival and antibody production of stable expression cells Aliquots of the cell culture were collected as samples at the start of the culture (day 0) and on days 2, 7, and 12 after the start. The cell viability was calculated and the antibody concentration in the supernatant was measured in the same manner as in Example 1.

[0091] (3) Results The viability (%) of the stably expressing strains of rituximab, the R3 mutant, and the R5 mutant is shown in Figures 33 to 35, and the antibody concentrations (mg / L) in the supernatant are shown in Figures 36 to 38. As shown in Figure 33, the viability of the stably expressing strain of rituximab on day 12 was lower with the addition of dextran than without polysaccharide. As shown in Figures 34 and 35, the viability of the stably expressing strains of the R3 mutant and the R5 mutant on day 12 was higher with the addition of dextran than without polysaccharide. For all stably expressing strains, the viability on day 12 was higher with the addition of dextran sodium sulfate than without polysaccharide.

[0092] As shown in Figures 36 to 38, the yields of rituximab, the R3 mutant, and the R5 mutant were all slightly reduced by the addition of dextran compared to when no polysaccharide was added. On the other hand, the yields of rituximab, the R3 mutant, and the R5 mutant were all increased by the addition of dextran sulfate sodium compared to when no polysaccharide was added. These results indicate that dextran, a neutral polysaccharide, does not affect the yields of rituximab, the R3 mutant, and the R5 mutant.

[0093] Example 7: Examination of the effects of anionic polysaccharides and neutral polyamino acids without sulfate groups Animal cells transfected with genes encoding rituximab and the R3 mutant were cultured in a medium containing alginate as a sulfate-free anionic polysaccharide or polyproline as a neutral polyamino acid to examine whether the antibody yield increased.

[0094] (1) Cultivation of stable expression strains Frozen stocks of stable expression strains of rituximab and the R3 mutant were thawed, cultured, and passaged as in Example 1. Test media were prepared by adding sodium alginate 80-120 (Fujifilm Wako Pure Chemical Industries, Ltd.) or poly-L-proline (Sigma) to a concentration of 1 mg / mL to CD FortiCHO™ medium containing 8 mM L-glutamine. As in Example 1, each stable expression strain was inoculated into each test medium and cultured for 12 days. For comparison, each stable expression strain was cultured for 12 days in medium without polysaccharides or polyamino acids.

[0095] (2) Evaluation of survival and antibody production of stable expression cells Aliquots of the cell culture were collected as samples at the start of the culture (day 0) and on days 5, 7, 9, and 12 after the start. The cell viability was calculated and the antibody concentration in the supernatant was measured in the same manner as in Example 1.

[0096] (3) Results The viability (%) of the stably expressing cells of rituximab and the R3 mutant is shown in Figures 39 and 40, and the antibody concentration (mg / L) in the supernatant is shown in Figures 41 and 42. As shown in Figure 39, the viability of the stably expressing cells of rituximab on day 12 was slightly higher with the addition of alginate or polyproline than without. As shown in Figure 40, the viability of the stably expressing cells of the R3 mutant on day 9 was higher with the addition of alginate or polyproline than without.

[0097] As shown in Figure 41, the yield of rituximab was reduced by the addition of alginate or polyproline compared to the absence of alginate or polyproline. As shown in Figure 42, the yield of the R3 mutant was almost unchanged with or without polyproline. On the other hand, the yield of the R3 mutant was significantly increased with the addition of alginate compared to the absence of alginate or polyproline. This improvement in the yield of the R3 mutant was significant even when the viability improvement due to the addition of alginate was taken into account. These results indicate that alginate, an anionic polysaccharide without sulfate groups, has the effect of improving the yield of rituximab and the R3 mutant, while polyproline, a neutral polyamino acid, does not.

[0098] Example 8: Examination of the effect of sulfated polysaccharides on the production of antibodies other than rituximab Animal cells transfected with genes encoding various antibody variants other than rituximab were cultured in a medium containing sulfated polysaccharides to examine whether antibody yields increased. In this example, a transient expression system was used. The affinity of each antibody for its antigen was also evaluated.

[0099] (1) Obtaining genes for various antibody mutants other than rituximab Polynucleotides encoding the light chains of each antibody and their mutants were synthesized by PCR using plasmid DNA containing genes encoding trastuzumab (humanized antibody), anti-lysozyme antibody (mouse antibody), anti-IL-6 antibody (mouse / human chimeric antibody), and anti-IL-8 antibody (mouse / human chimeric antibody) as templates, similar to the method described in U.S. Patent Application Publication No. 2018 / 0179298. Polynucleotides encoding the heavy chains of each antibody were also synthesized. The mutants of trastuzumab and anti-IL-8 antibody were R3 and R4 mutants. The mutants of anti-lysozyme antibody and anti-IL-6 antibody were R5 mutants.

[0100] The R3 mutant of trastuzumab was an antibody in which the amino acid residues at positions 65, 67, and 70 of the light chain defined by the Chothia method were substituted with arginine residues, and the R4 mutant was an antibody in which the amino acid residues at positions 63, 65, 67, and 70 of the light chain defined by the Chothia method were substituted with arginine residues. The R3 mutant of the anti-IL-8 antibody was an antibody in which the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Chothia method were substituted with arginine residues, and the R4 mutant was an antibody in which the amino acid residues at positions 63, 65, 67, and 70 of the light chain defined by the Chothia method were substituted with arginine residues. The R5 mutants of the anti-lysozyme antibody and anti-IL-6 antibody were antibodies in which the amino acid residues at positions 63, 65, 67, 70, and 72 of the light chain defined by the Chothia method were substituted with arginine residues.

[0101] Polynucleotides encoding the light chains of each antibody mutant were inserted into a pcDNA3.4 vector (Thermo Fisher Scientific). This resulted in the preparation of plasmid DNAs for animal cell expression (hereinafter also referred to as "light chain plasmids") containing genes encoding the light chains of various antibodies and their mutants. Furthermore, polynucleotides encoding the heavy chains of each antibody were inserted into a pcDNA3.4 vector to obtain plasmid DNAs for animal cell expression (hereinafter also referred to as "heavy chain plasmids") containing genes encoding the heavy chains of each antibody. To evaluate the affinity for antigens, light chain plasmids for rituximab, its R3 mutants, and its R5 mutants, as well as heavy chain plasmids for rituximab, were also prepared using the animal cell expression plasmid DNAs for rituximab, its R3 mutants, and its R5 mutants prepared in Example 1.

[0102] Based on the nucleotide sequence of the gene encoding each antibody, the amino acid sequences of the light and heavy chains of the antibody (IgG) were determined. These amino acid sequences are shown below. The amino acid sequences of the light chains of each mutant (IgG) prepared are also shown below. The underlined parts indicate the positions substituted with arginine residues.

[0103] Trastuzumab heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)

[0104] Trastuzumab light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRGEC (SEQ ID NO: 6)

[0105] Light chain of the R3 variant of trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSG R R R GT RFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRGEC (SEQ ID NO: 7)

[0106] -Trastuzumab R4 mutant light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF R G R R R GT R FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSLPVTKSFNRGEC (SEQ ID NO: 8)

[0107] Heavy chain of anti-lysozyme antibody DVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMGYVSYSGSTYYNPSLKSRISITRDTSKNQYYLDLNSVTTEDTATYYCANWDGDYWGQGTLV TVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 9)

[0108] Light chain of anti-lysozyme antibody DIVLTQSPATLSVTPGNSVSLSCRASQSIGNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 10)

[0109] Light chain of R5 mutant anti-lysozyme antibody DIVLTQSPATLSVTPGNSVSLSCRASQSIGNNLHWYQQKSHESPRLLIKYASQSISGIPSRF R G R G R GT R F R LSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 11)

[0110] Heavy chain of anti-IL-6 antibody EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAREGYGNLERDCWGQG TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12)

[0111] Light chain of anti-IL-6 antibody DIVLTQSPASLAVSLGQRATISCRASESVDGFGISFMNWFQQKPGQPPKLLIYVASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDSAMYFCQQSKEVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTMSFNRGEC (SEQ ID NO: 13)

[0112] Light chain of the R5 mutant of anti-IL-6 antibody DIVLTQSPASLAVSLGQRATISCRASESVDGFGISFMNWFQQKPGQPPKLLIYVASNQGSGVPARF R G R G R GT R F RLNIHPMEEDDSAMYFCQQSKEVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTMSFNRGEC (SEQ ID NO: 14)

[0113] Heavy chain of anti-IL-8 antibody EVKLVESGGGLVKPGGSLKLSCAASGFTFNNYAMSWVRQTPEKRLEWVASISSGGNTYYPDSVKGRFTLSRDNARNILYLQMSRLRSEDTAMYYCARDKLRLPNWYFDVWGA GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15)

[0114] Light chain of anti-IL-8 antibody DIVLTQSPPSLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKVLIYGASNLESGIPARFSGSGSGTDFTLNIYPVEEEDAATYYCQQSNEDPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTMSFNRGEC (SEQ ID NO: 16)

[0115] Light chain of R3 mutant of anti-IL-8 antibody DIVLTQSPPSLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKVLIYGASNLESGIPARF R G R G R GTDFTLNIYPVEEEDAATYYCQQSNEDPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTMSFNRGEC (SEQ ID NO: 17)

[0116] - Light chain of R4 mutant of anti-IL-8 antibody DIVLTQSPPSLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKVLIYGASNLESGIPARF R G R G R GT R FTLNIYPVEEEDAATYYCQQSNEDPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTMSFNRGEC (SEQ ID NO: 18)

[0117] (2) Introduction of antibody-encoding genes into animal cells and cell culture [reagent] Expi293(TM) cells (Thermo Fisher Scientific) Expi293™ Expression Medium (Thermo Fisher Scientific) ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific)

[0118] (2.1) Transfection Expi293™ cells were grown in a shaking culture (150 rpm) at 37°C in a 5% CO atmosphere. Depending on the number of samples, 25.5 mL of cell culture (3 x 10 6 A DNA solution of the following composition was prepared using the light chain plasmid and heavy chain plasmid, and allowed to stand for 5 minutes.

[0119] [DNA solution] Light chain plasmid solution equivalent to 15 μg (μL) Heavy chain plasmid solution equivalent to 15 μg (μL) Opti-MEM (trademark) appropriate amount (mL) Total 1.5 mL

[0120] A transfection reagent having the following composition was prepared and allowed to stand for 5 minutes. ExpiFectamine Reagent 80μL Opti-MEM(trademark) 1420μL Total 1.5 mL

[0121] The prepared DNA solution and transfection reagent were mixed and allowed to stand for 20 minutes. The resulting mixture (3 mL) was added to cell cultures (25.5 mL) for cell transfection. The transfected cells were cultured at 37°C under a 5% CO2 atmosphere with shaking (125 rpm) for 4 hours. Dextran sodium sulfate (molecular weight 5000) (Sigma) was added to the cell cultures to a concentration of 1 mg / mL, and further cultured with shaking. For comparison, cell cultures without dextran sodium sulfate were also cultured with shaking. 20 hours after transfection, 150 μL and 1.5 mL of ExpiFectamine™ Transfection Enhancer 1 and 2, respectively, were added to the cell cultures and cultured at 37°C under a 5% CO2 atmosphere with shaking (125 rpm) for 4 days. Five days after transfection, the culture supernatants were collected from each cell culture.

[0122] (3) Evaluation of antibody production A portion of the collected culture supernatant was taken and used as a sample for measuring antibody concentration. The concentration of each antibody in the culture supernatant was measured by sandwich ELISA. Rituximab, trastuzumab, anti-IL-6 antibody, and anti-IL-8 antibody were measured in the same manner as in Example 1. Anti-lysozyme antibody, a mouse antibody, was measured as follows.

[0123] A solution of goat anti-mouse IgG (H+L) Highly Cross-Adsorbed (Thermo Fisher Scientific) diluted to 5 μg / mL in KPL coating solution (SeraCare Life Sciences) was added to each well of a 96-well plate (Corning) and incubated overnight at 4°C. After removing the solution from each well, 1% BSA / PBS was added and incubated at room temperature for 3 hours. After removing the solution from each well, the plate was washed three times with PBST. Culture supernatant was added to each well and incubated at room temperature for 1 hour. After removing the solution from each well, the plate was washed three times with PBST. A solution of HRP-labeled goat F(ab')2 fragment anti-mouse IgG (H+L) (Beckman) diluted to 0.3 μg / mL in 1% BSA / PBS was added to each well and incubated at room temperature for 1 hour. After removing the solution from each well, the plate was washed three times with PBST. The color reaction was developed using KPL TMB peroxidase substrate (SeraCare Life Sciences) and stopped using KPL TMB peroxidase stop solution (SeraCare Life Sciences). The absorbance at 450 nm was measured using a SpectraMax® 190 microplate reader (Molecular Devices). The antibody concentration in each supernatant was obtained using a standard curve of a reference antibody.

[0124] (4) Evaluation of antibody affinity for antigen (4.1) Antibody purification Antibodies in the culture supernatant collected from cell cultures supplemented with dextran sulfate sodium were purified using MabSelect™ (Cytiva). The resulting antibody solution was further purified by gel filtration chromatography using Superdex® 200 Increase 10 / 300 GL (Cytiva). PBS was used as the mobile phase, and purification was performed at a flow rate of 0.75 mL / min. The antibody concentration in the purified antibody solution was calculated by measuring the absorbance at 280 nm.

[0125] (4.2) Biotinylation of Trastuzumab and Its Variants Trastuzumab and its R3 and R4 mutants were labeled with biotin using a Biotin Labeling Kit-NH2 (Dojindo Laboratories, Inc.) according to the instructions attached to the kit.

[0126] (4.3) Measurement of affinity for antigen The affinity of each antibody for its antigen was measured by ELISA. The antigens used for each antibody were full-length human CD20 / MS4A1 protein with a His tag (HEK293) (ACROBiosystems), recombinant human ErbB2 / Her2 Fc chimera (R&D Systems), chicken egg white lysozyme (Sigma), animal-free recombinant human IL-6 (PEPROTECH), and interleukin-8 (CXCL8) (Shenandoah Biotechnology). The specific procedure was as follows: A solution of each antigen diluted to 5 μg / mL in PBS was added to each well of a 96-well plate (Nunc) and incubated overnight at 4°C. After removing the solution from each well, 1% BSA / PBS was added and incubated at room temperature for 3 hours. After removing the solution from each well, the plate was washed three times with PBST. The antibody solution was added to each well and incubated at room temperature for 15 minutes. After removing the solution from each well, the plate was washed three times with PBST. A solution of HRP-conjugated anti-human IgG Fc antibody (Bethyl Laboratories), HRP-conjugated goat F(ab')2 fragment anti-mouse IgG (H+L) (Beckman), or HRP-conjugated streptavidin (Thermo Fisher Scientific) diluted to 0.3 μg / mL in 1% BSA / PBS was added to each well and incubated at room temperature for 1 hour. After removing the solution from each well, the plate was washed three times with PBST. The color reaction was developed using KPL TMB peroxidase substrate (SeraCare Life Sciences) and stopped with KPL TMB peroxidase stop solution (SeraCare Life Sciences). The absorbance at 450 nm was measured using a SpectraMax® 190 microplate reader (Molecular Devices).

[0127] (5) Results The expression levels and antigen affinities of each antibody other than rituximab and its mutants are shown in Tables 1 to 4 and Figures 43 to 46. The antigen affinities of rituximab and its mutants are shown in Table 5 and Figure 47. In the tables and figures, "WT" refers to an antibody without a substitution of amino acid residues in the light chain FR3. In the tables, "-DS" refers to the antibody concentration (mg / L) in the supernatant of a cell culture without the addition of dextran sulfate sodium. "+DS" refers to the antibody concentration (mg / L) in the supernatant of a cell culture with the addition of dextran sulfate sodium. "Ratio" refers to the ratio of the +DS value to the -DS value. "EC50" refers to the 50% effective concentration. "EC50 ratio" refers to the affinity ratio of each mutant when the EC50 of WT is set to 1.0 (EC50 ratio).

[0128] [Table 1]

[0129] [Table 2]

[0130] [Table 3]

[0131] [Table 4]

[0132] [Table 5]

[0133] As shown in Tables 1 to 4, the yields of trastuzumab, anti-lysozyme antibody, anti-IL-6 antibody, and anti-IL-8 antibody were almost unchanged with or without dextran sulfate sodium. On the other hand, the yields of each antibody mutant were significantly increased with the addition of dextran sulfate sodium compared to without its addition. Although the above antibody mutants share a common feature in which a specific amino acid residue in the light chain FR3 is substituted with an arginine residue, the amino acid sequences of the light and heavy chains are different. This suggests that the effect of polyanionic compounds on the yield of mutants can be applied to all antibody mutants. Furthermore, as shown in Tables 1 to 5 and Figures 43 to 47, all antibody mutants exhibited improved affinity for antigen compared to the WT. This suggests that the addition of dextran sulfate sodium to the culture medium does not affect the affinity of the antibody for the antigen.

Claims

1. culturing animal cells into which an antibody-encoding gene has been introduced in the presence of a polyanionic compound to cause the animal cells to produce the antibody; the polyanionic compound is at least one selected from the group consisting of anionic polysaccharides and anionic polyamino acids, the antibody has at least three amino acid residues in framework region 3 (FR3) substituted with arginine residues or lysine residues; the anionic polysaccharide is a sulfated polysaccharide or an alginate; the at least three amino acid residues of FR3 include at least three residues selected from the group consisting of positions 63, 65, 67, 70, and 72 of the light chain as defined by the Chothia method; Methods for producing antibodies.

2. 2. The method according to claim 1, wherein the sulfated polysaccharide is at least one selected from the group consisting of dextran sulfate and its salts, glycosaminoglycans, and proteoglycans containing sulfate groups.

3. 3. The method according to claim 2, wherein the glycosaminoglycan is at least one selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, pentosan sulfate, and salts thereof.

4. The method according to claim 2 or 3, wherein the proteoglycan containing a sulfate group is a protein covalently bound to at least one selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and salts thereof.

5. A method according to any one of claims 1 to 4, wherein the animal cells are cultured in the presence of an anionic polyamino acid, and the anionic polyamino acid is at least one selected from the group consisting of polyglutamic acid, polyaspartic acid, and salts thereof.

6. 6. The method according to claim 1, wherein the animal cells are cultured in a medium containing the polyanionic compound at a concentration of 0.05 mg / mL to 20 mg / mL.

7. The method according to any one of claims 1 to 6, wherein the antibody is an IgG.

8. The method according to any one of claims 1 to 7, wherein the antibody has improved affinity for an antigen compared to the antibody before the amino acid residue substitution.

9. The method according to claim 1, wherein the antibody is an antibody selected from the following 1) to 4), in which at least three amino acid residues in FR3 have been substituted with arginine residues or lysine residues: 1) An antibody comprising a light chain having the amino acid sequence represented by SEQ ID NO: 2 and a heavy chain having the amino acid sequence represented by SEQ ID NO: 1; 2) an antibody comprising a light chain having the amino acid sequence represented by SEQ ID NO: 6 and a heavy chain having the amino acid sequence represented by SEQ ID NO: 5; 3) an antibody comprising a light chain having the amino acid sequence represented by SEQ ID NO: 13 and a heavy chain having the amino acid sequence represented by SEQ ID NO: 12; and 4) An antibody comprising a light chain having the amino acid sequence represented by SEQ ID NO: 16 and a heavy chain having the amino acid sequence represented by SEQ ID NO:

15.

10. The method according to claim 1, wherein the antibody is any one of antibodies selected from the following 5) to 8): 5) An antibody comprising a light chain having the amino acid sequence represented by SEQ ID NO: 3 or 4 and a heavy chain having the amino acid sequence represented by SEQ ID NO: 1; 6) An antibody comprising a light chain having the amino acid sequence represented by SEQ ID NO: 7 or 8 and a heavy chain having the amino acid sequence represented by SEQ ID NO: 5; 7) An antibody comprising a light chain having the amino acid sequence represented by SEQ ID NO: 14 and a heavy chain having the amino acid sequence represented by SEQ ID NO: 12; and 8) An antibody comprising a light chain having the amino acid sequence represented by SEQ ID NO: 17 or 18 and a heavy chain having the amino acid sequence represented by SEQ ID NO: 15.

Images