Method for producing an antibody having improved antibody-dependent cellular cytotoxicity

Abstract

To provide a method for producing an antibody having high antibody-dependent cellular cytotoxicity.SOLUTION: A method for producing an antibody includes the steps of: culturing a mammalian cell that can express an antibody; and recovering an antibody expressed by the mammalian cell, contained in the resultant culture. The step of culturing is conducted in a medium with its pH controlled to be higher than 6.6 and lower than 7.0, to produce an antibody having improved antibody-dependent cellular cytotoxicity.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to a method for producing an antibody having high antibody-dependent cellular cytotoxicity (ADCC) activity, and more specifically to a method for improving the ADCC activity of an antibody expressed by a host by optimizing a process for culturing the host capable of expressing the antibody. [Background technology]

[0002] Recombinant proteins are currently used in a wide range of fields. Their importance has increased in recent years due to the growth of biopharmaceuticals, such as antibody drugs. Recombinant proteins are mainly produced using Escherichia coli, yeast, insect cells, and mammalian cells as hosts, but recombinant protein expression systems using mammalian cells as hosts are becoming increasingly important due to the three-dimensional structure of the expressed recombinant protein and post-translational modifications such as glycosylation. In particular, Chinese hamster ovary cells (CHO cells) are used as hosts for the expression of many recombinant proteins. Furthermore, recombinant proteins derived from recombinant CHO cells have been confirmed to be safe for use as pharmaceuticals, and are therefore the mammalian cells most commonly used as hosts for the production of antibody drugs.

[0003] Antibody drugs are drugs whose main component is a monoclonal antibody, and their therapeutic effects include signal transduction inhibition, induction of apoptosis, antibody-dependent cellular cytotoxicity (ADCC) action, and complement-dependent cytotoxicity (CDC) action. Of these, ADCC activity and CDC activity are called effector functions, and are important because they induce immune mechanisms to damage target cells such as cancer cells. ADCC activity in particular is an important activity for the cytotoxicity of antibody drugs.

[0004] Methods for improving the ADCC activity of an antibody can be broadly divided into methods for substituting specific amino acid residues in the Fc region of the antibody with other amino acid residues, and methods for modifying the sugar chains added to the Fc region of the antibody. Sugar chains added to the Fc region of an antibody include N-type sugar chains added to the side chains of asparagine (Asn) groups in specific amino acid sequences (e.g., Asn-X-Ser / Thr) (X represents any amino acid residue), and O-type sugar chains bound to the side chains of serine (Ser) or threonine (Thr) residues. Of these, it has been reported that ADCC activity can be improved by deleting the core fucose of N-type sugar chains (Non-Patent Document 1).

[0005] It is known that when mammalian cells capable of expressing an antibody are cultured and the antibody expressed by the mammalian cells contained in the resulting culture is collected to produce the antibody, changing the culture conditions of the mammalian cells changes the structure of the N-glycan attached to the Fc region of the antibody (Patent Document 1). However, the glycans actually attached to the Fc region of an antibody are heterogeneous, and it is difficult to completely link the relationship between the glycan structure and ADCC activity. Therefore, it has been difficult to establish conditions for producing an antibody with high ADCC activity by controlling the glycan structure, particularly conditions for culturing mammalian cells capable of expressing the antibody. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2017-506515 A [Non-patent literature]

[0007] [Non-Patent Document 1] Mori.K, et al.,Cytotechnology,55,109-114(2007) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for producing an antibody having high antibody-dependent cellular cytotoxicity, comprising the steps of culturing mammalian cells capable of expressing an antibody and recovering an antibody expressed by the mammalian cells contained in the obtained culture. [Means for solving the problem]

[0009] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that, in the process of culturing mammalian cells capable of expressing an antibody, culturing the mammalian cells in a medium controlled at an appropriate pH improves the antibody-dependent cellular cytotoxicity (ADCC) activity of the antibody expressed by the mammalian cells contained in the obtained culture, and thus completed the present invention.

[0010] That is, the present invention includes the aspects described below.

[0011] (1) A method for producing an antibody, comprising the steps of culturing mammalian cells capable of expressing an antibody and recovering the antibody expressed by the mammalian cells contained in the obtained culture, the method producing an antibody with improved ADCC activity by carrying out the culturing step in a medium whose pH is controlled to be higher than pH 6.6 and lower than pH 7.0.

[0012] (2) The method of (1), wherein the antibody comprises a human Fc region.

[0013] (3) A method for monitoring the culture step in the production method described in (2) by evaluating the affinity of an antibody obtained by the production method described in (2) to human FcγRIIIa.

[0014] (4) A method for evaluating the medium pH in the production method described in (2) by evaluating the affinity of an antibody obtained by the production method described in (2) to human FcγRIIIa.

[0015] (5) The method described in (3) or (4), in which the affinity of the antibody obtained by the manufacturing method described in (2) to human FcγRIIIa is evaluated based on the binding strength between the antibody obtained by the manufacturing method described in (2) and a human FcγRIIIa-immobilized separation agent.

[0016] The present invention will be described in detail below.

[0017] The production method of the present invention is characterized in that when mammalian cells capable of expressing an antibody are cultured, the cells are cultured in a medium whose pH is controlled to be higher than pH 6.6 and lower than pH 7.0. If the pH of the medium is within the above-mentioned range, the proliferation of the cells and the productivity of the antibody are not significantly inhibited. The pH of the medium can be controlled by adjusting the concentration of carbon dioxide in the gas phase and adding an alkaline solution to the medium. An example of the alkaline solution is sodium bicarbonate (NaHCO 3 The carbon dioxide concentration in the gas phase and the amount of the alkaline solution added may be adjusted so that the pH of the medium falls within the above-mentioned range, preferably from pH 6.65 to pH 6.95, more preferably from pH 6.75 to pH 6.95, and even more preferably from pH 6.80 to pH 6.90.

[0018] The mammalian cells used in the production method of the present invention are not particularly limited as long as they are capable of expressing the antibody to be produced. Examples include Chinese hamster ovary (CHO) cells (CHO-K1, CHO-S, CHO-DG44, and CHO-DXB11), mouse myeloma-derived cells (SP2 / 0, NS0), human embryonic kidney-derived cells (HEK cells), human leukemia-derived cells (HL-60 cells), human cervical cancer-derived cells (HeLa cells), and African green monkey kidney-derived cells (COS cells). Among these, it is preferable to use CHO cells, which are widely used for recombinant antibody production.

[0019] The medium used in the production method of the present invention is not particularly limited as long as it has a pH within the above-mentioned range and is a medium in which the host mammalian cells can grow and express the antibody. Examples include media that require animal serum (RPMI1640, D-MEM, etc.) and media with chemically defined components (BalanCD CHO Growth A [Irvine Scientific], FreeStyle CHO Expression MediumCD [Thermo Fisher], OptiCHO [Thermo Fisher], EX-CELL CD CHO Fusion / EX-CELL Advanced CHO Fed-batch Medium [Merck], and CHOgro [Mirus]). Furthermore, the above-mentioned medium may contain medium components such as nutrients, hormones, growth factors, specific ions (sodium, potassium, calcium, magnesium, etc.), vitamins, nucleosides, nucleotides, amino acids such as glutamine, inorganic salts (copper, zinc, cobalt, nickel, etc.), lipids, and glucose. Furthermore, antibiotics such as G418, puromycin, blasticidin, zeocin, hygromycin, phleomycin, kanamycin, and ampicillin may be further added.

[0020] The culture step in the production method of the present invention may be carried out appropriately depending on the mammalian cells used as the host and the antibody expressed by the cells. For example, the mammalian cells capable of expressing an antibody may be inoculated into a flask containing a medium having a pH in the aforementioned range, and the flask may be shaken to culture the cells. Alternatively, the mammalian cells capable of expressing an antibody may be inoculated into a bioreactor containing a medium whose pH has been controlled in the aforementioned range, and the cells may be cultured by batch culture, semi-batch culture (also called fed-batch culture), perfusion culture, or a combination thereof. When the mammalian cells are CHO cells, the cells may be cultured in a bioreactor containing a medium having a pH in the aforementioned range, and ... by batch culture, semi-batch culture (also called fed-batch culture), perfusion culture, or a combination thereof. 2 It is preferable to culture the bacteria at a temperature of 30°C to 37°C in the presence of the bacteria.

[0021] An example of an antibody produced by the method of the present invention is an antibody containing a human Fc region. Specific examples include human antibodies, humanized antibodies, chimeric antibodies between humans and other animals (such as mice), and human Fc fusion proteins. When the antibody containing a human Fc region is immunoglobulin G (IgG), four subclasses (IgG1, IgG2, IgG3, and IgG4) are known, of which IgG1 and IgG3 have high antibody-dependent cellular cytotoxicity (ADCC) activity and are therefore preferred embodiments of the antibody produced by the method of the present invention. Furthermore, IgG1 has particularly high ADCC activity and is therefore a particularly preferred embodiment of the antibody produced by the method of the present invention.

[0022] The mammalian cells capable of expressing an antibody used in the production method of the present invention may be prepared by transforming the mammalian cells with an expression vector containing a polynucleotide encoding the antibody. In addition to a promoter and a polynucleotide encoding the antibody, the expression vector may further contain polyA, a secretion signal required for secretion and expression of the recombinant antibody, a gene amplification marker gene, an antibiotic resistance gene used for host selection, and a replication origin in a host other than a mammalian cell used for gene recombination.

[0023] The poly A is not particularly limited as long as it contains a termination signal, and examples include poly A derived from an antibody to be expressed, poly A derived from the SV40 viral genome, poly A of herpes virus thymidine kinase, poly A derived from bovine growth hormone, and poly A derived from rabbit β-globin gene.

[0024] The secretory signal is not particularly limited as long as it secretes the expressed antibody, and examples include a secretory signal derived from the recombinant antibody to be expressed, a secretory signal of human interleukin 2 (IL-2), a secretory signal of an azurocidin precursor, and a secretory signal of human serum albumin.

[0025] The gene amplification marker gene may be a gene suitable for the gene amplification method, for example, the dhfr gene may be used when the dihydrofolate reductase (dhfr) / methotrexate (MTX) method is used, and the GS gene may be used when the glutamine synthetase (GS) / methionine sulfoximine (MSX) method is used.

[0026] The antibiotic resistance gene may be selected from the group consisting of a G418 resistance gene, a puromycin resistance gene, a blasticidin resistance gene, a zeocin resistance gene, a hygromycin resistance gene, and a phleomycin resistance gene.

[0027] When the host other than a mammalian cell is E. coli, the replication origin can be, for example, ColE1, which has a high copy number in E. coli and results in a high yield of plasmid DNA.

[0028] Furthermore, the expression vector may further contain an enhancer to enhance the function of the promoter. There is no particular limitation on the enhancer to be used, and it may be appropriately selected taking into consideration the antibody and mammalian cells to be expressed. One example is an enhancer derived from cytomegalovirus (CMV).

[0029] In addition, the expression vector may further contain a LoxP gene to facilitate expression of the gene (polynucleotide encoding an antibody) introduced into a mammal. When an expression vector is introduced into a host cell containing a LoxP gene in its genome, a polynucleotide encoding a recombinant protein can be site-specifically introduced into the genome of the host cell by performing homologous recombination using Cre recombinase. In addition, CRISPR / Cas9 or the like can be used as a method for site-specifically introducing a polynucleotide encoding a recombinant protein into the genome of the host cell.

[0030] To transform mammalian cells with the expression vector, a transformation method may be appropriately selected from those commonly used by those skilled in the art, such as electroporation or lipofection using cationic liposomes, depending on the mammalian cells to be used as a host.

[0031] An antibody with improved ADCC activity is produced by culturing mammalian cells capable of expressing an antibody using the above-mentioned method, and then recovering the antibody expressed by the mammalian cells contained in the obtained culture. One example of a method for recovering an antibody is a method for recovering an antibody from the obtained culture by performing a purification procedure using chromatography such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography, either alone or in combination. The above-mentioned method is preferable in that the antibody can be recovered with high efficiency and high purity.

[0032] When the antibody produced by the present invention is an antibody containing a human Fc region, the culture state (culture step) of mammalian cells capable of expressing the antibody can be monitored by evaluating the affinity of the antibody with human FcγRIIIa. When the N-glycan attached to an antibody containing a human Fc region is deleted, the affinity of the antibody with human FcγRIIIa is significantly reduced. It is also known that the ADCC activity of an antibody containing a human Fc region is related to the affinity (binding ability) of the Fc region with human FcγRIIIa on the surface of immune cells (Nordstrom. J, L, et al., Breast Cancer Res., 13, 6, (2011)). Therefore, by evaluating the affinity of an antibody containing a human Fc region with human FcγRIIIa, the ADCC activity of the produced antibody can be monitored, and the culture step of mammalian cells capable of expressing the antibody can be monitored. As an example, if the affinity of the obtained antibody containing a human Fc region with human FcγRIIIa is reduced, the ADCC activity of the antibody is reduced; therefore, in order to improve the ADCC activity of the antibody, the culture pH (carbon dioxide concentration in the gas phase, amount of alkaline solution added, etc.), temperature, stirring speed, dissolved oxygen concentration, medium components, concentration of added drugs, etc. are appropriately adjusted.

[0033] A preferred embodiment of the affinity evaluation between an antibody containing a human Fc region and human FcγRIIIa is based on the binding strength between the antibody containing a human Fc region and a human FcγRIIIa-immobilized separation agent. When an antibody containing a human Fc region is applied to a column packed with a human FcγRIIIa-immobilized separation agent obtained by immobilizing human FcγRIIIa on a carrier, the antibody is separated based on the difference in the sugar chain structure added to the antibody (JP 2015-086216 A, WO2018 / 150973 A), and is separated based on the difference in the ADCC activity of the antibody (JP 2016-023152 A, WO2018 / 150973 A). Therefore, based on the shape of the separation pattern, the culture state (culture process) of mammalian cells capable of expressing the antibody in the present invention can be monitored. Specifically, when antibodies containing a human Fc region are separated using a column packed with a human FcγRIIIa-immobilized separation agent, antibodies with high ADCC activity are eluted later than antibodies with low ADCC activity (i.e., they have stronger binding strength to the human FcγRIIIa-immobilized separation agent). Therefore, the amount and / or proportion of the peak (fraction) from which the antibody with high ADCC activity is eluted is calculated from the peak area or peak height of the elution pattern obtained by the separation. If the amount and / or proportion decreases, the ADCC activity of the antibody has decreased, and therefore, in order to improve the ADCC activity of the antibody, the culture pH (carbon dioxide concentration in the gas phase, amount of alkaline solution added, etc.), temperature, stirring speed, dissolved oxygen concentration, medium components, concentration of added drugs, etc. are appropriately adjusted.

[0034] In addition, by culturing the cells under the same culture conditions and cell lines while changing only the pH of the medium, it is possible to evaluate the pH of the medium that will increase the ADCC activity. Specifically, the antibodies obtained by culturing in media of different pH are evaluated using the human FcγRIIIa-immobilized separation agent, and the ADCC activity of the obtained antibodies is compared based on the shape of the separation pattern and the amount and / or proportion of the peaks (fractions), thereby evaluating the medium against the antibody.

[0035] In the present specification, human FcγRIIIa refers to (A) an Fc binding protein comprising at least the amino acid residues from glycine at position 17 to glutamine at position 192 of the amino acid sequence of human FcγRIIIa (UniProt No. P08637); or (B) an Fc binding protein, comprising a polypeptide comprising at least the amino acid residues from glycine at position 17 to glutamine at position 192 of the amino acid sequence of human FcγRIIIa (UniProt No. P08637), with the proviso that one or more amino acid residues in the amino acid sequence from position 17 to position 192 have been deleted, substituted with other amino acid residues, or added; In addition, a preferred embodiment of the above (B) is as follows: Fc-binding proteins disclosed in JP2015-086216A; Fc-binding proteins disclosed in JP2016-169197A; Fc-binding proteins disclosed in JP2017-118871A; Fc binding proteins disclosed in WO2018 / 150973; Fc binding proteins disclosed in WO2019 / 083048; Examples include:

[0036] Furthermore, in the present invention, an antibody with improved ADCC activity means, for example, an antibody in which the proportion of the peak area or peak height at which an antibody with high ADCC activity is located in the results (elution pattern) obtained by separation using a column packed with a human FcγRIIIa-immobilized separation agent is improved by 2% or more, preferably 5% or more, more preferably 10% or more, even more preferably 15% or more, and even more preferably 20% or more, compared to the said proportion when the medium pH is controlled outside the aforementioned range (e.g., pH 7.1). Effect of the Invention

[0037] The present invention relates to a method for producing an antibody, which comprises the steps of culturing mammalian cells capable of expressing an antibody and recovering the antibody expressed by the mammalian cells contained in the obtained culture, and is characterized in that the culturing step is carried out in a medium whose pH is controlled to be higher than pH 6.6 and lower than pH 7.0. The present invention enables efficient production of antibodies that require antibody-dependent cellular cytotoxicity, such as anticancer agents.

[0038] Furthermore, when the antibody produced by the present invention contains a human Fc region, the culture step in the production method of the present invention can be monitored and the medium components can be evaluated by assessing the affinity of the antibody with human FcγRIIIa. [Brief description of the drawings]

[0039] [Figure 1] 1 shows the plasmid map of the mammalian expression plasmid pEFd. [Diagram 2] This shows the time course of antibody productivity when anti-IL-6R antibody expressing cells were batch cultured in a jar fermenter in BalanCD CHO Growth A medium controlled at pH 6.8, pH 6.9, pH 7.0, and pH 7.1. [Diagram 3] This shows the time course of antibody productivity when anti-IL-6R antibody expressing cells were batch cultured in a jar fermenter in BalanCD CHO Growth A medium controlled at pH 6.6, pH 7.0, and pH 7.4. [Figure 4] This shows the transition of viable cell density when anti-IL-6R antibody expressing cells were batch cultured in a jar fermenter in BalanCD CHO Growth A medium controlled at pH 6.8, pH 6.9, pH 7.0, and pH 7.1. [Diagram 5] This shows the transition of viable cell density when anti-IL-6R antibody expressing cells were batch cultured in a jar fermenter in BalanCD CHO Growth A medium controlled at pH 6.6, pH 7.0, and pH 7.4. [Figure 6]The figure shows chromatograms by FcR9_F column analysis of anti-IL-6R antibody obtained when anti-IL-6R antibody expressing cells were batch-cultured in a jar fermenter in BalanCD CHO Growth A medium controlled at pH 6.8, pH 6.9, pH 7.0, and pH 7.1. [Figure 7] This shows the area percentage of each peak in FcR9_F column analysis of anti-IL-6R antibody obtained when anti-IL-6R antibody-expressing cells were batch-cultured in a jar fermenter in BalanCD CHO Growth A medium controlled at pH 6.8, pH 6.9, pH 7.0, and pH 7.1. [Figure 8] This shows the change over time in antibody productivity when anti-IL-6R antibody expressing cells were batch cultured in a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled at pH 6.8, pH 6.9, pH 7.0, and pH 7.1. [Figure 9] This shows the change over time in antibody productivity when anti-IL-6R antibody expressing cells were batch cultured in a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled at pH 6.6, pH 7.0, and pH 7.4. [Figure 10] 1 shows the transition of viable cell density when anti-IL-6R antibody expressing cells were batch cultured in a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled at pH 6.8, pH 6.9, pH 7.0, and pH 7.1. [Figure 11] 1 shows the transition of viable cell density when anti-IL-6R antibody expressing cells were batch cultured in a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled at pH 6.6, pH 7.0, and pH 7.4. [Figure 12]The figure shows chromatograms by FcR9_F column analysis of anti-IL-6R antibody obtained when anti-IL-6R antibody-expressing cells were batch-cultured in a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled at pH 6.8, pH 6.9, pH 7.0, and pH 7.1. [Figure 13] This shows the area percentage of each peak in FcR9_F column analysis of anti-IL-6R antibody obtained when anti-IL-6R antibody-expressing cells were batch-cultured in a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled at pH 6.8, pH 6.9, pH 7.0, and pH 7.1. EXAMPLES

[0040] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0041] Example 1 Construction of anti-interleukin 6 receptor (IL-6R) antibody expressing cells (1) A vector capable of expressing anti-IL-6R antibody in mammalian cells was constructed as follows. (1-1) A gene encoding dihydrofolate reductase (dhfr) set forth in SEQ ID NO:1 and SV40 PolyA was synthesized by adding restriction enzyme SacII recognition sequences (CCGCGG) to both the 5' and 3' ends of the gene (commissioned to Integrated DNA Technologies) and cloned into a plasmid. (1-2) The E. coli JM109 strain was transformed with the plasmid prepared in (1-1). The resulting transformant was cultured, and the plasmid was extracted and digested with the restriction enzyme SacII to prepare a gene encoding dhfr-SV40PolyA, which was named dhfr-P1. (1-3) PCR was performed using oligonucleotide primers consisting of the sequences described in SEQ ID NO: 2 (5'-TCC[CCGCGG]GCGGGACTCTGGGGTTCGAAATGACCG-3') and SEQ ID NO: 3 (5'-TCC[CCGCGG]GGTGGCTCTAGCCTTAAGTTCGAGACTG-3') (the brackets in SEQ ID NO: 2 and 3 indicate the restriction enzyme SacII recognition sequence). Specifically, a reaction solution having the composition shown in Table 1 was prepared, and the reaction solution was heat-treated at 98°C for 30 seconds, and then 25 cycles of reaction were repeated, with one cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 5 minutes. By this PCR, a region of the pIRES vector excluding the neomycin resistance gene was amplified.

[0042] [Table 1]

[0043] (1-4) The PCR product prepared in (1-3) was purified, digested with the restriction enzyme SacII, and ligated with the dhfr-P1 prepared in (1-2). The ligation product was used to transform E. coli JM109 strain, and the plasmid was extracted from the cultured transformant to obtain the expression vector pIRES-dhfr containing the dhfr gene.

[0044] (2) Using the pIRES-dhfr prepared in (1) as a template, PCR was performed using oligonucleotide primers consisting of the sequences described in SEQ ID NO: 4 (5'-TTTAAATCA[GCGGCCGC]GCAGCACCATGGCCTGAAATAACCTCTG-3') and SEQ ID NO: 5 (5'-GCAAGTAAAACCTCTACAAATGTGGTAAA[CGATCG]CTCCGGTGCCCGT-3') (the brackets in SEQ ID NO: 4 indicate the restriction enzyme NotI recognition sequence, and the brackets in SEQ ID NO: 5 indicate the restriction enzyme PvuI recognition sequence). Specifically, a reaction solution having the composition shown in Table 2 was prepared, and the reaction solution was heat-treated at 98°C for 1 minute, and then the reaction was performed by repeating 30 cycles of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute. The PCR product amplified by this PCR (the region from the SV40 promoter, dhfr, to the SV40 PolyA) was named dhfr-P2.

[0045] [Table 2]

[0046] (3) pFUSEss-CHIg-hG1 (InvivoGen) containing the heavy chain constant region of a human antibody, pFUSE2ss-CLIg-hk (InvivoGen) containing the light chain constant region of a human antibody, and dhfr-P2 prepared in (2) were digested with the restriction enzymes NotI and PvuI, respectively, and then purified and ligated. The ligation product was used to transform E. coli JM109 strain, and the plasmid was extracted from the cultured transformant to obtain pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hk containing the SV40 promoter, dhfr, and SV40 PolyA. The plasmid obtained by incorporating the SV40 promoter, dhfr and SV40 PolyA into pFUSEss-CHIg-hG1 was named pFU-CHIg-dhfr, and the plasmid obtained by incorporating the SV40 promoter, dhfr and SV40 PolyA into pFUSE2ss-CLIg-hk was named pFU-CLIg-dhfr.

[0047] (4) The polynucleotide described in SEQ ID NO: 7, which encodes the heavy chain variable region of the anti-interleukin 6 receptor (hereinafter, IL-6R) antibody consisting of the amino acid sequence described in SEQ ID NO: 6, was added with a restriction enzyme EcoRI recognition sequence (GAATTC) and a guanine (G) to suppress frameshifting, and the restriction enzyme NheI recognition sequence (GCTAGC) was added to the 3' end of the polynucleotide described in SEQ ID NO: 7, which encodes the heavy chain variable region of the anti-interleukin 6 receptor (hereinafter, IL-6R) antibody consisting of the amino acid sequence described in SEQ ID NO: 6. The gene was then totally synthesized and cloned into a plasmid (consigned to FASMAC). The plasmid thus prepared was named pUC-VH6R. In addition, the polynucleotide described in SEQ ID NO: 9, which encodes the light chain variable region of the anti-IL-6R antibody consisting of the amino acid sequence described in SEQ ID NO: 8, was added with a restriction enzyme EcoRI recognition sequence (GAATTC) and a guanine (G) to suppress frameshifting, and the gene was then totally synthesized and cloned into a plasmid (consigned to FASMAC). The plasmid thus prepared was named pUC-VL6R.

[0048] (5) pUC-VH6R prepared in (4) and pFU-CHIg-dhfr prepared in (3) were digested with restriction enzymes EcoRI and NheI, respectively, and then purified and ligated. The ligation product was used to transform E. coli JM109 strain, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-6RH-dhfr expressing the heavy chain (H chain) of an anti-IL-6R antibody. In addition, pUC-VL6R prepared in (4) and pFU-CLIg-dhfr prepared in (3) were digested with restriction enzymes EcoRI and BsiWI, respectively, and then purified and ligated. The ligation product was used to transform E. coli JM109 strain, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-6RL-dhfr expressing the light chain (L chain) of an anti-IL-6R antibody.

[0049] Example 2 Construction of cells highly expressing anti-IL-6R antibody (1) pFU-6RH-dhfr and pFU-6RL-dhfr prepared in Example 1 were transfected into CHO cells (DG44 strain) using the Neon Transfection System (Thermo Fisher Scientific). The transformed cells were then cultured in CD OptiCHO Medium (Thermo Fisher Scientific) containing 50 μg / mL kanamycin and 40 mL / L GlutaMAX (Thermo Fisher Scientific) to obtain anti-IL-6R antibody-expressing cells. Then, the medium was supplemented with 50 ng / mL methotrexate. Gene amplification was performed by adding MTX.

[0050] (2) The cells treated with MTX in (1) were cloned by limiting dilution, and cells capable of stably and highly producing anti-IL-6R antibodies were selected using the ELISA (Enzyme-Linked ImmunoSorbent Assay) described below. (2-1) Anti-human Fab antibody (Bethyl) was immobilized at 1 μg / well onto the wells of a 96-well microplate (overnight at 4° C.). After immobilization, the wells were blocked with 20 mM Tris-HCl buffer (pH 7.4) containing 2% (w / v) SKIM MILK (Becton Dickinson) and 150 mM sodium chloride. (2-2) After washing with a washing buffer (20 mM Tris-HCl buffer (pH 8.0) containing 0.05% [w / v] Tween 20 (trade name) and 150 mM NaCl), the culture supernatant containing the antibody was added and the antibody was reacted with the immobilized protein (at 30°C for 1 hour). (2-3) After the reaction was completed, the plate was washed with the washing buffer, and peroxidase-labeled anti-human Fc antibody (Bethyl) diluted to 100 ng / mL was added at 100 μL / well. (2-4) After reacting at 30°C for 1 hour and washing with the washing buffer, TMB Peroxidase Substrate (KPL) was added at 50 μL / well. Then, 1 M phosphoric acid was added at 50 μL / well to stop color development, and the absorbance at 450 nm was measured using a microplate reader (Tecan) to select cell lines with high anti-IL-6R antibody production values.

[0051] (3) While increasing the MTX concentration stepwise (50 nM, 500 nM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM), limiting dilution was performed and clone selection was repeated by ELISA as described in (2). As a result, a cell line highly producing anti-IL-6R antibody was obtained.

[0052] Example 3: Effect of pH control in batch culture using a jar fermenter (part 1) (1) The anti-IL-6R antibody highly expressing cells prepared in Example 2 were inoculated into a 250 mL Erlenmeyer flask (Corning) containing 50 mL of BalanCD CHO Growth A medium (Irvine Scientific) containing 50 μg / mL kanamycin and 30 mL / L GlutaMAX (Thermo Fisher Scientific), and incubated at 130 rpm, 37° C., and 8% CO. 2 The culture was incubated with shaking under the following conditions.

[0053] (2) In three 250 mL sterilized jar fermenters (Biotec) equipped with a calibrated pH meter and dissolved oxygen (DO) meter, 100 mL of BalanCD CHO Growth A medium containing 50 μg / mL kanamycin and 30 mL / L GlutaMAX was placed, and 0.2 × 10 6 After inoculation to give a total volume of 110 mL, the above-mentioned medium was added.

[0054] (3) The jar fermenter containing the medium and cells was set in a control device (Bio Jr.8: Biot Co., Ltd.), and batch culture was performed for 12 days at 37°C and 130 rpm while flowing air into the gas phase at 100 mL / min. During the culture, the pH was controlled to a specified value between pH 6.6 and pH 7.4 by adjusting the carbon dioxide concentration in the gas phase and adding 0.5 M sodium bicarbonate solution at the same time, and the DO was controlled to maintain 50% of the saturated dissolved oxygen amount at 37°C. During the culture, 1 to 2 mL of the culture solution was sampled, and the viable cell density was measured using Vi-CELL XR (Beckman Coulter, Inc.), and the antibody productivity was measured using Cedex Bio (Roche Diagnostics, Inc.).

[0055] (4) After the culture was completed, the culture medium was centrifuged to remove cells and impurities, and the obtained supernatant was applied to a separation column (equilibrated with 20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride) prepared by packing 1.0 mL of MabSelect SuRe LX (GE Healthcare) into an open column.

[0056] (5) After washing the separation column with 10 mL of the buffer used for the equilibration, the antibody adsorbed on the separation column was eluted with 5 mL of 0.1 M glycine hydrochloride buffer (pH 3.0). The pH was returned to the neutral range by adding 1 mL of 1 M Tris-HCl (pH 8.0) to the eluate, and the buffer was exchanged with 50 mM citrate buffer (pH 6.5) containing 150 mM sodium chloride while concentrating with an ultrafiltration membrane, thereby obtaining highly pure anti-IL-6R antibodies with different controlled pH of the medium.

[0057] The change in the productivity of anti-IL-6R antibodies due to differences in the controlled pH of the medium is shown in Figures 2 and 3, while the change in the viable cell density of anti-IL-6R antibody-expressing cells is shown in Figures 4 and 5. It can be seen from Figures 2 to 5 that there is no effect on antibody productivity and cell proliferation if the medium pH is controlled to a value higher than pH 6.6 and lower than pH 7.4.

[0058] Example 4 Effect of pH control in batch culture using a jar fermenter (part 2) Of the antibodies obtained in Example 3(5), the medium pH was controlled to pH 6.8, pH 6.9, pH 7.0, and pH 7.1 for culture, and the obtained antibodies were analyzed by the method described below using a column packed with a carrier (separation agent) on which human FcγRIIIa was immobilized (FcR9_F column, described in Example 5 of WO2018 / 150973).

[0059] (1) The FcR9_F column was connected to a high-performance liquid chromatography system (Shimadzu Corporation), and the column was kept at a constant temperature of 25°C using a column oven. The column was equilibrated by passing 50 mM citrate buffer (pH 6.5) containing 150 mM sodium chloride through the column at a flow rate of 1.0 mL / min for 10 minutes.

[0060] (2) The anti-IL-6R antibody obtained in Example 3(5) was diluted to 1.0 mg / mL with the buffer used in (1), and 10 μL of the diluted antibody solution was added at a flow rate of 1.0 mL / min.

[0061] (3) After the buffer used in (1) was allowed to flow for 2 minutes at a flow rate of 1.0 mL / min, the antibody adsorbed to the FcR9_F column was eluted with a pH gradient of 50 mM citrate buffer (pH 4.5) containing 150 mM sodium chloride (a gradient in which 50 mM citrate buffer (pH 4.5) containing 150 mM sodium chloride became 100% in 18 minutes).

[0062] The obtained chromatogram is shown in FIG. 6, and the area ratios of each peak calculated from the chromatogram are shown in FIG. 7 and Table 3, respectively. The higher the ADCC (antibody-dependent cellular cytotoxicity) activity of an antibody, the stronger the binding strength with the FcγRIIIa-immobilized separation agent (JP Patent Publication 2016-023152, WO2018 / 150973). In other words, the higher the area ratio of peak 3 in FIG. 6, the higher the ADCC activity of the antibody. When the medium pH was controlled to pH 6.8 and pH 6.9 and cultured, the area ratio of peak 3 exceeded 30% (pH 6.8: 33.9%, pH 6.9: 34.7%). On the other hand, when the medium pH was controlled to pH 7.0 and pH 7.1 and cultured, the area ratio of peak 3 was below 30% (pH 7.0: 22.6%, pH 7.1: 26.7%).

[0063] These results demonstrate that the ADCC activity of antibodies expressed in mammalian cells capable of expressing antibodies is improved by culturing the cells while controlling the medium pH at pH 6.8 and pH 6.9.

[0064] [Table 3]

[0065] Example 5 Effect of pH control in batch culture using a jar fermenter (part 3) The cells highly expressing anti-IL-6R antibody prepared in Example 2 were cultured and the antibody productivity and viable cell count were measured in the same manner as in Example 3, except that EX-CELL Advanced CHO Fed-batch Medium (Merck) was used as the medium. The antibody expressed by the cells was analyzed using an FcR9_F column in the same manner as in Example 4.

[0066] The change in anti-IL-6R antibody productivity due to differences in the controlled pH of the medium is shown in Figures 8 and 9, and the change in viable cell density of anti-IL-6R antibody-expressing cells is shown in Figures 10 and 11. As with the use of Balan CD as the medium (Example 3), there is no effect on antibody productivity and cell proliferation if the medium pH is controlled to a value higher than pH 6.6 and lower than pH 7.4.

[0067] The chromatogram obtained by the analysis using the FcR9_F column is shown in FIG. 12, and the area ratio of each peak calculated from the chromatogram is shown in FIG. 13 and Table 4, respectively. When the medium pH was controlled to pH 6.8 and pH 6.9 for culture, the area ratio of peak 3 was about 40% (pH 6.8: 40.0%, pH 6.9: 39.2%). On the other hand, when the medium pH was controlled to pH 7.0 and pH 7.1 for culture, the area ratio of peak 3 decreased compared to when the medium pH was controlled to pH 6.8 and pH 6.9 (pH 7.0: 36.5%, pH 7.1: 31.4%).

[0068] From the above results, it can be seen that, as when BalanCD was used as the medium (Example 4), the ADCC activity of antibodies expressed by mammalian cells capable of expressing antibodies is improved by culturing the medium pH at controlled pH 6.8 and pH 6.9.

[0069] [Table 4]

Claims

1. A method for producing an antibody comprising the steps of: culturing mammalian cells capable of expressing an antibody comprising a human Fc region; and recovering the antibody expressed by the mammalian cells contained in the resulting culture, a step of producing the antibody having improved antibody-dependent cytotoxicity by carrying out the culture step in a medium controlled at a pH higher than 6.6 and lower than 7.0; monitoring the culturing step to assess the affinity of the resulting antibody for human FcγRIIIa; and adjusting the pH of the culture step based on the affinity evaluation. A manufacturing method comprising:

2. A method for evaluating the medium pH in the production method according to claim 1 by evaluating the affinity of an antibody obtained by the production method according to claim 1 for human FcγRIIIa.

3. The method according to claim 1 or claim 2, wherein the affinity between the antibody obtained by the manufacturing method described in claim 1 and human FcγRIIIa is evaluated based on the binding strength between the antibody obtained by the manufacturing method described in claim 1 and a human FcγRIIIa immobilized separation agent.

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