JP7669682B2 - Method for producing an antibody having improved antibody-dependent cellular cytotoxicity - Google Patents

Description

The present invention relates to a method for producing an antibody having high antibody-dependent cellular cytotoxicity (ADCC) activity. More specifically, the present invention relates 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.

Recombinant proteins are currently used in a wide range of fields. Their importance has increased with the recent 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 when producing antibody pharmaceuticals.

Antibody drugs are medicines whose main ingredient is a monoclonal antibody, and their therapeutic effects include inhibition of signal transduction, induction of cell death (apoptosis), antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). 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 cytotoxic effect of antibody drugs.

Methods for improving the ADCC activity of antibodies can be broadly divided into methods for substituting specific amino acid residues in the Fc region of an antibody with other amino acid residues, and methods for modifying the glycan attached to the Fc region of an antibody. Glycans attached to the Fc region of an antibody include N-glycans attached to the side chain of the asparagine (Asn) group of a specific amino acid sequence (e.g., Asn-X-Ser/Thr) (X represents any amino acid residue), and O-glycans attached to the side chain of a serine (Ser) or threonine (Thr) residue. Of these, it has been reported that ADCC activity can be improved by deleting the core fucose of the N-glycan (Non-Patent Document 1).

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, the structure of the N-glycan attached to the Fc region of the antibody changes when the culture conditions of the mammalian cells are changed (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 controlling the glycan structure to produce an antibody with high ADCC activity, particularly conditions for culturing mammalian cells capable of expressing the antibody.

JP 2017-506515 A

Mori. K, et al. , Cytotechnology, 55, 109-114 (2007)

The objective of the present invention is to provide a method for producing an antibody having high antibody-dependent cellular cytotoxicity in a method for producing an antibody, the method 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.

As a result of intensive research conducted by the present inventors to solve the above 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 resulting culture, and thus completed the present invention.

That is, the present invention includes the following aspects:

(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 resulting 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.

(2) The method according to (1), wherein the antibody is an antibody containing a human Fc region.

(3) A method for monitoring the culture process in the manufacturing method described in (2) by evaluating the affinity of an antibody obtained by the manufacturing method described in (2) with human FcγRIIIa.

(4) A method for evaluating the medium pH in the production method described in (2) by evaluating the affinity between an antibody obtained by the production method described in (2) and human FcγRIIIa.

(5) The method according to (3) or (4), in which the affinity between the antibody obtained by the manufacturing method described in (2) and 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.

The present invention is described in detail below.

The production method of the present invention is characterized in that when mammalian cells capable of expressing an antibody 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 carbon dioxide concentration in the gas phase and adding an alkaline solution to the medium. An example of the alkaline solution is a sodium bicarbonate (NaHCO ₃ ) solution. The carbon dioxide concentration in the gas phase and the amount of the alkaline solution added can be adjusted so that the pH of the medium is within the above-mentioned range, and it is preferable to adjust the pH to 6.65 or more and 6.95 or less, more preferably to adjust the pH to 6.75 or more and 6.95 or less, and even more preferably to adjust the pH to 6.80 or more and 6.90 or less.

There are no particular limitations on the mammalian cells used in the production method of the present invention, so 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). Of these, it is preferable to use CHO cells, which are widely used in recombinant antibody production.

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 allows the host mammalian cells to grow and express the antibody. Examples include media that require animal serum (RPMI1640, D-MEM, etc.) and media with chemically determined 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 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. Antibiotics such as G418, puromycin, blasticidin, zeocin, hygromycin, phleomycin, kanamycin, and ampicillin may also be added.

The culture step in the production method of the present invention may be appropriately performed depending on the mammalian cells used as the host and the antibody expressed by the cells. For example, the mammalian cells capable of expressing the antibody may be inoculated into a flask containing a medium having a pH in the above-mentioned range, and then the flask may be shaken to culture, or the mammalian cells capable of expressing the antibody may be inoculated into a bioreactor containing a medium controlled to a pH in the above-mentioned range, and then 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, they are preferably cultured in the presence of 5% to 8% _CO2 at a temperature of 30°C to 37°C.

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, 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.

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 the promoter and the 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.

There are no particular limitations on the poly A as long as it contains a termination signal. Examples include poly A derived from the antibody to be expressed, poly A derived from the SV40 virus genome, poly A from herpes virus thymidine kinase, poly A derived from bovine growth hormone, and poly A derived from rabbit β-globin gene.

The secretion signal is not particularly limited as long as it secretes the expressed antibody. Examples include a secretion signal derived from the recombinant antibody to be expressed, a secretion signal for human interleukin 2 (IL-2), a secretion signal for an azurocidin precursor, and a secretion signal for human serum albumin.

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

The antibiotic resistance gene may be selected from resistance genes corresponding to the antibiotic used in host selection, and examples include the G418 resistance gene, puromycin resistance gene, blasticidin resistance gene, zeocin resistance gene, hygromycin resistance gene, and phleomycin resistance gene.

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

Furthermore, the expression vector may further contain an enhancer to enhance the function of the promoter. There are no particular limitations 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).

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

To transform mammalian cells with the expression vector, a transformation method that is normally used by those skilled in the art, such as electroporation or lipofection using cationic liposomes, may be appropriately selected according to the mammalian cells to be used as a host.

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

When the antibody produced by the present invention is an antibody containing a human Fc region, the affinity of the antibody with human FcγRIIIa can be evaluated to monitor the culture state (culture process) of mammalian cells capable of expressing the antibody. 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 antibody produced can be monitored, and the culture process of mammalian cells capable of expressing the antibody can be monitored. As an example, if the affinity of the obtained antibody containing the human Fc region with human FcγRIIIa is reduced, the ADCC activity of the antibody is reduced, 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.

A preferred embodiment of the affinity evaluation between an antibody containing a human Fc region and human FcγRIIIa is an evaluation 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 Patent Publication No. 2015-086216, WO2018/150973), and also based on the difference in the ADCC activity of the antibody (JP Patent Publication No. 2016-023152, WO2018/150973). 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 a 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, and if the amount and/or proportion decreases, the ADCC activity of the antibody has decreased, so 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, added drug concentration, etc. are appropriately adjusted.

In addition, by culturing the cells under the same culture conditions and cell lines but 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 obtained and the amount and/or proportion of the peaks (fractions), thereby evaluating the medium for the antibodies.

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 as disclosed in WO2018/150973;
Fc binding proteins as disclosed in WO2019/083048;
Examples include:

In the present invention, an antibody with improved ADCC activity means, for example, an antibody in which the ratio 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 ratio when the medium is cultured while controlling the pH outside the aforementioned range (for example, pH 7.1).

The present invention is a method for producing an antibody, which includes a step of culturing mammalian cells capable of expressing an antibody and a step of 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 makes it possible to efficiently produce antibodies that require antibody-dependent cellular cytotoxicity, such as anticancer agents.

Furthermore, when the antibody produced by the present invention contains a human Fc region, the affinity of the antibody with human FcγRIIIa can be evaluated to monitor the culture step in the production method of the present invention and evaluate the medium components.

1 shows the plasmid map of mammalian expression plasmid pEFd. 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. 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. 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. 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. 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. The figure shows the area percentages 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. This shows the time course of 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. This shows the time course of 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. This 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. This 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. 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. The figure shows the area percentages 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 using EX-CELL Advanced CHO Fed-batch Medium adjusted to pH 6.8, pH 6.9, pH 7.0, and pH 7.1.

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

Example 1 Construction of cells expressing anti-interleukin-6 receptor (IL-6R) antibody (1) A vector capable of expressing anti-IL-6R antibody in mammalian cells was constructed by the following method.
(1-1) A gene encoding dihydrofolate reductase (dhfr) and SV40 PolyA as set forth in SEQ ID NO:1 with restriction enzyme SacII recognition sequences (CCGCGG) added to both the 5' and 3' ends was totally synthesized (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 carried out 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') (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.

(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.

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

(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 in which the SV40 promoter, dhfr and SV40 PolyA were incorporated into pFUSEss-CHIg-hG1 was named pFU-CHIg-dhfr, and the plasmid in which the SV40 promoter, dhfr and SV40 PolyA were incorporated into pFUSE2ss-CLIg-hk was named pFU-CLIg-dhfr.

(4) The polynucleotide described in SEQ ID NO: 7, which encodes the heavy chain variable region of an anti-interleukin 6 receptor (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 to the 5' end, and the restriction enzyme NheI recognition sequence (GCTAGC) was added to the 3' end, and the gene was 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 an 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 to the 5' end, and the restriction enzyme BsiWI recognition sequence (CGTACG) was added to the 3' end, and the gene was totally synthesized and cloned into a plasmid (consigned to FASMAC). The resulting plasmid was named pUC-VL6R.

(5) pUC-VH6R prepared in (4) and pFU-CHIg-dhfr prepared in (3) were digested with the 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 the 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 the restriction enzymes EcoRI and BsiWI, 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 the plasmid pFU-6RL-dhfr that expresses the light chain (L chain) of the anti-IL-6R antibody.

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 a 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. Gene amplification was then performed by adding 50 ng/mL methotrexate (MTX) to the medium.

(2) The cells treated with MTX in (1) were cloned by limiting dilution, and cells capable of stably and highly producing anti-IL-6R antibody were selected using the ELISA (Enzyme-Linked Immunosorbent Assay) described below.
(2-1) Anti-human Fab antibody (Bethyl) was immobilized at 1 μg/well on 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), a 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 a 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 a cell line with high anti-IL-6R antibody production and high measured value.

(3) The MTX concentration was increased stepwise (50 nM, 500 nM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM), and the limiting dilution was repeated to select clones by the ELISA described in (2). As a result, a cell line highly producing anti-IL-6R antibody was obtained.

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 cultured with shaking at 130 rpm, 37° C., and 8% _CO2 .

(2) 100 mL of BalanCD CHO Growth A medium containing 50 μg/mL kanamycin and 30 mL/L GlutaMAX was placed in three 250 mL sterilized jar fermenters (BioT) equipped with calibrated pH meters and dissolved oxygen (DO) meters. The anti-IL-6R antibody highly expressing cells cultured in (1) were inoculated at 0.2 × ¹⁰ cells/mL, and the above-mentioned medium was added to make the total volume 110 mL.

(3) The jar fermenter containing the medium and cells was set in a control device (Bio Jr. 8: Biot Co.), 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 a 0.5 M aqueous solution of sodium bicarbonate, and the DO was controlled to maintain 50% of the saturated dissolved oxygen amount at 37°C. 1 to 2 mL of the culture medium was sampled during the culture, 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).

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

(5) After washing the separation column with 10 mL of the buffer used for the equilibration, the antibody adsorbed to 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 antibody with a different controlled pH of the medium.

Figures 2 and 3 show the change in productivity of anti-IL-6R antibodies due to differences in the controlled pH of the medium, and Figures 4 and 5 show the change in viable cell density of anti-IL-6R antibody-expressing cells. From Figures 2 to 5, it can be said that there is no effect on antibody productivity and cell proliferation if the medium pH is controlled to a pH higher than 6.6 and lower than 7.4.

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).

(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 in a column oven. The column was equilibrated by passing 50 mM citrate buffer (pH 6.5) containing 150 mM sodium chloride at a flow rate of 1.0 mL/min for 10 minutes.

(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.

(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).

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 fell below 30% (pH 7.0: 22.6%, pH 7.1: 26.7%).

These results show that the ADCC activity of antibodies expressed by mammalian cells capable of expressing antibodies is improved by culturing the cells while controlling the medium pH to pH 6.8 and pH 6.9.

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 the medium was EX-CELL Advanced CHO Fed-batch Medium (Merck). The antibody expressed by the cells was analyzed using an FcR9_F column in the same manner as in Example 4.

Figures 8 and 9 show the change in productivity of anti-IL-6R antibodies due to differences in the controlled pH of the medium, and Figures 10 and 11 show the change in viable cell density of anti-IL-6R antibody-expressing cells. As with the case of using Balan CD as the medium (Example 3), it can be said that there is no effect on antibody productivity and cell proliferation if the medium pH is controlled to a pH higher than 6.6 and lower than 7.4.

The chromatogram obtained by analysis using the FcR9_F column is shown in Figure 12, and the area ratio of each peak calculated from the chromatogram is shown in Figure 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 approximately 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%).

These results show that, as in the case of using BalanCD as the medium (Example 4), the ADCC activity of the antibody expressed by mammalian cells capable of expressing antibodies can be improved by culturing the cells while controlling the medium pH to pH 6.8 and pH 6.9.

Claims (3)

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 : 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. 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.