JP2022094382A - Method for producing antibody having improved antibody-dependent cellular cytotoxicity - Google Patents

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

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 ADCC activity of an antibody expressed by the host by optimizing the step of culturing the host capable of expressing the antibody.

Currently, recombinant proteins are used in a wide range of fields. Its importance is further increasing due to the growth of biopharmacy represented by antibody drugs in recent years. Recombinant proteins are mainly produced using Escherichia coli, yeast, insect cells, and mammalian cells as hosts. However, due to the three-dimensional structure of the expressed recombinant proteins and post-translational modifications such as sugar chain addition, mammalian cells are used. The importance of the recombinant protein expression system used as a host is increasing. In particular, Chinese hamster ovary cells (hereinafter referred to as CHO cells) are used as hosts for expression of many recombinant proteins. Further, since the recombinant protein derived from the recombinant CHO cell has been confirmed to be safe for use as a pharmaceutical product, it is the most commonly used mammalian cell as a host for producing an antibody pharmaceutical product.

Antibody drugs are drugs containing monoclonal antibodies as the main component, but their therapeutic effects include signal transduction inhibition, induction of cell death (Apoptosis), antibody-dependent cellular cytotoxicity (ADCC) action, and complement-dependent cellular cytotoxicity (CDC). ) There is an action. Of these, ADCC activity and CDC activity are called effector functions and are important because they damage target cells such as cancer by inducing an immune mechanism. In particular, ADCC activity is an important activity for the cytotoxic action of antibody drugs.

Methods for improving ADCC activity of an antibody are roughly divided into a method of substituting a specific amino acid residue in the Fc region of an antibody with another amino acid residue and a method of modifying a sugar chain added to the Fc region of an antibody. Will be done. The sugar chain added to the Fc region of the antibody is added to the side chain of the asparagine (Asn) group of a specific amino acid sequence (eg, Asn-X-Ser / Thr) (where X indicates an arbitrary amino acid residue). There are N-type sugar chains and O-type sugar chains that bind to the side chains of serine (Ser) or threonine (Thr) residues. Among these, it has been reported that ADCC activity is improved by deleting core fucose of N-type sugar chain (Non-Patent Document 1).

By culturing a mammalian cell capable of expressing an antibody and recovering the antibody expressed by the mammalian cell contained in the obtained culture, the culture conditions of the mammalian cell are set when the antibody is produced. It is known that when the antibody is changed, the structure of the N-type sugar chain added to the Fc region of the antibody changes (Patent Document 1). However, the sugar chain actually added to the Fc region of the antibody is heterogeneous, and it is difficult to completely link the relationship between the structure of the sugar chain and ADCC activity. Therefore, it has been difficult to establish conditions for producing an antibody having high ADCC activity by controlling the sugar chain structure, particularly conditions for culturing mammalian cells capable of expressing the antibody.

Japanese Unexamined Patent Publication No. 2017-506515

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

An object of the present invention is a method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody and a step of recovering the antibody expressed by the mammalian cell contained in the obtained culture. , To provide a method for producing an antibody having high antibody-dependent cellular cytotoxicity.

As a result of diligent studies to solve the above problems, the present inventors obtained by culturing the mammalian cells in a medium controlled to an appropriate pH in the step of culturing the mammalian cells capable of expressing the antibody. It has been found that the antibody-dependent cytotoxicity (ADCC) activity of the antibody expressed by the mammalian cells contained in the obtained culture is improved, and the present invention has been completed.

That is, the present invention includes the aspects described below.

(1) The culture step is carried out in a method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody and a step of recovering the antibody expressed by the mammalian cell contained in the obtained culture. A method for producing an antibody having improved ADCC activity by using a medium controlled to have a pH higher than pH 6.6 and lower than pH 7.0.

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

(3) A method for monitoring the culture step in the production method according to (2) by evaluating the affinity between the antibody obtained by the production method according to (2) and human FcγRIIIa.

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

(5) The affinity between the antibody obtained by the production method described in (2) and human FcγRIIIa was evaluated, and the binding force between the antibody obtained by the production method described in (2) and the human FcγRIIIa immobilization separating agent. The method according to (3) or (4), which is carried out based on the above.

Hereinafter, the present invention will be described in detail.

The production method of the present invention is characterized in that when a mammalian cell capable of expressing an antibody is cultured, it is cultured in a medium in which the pH is controlled to be higher than pH 6.6 and lower than pH 7.0. As long as the pH of the medium is in the above range, cell proliferation and antibody productivity are not significantly impaired. To control the pH of the medium, the carbon dioxide concentration in the gas phase may be adjusted and an alkaline solution may be added to the medium. As an example of the alkaline solution, a sodium hydrogen carbonate (NaHCO ₃ ) solution can be exemplified. 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 is in the above-mentioned range, preferably pH 6.65 or more and pH 6.95 or less, and pH 6.75 or more and pH 6.95. It is more preferable to adjust the pH to the following, and even more preferable to adjust the pH to 6.80 or more and 6.90 or less.

The mammalian cells used in the production method of the present invention are not particularly limited as long as they can express the antibody to be produced. As an example, Chinese hamster ovary (CHO) cells (CHO-K1, CHO-S, CHO-DG44 and CHO-DXB11), mouse myeloma-derived cells (SP2 / 0, NS0), human fetal kidney-derived cells (HEK). Cells), human leukemia cell-derived cells (HL-60 cells), human cervical cancer-derived cells (HeLa cells), and African green monkey kidney cell-derived cells (COS cells). Of these, the use of CHO cells, which are widely used for producing recombinant antibodies, is preferable.

The medium used in the production method of the present invention is not particularly limited as long as the pH is in the above-mentioned range and the host mammalian cells can grow and express the antibody. As an example, a medium requiring animal-derived serum (RPMI1640, D-MEM, etc.), a medium whose composition is chemically determined (BalanCD CHO Growth A [Irbine Scientific], FreeStyle CHO Expression MediumCD [ThermoF]. , OptiCHO [Thermo Fisher], EX-CELL CD CHO Fusion / EX-CELL Advanced CHO Fed-batch Medium [Merck] and CHOgro [Mirus]). Furthermore, in the above-mentioned medium, 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, glucose and other medium constituents may be included. Further, antibiotics such as G418, puromycin, blastsidedin, zeocin, hygromycin, phleomycin, kanamycin and ampicillin may be further added.

The culture step in the production method of the present invention may be appropriately performed depending on the mammalian cell used as a host and the antibody expressed in the cell. As an example, a flask containing a medium having a pH in the above-mentioned range may be inoculated with mammalian cells capable of expressing an antibody, and then the flask may be shaken for culturing. The bioreactor placed may be inoculated with mammalian cells capable of expressing an antibody, and then cultured by batch culture, semi-batch culture (also referred to as culture medium), perfusion culture, or a combination thereof. When the mammalian cells are CHO cells, it is preferable to culture them at a temperature of 30 ° C. to 37 ° C. in the presence of 5% to 8% CO ₂ .

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

The animal cell capable of expressing the antibody used in the production method of the present invention may be produced by transforming the animal cell with an expression vector containing a polynucleotide encoding the antibody. In addition to the promoter and the polynucleotide encoding the antibody, the expression vector includes polyA, a secretory signal required for secreted expression of recombinant antibody, a gene amplification marker gene, and an antibiotic resistance gene used for host selection. Or, it may further include a replication initiation site in a host other than the mammalian cell used for gene recombination.

The poly A is not particularly limited as long as it contains a termination signal, and as an example, poly A derived from an antibody to be expressed, poly A derived from the SV40 virus genome, poly A derived from herpesvirus thymidine kinase, and poly A derived from bovine growth hormone. , Poly A derived from the β-globin gene of rabbits.

The secretory signal is not particularly limited as long as it secretes the expressed antibody. The secretory signal of human serum albumin can be mentioned.

As the gene amplification marker gene, a gene suitable for the method of gene amplification may be used. 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 sulfoxymin (MSX) method is used.

For the antibiotic resistance gene, a resistance gene corresponding to the antibiotic used for host selection may be selected. As an example, a G418 resistance gene, a puromycin resistance gene, a blastsaidin resistance gene, a zeosin resistance gene, and a hyglomycin resistance gene may be selected. , Freomycin resistance gene.

The origin of replication can be exemplified by ColE1, which has a high number of copies in Escherichia coli and a high yield of plasmid DNA when the host other than the mammalian cell is Escherichia coli.

Further, the expression vector may further contain an enhancer for enhancing the action of the promoter. The enhancer to be used is not particularly limited, and may be appropriately selected in consideration of the antibody to be expressed and mammalian cells. An example is an enhancer derived from cytomegalovirus (CMV).

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

In order to transform a mammalian cell with the expression vector, among the transformation methods usually used by those skilled in the art, such as electroporation and lipofection using a cationic liposome, it is appropriate according to the mammalian cell used as a host. You can select it.

After culturing the mammalian cells capable of expressing the antibody by the method described above, the antibody expressed by the mammalian cells contained in the obtained culture is recovered to produce an antibody having improved ADCC activity. As an example of the method for recovering an antibody, a method for recovering an antibody from the obtained culture by a purification operation by chromatography such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography or the like alone or in combination. Can be given. The above method is preferable in that the antibody can be recovered with high efficiency and high purity.

When the antibody produced in the present invention is an antibody containing a human Fc region, the culture state (culture step) of mammalian cells capable of expressing the antibody is monitored by evaluating the affinity between the antibody and human FcγRIIIa. can. Deletion of the N-type sugar chain added to an antibody containing a human Fc region significantly reduces the affinity between the antibody and human FcγRIIIa. It is also known that the ADCC activity of an antibody containing a human Fc region is associated with the affinity (binding) between the Fc region and human FcγRIIIa on the surface of immune cells (Nordstrom.J, L, et al). ., Breast Cancer Res., 13, 6, (2011)). Therefore, by evaluating the affinity between the antibody containing the human Fc region and human FcγRIIIa, the ADCC activity of the produced antibody can be monitored, and the culture process of mammalian cells capable of expressing the antibody can be monitored. As an example, when the affinity between the obtained antibody containing the human Fc region and human FcγRIIIa is decreased, the ADCC activity of the antibody is decreased, so that the culture pH is improved in order to improve the ADCC activity of the antibody. (Carbon dioxide concentration in the gas phase, amount of alkaline solution added, etc.), temperature, stirring rotation speed, dissolved oxygen concentration, medium component, additive drug concentration, etc. are adjusted as appropriate.

A preferred embodiment of the affinity evaluation between the antibody containing the human Fc region and the human FcγRIIIa is the evaluation based on the binding force between the antibody containing the human Fc region and the human FcγRIIIa immobilization separating agent. When an antibody containing a human Fc region is applied to a column packed with a human FcγRIIIa-immobilized separator obtained by immobilizing human FcγRIIIa on a carrier, the antibody is separated based on the difference in sugar chain structure added to the antibody (Japanese Patent Laid-Open No. 2015). -086216, WO2018 / 150973), and separated based on the difference in ADCC activity of the antibody (Japanese Patent Laid-Open No. 2016-0223152, WO2018 / 150973). Therefore, based on the shape of the separation pattern, it is possible to monitor the culture state (culture step) of the mammalian cells capable of expressing the antibody in the present invention. Specifically, when an antibody containing a human Fc region is separated using a column packed with a human FcγRIIIa immobilized separating agent, an antibody having high ADCC activity is eluted later than an antibody having low ADCC activity (that is, human). Strong binding force to FcγRIIIa immobilization separator). Therefore, the amount and / or the ratio of the peak (fraction) in which the antibody having high ADCC activity is eluted is calculated from the peak area or the peak height of the elution pattern obtained by the separation, and the amount and / or the ratio is calculated. If it decreases, the ADCC activity of the antibody is 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, and stirring rotation speed are reduced. , Dissolved oxygen concentration, medium component, additive drug concentration, etc. are adjusted as appropriate.

Further, by culturing under the same culture conditions and cell lines and changing only the medium pH, it is possible to evaluate the medium pH at which ADCC activity becomes higher. Specifically, the antibody obtained by culturing in a medium having a different pH was evaluated by the human FcγRIIIa immobilized separator, and the shape and peak (fraction) of the obtained separation pattern were determined by the amount and / or ratio. The ADCC activity of the obtained antibody is compared to evaluate the antibody in the medium.

In this specification, human FcγRIIIa is referred to as human FcγRIIIa.
(A) an Fc-binding protein containing at least the amino acid residue from the 17th glycine to the 192nd glutamine in the amino acid sequence of human FcγRIIIa (UniProt No. P08637), or (B) human FcγRIIIa (UniProt No. P08637). ) Contains at least the amino acid residues from the 17th glycine to the 192nd glutamine, but in the 17th to 192nd amino acid residues, one or more amino acid residues are deleted, and other amino acid residues are deleted. Fc-binding proteins, including polypeptides substituted or added to amino acid residues,
Means that. Further, as a preferred embodiment of the above (B),
Fc-binding protein disclosed in Japanese Patent Application Laid-Open No. 2015-08626,
Fc-binding protein disclosed in JP-A-2016-169197,
Fc-binding protein disclosed in JP-A-2017-118871
Fc-binding protein disclosed in WO2018 / 150973,
Fc-binding protein disclosed in WO2019 / 083048,
Can be given.

Further, in the present invention, the antibody having improved ADCC activity is defined as, for example, an antibody having high ADCC activity among the results (elution pattern) obtained by separation using a column packed with a human FcγRIIIa immobilized separating agent. The ratio of the peak area or the peak height is 2% or more, preferably 5% or more, more than the ratio when the culture is performed by controlling the pH of the medium outside the above range (for example, pH 7.1). It means an antibody that is preferably improved by 10% or more, more preferably 15% or more, and even more preferably 20% or more.

The present invention is a method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody and a step of recovering the antibody expressed by the mammalian cell contained in the obtained culture. Is characterized in that the medium is controlled to have a pH higher than pH 6.6 and lower than pH 7.0. INDUSTRIAL APPLICABILITY According to the present invention, an antibody requiring antibody-dependent cellular cytotoxicity such as an anticancer agent can be efficiently produced.

When the antibody produced by the present invention is an antibody containing a human Fc region, the affinity between the antibody and human FcγRIIIa can be evaluated to monitor the culture process and evaluate the medium components in the production method of the present invention. ..

A plasmid map of the expression plasmid pEFd for mammals is shown. It shows the transition of antibody productivity when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in BalanCD CHO Growth A medium controlled to pH 6.8, pH 6.9, pH 7.0 and pH 7.1. .. It shows the transition of antibody productivity when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in BalanCD CHO Growth A medium controlled to pH 6.6, pH 7.0, and pH 7.4. It shows the transition of the viable cell density when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in BalanCD CHO Growth A medium controlled to pH 6.8, pH 6.9, pH 7.0 and pH 7.1. .. It shows the transition of the viable cell density when the anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in BalanCD CHO Growth A medium controlled to pH 6.6, pH 7.0, and pH 7.4. Anti-IL-6R antibody obtained when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in BalanCD CHO Growth A medium controlled to pH 6.8, pH 6.9, pH 7.0 and pH 7.1. The chromatogram by FcR9_F column analysis is shown. Anti-IL-6R antibody obtained when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in BalanCD CHO Growth A medium controlled to pH 6.8, pH 6.9, pH 7.0 and pH 7.1. The area ratio of each peak by FcR9_F column analysis is shown. Changes in antibody productivity when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled to pH 6.8, pH 6.9, pH 7.0 and pH 7.1. Is shown. It shows the transition of antibody productivity when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled to pH 6.6, pH 7.0 and pH 7.4. .. Changes in viable cell density when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled to pH 6.8, pH 6.9, pH 7.0 and pH 7.1. Is shown. It shows the transition of the viable cell density when anti-IL-6R antibody-expressing cells were batch-cultured by a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled to pH 6.6, pH 7.0 and pH 7.4. .. Anti-IL obtained when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled to pH 6.8, pH 6.9, pH 7.0 and pH 7.1. The chromatogram by FcR9_F column analysis of -6R antibody is shown. Anti-IL obtained when anti-IL-6R antibody-expressing cells were batch-cultured with a jar fermenter in EX-CELL Advanced CHO Fed-batch Medium controlled to pH 6.8, pH 6.9, pH 7.0 and pH 7.1. The area ratio of each peak by FcR9_F column analysis of -6R antibody is shown.

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

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 by the following method.
(1-1) Addion of restriction enzyme SacII recognition sequence (CCGCGG) to both the 5'end and 3'end to the gene encoding dihydrofolate reductase (dhfr) and SV40's PolyA set forth in SEQ ID NO: 1. All the genes were synthesized (consigned to Integrated DNA Technologies) and cloned into a plasmid.
(1-2) Escherichia coli JM109 strain was transformed with the plasmid prepared in (1-1). The obtained transformant was cultured, a plasmid was extracted, and then digested with the restriction enzyme SacII to prepare a gene encoding dhfr-SV40PolyA and named dhfr-P1.
(1-3) Described in SEQ ID NO: 2 (5'-TCC [CCGCGG] GCGGGACTCGGGGTTCGAAAATTGACCG-3') and SEQ ID NO: 3 (5'-TCC [CCGCGG] GGTGGCTAGCCTAAGTCGACTG-3') using a pIRES vector (Clontech) as a template. PCR was performed using an oligonucleotide primer consisting of the sequences of (the square brackets in SEQ ID NOs: 2 and 3 indicate the restriction enzyme SacII recognition sequence). Specifically, a reaction solution having the composition shown in Table 1 is prepared, the reaction solution is heat-treated at 98 ° C. for 30 seconds, then the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, 72. The reaction was carried out by repeating 25 cycles with the third step of 5 minutes at ° C as one cycle. By this PCR, the 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 dhfr-P1 prepared in (1-2). Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain an expression vector pIRES-dhfr containing the dhfr gene.

(2) Described in SEQ ID NO: 4 (5'-TTTAAATCA [GCGGCCGC] GCAGCACCATGGCCTGAATAACCTCTG-3') and SEQ ID NO: 5 (5'-GCAAGTAAAACCTACCAAAATGTGGTACGC) [CGGTCGTAC] using pIRES-dhfr prepared in (1) as a template. PCR was performed using an oligonucleotide primer consisting of the above sequences (the square brackets in SEQ ID NO: 4 indicate the limiting enzyme NotI recognition sequence, and the square brackets in SEQ ID NO: 5 indicate the limiting enzyme PvuI recognition sequence). .. Specifically, a reaction solution having the composition shown in Table 2 is prepared, the reaction solution is heat-treated at 98 ° C. for 1 minute, then the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, 72. The reaction was carried out by repeating 30 cycles with the third step of 1 minute at ° C as one cycle. The PCR product amplified by this PCR (SV40 promoter, dhfr, region up to PolyA of SV40) was named dhfr-P2.

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

(4) Restriction enzyme EcoRI at the 5'end of the polynucleotide set forth in SEQ ID NO: 7, which encodes the heavy chain variable region of an anti-interleukin 6 receptor (hereinafter, IL-6R) antibody consisting of the amino acid sequence set forth in SEQ ID NO: 6. A gene with a recognition sequence (GAATTC) and guanine (G) added to suppress the frame shift and a restriction enzyme NheI recognition sequence (GCTAGC) added to the 3'end was totally synthesized and cloned into a plasmid (consigned to FASMAC). The prepared plasmid was named pUC-VH6R. In addition, a restriction enzyme EcoRI recognition sequence (GAATTC) and a frame shift inhibition were added to the 5'end of the polynucleotide set forth in SEQ ID NO: 9, which encodes the light chain variable region of the anti-IL-6R antibody consisting of the amino acid sequence set forth in SEQ ID NO: 8. Therefore, a gene with guanine (G) added and a restriction enzyme BsiWI recognition sequence (CGTACG) added to the 3'end was totally synthesized and cloned into a plasmid (consigned to FASMAC). The prepared plasmid was named pUC-VL6R.

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

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

(2) The cells treated with MTX in (1) can be single-cloned by the ultra-dilution method, and the anti-IL-6R antibody can be stably and highly produced by using the ELISA (Enzyme-Linked Immunosorbent Assay) described below. Cells were selected.
(2-1) Anti-human Fab antibody (Bethyl) was immobilized at 1 μg / well in a well of a 96-well microplate (overnight at 4 ° C.). After completion of immobilization, blocking was performed with 20 mM Tris-hydrochloric acid 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] Protein 20 (trade name) and 150 mM NaCl), the antibody is used. The containing culture supernatant was added, and the antibody was reacted with the immobilized protein (at 30 ° C. for 1 hour).
(2-3) After completion of the reaction, the reaction was washed with the washing buffer, and an anti-human Fc antibody (Bethyl) labeled with peroxidase 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. After that, color development was stopped by adding 1 M phosphoric acid at 50 μL / well, and the absorbance at 450 nm was measured using a microplate reader (Tekan) to obtain a highly measured anti-IL-6R antibody-producing cell line. Selected.

(3) Extradilution is performed while increasing the MTX concentration stepwise (50 nM, 500 nM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM), and clone selection is performed by the ELISA described in (2). Was repeated. As a result, an anti-IL-6R antibody high-producing cell line was obtained.

Example 3 Effect of pH control in batch culture using jar fermenter (Part 1)
(1) A 250 mL Erlenmeyer flask (C.) containing 50 mL of BalanCD CHO Grotth A medium (Irvine Scientific) containing 50 μg / mL canamycin and 30 mL / L of GlutaMAX (Thermo Fisher Scientific). The anti-IL-6R antibody high-expressing cells prepared in No. 2 were inoculated and cultured with shaking under the conditions of 130 rpm, 37 ° C., and 8% CO ₂ .

(2) 100 mL BalanCD containing 50 μg / mL canamycin and 30 mL / L GlutaMAX in 3 250 mL sterilized Jarfermenters (Biot) set with a calibrated pH meter and dissolved oxygen (DO) meter. CHO Growth A medium was added, and the anti-IL-6R antibody high-expressing cells cultured in (1) were inoculated to 0.2 × 10 ⁶ cells / mL, and then the above-mentioned medium was added so that the total volume was 110 mL.

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

(4) Separation column prepared by centrifuging the culture solution after completion of culture to remove cells and impurities, and filling the obtained supernatant with 1.0 mL of MabSelect SuRe LX (GE Healthcare) in an open column. Applied to (equalized with 20 mM Tris-HCl (pH 7.4) containing 150 mM sodium chloride).

(5) The separation column was washed with 10 mL of the buffer used for the equilibration, and then the antibody adsorbed on the separation column was eluted with 5 mL of 0.1 M glycine-hydrochloric acid buffer (pH 3.0). The pH was returned to the neutral region by adding 1 mL of 1 M Tris-HCl (pH 8.0) to the eluent, and 50 mM citric acid buffer (pH 6.) containing 150 mM sodium chloride while concentrating with an ultrafiltration membrane. By exchanging the buffer solution in 5), a high-purity anti-IL-6R antibody having a different controlled pH of the medium was obtained.

The changes in the productivity of the anti-IL-6R antibody due to the difference in the controlled pH of the medium are shown in FIGS. 2 and 3, and the changes in the viable cell density of the anti-IL-6R antibody-expressing cells are shown in FIGS. 4 and 5, respectively. From FIGS. 2 to 5, it can be said that if the medium pH is controlled to be higher than pH 6.6 and lower than pH 7.4, there is no effect on antibody productivity and cell proliferation.

Example 4 Effect of pH control in batch culture using jar fermenter (Part 2)
Among 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 culturing, and human FcγRIIIa was immobilized on the obtained antibodies. Analysis was performed by the following method using a column (FcR9_F column, described in Example 5 of WO2018 / 150973) packed with the carrier (separator).

(1) The FcR9_F column was connected to a high performance liquid chromatography device (Shimadzu Corporation), the column was maintained at a constant temperature of 25 ° C. in a column oven, and a 50 mM citric acid buffer (pH 6) containing 150 mM sodium chloride was added. The column was equilibrated by running (5) at a flow rate of 1.0 mL / min for 10 minutes.

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

(3) After flowing the buffer solution used in (1) at a flow rate of 1.0 mL / min for 2 minutes, a pH gradient (at 18 minutes) with a 50 mM citric acid buffer solution (pH 4.5) containing 150 mM sodium chloride. The antibody adsorbed on the FcR9_F column was eluted with a 50 mM citric acid buffer (pH 4.5) containing 150 mM sodium chloride as a 100% gradient).

The obtained chromatogram is shown in FIG. 6, and the area ratio of each peak calculated from the chromatogram is shown in FIG. 7 and Table 3, respectively. The higher the ADCC (antibody-dependent cellular cytotoxicity) activity, the stronger the binding force to the FcγRIIIa-immobilized separating agent (Japanese Patent Laid-Open No. 2016-0223152, WO2018 / 150973). That is, it can be said that the higher the area ratio of the peak 3 in FIG. 6, the higher the ADCC activity. When the culture medium was cultured with the pH controlled to pH 6.8 and pH 6.9, 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 for culturing, the area ratio of peak 3 was less than 30% (pH 7.0: 22.6%, pH 7.1: 26.7%).

From the above results, it can be seen that the ADCC activity of the antibody expressed by the mammalian cell capable of expressing the antibody is improved by controlling the medium pH to pH 6.8 and pH 6.9 and culturing.

Example 5 Effect of pH control in batch culture using jar fermenter (Part 3)
Culturing and antibody productivity of anti-IL-6R antibody high-expressing cells prepared in Example 2 by the same method as in Example 3 except that the medium was EX-CELL Advanced CHO Fed-batch Medium (Merck). The number of living cells was measured, and the antibody expressed by the cells was analyzed by the FcR9_F column in the same manner as in Example 4.

The changes in the productivity of the anti-IL-6R antibody due to the difference in the controlled pH of the medium are shown in FIGS. 8 and 9, and the changes in the viable cell density of the anti-IL-6R antibody-expressing cells are shown in FIGS. 10 and 11, respectively. As in the case of using BalanCD as a medium (Example 3), if the medium pH is controlled to a pH higher than pH 6.6 and lower than pH 7.4, it can be said that there is no effect on antibody productivity and cell proliferation.

The chromatogram obtained by the analysis by the FcR9_F column is shown in FIG. 12, and the area ratio of each peak calculated from the chromatogram is shown in FIGS. 13 and 4, respectively. When culturing with the medium pH controlled to pH 6.8 and pH 6.9, the area ratio of peak 3 occupied about 40% (pH 6.8: 40.0%, pH 6.9: 39.2%). ). On the other hand, when the culture medium pH was controlled to pH 7.0 and pH 7.1 and cultured, the area ratio of peak 3 was reduced as compared with the case where the culture medium was controlled to pH 6.8 and pH 6.9 (pH 7.0 :). 36.5%, pH 7.1: 31.4%).

From the above results, as in the case of using BalanCD as a medium (Example 4), the antibody expressed by mammalian cells capable of expressing the antibody by controlling the medium pH to pH 6.8 and pH 6.9 and culturing the medium. It can be seen that the ADCC activity of the cells is improved.

Claims (5)

In a method for producing an antibody, which comprises a step of culturing a mammalian cell capable of expressing an antibody and a step of recovering the antibody expressed by the mammalian cell contained in the obtained culture.
A method for producing an antibody having improved antibody-dependent cellular cytotoxicity by performing the culture step in a medium controlled to have a pH higher than pH 6.6 and lower than pH 7.0. The production method according to claim 1, wherein the antibody is an antibody containing a human Fc region. A method for monitoring a culture step in the production method according to claim 2, by evaluating the affinity between the antibody obtained by the production method according to claim 2 and human FcγRIIIa. A method for evaluating a medium pH in the production method according to claim 2, by evaluating the affinity between the antibody obtained by the production method according to claim 2 and human FcγRIIIa. The affinity between the antibody obtained by the production method according to claim 2 and human FcγRIIIa is evaluated based on the binding force between the antibody obtained by the production method according to claim 2 and the human FcγRIIIa immobilization separating agent. , The method according to claim 3 or 4.

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