RU2537266C1 - Optimised dna sequences, coding light and heavy chains of monoclonal antibody, binding tumour necrosis factor alpha, intended for antibody biosynthesis, method of obtaining antibody by recombinant cell line cho-s - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, in particular to molecular biology, biotechnology, genetic engineering. The invention represents optimised DNA sequences, coding a light and heavy chain of a monoclonal antibody, intended for the synthesis of the antibody in recombinant CHO-S cells, and a method of obtaining the said antibody in the said cells. The performed work resulted in the elaboration of several conditions making it possible to obtain the monoclonal antibody with high productivity.

EFFECT: invention makes it possible to maximise the cell productivity and optimise post-translational modifications of the antibody, in particular glycosylation of heavy chains of the antibody.

3 cl, 2 dwg, 6 tbl

Description

The invention relates to a pharmaceutical substance of a therapeutic monoclonal antibody that neutralizes human TNF-α activity. An optimized DNA sequence, an antibody biosynthesis method based on a recombinant CHO-S cell line with a description of important parameters of the final product are presented. The invention can be used to create a pharmaceutical product used for autoimmune diseases, in particular rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, psoriatic arthritis, etc.

Antibodies (immunoglobulins) are a class of biological molecules synthesized by the B-lymphocytes of the immune system. They have the property of highly specific binding to foreign objects, thereby neutralizing and inducing their destruction by killer cells. With the development of hybridoma cell technologies and molecular biological methods, it became possible to artificially create and produce antibodies specific for only a given molecular target (monoclonal antibodies). Currently, this property of monoclonal antibodies is actively used to treat various oncological and autoimmune diseases. Autoimmune diseases, among which rheumatoid arthritis dominates, are a serious social and economic problem. The gold standard for the treatment of rheumatoid arthritis is the use of recombinant drugs based on monoclonal antibodies to tumor necrosis factor (TNF). Blocking the binding of TNF to immunocompetent cells by monoclonal antibodies provides the anti-inflammatory effect of such drugs and stable remission in patients.

For the purpose of industrial production of proteins of pharmaceutical importance, in particular monoclonal antibodies, CHO cells are widely used. An important property of CHO cells for use in cell biotechnology is their wide adaptability to various cultivation conditions and high genetic plasticity, which makes it possible to carry out various genetic manipulations with these cells with relative ease. In addition, these cells are capable of growth at a very high cell density in large large-sized fermenters up to 10 thousand liters. Moreover, CHO cells comply with the principles of biosafety, since the vast majority of human pathogenic viruses, including immunodeficiency, polio, influenza and herpes viruses, are unable to reproduce.

The aforementioned qualities have made CHO cells the main cell culture in world biotechnology. Currently, about 70% of recombinant pharmaceutical biologics is made on the basis of CHO cells.

The monoclonal antibody (Remicade®) monoclonal antibody based on the monoclonal anti-human TNF-α antibody that is already on the market is still produced in the murine melanoma cell line SP2 / 0, developed about 20 years ago, while the developed strain producing monoclonal anti-TNF antibody α-human is produced in CHO-S cells, which are today the "gold standard" in the ratio of efficiency and safety in the production of biopharmaceuticals, and allows you to minimize the risks of contamination of the product with endogenous viruses and producer cell proteins during the manufacturing process. Obviously, the transition to a fundamentally new cellular expression system requires the creation and development of each stage of a new technological process - from cultivation to the stages of final purification and obtaining GLF.

Effective expression requires optimization of the DNA sequence of the target protein. It was shown that effective expression is influenced by factors such as the percentage of G-pairs, the secondary structure of RNA, the presence of internal splicing signals, the presence of short reading frames, long homopolymer sequences, the use of codons and pairs of codons with different degrees of occurrence. It has been shown that for antibodies, DNA sequence optimization can significantly increase expression. Thus, DNA sequences encoding the heavy and light chains of a monoclonal antibody to TNF have been optimized to obtain a high level of expression.

The development of a cell line producing a monoclonal antibody to human TNF-α included the following steps:

- optimization of DNA sequences encoding the light and heavy chain of the antibody (figure 1);

- the creation of two expression constructs encoding the heavy and light chains of a recombinant monoclonal antibody (figure 2);

- carrying out transfection of CHO cells with the obtained expression plasmids;

- selection of clones and the creation of a cell bank of the producer strain.

Expression plasmids v240 and v241 containing cDNAs encoding a recombinant monoclonal antibody to human TNF-α were constructed under the control of the cytomegalovirus promoter. Expression plasmids were synthesized using standard molecular biological methods.

The transfection of the CHO-S cell line was carried out by chemical method. Successfully transfected cells were selected using antibiotics: zeocin and blasticidin. To select clones with maximum antibody performance, the method of limiting (limiting) dilutions was used, followed by assessment of the concentration of monoclonal antibodies using ELISA. Selected clones were amplified under normal cell growth and cell banks were created.

The technical result of the invention is to maximize cell productivity and optimize post-translational modifications of the antibody, in particular glycosylation of antibody heavy chains. As a result of the work done, several conditions were developed that made it possible to obtain a monoclonal antibody with high productivity and various heavy chain glycosylation depending on the cultivation conditions.

Examples

ActiCHO P CD medium (GE Healthcare) supplemented with 6 mM L-glutamine and 0.1% Pluronic F-68 was chosen as the main culture medium. UMG (300 mM uridine, 1500 mM galactose, 0.6 mM MnCl ₂ ), ActiCHO FeedA CD (GE Healthcare) together with ActiCHO FeedB CD (GE Healthcare) (in a volume ratio of 10: 1, added separately) were used as additives glucose 250 g / l; and FunctionMAX Titer Enhancer (Life Technologies). The cultivation schemes developed that describe the order of application of the various components are presented in Table 1. Cells were cultured in shaker flasks on an orbital shaker at ~ 110 rpm at 36.5 ° C, 5% CO ₂ in an incubator. For cultivation used cells deposited in the All-Russian Collection of Industrial Microorganisms under registration number H-145.

Table 1 The scheme of making components during cultivation Components Day 0 3 four 5 6 7 8 9 10 eleven 12 13 fourteen ActiCHO Feed-A + B 1.76% 1.7 6% 1.7 6% 1.7 6% 1.7 6% 1.7 6% 1.7 6% 1.7 6% 1.7 6% 1.7 6% 1.7 6% 1.7 6% 1.7 6% FM 3.5% 3.5% 3.5% 3.5% Umg 4x 4x 4x 4x 4x 4x

It was experimentally found that the addition of NaCl to a concentration of 0.5 M and 1M increases the yield of the target antibody after the first stage of affinity purification. As an affinity sorbent, Mab Select (GE Healthcare) was used. To optimize the conditions used 5 ml of sorbent. Before applying the samples, the column was balanced with 4-5 volumes (relative to the column volume) of Mab 1 buffer (20 mM PhNa, 0.5 M NaCl, pH 7.0). Elution from the column is carried out with 4-5 volumes of Mab 2 buffer (100 mM Gly-HCl, pH 3.5). The yield was estimated by the amount of protein measured spectrophotometrically after elution. It was proved that the introduction of sodium chloride before the stage of affinity chromatography at a concentration of 0.5 M and 1 M increases the yield of the antibody (table 2). It was decided to add sodium chloride to the culture fluid at a concentration of 0.5 M immediately before the stage of filtration of the culture fluid.

table 2 The yield of antibodies to TNF-α with MabSelect depending on the concentration of sodium chloride in the culture fluid The concentration of NaCl in the culture fluid, M MabSelect sorbent capacity, mg / ml 0 17.7 0.5 26.28 one 26.3

Before antibody purification, the culture fluid is thawed, NaCl is added to a final concentration of 0.5 M and filtered. The first stage of purification is carried out at a temperature of 2-8 ° C. The cleaning conditions are the same as described above, taking into account the larger volume of the sorbent. During the elution, the protein solution is titrated to pH 5.5 ± 0.2 with a solution of 1 M Tris-HCl pH 8.0. The second stage of purification of monoclonal antibodies is carried out on a sorbent Fractogel SO ₃ (Merck) at a temperature of 2-8 ° C. The column was equilibrated with Frac 1 buffer (20 mM NaAc, pH 5.5). The monoclonal antibody solution is diluted with Frac 1 equilibration buffer (20 mM NaAc, pH 5.5) to a conductivity value of no higher than 5 mS / cm (dilution factor 3-4 times), adjusted pH to 5.5 ± 0.2 0.25 M acetic acid is applied to the column. The column was washed with 300 ml of Frac 1 buffer. The column was eluted with a stepwise gradient of 20% Frac 2 buffer (20 mM NaAc, 1 M NaCl, pH 5.5) in Frac 1 buffer (20 mM NaAc, pH 5.5). Starting at an optical density of 10 mAU (UV 280 nm), fractions were collected and analyzed by HPLC-HPLC. According to the analysis results, fractions with a purity of 98.5% and higher are combined. The resulting protein solution is filtered through a filter head onto a Steritop 0.22 μm vial. The antibody solution is then concentrated using tangential filtration membranes with a cutoff limit of 50 kDa, balanced with GLF buffer (5 mM sodium hydrogen phosphate, 1.6 mM sodium dihydrogen phosphate, 142 mM sucrose, 0.05% polysorbate-80). Next, the solution is subjected to the last stage - gel filtration chromatography on a Sephadex G25 sorbent (GE Healthcare). The process is carried out at a temperature of 2-8 ° C. A solution of monoclonal antibody was applied to the column, previously equilibrated with GLF buffer, and elution from the column was carried out in a volume of GLF buffer. The resulting antibody solution is filtered through a 0.2 μm filter and at -20 ° C.

The composition of a stable pharmaceutical composition in the form of a lyphilisate, which includes, mg, was revealed: developed antibody against human TNF-α - 100; sodium hydrogen phosphate dihydrate - 6.1; sodium dihydrogen phosphate monohydrate - 2.2; sucrose - 500 and polysorbate - 80-0.5. To obtain this composition, the following drying conditions were used:

Freezing - 2 hours at -30 - -40 ° C,

Sublimation at -20 ° C, 6 hours, 70-110 mTorr,

Step transition to desorption - from -20 to + 25 ° С for 2 hours at 70-110 mTorr,

Desorption at + 25 ° C, 5 hours, 70-110 mTorr.

In vitro and in vivo studies were performed to evaluate biological activity. In particular, a number of studies were conducted to comprehensively evaluate the binding of an antibody to an antigen: by ELISA, binding to human TNF-α in soluble monomeric and trimeric forms was studied, binding to transmembrane insoluble TNF-α on the cell surface was studied by sorting fluorescently activated cells. and, moreover, using the method of surface plasma resonance, dissociation constants were determined (for a complex of antibodies with the monomeric and trimeric form of TNF-α). To evaluate biological activity, a study of neutralizing activity against WEHI-164 was conducted. The results are presented in table 3. The data confirm the presence of biological activity of the obtained antibodies in vitro.

Table 3 The results of the evaluation of the biological activity of in vitro monoclonal antibodies to human TNF-α Study Value Standard deviation Binding to soluble human TNF-a in monomeric form, EC50 (nM) 0.1335 0.0262 Binding to soluble human TNF-a in a homotrimeric form, EC50 (nM) 0.0251 0.0037 Binding to human transmembrane TNF-a, EC50 (nM) 5.632 0.5928 Neutralizing activity against WEHI-164 cells, EC50 (nM) 0.4854 0.0888

The effectiveness of the developed monoclonal antibody to human TNF-α was proved in vivo on transgenic Tg197 mice with overexpression of human TNF-α, leading to the development of polyarthritis, starting from the 3-4th week of the animal’s life. The therapeutic efficacy of the developed antibody was evaluated in preventing the onset of arthritis symptoms in genetically modified Tg197 mice. To conduct this study, mice (12 mice in each group, an equal number of males and females) received the test antibody bolus, once (at doses of 10 mg / kg and 30 mg / kg), intraperitoneally in the fourth week of life after the onset of arthritis, but before development of these manifestations, and then assessed the state of these mice over the age of 5 weeks until reaching the 9th weeks of life. As the results of the study showed, treatment with the studied drug at a dosage of 10 mg / kg had an overwhelming effect on the development of manifestations of polyarthritis, but did not provide complete protection against the development of this disease. In the case of a dosage of 30 mg / kg, the studied drug had an overwhelming effect on the development of manifestations of polyarthritis and kept them statistically uniform compared to mice aged 3 weeks from the control group who did not receive the drug. Tables 4-6 show the data of the study of the effectiveness of the developed drug on Tg197 mice.

Table 4 The average body weight in the group Average body weight (g) Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Age 4 weeks, control group not receiving the drug 10.93 - - - - - G1-PRM-002 10 mg / kg 12.24 15.28 16.62 17.36 18.02 18.88 G2-PRM-002 30 mg / kg 12.27 15.75 17.51 18.21 19.78 20.86 G3-physiological 12.26 14.34 15.23 15,58 15.71 16.40

Table 5 Average in-vivo arthritis score The average in-vivo arthritis score Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Age 4 weeks, control group not receiving the drug 0.38 - - - - - G1-PRM-002 10 mg / kg 0.40 0.43 0.66 0.71 0.82 0.91 G2-PRM-002 30 mg / kg 0.38 0.28 0.36 0.32 0.35 0.45 G3 saline 0.29 0.70 0.88 0.99 1.27 1.25

Table 6 The average histopathological assessment of the level of development of arthritis, ex-vivo Average histopathological assessment of arthritis Week 4 Week 9 Age 4 weeks, control group not receiving the drug 1,50 - G1-PRM-002 10 mg / kg - 2.83 G2-PRM-002 30 mg / kg - 1.42 G3 saline - 3.27

Claims (3)

1. DNA encoding the heavy chain of an antibody that binds human TNF-α, represented by SEQ ID: 1 and intended for the synthesis of this antibody in recombinant CHO-S cells. 2. DNA encoding the light chain of an antibody that binds human TNF-α, represented by SEQ ID: 2 and intended for the synthesis of this antibody in recombinant CHO-S cells. 3. A method for producing a monoclonal antibody that binds human TNF-α, comprising introducing DNA according to claims 1 and 2 into recombinant CHO-S cells, their cultivation and subsequent purification of antibodies to TNF-α.

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