RU2549692C1 - Infliximab, optimised gene of monoclonal antibody (versions), recombinant cell line, antibody producer and method for biosynthesis thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented optimised genes of light and heavy chains of Infliximab, an anti-tumour necrosis factor alpha (TNF-alpha) antibody, as well as a cell line VKPM-N-131, and a method for antibody biosynthesis. Nucleotide sequences of the genes coding the light and heavy chains of Infliximab are optimised in order to provide the content of codones most specific for mammals; the G/C content is expected to make 50-60% of the total composition; the absence of expanded tracts of a degenerate composition and the absence of RNA secondary structures.

EFFECT: Chinese hamster ovary cell line (CHO) produced by transfection by expression structures containing the genetic sequences according to the invention, enables producing at least 50 mg/l of the monoclonal antibody Infliximab.

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Description

The invention relates to biotechnology and is intended to produce a recombinant monoclonal antibody infliximab. The biosynthesis of the target protein is carried out by suspension cultivation of a recombinant VKPM-H-131 cell line obtained by introducing an expression vector containing optimized infliximab heavy and light chain genes into the ovary cells of the Chinese hamster CHO.

The recombinant VKPM-H-131 cell line was obtained by transfection of the CHO-S suspension cell line with the pVitroneo-Infliximab-LC-HC vector containing the optimized infliximab antibody light chain gene and the optimized heavy chain gene coupled to the gene encoding the antibiotic resistance marker geneticin (G418) using the site of the internal landing of the ribosome (IRES element).

Infliximab is a chimeric monoclonal antibody specific for tumor necrosis factor alpha (TNF-alpha). It is used to treat rheumatoid arthritis and other inflammatory diseases. Infliximab has a high degree of affinity for TNF-alpha-cytokine with a wide range of biological effects [1]. TNF-alpha is a mediator of the inflammatory response, triggering a cascade of various immunological reactions. Infliximab quickly binds and forms a stable compound with both forms of human TNF-alpha (soluble and transmembrane), reducing its functional activity [2]. Blocking the binding of TNF-alpha to immunocompetent cells by this antibody provides an anti-inflammatory effect and stable remission in patients [1, 3].

Infliximab is an IgG antibody of subclass 1 and consists of 2 identical light chains and 2 identical heavy chains. The mass of a full-sized antibody is 145.6 kDa, light and heavy chains 23.4 kDa and 49.4 kDa, respectively [1]. Infliximab, like other antibodies, is characterized by N-glycosylation, which consists in the attachment of two-antenna carbohydrate chains to asparagine residues located in the constant regions of the molecules. The main glycoforms of the antibody are G0F (fucosylated, without galactose residues - 50-60%) and G1F (fucosylated, with one galactose residue - 30-40%). Glycosylation is the most important characteristic of monoclonal antibodies used in biopharmaceuticals, since molecular packing, stability, solubility, as well as biological activity and pharmacokinetic parameters depend on it [4-6].

Full-sized monoclonal antibodies are produced exclusively using mammalian cells, since only the mammalian cell apparatus is able to ensure the correct assembly of the whole antibody and its correct glycosylation, which is a necessary condition for the manifestation of its biological activity and stability in vivo [7, 8]. The well-known drug monoclonal antibody infliximab (Remicade) was obtained by the company in Sp2 / 0 mouse myeloma cells [9]. However, the use of cell lines having a tumor origin in the production of biotechnological drugs does not meet the quality requirements for drugs of this type.

Object of the invention: expanding the arsenal of methods for the biosynthesis of monoclonal antibodies infliximab.

The problem is solved by:

- obtaining optimized genes encoding the light and heavy chains of the monoclonal antibody infliximab;

- constructing the VKPM-H-131 cell line by maintaining the pVitroneo-Infliximab-LC-HC vector containing optimized infliximab light and heavy chain genes into the ovary cells of the Chinese hamster of the CHO-S line;

- development of a method for biosynthesis of infliximab monoclonal antibody by suspension cultivation of a recombinant VKPM-H-131 cell line.

For the production of complex recombinant proteins, such as monoclonal antibodies, the Chinese hamster ovary CHO cell culture is mainly used [10]. The advantages of using CHO cells are their high proliferation rate, ease of cultivation and a good degree of characterization, which greatly simplifies the registration of recombinant drugs [7]. When creating a stable cell line, the optimal choice for further use in production is the suspension variant of CHO-S cell culture [8, 11, 12]. Suspension cultivation allows cells to grow to high densities (10 ⁷ -10 ⁸ cells / ml medium) without the use of blood serum, which increases their biological safety and facilitates the subsequent stages of purification [11, 13].

In the production of glycosylated recombinant human proteins, CHO cell lines are used that carry the target protein genes integrated into the genome under the control of strong promoters of cellular or viral origin. The most effective ones include the CMV promoters (promoter-enhancer element of the early human cytomegalovirus genes), EF1α (promoter of the alpha subunit of translation elongation factor 1) [14–16], or the CMV-EF1α hybrid promoter containing the core region of the EF1α promoter constructed on their basis and an enhancer of the promoter-enhancer region of the CMV gene. It was shown that for some transgenes, CMV-EF1α provides a higher level of expression compared with CMV [15, 17]. The above elements are used in the construction of the inventive cell line.

In order to optimize expression, in addition to choosing effective promoters, methods have been developed that lead to an increase in the biosynthesis of the target protein. An important optimizable parameter is the frequency of use of synonymous codons in the gene of interest [18]. Numerous programs and strategies for such optimization are based on the well-known positive correlation between the level of expression of interest genes and the correspondence of codon usage frequencies in the gene of interest and the body as a whole [18, 19]. Moreover, in order to optimize the codon composition, the primary structure of the gene of interest is changed. However, the result of such a change is not only an improvement in the codon composition of the gene, but also the appearance of secondary structures in the transcripts of this gene that contribute to the improvement of its translation [20]. In the claimed invention, the optimization of the codon composition of genes encoding the target recombinant protein is carried out.

The method in General

To obtain the infliximab monoclonal antibody, cells of the Chinese hamster VKPM-H-131 recombinant ovary cell line are seeded in ventilated flasks with a volume of 125 ml containing 20-30 ml of medium to a density of 0.2-0.5 million cells / ml. For cultivation using serum-free Power CHO-2 medium (Lonza, Switzerland) with the addition of L-glutamine, sodium hypoxanthine and thymidine to a final concentration of 2-8 mm, 50-150 μm and 12-20 μm, respectively. Cultivation is carried out at a temperature of 37 ° C in a humid environment (humidity not less than 70-90%) with a 5% CO ₂ content with stirring at a speed of 100-130 rpm. The process is stopped after reducing the content of living cells to the level of 50-70%. After 7-9 days, the concentration of the target product in the culture fluid is determined by enzyme-linked immunosorbent assay, in particular, using a set of reagents Serazym IgG ELISA kit (Seramun, Germany). It is at least 50 mg / l of the monoclonal antibody infliximab, which corresponds to a specific productivity of at least 15 pg per cell per day.

The invention is illustrated by the following figures.

Figure 1. PVitroneo-Infliximab-LC-HC expression vector map: LC infliximab - mAb light chain, HC infliximab - mAb heavy chain, CMV promoter - human cytomegalovirus early gene promoter, EM7 promoter - bacterial antibiotic resistance gene promoter, rEF1, mEF1 - alpha promoter subunits of translation elongation factor 1, CMV enhancer — enhancer of the early human cytomegalovirus genes, SV40 enhancer — enhancer of the early human polyomavirus genes, Neo — neomycin phosphor transferase gene, pMB1 ori — plasmid replication start sites, SV40 polyV, A, E, EF, EF ES is the site of internal planting of the ribosome of the FMDV virus.

Figure 2. Electrophoregram of plasmid pVitroneo-Infliximab-LC-HC before and after digestion with restriction enzymes BamHI and Kpn2I. Expected fragments 6988, 1422 bp (along tracks: 1 — undigested plasmid, 2 — molecular weight marker, 3 — cleaved plasmid).

Figure 3. Electrophoregram of the culture fluid of clones producing monoclonal antibodies infliximab. SDS-PAGE electrophoresis was carried out under reducing conditions, the culture fluid of clones 3, 5, 8 and 14 was concentrated 10 times. St - standard, Remicade drug (Janssen Biotech., USA): 0.15, 0.3 and 1 μg per lane, respectively.

Example 1. Construction of expression vector pVitroneo-Infliximab-LC-HC containing optimized genes for the heavy and light chain infliximab

The nucleotide sequences of genes encoding the light and heavy chains of infliximab are selected based on their amino acid sequences.

Gene optimization is carried out in accordance with the following parameters:

- the content of codons most typical for mammals;

- the content of G / C should be 50-60% of the total composition;

- the absence of extended tracts of degenerate composition;

- lack of secondary RNA structures.

The optimized nucleotide sequences of the infliximab light chain SEQ ID NO: 1 and the infliximab heavy chain SEQ ID NO: 2 were synthesized by Service Gene (St. Petersburg). For constructing the expression vector, the base expression vector pVitro-neo (InvivoGen, USA) is used. The pVITROneo vector contains the neomycin phosphotransferase gene, which provides resistance to antibiotics of the neomycin group, as well as two chimeric promoters, one of which is a hybrid of the promoter region of the rat EF1α gene and the enhancer region of CMV, and the second is a hybrid of the promoter region of the mouse EF1α gene and enhancer region SV40 (FIG. 1).

The genes encoding the infliximab light and heavy chains are cloned into the pVitro-neo vector to obtain the pVitroneo-Infliximab-LC-HC expression vector (FIG. 1). In this case, the heavy chain gene of SEQ ID NO: 2 is connected via an IRES element to the gene encoding the antibiotic resistance marker G418. Thus, these two genes are transcribed into one mRNA, from which two proteins are translated, due to which stricter control of heavy chain expression is achieved. The infliximab antibody light chain gene SEQ ID NO: 1 was introduced into the plasmid at the BglII and NheI restriction sites, the infliximab antibody heavy chain gene SEQ ID NO: 2 was introduced at the Kpn2I and BamHI restriction sites. Cloning yields the pVitroneo-Infliximab-LC-HC vector, in which the light chain gene is controlled by the SV40 / mEF1 chimeric promoter, and the heavy chain gene is controlled by the CMVenh / rEF1 promoter (Fig. 1). The correct assembly of the expression vector is checked using extended restriction analysis (figure 2). The nucleotide sequences of both genes and adjacent sites are confirmed by sequencing.

Example 2. Transient transfection and analysis of the level of expression of infliximab by ELISA

For transient (temporary) transfection with expression constructs containing the infliximab heavy and light chain genes, CHO-S cells (Invitrogen, USA) are used, which are cultured in 125 ml ventilated flasks at a temperature of 37 ° C in a humid environment (80% humidity) ) with 5% CO ₂ in serum-free Power CHO-2 medium (Lonza, Switzerland) with the addition of L-glutamine, sodium hypoxanthine and thymidine to a final concentration of 4 mM, 100 μM and 16 μM, respectively. The cultivation is carried out with a stirring speed of 120 rpm After the suspension culture reaches a density of ~ 1 * 10 ⁶ cells / ml, Power CHO-2 medium is replaced with an equal volume of CD CHO medium (Invitrogen, USA) for transfection.

Transfection with the expression vector is performed using the FreeStyle MAX reagent (Invitrogen, USA) in accordance with the standard protocol of the Invitrogen company for 6-well plates. 24 hours after transfection, CD CHO medium was replaced with an equal volume of Power CHO-2 medium. To assess the effectiveness of transfection, the control vector pEYFP-N1 (Clontech, USA) containing the green fluorescent protein gene is used.

To measure the concentration of infliximab synthesized by cells, an enzyme-linked immunosorbent assay (ELISA) is used, in particular, using the Serazym IgG ELISA kit (Seramun, Germany). To construct the calibration curve of ELISA using the original drug Remicade (Janssen Biotech., USA).

The concentration of the target antibody is measured 4 days after transfection. The concentration of infliximab in the culture fluid is at least 7 mg / L.

Example 3. Obtaining a stable clone of the cell line VKPM-H-131, producer of the monoclonal antibody infliximab

To select stable clones, the day after transfection, the cells obtained in Example 2 are transferred to Power CHO-2 medium containing the G418 antibiotic to a concentration of 400 μg / ml. An antibiotic cell suspension is transferred to the wells of 96-well plates at a concentration of 300 cells per well. The total number of tablets is 10 pieces. The cultivation of tablets with cells is carried out in a stationary position for 20 days at a temperature of 37 ° C in a humid environment with 5% CO ₂ content. Every 7 days, 100 μl of fresh medium is added to the wells of the plates.

Starting from 12 days, the growth of cells in the wells is monitored under a microscope, noting those wells in which the growth of colonies from one cell is observed. Since the number of cells that enter the well is subject to the Poisson distribution, the probability of the absence of clonality (getting 2 or more cells into one well) can be calculated based on the proportion of positive holes according to the formula:

P (n> 1) = 1-Σ (e ^-λ λ ⁿ / n!),

where λ = -ln (P (0)) is the fraction of wells in which cell growth is observed,

n is 0; 1 - the number of cells in the hole.

Upon reaching 80-100% confluency, individual clones are transferred to new 96-well plates. After 5 days, culture fluid samples are analyzed for the presence of infliximab. The clones that showed the highest productivity when cultured in 96-well plates were transferred to 24-well plates and cultured for 5 days, and then transferred to 125 ml flasks for suspension cultivation. According to the results of the production cycle in flasks, the productivity of the clones is evaluated. Based on the criteria of growth, viability and productivity, the best clones are selected for which a cell bank is created.

The selection of stable clones by the method of limiting dilution is carried out within 20 days. According to the results of productivity analysis, the best clones for scaling the process are transferred sequentially to 24-well, 6-well plates, then to culture bottles and flasks. In the process of cultivation analyze cell density and viability.

According to the results of the exponential growth phase (first 4 days of cultivation), the cell doubling time is calculated by the formula:

T ₂ = T / log ₂ (N _con / N _beg ),

where T is the duration of cultivation (96 h), N _con and N _beg is the final and initial cell density, respectively.

After completion of cultivation (on day 7), the concentration of infliximab in the culture fluid samples was measured by ELISA. For the clone producer selected in this way, the deposit was made to the All-Russian Collection of Industrial Microorganisms of the Federal State Unitary Enterprise GosNIIgenetika (VKPM) under the number VKPM-N-131.

Example 4. Characterization of the resulting monoclonal antibody infliximab

The analysis of the productivity of the claimed cell line is performed using the ELISA kit Serazym IgG ELISA kit (Seramun, Germany). To assess the ratio of light and heavy chains, as well as the level of antibody synthesis by the obtained clones, the culture fluid was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis) under denaturing conditions (Fig. 3).

SDS-PAGE electrophoresis under reducing conditions was carried out according to the standard Laemmli technique in 12% gel, followed by Coomassie-R250 staining [21]. As a standard, the drug Remicade (Janssen Biotech., USA) is used. The intensity of the bands on the gels and membranes is calculated using the Quantity One program (BioRad, USA). From the results shown in figure 3, it follows that the synthesized monoclonal antibody infliximab in the ratio of the content of light and heavy chains corresponds to the standard.

Thus, a method has been developed for the production of infliximab monoclonal antibody based on ovary cells of the Chinese hamster of the CHO line, which are preferred for the production of biopharmaceuticals, in contrast to the method for producing the closest analogue, using murine myeloma cells as a producer. The cultivation of the producer, according to the claimed method, in contrast to the method of the closest analogue, is carried out in a serum-free nutrient medium, which meets modern safety requirements for the quality of biopharmaceutical preparations. Using this method allows you to achieve a specific productivity of at least 15 picograms per cell per day. This value of specific productivity is comparable to or exceeds the values achieved by the classical method of selection or amplification of highly productive clones of monoclonal antibodies using methotrexate or other selection methods [22-26].

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Claims (4)

1. The light chain gene of the monoclonal antibody infliximab having the optimized nucleotide sequence of SEQ ID NO: 1. 2. The heavy chain gene of the monoclonal antibody infliximab having the optimized nucleotide sequence of SEQ ID NO: 2. 3. The recombinant cell line VKPM-H-131 is a producer of the infliximab monoclonal antibody, comprising two identical light chains encoded by the optimized gene according to claim 1, and two identical heavy chains encoded by the optimized gene, according to claim 2. 4. The method of biosynthesis of a monoclonal antibody infliximab by suspension cultivation of a recombinant cell line according to claim 3.

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