RU2556816C1 - Strain of cells of chinese hamster ovaries - producer of recombinant antibody against tumour necrosis factor alpha of human being - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain of cells of the Chinese hamster ovaries CHO-Inflix 20/5 is produced, transfected by vectors, expressing light and heavy chains of chimeric antibody against tumour necrosis factor alpha (TNF-α) of human being is produced. The strain is deposited into the Russian collection of cellular cultures under No. RKKK(P)760D.

EFFECT: invention allows to produce chimeric antibody with the specific efficiency no less than 33,5 picograms per cell a day.

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Description

The invention relates to biotechnology, in particular the production of chimeric antibodies to human tumor necrosis factor.

Tumor necrosis factor (currently referred to as tumor necrosis factor alpha) (TNF-α) is a cytokine produced by a number of cells and tissues, primarily macrophages, monocytes, CD4 + and CD8 + T-lymphocytes in response to stimulation by bacterial endotoxin ( Ketlinsky S.A., Simbirtsev A.S. Cytokines, St. Petersburg, 2008). TNF-α in its biologically active form is a trimer, and the groove formed by neighboring subunits is important for the interaction of the cytokine receptor. Having a pleiotropic effect, TNF-α is a mediator of the inflammatory process. In pathological conditions, increased production of TNF can be accompanied by tissue damage, vascular and cellular reactions that develop and support chronic inflammation. In particular, increased production of TNF-α plays an important role in sepsis, transplant rejection and a number of autoimmune diseases, such as rheumatoid arthritis, Crohn’s disease, systemic lupus erythematosus, ulcerative colitis, multiple sclerosis, autoimmune diabetes, ulcerative colitis and many other diseases and pathological conditions.

TNF-α inhibitors are molecules, primarily artificially synthesized pharmaceutical agents that can inhibit the activity of the pro-inflammatory cytokine TNF-α and thereby affect the symptoms of a number of diseases. In this regard, the search for TNF-α inhibitors is of great practical importance, since a substance with sufficient specificity and selectivity for TNF-α can be an effective prophylactic or therapeutic pharmaceutical compound for the prevention or treatment of disorders in which TNF-α is involved as a causative factor. The most promising direction in this case is the use of antibodies for these purposes. Antibody-based therapies have significant advantages over chemical pharmaceuticals because they have perfect selectivity for their target and low intrinsic toxicity.

Methods for treating toxic shock, regression of tumors, inhibiting cytotoxicity, autoimmune disease such as rheumatoid arthritis and Crohn’s disease, graft versus host disease, bacterial meningitis using antibodies to TNF-α (RU 2455312, 2012; US 6448380) are currently described. , 2002; US 6451983, 2002; US 6498237, 2002; US 5672347, 1997; US 5656272, 1997).

However, so far none of the currently available drugs is effective enough for the treatment of autoimmune diseases and most have limitations due to severe toxicity. In addition, traditional antibodies have a binding activity that depends on pH, and therefore, they are not suitable for use in media outside the physiological pH range, for example, in the treatment of gastric bleeding, stomach surgery, etc.

Among antibodies capable of inhibiting TNF-α, the most famous are such as infliximab (Remicade) - a chimeric immunoglobulin monoclonal antibody to TNF-α, adalimumab (Humira) - a human monoclonal antibody to TNF-α, as well as hybrid proteins such as etanercept (Enbrel ) - human recombinant protein receptor for TNF-α and certolizumab pegol (Symzia) - pegylated monoclonal antibody to TNF-α, free of the Fc fragment (www.rheumatology.kiev.ua/wp-content/uploads/magazine/38/ 21.pd).

Moreover, the most extensive (more than 300,000 patients) and long-term (more than 6 years) clinical experience has been accumulated in relation to the drug infliximab (Remicade, Schering-Plow) - chimeric monoclonal antibodies to the tumor necrosis factor, which is widely used in almost all countries of the world, including number in Russia (medline.uz/article/rheumatology/1177.htm). The drug showed high efficacy in the treatment of rheumatoid arthritis (after a single injection, the effect lasted for about 6-12 weeks, and repeated injections made it possible to maintain remission in about half of patients), with Crohn's disease, with seronegative spondylarthropathy (Nasonov E.L. Tumor necrosis factor - new target for anti-inflammatory treatment of rheumatoid arthritis Russian Medical Journal, 2000; 8 (17): 718-722; Furst DE, Keystone E., Maini RN, Smolen JS Recapulation of the round-table discussion - assessing the role of anti-tumor necrosis factor therapy in the treatment of rheumatoid arthritis. Rheumatol., 1999 ; 38 (suppl.): 50-53 .; Bosch van den F., Kruithof E., Baeten D., et al. Effects of a loading dose regimen of three infusions of chimeruic monoclonal antibody to tumor necrosis factor a (infliximab) in spondyloarthropathy: an open pilot study. Ann. Rheum. Dis., 2000; 59: 428-433).

The chimeric (mouse-human) antibody infliximab (Remicade) consists of the variable (Fv) region of high affinity neutralizing murine monoclonal anti-TNF antibodies coupled to a fragment of the human IgG1k molecule, which generally occupies two-thirds of the antibody molecule and provides its effector functions. Infliximab binds TNF-α with Ka = 1.8 × 10 ⁹ l / mol and effectively neutralizes its biological activity. According to in vitro experiments, the drug, forming stable complexes with TNF-α, effectively inhibits the biological activity of secreted and membrane-associated TNF-α and induces the lysis of TNF-producing cells in a complement-dependent way or due to antibody-dependent cellular cytotoxicity (www .mednovosti.by. Archive, No. 8, 1995).

Infliximab is produced by culturing mouse SP2 / 0 myeloma cells transformed with plasmid vectors expressing light and heavy chains, followed by purification of the secreted antibody from the cell culture medium (WO 9216553, 1992). The line based on mouse cells SP2 / 0 - producer of infliximab was obtained more than 20 years ago using the technologies that existed at that time, and does not allow to obtain an antibody with a sufficiently high yield. In addition, it has now been established that glycosylation of proteins, in particular antibodies, in mouse cells has a number of differences from glycosylation in human cells, which indicates a potential threat of the development of an immune response when introducing recombinant antibodies produced by mouse cells into the human body. In this regard, it is more promising to obtain recombinant antibodies for therapeutic use in Chinese hamster cells (e.g., CHO cells), where the glycosylation pattern of antibodies is closer to human (Raju TS Glycosylation variations with expression systems and their impact on biological activity of therapeutic immunoglobulins. BioProcess International, 2003: 44-52).

The closest in technical essence to the claimed invention is the technology for producing infliximab antibodies in Chinese hamster ovary cells using genes encoding the light and heavy chains of the chimeric (mouse-human) antibody infliximab (WO 2011015916, 2011)., Where a vector containing S / MAR sequence.

The disadvantage of this technology is the rather complicated technology of isolating the target product (WO 2011015919, 2011) and, according to preliminary data, the yield of the target product is not high enough (the patent does not contain information about the results of using the invention).

The problem solved by the authors was to expand the assortment of used strains of cultured Chinese hamster ovary cells (CHO) in order to obtain a strain that provides a higher and more stable yield of chimeric anti-human TNF-α antibody.

The technical task was to develop a new, more efficient strain of CHO cells, a producer of a chimeric anti-human TNF-α antibody

The technical result was achieved by creating a strain of Chinese hamster ovary cells CHO-Inflix 20/5, a producer of a chimeric antibody with a specific productivity of 33.5 pg of antibody per cell per day, by transforming cells with expression vectors containing Seq IDs 1 and 2, physical maps of which presented in FIG. 1 and 2.

This strain of ovarian cells of the Chinese hamster CHO-Inflix 20/5, a producer of chimeric antibodies against human TNF-α, was placed in the Russian collection of cell cultures on September 16, 2013 under the number RKKK (P) 760D.

The strain has the following characteristics:

Registration number: RKKK (P) 760D,

Strain copyright: CHO-Inflix 20/5

Reasons for deposit: Producer of recombinant chimeric antibodies against tumor necrosis factor (TNF) -α human

Pedigree strain: Parent cell line CHO ^dhfr- . Cotransfection with pIRES-DHFR / InflixH and pIRES-NEO / InflixL plasmids, selection of a stable, highly productive clone in a medium without hypoxanthine / thymidine with 500 μg / ml G-418 and a gradual increase in methotrexate concentration.

The number of passages at the time of certification and deposit: 20

Standard growing conditions: α-MEM medium with 4 nM glutamine, 10% fetal serum, 500 μg / ml G-418, 500 nM methotrexate, 37 ° C, 5% CO ₂ .

Cultural properties: Adhesive type of growth, cultivation in mattresses, sowing dose of 10,000 cells / cm ² , reaching a monolayer after 4 days. To remove from plastic, a Versene-trypsin solution (1: 1) is used.

Characterization of cell cultivation in the animal body: cultivation in the animal body is not applied

Species data: Cricetulus griseus (PCR analysis with species-specific primers).

Marker signs of the strain and methods for their assessment: the ability to grow in the presence of 500 μg / ml G-418 and 500 nM methotrexate

Contamination by bacteria, fungi, mycoplasmas and viruses: no contamination

Characteristics of the useful substance produced by the strain: Recombinant chimeric antibody (IgG1 isotype, kappa) to human alpha tumor necrosis factor, neutralizing the biological activity of TNF-alpha (enzyme-linked immunosorbent assay, Western blot, biological test on cell line L-929)

Characterization of the biosynthesis of useful products (product yield on medium, activity level and method for its determination): a recombinant antibody is secreted into the culture medium in an amount of at least 33.5 pg per cell per day. Cultivation stability - at least 30 passages.

Cryopreservation method: fetal serum with 10% DMSO, 3-4 × 10 ⁶ cells / ml, freezing to -70 ° C at a rate of 1 ° C / min, then placing in liquid nitrogen, the viability after thawing and washing from the cryopreservative 85% ( with trypan blue).

The essence of the invention is illustrated by the accompanying drawings:

FIG. 1. Map of the expression plasmid pIRES-DHFR / InflixH, where CMV Promoter is a promoter of the early cytomegalovirus proteins;

a-TNF heavy chain - gene of the heavy chain of a chimeric antibody against human TNF-alpha;

IRES - internal ribosome landing site;

DHFR - dihydrofolate reductase gene;

PolyA — RNA polyadenylation site from a bovine growth hormone gene;

pUC Ori — replication start point;

AmpR - ampicillin resistance gene;

FIG. 2. Map of the expression plasmid pIRES-NEO / InflixL, where

CMV promoter - promoter of early cytomegalovirus proteins;

a-TNF light chain - light chain gene of the chimeric antibody against human TNF-alpha;

Synthetic intron - intron;

IRES - internal ribosome landing site;

NEO - neomycin resistance gene;

Poly A — RNA polyadenylation site from a bovine growth hormone gene;

pUC Ori — replication start point;

AmpR - ampicillin resistance gene;

FIG. 3. The results of suspension cultivation of cells of strain CHO-Inflix 20/5 in serum-free medium, where fatty acids are the concentration of viable cells;

FIG. 4. The neutralizing activity of antibodies produced by cells of the clone CHO-Inflix 20/5, against human TNF-α.

The list of attached sequences:

Seq ID No. 1 - DNA sequence encoding the light chain of a chimeric antibody against human TNF-α

Seq ID No. 2 - DNA sequence encoding the heavy chain of a chimeric antibody against human TNF-α

Seq ID No. 3 - amino acid sequence of the light chain of the chimeric antibody against human TNF-α

Seq ID No. 4 is the amino acid sequence of the heavy chain of a chimeric anti-human TNF-α antibody.

The essence and advantages of the invention are illustrated by the following examples:

Example 1. Construction of artificial genes encoding the light and heavy chains of the chimeric (mouse-human) antibodies against human TNF-α and vector constructs for their expression

In order to increase the productivity of the strain of the nucleotide sequence in the genes encoding the light (Seq ID No. 1) and heavy (Seq ID No. 2) chains of the known variant (RU 2004117915, WO 03042247) of the chimeric anti-human TNF-α antibody, it was designed taking into account codon frequencies in the genome of the Chinese hamster (Cricetulus griseus) from the codon usage database [2], and sequences encoding the signal peptide from mouse immunoglobulin (MDFQVQIFSFLLISASVIISRG) and Kozak sequences were placed at the 5′-ends of these genes. DNA fragments containing these genes with the addition of flanking restriction sites (BamHI from the 5′-end and NotI from the 3′-end) were obtained using automated chemical synthesis and ligation. The resulting fragments were cloned into the expression vectors pIRES-DHFR and pIRES-NEO to obtain two expression plasmids pIRES-DHFR / InflixH (Fig. 1) and pIRES-NEO / InflixL (Fig. 2). The correct assembly of plasmids and the absence of mutations in the synthesized genes were verified by sequencing.

Example 2. Obtaining a strain of cells CHO-Inflix 20/5 - producer of a chimeric antibody against human TNF-α

Dihydrofolate reductase (CHO ^{dhfr-defective} ) ovary cells of the Chinese hamster line were transfected with a mixture of plasmids pIRES-DHFR / InflixH and pIRES-NEO / InflixL taken in a 2: 3 ratio and pretreated with PvuIo restriction enzyme reagent. 48 hours after transfection, 142 ± 30 ng / ml of human immunoglobulins were determined by ELISA using supernatants. Then the cells were transferred to a selective medium without thymidine and hypoxanthine containing 500 μg / ml G-418. In the following days, growth cessation was observed, and then the active death of the bulk of the cells was observed. After 12-14 days, stable growth of colonies resistant to growth in a selective medium was recorded. Colonies were pooled, transferred to 6-well culture plates, and then 10 ⁵ cells per well were seeded in 4 ml of medium. After six days, the concentration of human antibodies in the supernatants was 507 ± 17 ng / ml. The resulting pool of cells was cryopreserved, after which cloning was performed to select individual clones with high production of recombinant antibodies. Cloning was performed by limiting dilution in 96-well plates. After 10 days, wells containing a single clone were visually selected. A total of 295 such clones were selected. Then, using ELISA, the content of human immunoglobulins in the supernatant was analyzed. As a result of screening, 36 clones were selected, in the supernatants of which the concentration of human IgG exceeded 230 ng / ml.

These clones were transferred to 12-well plates, and then, upon reaching the monolayer, 10 ⁵ / well were seeded in 6-well plates in a volume of 4 ml. After 6 days, the concentration of immunoglobulins in supernatants was re-determined. Based on the results of this screening, 9 clones with persistent high production of human IgG (3 μg / ml and above) were selected. Further, the selected candidate clones were cryopreserved.

Subsequent cultivation of the cells of the selected clones and a comparison of their productivity showed that clones No. 7, 11, 19, 20, and 24 turned out to be clones with the maximum specific and volumetric productivity and minimum growth rate. The indicators of these clones are shown in Table 1.

Next, the cells of the five candidate clones were cultured in the presence of gradually increasing concentrations of methotrexate (20 nM - 100 nM - 500 nM) for amplification of the target genes. After successful adaptation to 500 nM methotrexate, a volumetric production of the resulting clones was compared. The maximum concentration of human IgG in the supernatant was found in clone No. 20 - 35.8 μg / ml. The specific productivity in this case was 22.3 ± 6.0 pg / cell / day.

The cells of this clone were subcloned using limiting dilutions; 12 subclones were analyzed. Cells were plated at 10 ⁵ / well in 6-well plates in a volume of 4 ml and the concentration of immunoglobulins in the supernatant was measured after 5, 6 and 7 days. The results are presented in table 2.

Maximum volumetric production was recorded in the supernatant of subclone 20/5. The specific productivity of this subclone was 33.5 pg / cell / day. Cells of the subclone, called CHO-Inflix 20/5, were propagated, cryopreserved and deposited in RKKK under the number of RKKK (P) 760D.

Example 3. Production of recombinant antibodies by CHO-Inflix 20/5 cells in serum-free medium.

Cells of the CHO-Inflix 20/5 clone previously adapted for suspension cultivation were seeded in 150 ml of serum-free CDM4CHO medium (HyClone), with 6 mM alanylglutamine in a 500 ml Erlenmeyer flask (culture density 5 × 10 ⁵ cells / ml). Then the cells were cultured on a shaker (130 rpm, 37 ° C, 5% CO ₂ ) and daily the density and viability of the cells, as well as the content of recombinant immunoglobulins, were evaluated. The results presented in FIG. 3 show that the maximum density of viable cells was observed on day 6 and amounted to 5.0 × 10 ⁶ / ml. On day 7, viability decreased to 75%, while the concentration of immunoglobulins was more than 0.2 g / l.

Example 4. Purification of a recombinant antibody and analysis of biological activity.

On the 7th day of cultivation (example 3), cells of strain CHO-Inflix 20/5 were removed by microfiltration and the resulting culture liquid was subjected to three-stage chromatographic purification on an affinity sorbent with immobilized protein A, anion exchange resin Q-Sepharose XL and cation exchange resin SP-Sepharose FF, then concentrated and sterilized by ultrafiltration. A total of 18 mg of highly purified antibody was obtained in 70% yield.

Testing of the neutralizing activity of the obtained antibodies was carried out in a cytotoxic test on the cell line L-929 (collection of the Russian Academy of Sciences). Cells were plated at a density of 3 × 10 ⁴ cells per well in a 96-well flat-bottomed plate. The next day, the medium in the cells was completely replaced with fresh one with the addition of actinomycin D (Sigma) at a final concentration of 1 μg / ml.

The antibody obtained from clone CHO-Inflix 20/5, the well-known chimeric antibody to human TNF-alpha (the drug "Remicade"), as well as control recombinant human antibodies that do not interact with TNF-α, were titrated in a concentration range from 200 to 0, 78 ng / ml and incubated for 1 hour at room temperature with human TNF-α (ProSpec) at a concentration of 1 ng / ml. Next, a mixture of antibodies and human TNF-α was introduced into a tablet with cells.

After 24 hours, the medium was removed, the cells were stained with a crystal violet solution and dried. Then, the colored precipitate was dissolved in a solution of sodium dodecyl sulfate with glycerol and photometric at 595 nm on a Victor ² tablet counter. The results were expressed in units of optical density. The results presented in FIG. 4 show that the antibody produced by the cells of the strain CHO-Inflix 20/5 inhibits the activity of human TNF-α identical to the commercial remixade chimeric antibody infliximab.

Thus, we obtained a strain of CHO-Inflix MG20 / 5 cells, a producer of a chimeric recombinant antibody against human TNF-α, whose neutralizing properties are identical to the drug infliximab (Remicade).

Claims (1)

The strain of ovary cells of the Chinese hamster CHO-Inflix 20/5, transfected with expression vectors containing DNA sequences SEQ ID NO: 1 and SEQ ID NO: 2 - producer of a chimeric antibody to human tumor necrosis factor alpha, deposited in the Russian collection of cell cultures under No. RACC (P) 760D.

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