RU2562169C2 - Culture cell strain cho-il7/13 producing human interleukin-7 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and concerns the cell strain CHO-IL7/13. The characterised strain is produced by transfection of the cells CHO^dhfr by pIPvES-DHFR/IL7 plasmid containing human interleukin-7 gene. pIPvES-DHFR/IL7 plasmid has been synthesised by primer amplification of human interleukin-7 complementary DNA: ecoIL CCTGAATTCCACCATGTTCCATGTTTCTTTTAG containing EcoRI restriction site and Kozak sequence, and bamIL CCAGGATCCTCAGTGTTCTTTAGTGCCCATC containing BamHI restriction site that is followed by cloning according to the restriction site data into pIRES-DHFR vector.

EFFECT: presented strain has a higher producing ability of human recombinant interleukin-7 and can be used for treating the number of pathological conditions associated with the low content of B- and T-lymphocytes.

4 dwg, 3 ex

Description

The invention relates to biotechnology, in particular to a technology for producing recombinant human interleukin-7.

Interleukin-7 (IL-7) is a cytokine that stimulates the proliferation of B and T lymphocytes and their precursors. Loss of the IL-7 gene (knockout) by the body causes emptying of the thymus, total lymphopenia, severe immunodeficiency, and a decrease in the intensity (7-10 times) of the response of immunocompetent cells to the action of stimulants. In this case, the formation of gamma / delta T-lymphocytes is completely blocked, the process of formation of alpha / beta T-cells is weakened. With an excess of IL-7, the level of B- and T-lymphocytes in the blood is increased.

It is assumed that IL-7 can be successfully used to treat a number of pathological conditions associated with a low content of B and T lymphocytes, in particular:

- to restore the content of T cells, mainly CD4 +, in combination with highly active antiretroviral therapy for HIV-infected people;

- for the treatment of lymphopenia associated with the use of cytostatics and radiation therapy in the treatment of cancer;

- for the treatment of cancer in combination with local hyperthermia;

- for the regeneration of T cells after transplantation of hematopoietic stem cells.

The interleukin-7 molecule is a glycosylated polypeptide chain with a length of 152 amino acid residues. The molecule contains 6 cysteine residues forming three disulfide bridges, as well as three N-glycosylation sites and one O-glycosylation site. The secretion of the synthesized protein is directed by a signal sequence containing 25 amino acid residues [ru.wikipedia.org/wiki/Interleukin_7; Ketlinsky S.A., Simbirtsev A.S. Cytokines. S.-Pb., 2008].

To obtain IL-7, recombinant cells of bacteria, yeast, and higher eukaryotes are used, transformed with the corresponding vectors expressing human IL-7. However, the non-glycosylated form of IL-7 is synthesized in bacterial cells, which is characterized by instability when administered in vivo compared to the native (glycosylated) form. IL-7, synthesized in yeast cells, contains carbohydrate chains that differ from the carbohydrate chains of the native protein, which leads to a change in its antigenic properties.

The most promising human IL-7 producers for use as a medicine can be recombinant cultured higher eukaryotic cells, in particular BHK, COS, CHO, RUL-293 cells, which provide synthesis, glycosylation, folding, cleavage of the signal sequence and secretion into the medium culturing proteins identical to native human proteins. In particular, the synthesis of human IL-7 in a number of cultured mammalian cells has been described (WO 2004018681, WO 8903884). As a result, HEK-293, COS-7, BHK cells were synthesized that synthesized human IL-7 during cultivation for 10 days. The identity of the obtained products of human interleukin-7 was established, however, no data on the productivity of the obtained producer cells were provided.

The closest in technical essence to the claimed invention is the technology described in patent WO 2004018681, which describes the receipt of IL-7 produced by Chinese hamster ovary cells (CHO), HEK-293. The disadvantage of this technology is the relatively low yield of IL-7.

The challenge facing the authors was to expand the range of producer strains with the goal of creating higher-performance cell lines producing human IL-7.

This problem is solved by the fact that a strain of cultured Chinese hamster ovarian cells CHO-IL 7/13, stably expressing highly active recombinant human interleukin-7 (IL-7) obtained by transfection of CHO ^dhfr cells ^with plasmid pIRES-DHFR / IL7 containing the IL-gene 7, the circuit of which is shown in FIG. 1. The strain is characterized by the following properties.

Morphological characters: Cells of regular round shape, single or grouped into clusters.

Cultural traits: Type of growth - suspension.

Resistance to selective factors: Grows on medium with 500 ng / ml metatrexate.

Cryopreservation. Cryopreservation medium - 90% fetal serum and 10% dimethyl sulfoxide. Storage temperature -196 ° C (liquid nitrogen).

Contamination. Cells of strain CHO-IL7 / 13 are free of mycoplasma.

Pedigree strain: Parent cell line CHO ^dhfr- . Transfection with plasmid pIRES-DHFP / IL7, selection of a stable highly productive clone in a medium without hypoxanthine / thymidine with an increase in methotrexate concentration.

Standard growing conditions: PF-CHO LS medium without hypoxanthine / thymidine with 4 nM glutamine, 500 nM metatrexate, 37 ° C, 5% CO ₂ .

Cultural properties: Suspension cultivation in test tubes, flasks or 6-well plates on an orbital shaker with a rotation speed of 150-180 rpm. Sowing dose 0.3-0.5 × 10 ⁶ / ml, reseeding every 3-4 days. The doubling time is 22-24 hours.

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

Characteristics of the useful substance produced by the strain: recombinant human interleukin-7 (evaluation using solid-phase enzyme-linked immunosorbent assay, Western blot, biological test).

Characterization of biosynthesis of useful products (product yield on medium, activity level and method for its determination): Recombinant human interleukin-7 is secreted into the culture medium at a concentration of at least 10 μg / ml for 4 days. The cultivation stability is 15 passages. Activity Determination Method - Conlon P.J. et al. Murine thymocytes proliferate in direct response to interleukin-7 // Blood. - 1989 .-- V.74 (4). - P.1368-73.

Cryopreservation method: fetal serum with 10% DMSO, 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).

Other features of the strain: It is possible to cultivate cells in DMEM / F12 medium with 10% fetal serum (adhesive growth type) without additional adaptation.

The invention is illustrated by the following figures. Figure 1 - Map of the expression plasmid pIRES-DHFR / IL

The ring diagram of the plasmid pIRES-DHFR / IL7, where the following notation is used: Amp R - beta-lactamase gene: P CMV IE - promoter of early cytomegalovirus proteins; IL7 - human interleukin-7 gene; IVS - synthetic intron; mDHFR - modified dihydrofolate reductase gene; pA BGH - signal polyadenylation and termination of transcription of the gene for growth hormone bull; Ori is a bacterial replicator.

Fig 2. The results of screening for the production of 24 candidate CHO-L7 clones 72 and 96 hours after the start of cultivation.

Figure 3 - Binding of proteins of the culture medium of pIRES-DHFR / IL7 cells with an antibody to human interleukin-7 in an immunoblot.

1 - Medium of cell culture of clone 11, 10 μl

2 - Medium of cell culture of clone 13, 10 μl

3 - A sample of human IL-7 expressed in E. coli cells (non-glycosylated) (R&D Systems, Cat. No. 207-IL-005 / CF) - 1 μl

4 - Molecular weight markers.

Figure 4. Determination of the biological activity of IL-7 secreted by cells of clone 7/13.

Seq ID No. 1 Sequence List - DNA encoding human interleukin-7 (Appendix)

The invention is illustrated by the following examples:

Example 1. Isolation of human interleukin-7 cDNA and expression plasmid. To obtain human IL-7 cDNA, a cDNA preparation was used on an RNA template from human uterine adenocarcinoma. Next, human IL-7 cDNA was amplified using primers

IL7-for (5′-TGCTCCAGTTGCGGTCATCATGACTA-3 ′) and

IL7-rev (5′-CTGCATAAGCAGATAGATTCTTGGAGGATGC-3 ′),

amplification parameters: 95 ° C × 5 min; 35 cycles: 95 ° C × 15 sec, 55 ° C × 15 sec, 72 ° C × 20 sec and an additional completion cycle of 2 min × 72 ° C. The amplification product of 649 nucleotides in length was purified by electrophoresis on a 2% agarose gel and cloned into pCD2.1 vector (Invitrogen). Plasmid DNA from several clones was isolated and sequenced in two directions using the above primers. Comparison of the sequence of the insert contained in one of the obtained plasmids, pCR2.1-IL7-5, with the reference sequence of variant 1 of the human IL-7 cDNA annotated in GenBank (NM_000880.3), showed their complete identity (Seq ID No. 1.) .

To create an expression plasmid, the IL-7 cDNA sequence was amplified using ecoIL primers (CCTGAATTCCACCATGTTCCATGTTTCTTTTAG) containing the EcoRI restriction site and the Kozak sequence, and bamIL (CCAGGATCCTCAGTGTTCTT restriction site containing the RAM site containing the RAM site containing the expression plasmid pIRES-DHFR / IL7. The scheme of the plasmid pIRES-DHFR / IL7 is presented in Figure 1.

Example 2. Obtaining a strain of cultured producer cells of human IL-7. The expression plasmid pIRES-DHFR / IL7 was linearized by restriction enzyme PvuI. The CHO ^dhfr- cells adapted for suspension growth were cultured in PF-CHO LS (HyClone) medium with 4 mM glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin, 0.1 mM hypoxanthine and 0.016 mM thymidine (Sigma) ( full environment). Cells were grown in 50 ml centrifuge tubes on a shaker at a temperature of 37 ° C and a platform rotation speed of 180 rpm. Permanent transfection of CHO ^dhfr- suspension cells was performed in 24-well plates in serum-free medium using the FreeStyle ™ MAX Reagent liposome reagent (Invitrogen) according to the manufacturer's instructions. One day after transfection, the medium was replaced with complete medium, and another day later it was transferred to selective conditions and cultivated further in a medium without hypoxanthine and thymidine and with 20 nM methotrexate (MTX, Sigma), replacing the medium every 3 days.

The concentration of IL-7 was determined using enzyme immunoassay. Pools of stably transfected cells were plated on PF-CHO LS medium without hypoxanthine / thymidine with 1% serum and 20 nM metatrexate with a density of 1.5 cells per well in 96-well flat-bottom plates in a volume of 200 μl per well. On days 7 and 11, half of the medium was replaced by adding fresh serum-free medium. Before the first medium change, all boards were visually scanned and wells containing single clones were marked. On day 14, screening was performed to determine the concentration of a specific protein in the supernatants of wells with single clones. Then, the selected clones were first transferred to 24-well plates (600 μl of medium per well), then to 6-well plates (3 ml of medium per well), and then to 50-ml centrifuge tubes. Cell cultivation was performed on a shaker at a platform rotation speed of 180 rpm. As a result of screening 24 clones cultured in a medium with successively increasing concentrations of methotrexate (20-50-100-250-500 nM), a strain of Chinese hamster CHO-IL7 / 13 ovarian cells with a specific productivity (qP) of 6.5 was obtained PG IL-7 / cells / day (Fig 2). The maximum concentration of human IL-7 during cultivation of the cells of this clone was 10,850 ng / ml. Cryopreserved cells of clone CHO-IL7 / 13 are placed in the collection.

Example 3. Analysis of the protein produced by cells of strain CHO-IL 7/13.

Cells of strain CHO-IL 7/13 were cultured in a medium with 20 nM methotrexate, as described in example 2, for 72 hours. Cells were besieged by centrifugation and samples of the culture medium were subjected to polyacridamide gel electrophoresis with DDS-Na. The separated proteins were transferred onto a nitrocellulose membrane, which was incubated with known monoclonal antibodies against human interleukin-7 (Human IL-7 DuoSet, manufactured by R&D Systems, USA). The result obtained after treatment with the second antibodies and the manifestations shown in FIG. 3 shows the presence of a protein band with a molecular weight of 29 kilodaltons, which binds to antibodies to human IL-7 and is absent in the control cell culture medium. This molecular weight corresponds to the glycosylated form of human interleukin-7. The productivity of the strain CHO-IL7 / 13 was evaluated using enzyme-linked immunosorbent assay with antibodies Human IL-7 DuoSet. The measurement results are shown in Fig.4.

The determination of the biological activity of recombinant human IL-7 was performed on the primary culture of mouse splenocytes, by the ability of this cytokine to stimulate the proliferation of various cells of the lymphocytic series [2-4]. Splenocytes were isolated from the spleen of F1 hybrid mice (C57BL / 6 × CBA) as follows. Under aseptic conditions, the spleens were crushed, homogenized by repeatedly passing a syringe through a needle (d = 1.2 mm). The cell suspension in sterile buffered saline (PBS) was filtered through a double layer of gauze and centrifuged. The red blood cells were then lysed with a 0.86% NH ₄ Cl solution, then the cells were washed twice in PBS and resuspended in RPMI-1640 medium (Sigma) with 2.0 mM L-glutamine, 50 μM 2-mercaptoethanol and 80 mg / L gentamicin (complete Wednesday). The obtained cells (5 × 10 ⁵ cells per well) were incubated in 4 parallels in the presence of different concentrations of a standard sample of IL-7 protein (R&D Systems, Cat. No. 207-IL-005) or various dilutions of the supernatants of the culture medium of the cultured cell strain IL -7/13, in which the concentration of IL-7 was previously determined using enzyme-linked immunosorbent assay (R&D Systems). Next, samples of supernatants were diluted so that the resulting dilutions contained 100, 10, or 1 ng / ml of recombinant protein. Cell incubation with the samples was performed in complete medium with the addition of 10% calf embryonic serum (TES) (Sigma) in 96-well flat-bottom culture plates (Costar) for 72 hours in a CO ₂ incubator at 37 ° C under conditions of absolute humidity. 20 hours before the end of the incubation, ³ H-thymidine (Isotope, St. Petersburg) was added to the cells at a final concentration of 5 μCi / ml. Next, the cells were transferred to glass fiber filters using a FilterMate 96 harvester (Perkin Elmer) and thymidine incorporation intensity was determined on a MicroBeta TriLux 1450 β-counter (Perkin-Elmer), which was expressed in pulses per minute. The averaged result of four parallel experiments is presented in Fig. 4, from which it follows that the efficiency of stimulation of splenocyte proliferation by the supernatant of clone cells of IL-7/13 practically coincides with the efficiency of a standard sample of IL-7 in appropriate doses, which indicates the presence of recombinant IL-7 adequate biological activity.

Claims (1)

The strain of cultured CHO-IL7 / 13 cells producing human interleukin-7 obtained by transfection of CHO ^dhfr cells ^with plasmid pIPvES-DHFR / IL7 containing the human interleukin-7 gene, the general scheme of which is shown in FIG. 1, which was synthesized by amplification of human interleukin-7 cDNA using primers: ecoIL CCTGAATTCCACCATGTTCCATGTTTCTTTTAG containing the EcoRI restriction site and the Kozak sequence, and bamIL CCAGGATCCTCAGTGTTCTTTTAGTGCCCATC containing the DHF restriction site for the RR restriction site.

Images