RU2627181C2 - Expressional plasmid lentiviral vector for heterological expression of recombinant human protein cd47 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a plasmid lentiviral vector for heterologous expression of a recombinant human CD47 protein in mammalian cells. The vector contains a highly functional cytomegalovirus promoter (CMV), a CD47 receptor extracellular domain sequence, a TEV cysteine protease recognition sequence, a tagRFP fluorescent protein sequence acting as a selectable marker. All three sequences are in the same reading frame under the control of this promoter.

EFFECT: invention allows to obtain a highly productive and stable protein expression system for the further production of its purified form.

2 cl, 1 ex, 4 dwg

Description

The invention relates to biotechnology and pharmaceuticals and relates to a new lentiviral vector for the expression of CD47 protein in mammalian cells.

The current production of recombinant proteins is mainly based on prokaryotic expression systems, such as, for example, various modifications of the bacterial strain of E. coli. However, in modern scientific studies, proteins expressed in eukaryotic cells, in particular in mammalian cells, are increasingly used. The choice of such a system is explained by the presence of molecular mechanisms of post-translational modification of protein molecules in eukaryotic cells, as well as by more accurate folding processes of complex polypeptides. The need to use the eukaryotic system also arises in the case of expression of the human CD47 receptor, which contains a sufficiently large glycosylated domain. The CD47 receptor is expressed on many types of cells and is involved in a variety of cellular processes, including mechanisms such as proliferation, differentiation, cell migration, cell adhesion, and apoptosis signaling [Brown E.J., Frazier W.A. Integrin-associated protein (CD47) and its ligands // Trends. Cell Biol. - 2001. - Vol. 11, N. 3. - P. 130-135]. On the other hand, recent studies have shown that CD47 is overexpressed in various types of tumor cells such as, for example, cancer cells of the bladder or cells of acute myeloid leukemia [B. Edris et al., Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma, 2012]. Thus, CD47 may act as a potential target in anticancer therapy.

Single chain antibodies can be used as effective antitumor therapy. However, a significant amount of the corresponding protein is required to create suitable monoclonal antibodies by phage or ribosome display methods. This problem can be solved by creating an expression plasmid lentiviral vector, followed by obtaining a high-performance cell line of the CD47 expressor.

Among the mammalian cell lines used in biopharmaceutical production and scientific research, the most common cell lines are HEK293 (human embrionic kidney 293 cells), CHO (Chinese hamster ovary cells), PER.C6 (protein manufacturing adopted human cells) [http: //www.science-education.ru/pdf72012/5/237.pdf]. The choice of the producer line is determined by the specificity of the synthesized protein and the general purpose of its production. Obtaining a stable cell line expressing a given genetic sequence is not difficult due to standard techniques and a wide range of chemical transfection drugs available on the market. But, it is worth noting that the creation of an effective producer line is determined not only by the successful introduction of the DNA sequence into the genome of the host cell, but mainly by the level of the target mRNA and, ultimately, the number of stable molecules of the target protein.

The efficiency of expression largely depends on the choice of the promoter region that controls the transcription of the introduced DNA sequence. Different promoter regions differ not only in their ability to attract the transcriptional apparatus of the cell, but also in different levels of methylation. So, viral promoters are more often inactivated by methylation compared to cell promoters. A significant role is played by mutations that occur during cell proliferation. The occurrence of spontaneous deletions and translocations can significantly reduce the expression level of the target cassette. Recent studies have shown that methylation and chromosomal rearrangements can be reduced by creating recombination hotspots in the target sequence that would be homologous to certain sequences in the transcriptionally active part of the cell genome. Also, the likelihood of deletions or methylation can be reduced by bringing the target protein sequence closer to the selection marker sequence. Thus, the creation of an effective producer is achieved using the following approaches:

- use of the most suitable promoter region

- reducing the likelihood of methylation and mutations of the target sequence by directed integration into transcriptionally active parts of the genome.

- maximum approximation of the target sequence and selection marker

Also, the choice of the marker itself, which determines the selection of cells that have received a genetic construct, plays an important role. Increasingly, to obtain superproducers, recombinant plasmids are used that contain markers that lead to the amplification of the target gene. Such systems include constructs containing the sequence of DHFR (dehydrofolate reductase) and GS (glutathione synthetase) (for example, RU 2488633, EA 018099). However, the use of such systems requires special cell lines deficient in these genes. In addition, obtaining cells with a high level of expression requires a significant amount of time and materials, which is associated with a long, gradual increase in MTX (methotrexate) in the culture medium. The use of antibiotics as a marker of selection also requires a significant amount of time spent on selection. In this case, serious difficulties arise with the selection of the most effective producers from a large number of clones that have received resistance to a specific antibiotic.

The technical solution to such problems can be associated with the use of recombinant plasmid DNA with the following properties:

- a strong promoter region, providing a high and stable level of transcription of the DNA sequence of the target cassette

- the use of a lentiviral system for delivering the target sequence to the genome of the host cell. Using a lentiviral system allows you to get an almost unlimited number of events of integration of a given sequence into the cell chromosome. Sometimes this property has negative consequences, reflected in a decrease in the overall metabolism of the cell, which, ultimately, leads to its death. However, the number of integrations is easily controlled by a decrease (or increase) in the titer of viral particles. Thus, the probability of getting the desired gene in a region with high transcriptional activity is very high.

- selection of the most optimal selection marker, as close as possible to the target sequence. To reduce the distance between the gene sequence of the target protein and the selection marker, it is mainly customary to use the IRES sequence (internal ribosome landing site). This sequence allows the ribosome to transfer almost continuously from one translated region to another without spending time on complete disassembly of the complex and the process of scanning mRNA. An alternative method for maximizing the convergence of sequences can be the creation of a fused DNA construct, the expression product of which is a more complex polypeptide molecule.

US Pat. No. 5,562,997 describes a method for producing a CD47-soluble protein using expression vectors with the sequence CD47 using a host cell e. coli followed by purification of the isolated protein. It is noted that transcription cassettes can be introduced into many vectors, for example, plasmids, retrovirus, lentivirus; adenovirus. But specific designs are not disclosed.

US 8,329,462 relates to lentiviral vectors that are safe, effective, and very powerful for the expression of transgenes, for example, erythropoietin, integrin for human gene therapy in blood cells. Lentiviral vectors include a promoter such as EF1.alpha, PGK, gp91hox, clotting Factor IX, a clotting Factor V111, insulin, a PDX1, CD11, CD4, CD2mra gp47. He revealed the possibility of expression of CD47.

The technical task was to create a vector for obtaining highly productive and stable expression systems of recombinant proteins in mammalian cells, in particular in HEK293 cells.

The invention is based on the plasmid lentiviral DNA vector developed by the authors (Fig. 1), which includes a cytomegalovirus promoter that provides a high level of mRNA production, a CD47 receptor extracellular domain sequence, a TEV protease recognition and cleavage site, tobacco etch virus tagRFP gene sequence - red fluorescence a protein that allows you to sort only cells containing inserts of the target sequence. All three sequences are in the same reading frame under the control of this promoter.

The sequence of CD47 (SEQ ID NO 1):

The sequence of the cytomegalovirus promoter (SEQ ID NO 2):

The sequence of the TEV protease recognition site (SEQ ID NO 3):

The nucleotide sequence encoding a red fluorescent tagRFP protein (SEQ ID NO 4):

The nucleotide sequence encoding 10 histidine residues (SEQ ID NO 5):

The nucleotide sequence encoding a fusion protein, including sections of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5 (SEQ ID NO 6):

The term "expression vector" means plasmid DNA containing all the necessary genetic elements for the expression of the target gene embedded in it, for example, a promoter and terminator, and elements for amplification of the expression cassette and selection of clones with multiple copies of the expression cassette in the genome. The vector size is 8015 bp (base pairs).

The complete sequence of the fragment inserted into the chromosome of the cell includes the following elements:

- 5 'and 3' LTR - long terminal repeats - sequences used by the virus to integrate its own genome into the host genome by homologous recombination.

- Central polypurine tract (sRPT) - ensures the penetration of the target sequence into the nuclei of mitotically inactive cells.

- Post-transcriptional regulatory element (WPRE) is a regulatory region that enhances the expression of the target sequence.

- Kozak - the consensus sequence necessary for landing ribosomes and initiating translation of mRNA.

- CD47 - the sequence of the extracellular domain of CD47

- TEV - TEV protease cleavage site (tobacco etch virus)

- tagRFP - red fluorescent protein, acts as a selective marker

- NT - a sequence including 10 histidine residues necessary for chromatographic purification of the obtained protein

The vector was obtained using standard genetic engineering methods, commercially available plasmids and chemically synthesized oligonucleotides (Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. 2nd ed. New York, NY: Cold Spring Harbor Laboratory Press; 1989) by constructing a new expression lentiviral plasmid DNA (Fig. 1),

The present invention is intended to provide highly productive and stable expression systems for the recombinant human CD47 receptor in mammalian cells with the goal of further producing purified CD47 protein. The technical result is achieved by introducing into the cells the obtained lentiviral construct containing regulatory elements for heterologous expression of the recombinant protein, high-frequency integration of a given sequence and a selective marker that allows you to track the quality and quantity of such integrations. This vector system allows you to almost instantly select the desired clones - cells containing the insert. The number of inserts and, consequently, the total amount of synthesized protein can be determined by the intensity of the glow of the red fluorescent tagRFP protein. Cell sorting is performed by cell sorting using any suitable equipment. Subsequently, during the purification of protein molecules, the fusion sequences of CD47 and fluorescent protein can be separated by treating the purified preparation with TEV protease. Then the target protein can be purified from impurities by chromatographic separation.

Cell growth, isolation and purification of the target protein from a culture or similar fluid can be carried out by a standard cultivation method. The culture medium used for cultivation can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, an appropriate amount of nutritional supplements necessary for cell growth. Various carbohydrates such as glucose or sucrose and other organic acids can be used as a carbon source. As the nitrogen source, various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural sources of nitrogen, can be used. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. As vitamins, thiamine, yeast extract, and the like can be used.

Cultivation can be carried out on standard DMEM medium containing 10% bovine fetal serum, 4 mM L-glutamine, 1 mM sodium pyruvate, streptomycin / penicillin at a concentration of 100 μg / ml and 100 u / ml, respectively, pH 8.1 ± 0 , 1 under aerobic conditions, preferably with a high CO2 content (5%), such as stirring the culture fluid in the flasks, at a temperature of 37 ° C. For the production of a sufficient amount of protein, an average of 3 to 5 days of cultivation is required.

To test the invention presented in paragraph 1 of the claims, the vector was introduced into transformed human kidney epithelial cells (HEK293T cells) using Invitrogene lipofection kits (LipofectAMINE Plus). Three packaging plasmids were introduced simultaneously with lentiviral constructs: pREV, pGAG and pVSV-G, which encode, respectively, reverse transcriptase, human immunodeficiency virus (GAG1) GAG protein and vesicular stomatitis virus G-protein. The optimal ratio of plasmids during transfection, giving the maximum titers of recombinant virions, was determined experimentally (vector: pREV: pGAG: pVSV - G = 2: 4: 2: 1), after which the efficiency of transfection was visually evaluated using a fluorescence microscope (figure 2) . Recombinant virions were collected for 72 hours at 8-hour intervals. The supernatants were combined and the virions were precipitated with 12% polyethylene glycol at 4 ° C for 12 hours, followed by centrifugation for 10 minutes at 5000 rpm. The virions were then suspended in fresh medium (1/10 of the original volume) and filtered through a sterilizing nozzle for a syringe. Sterile virions were packaged and stored frozen at -70 ° C. The HEK293T human embryo kidney cells were used to introduce the vector into cells for expression of the CD47 protein. After 72 hours, the infected cells were sorted on a cell sorter, as a result of which a fraction of the most brightly luminous cells was selected, into which the largest number of copies of the construct was embedded. The selected cells were grown in the required amount, after which CD47 protein was isolated and purified on a chromatographic column (Fig. 3). The resulting cell line with the introduced genetic construct can be frozen and used to obtain the following batches of protein, because, in contrast to the usual expression constructs, after the introduction of which the expression of the target protein gradually fades, the cells expressing the lentiviral vector described in paragraph 1 of the Formula the inventions retain the initial, stably high level of expression of the target protein. The expression level of CD47 in cells with the introduced vector pLCMV-CD47-TEV-tRFP was evaluated by ELISA every 10 days for 50 days in comparison with cells where the construct pLCMV-CD47-tRFP was expressed (Fig. 4).

Claims (10)

1. Expression plasmid lentiviral vector for heterologous expression of recombinant human CD47 protein in mammalian cells, characterized in that it includes a cytomegalovirus promoter that provides a high level of mRNA production, followed by a nucleotide sequence encoding the extracellular domain of the CD47 receptor (SEQ ID NO: 1) linked to a sequence encoding a peptide region for the recognition and cleavage of the TEV protease (tobacco etch virus) linked to the nucleotide sequence of the tagRFP gene encoding a red fluorescent protein, which allows you to sort only cells containing inserts of the target sequence. 2. Plasmid lentiviral DNA vector according to claim 1, characterized in that the complete sequence of the fragment inserted into the chromosome of the cell includes the following elements: 5 'and 3' LTR — long terminal repeats — sequences used by the virus to integrate its own genome into the host genome using viral integrase; central polypurine tract (cPPT), which ensures the penetration of the target sequence into the nuclei of mitotically inactive cells; post-transcriptional regulatory element (WPRE) - a regulatory region that enhances the expression of the target sequence; Kozak - the consensus sequence necessary for landing ribosomes and initiating translation of mRNA; CD47 — CD47 extracellular domain sequence; TEV — TEV protease cleavage site (tobacco etch virus); tagRFP is a red fluorescent protein that acts as a selective marker; NT is a sequence of 10 histidine residues necessary for chromatographic purification of the obtained protein.

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