RU2841398C1 - Recombinant plasmid dna pfuse 1-68 mar tsh, a coding sequence of thyrotropin alpha protein, a cell line rhtshcho, transformed by plasmid dna pfuse 1-68 mar tsh by nucleofection and secreting recombinant human thyrotrophic hormone - Google Patents

Abstract

FIELD: genetic engineering; biotechnology.

SUBSTANCE: recombinant plasmid DNA pFUSE 1-68 MAR TSH with SEQ ID NO: 1, coding cDNA genes of alpha and beta subunits of recombinant thyrotropin alpha, and a cell line of Chinese hamster ovary rhTSHCHO cells producing recombinant human thyrotropin alpha and created by transformation of the CHO DG44 line by nucleofection of said recombinant plasmid DNA.

EFFECT: invention enables to obtain a highly productive stable producer line of recombinant human thyrotropin alpha, as well as synthesise both subunits of thyrotropin alpha, which reduces heterogeneity of the pool of transformed cells.

2 cl, 3 dwg, 2 ex

Description

The object of the invention is the recombinant plasmid DNA pFUSE 1-68 MAR TSH, encoding cDNA of the alpha and beta subunit genes of recombinant thyrotropin alpha, and the rhTSHCHO cell line obtained by transforming the Chinese hamster ovary cell culture CHO DG44 using the nucleofection method of the recombinant plasmid DNA pFUSE 1-68 MAR TSH.

Thyroid-stimulating hormone (thyrotropin alpha, TSH) is a glycoprotein; the carbohydrate portion of the subunits makes up more than 10% of their mass and is necessary for normal thyroid-stimulating action and intravascular kinetics.

TSH binds to specific receptors on the surface of thyroid epithelial cells and stimulates the production and activation of thyroxine. The TSH receptor is a membrane GPCR receptor coupled with a G protein, which in turn activates adenylate cyclase and increases iodine consumption by the cells of the gland. A further increase in cAMP levels causes the effect of TSH on the biosynthesis of triiodothyronine (T3) and thyroxine (T4), which are the most important hormones regulating growth and development.

Previously, TSH isolated from the pituitary gland of cattle was used in clinical practice. Thus, bovine TSH was used to check the function of the thyroid gland and increase the absorption of radioactive iodine in patients with thyroid cancer. However, it caused adverse allergic reactions, so after the development of recombinant DNA technologies and the emergence of the possibility of artificially obtaining the hormone, the use of bovine TSH was abandoned. Recombinant human TSH (rhTSH) has an amino acid structure identical to human pituitary TSH and is produced by modified cell lines based on Chinese hamster CHO cells.

One of the best known recombinant human TSH preparations is Thyrogen (rhTSH; Thyrogen®). It has been approved by the US FDA for diagnostic use in patients with thyroid cancer. The alpha and beta subunits of Thyrogen® are identical to the human pituitary TSH subunits. Thyrogen® has a specific activity of approximately 4 IU/mg and is a potent stimulator of T4, T3, and thyroglobulin (Tg) secretion in healthy volunteers. It also increases thyroid iodide uptake in patients with thyroid cancer or multinodular goiter and in volunteers, even those exposed to large amounts of stable iodide. Patients with thyroid cancer who have undergone thyroidectomy and radioactive iodine ablation but who are at risk for developing residual thyroid cancer are candidates for rhTSH in preparation for whole-body iodide scanning and serum TSH measurement. In patients with thyroidectomized thyroid cancer who are unable to secrete TSH from the pituitary gland after thyroid hormone withdrawal, rhTSH is the only acceptable method of preparing them for these procedures. rhTSH, in addition to being useful in the management of patients with thyroid cancer, is potentially useful for testing thyroid reserve and for assisting in thyroid-related nuclear medicine procedures.

Patent US 6,455,282 (Genzyme Corporation, USA) describes biologically active heteropolymeric proteins consisting of multiple subunits, including TSH, and methods for their production. It is shown that both subunits are synthesized in a single cell with expression vectors containing heterologous DNA encoding the subunits both as part of a single plasmid DNA and as part of different plasmid DNA. Both options involve the use of the SV40 virus late promoter sequence to express the alpha subunit gene and the mouse metallothionein protein promoter. The use of the BPV bovine papillomavirus promoter is also mentioned. The patent expired in 2019.

Patent US 7,479,549 describes a nucleic acid having a sequence at least 98% homologous to a sequence encoding the α-subunit of canine thyroid stimulating hormone. The invention in the patent also includes a method for producing a recombinant canine thyroid stimulating hormone (rcTSH) subunit by expressing the sequences in a transgenic insect cell modified with sialylated proteins and producing a sialylated rcTSH subunit for use in the diagnosis and treatment of hypothyroidism in dogs.

US Patent 20110052591 describes pure recombinant TSH obtained from a clone containing the complete nucleotide sequence for TSH expression. Polynucleotide and polypeptide sequences are described that encode novel variants of mouse or human beta-thyroid stimulating hormone proteins that can be used in therapeutic, diagnostic and pharmacogenomic applications for the prevention, treatment or reduction of the severity of thyroid stimulating hormone-beta-related diseases. Variants of constructs using the human cytomegalovirus promoter are presented.

US Patent 20190062396 presents a method for producing recombinant human thyroid hormone. The patent presents a genetic construct containing sequences of the beta and alpha subunit genes of TSH, separated by an IRES (Internal Ribosome Entry Site) sequence, under the control of a single human cytomegalovirus promoter and a bovine growth hormone (BGH) terminator, as well as a sequence of a selection protein - dehydrofolate reductase (DHFR).

The use of classical approaches using the DHFR gene requires a long time for the selection of the producer clone. For a technical solution to the problem of a long time for the selection of the producer clone, a strategy was chosen using the nucleotide sequence of the extended region of the internal ribosome landing and the PEST sequence in the hDHFR-PEST fusion protein, which provides a reduced level of dehydrofolate reductase inside the cell and leads to more effective selection in the presence of methotrexate, which allows obtaining producer lines with the maximum level of secretion of the recombinant protein.

The invention is illustrated by the following graphic materials.

Fig. 1. Scheme of the genetic construct - recombinant plasmid DNA pFUSE 1-68 MAR TSH, with the main genetic elements and key sites of restriction endonuclease hydrolysis.

Fig. 2. Scheme of selection of a pool of cells transformed with recombinant plasmid DNA pFUSE 1-68 MAR TSH.

Fig. 3. Electrophoretic analysis of the purified thyrotropin alpha preparation obtained as a result of culturing the rhTSHCHO cell line

As a result of solving the stated technical problem, the following technical result is obtained:

1. The recombinant plasmid DNA pFUSE 1-68 MAR TSH SEQ ID NO: 1, containing the nucleotide sequences of both subunits of human thyrotropin alpha in a single mRNA, is preferred in the present invention, since it allows each transformed single cell to synthesize both subunits of thyrotropin alpha, which reduces the heterogeneity of the pool of transformed cells, and contains:

- the nucleotide sequence of attachment to the nuclear matrix MAR1-68, which allows for an increase in the production of the target protein due to both an increase in the level of transcription of the gene located in close proximity and due to greater stability of the ribosome-transcript complex,

- highly efficient synthetic hEF-HTLV promoter,

- the nucleotide sequence of the cDNA of the human thyrotropin alpha beta subunit gene (TSH beta),

- the nucleotide sequence of the internal ribosome entry site (IRES), which ensures cap-independent or internal initiation of translation in eukaryotic cells,

- the nucleotide sequence of the cDNA of the human thyrotropin alpha subunit gene (TSH alpha),

- the nucleotide sequence of the extended internal ribosome entry site (IRESatt), which provides a lower level of internal translation initiation in eukaryotic cells,

- the nucleotide sequence of the cDNA of the human dehydrofolate reductase gene (hDHFR), which ensures the viability of CHO DG44 cells when grown on synthetic media that do not contain hypoxanthine and thymidine, and is in the same reading frame with the nucleotide sequence of the 39 C-terminal amino acids of mouse ornithine decarboxylase (PEST), which ensures rapid degradation of the hDHFR-PEST fusion protein.

2. The rhTSHCHO cell line is a highly productive stable producer of recombinant human thyroid-stimulating hormone (thyrotropin alpha), created by transforming the CHO DG44 line using the nucleofection method with recombinant plasmid DNA pFUSE 1-68 MAR TSH, the genome of which contains the expression plasmid DNA pFUSE 1-68 MAR TSH encoding the cDNA of the genes for the alpha and beta subunits of recombinant thyrotropin alpha.

The invention is illustrated by the following examples.

EXAMPLE 1

Creation of the genetic construct pFUSE 1-68 MAR TSH SEQ ID NO: 1

A DNA fragment containing the nucleotide sequence of MAR1-68 was obtained by PCR using the genomic DNA of HEK293 cells as a template and specific oligonucleotides 5-aatacgatcgccctcttgcagatccctgaactgaggag and 5-aatacgatcggcccagtcttgtttctcttaaatcgtccaaacca. The resulting PCR product was cloned into the plasmid DNA pFuse-hIgG1-Fc2 using the restriction endonuclease PvuI to obtain the genetic construct pFuse-hIgG1-Fc2-1-68 MAR. The synthetically obtained DNA fragment containing the nucleotide sequence encoding the cDNA of the alpha and beta subunit genes of recombinant thyrotropin alpha and the nucleotide sequence encoding the hDHFR-PEST fusion protein was cloned into the plasmid DNA pFuse-hIgG1-Fc2-1-68 MAR using restriction endonucleases SfoI and NheI to obtain the genetic construct pFUSE 1-68 MAR TSH (Fig. 1).

EXAMPLE 2

Obtaining a CHO DG44 cell line producing recombinant human thyrotropin alpha

The pFUSE 1-68 MAR TSH genetic construct was transformed into CHO DG44 cells by nucleofection using the Amaxa Cell Line Nucleofector Kit V reagent kit (Lonza, Germany) on a Nucleofector 2b device (Lonza, Germany). For this purpose, according to the manufacturer's protocol, 3 nucleofection reactions were performed containing 2, 5 and 10 μg of pFUSE 1-68 MAR TSH plasmid DNA and 1 million DG44 cells each. Transfected cells were pooled into one 125 ml Erlenmeyer flask (Corning, USA) containing 30 ml of the basic growth medium without hypoxanthine and thymidine (ProCHO5, 4 mM glutamine, 4 mM GlutaMAX) preheated to 37 °C and cultured on an orbital shaker in an incubator (37 °C, 5% CO ₂ , humidity 95%, 125 rpm). On the 2nd day after transfection, a sample of the culture fluid was taken to assess thyrotropin production by ELISA. Pool production was 0.070 pg/cell/day (0.04 mg/l of culture).

To select the most productive cells and maximize the amplification of the genetic cassette of the pFUSE 1-68 MAR TSH construct in the CHO DG44 genome, the cell pool after transfection was passaged according to the scheme shown in Fig. 2. For this purpose, the cells were passaged in the above-mentioned medium and conditions for 14 days until the culture viability was restored to more than 90%. Subculture was performed every 3-6 days by centrifuging the culture at 200g, collecting 90% of the supernatant, and adding an equal volume of fresh medium to the remaining conditioned medium with the cells in the sediment. After this time, the cell pool viability was 93%, the pool production was 0.18 pg/cell/day (0.504 mg/l of culture).

The cells were then passaged as described above into the same medium but now containing 250 nM methotrexate (MTX, Sigma-Aldrich). Methotrexate is an inhibitor of human dehydrofolate reductase (hDHFR), which creates additional metabolic pressure on the cells and leads to the accumulation of the entire genetic cassette of the pFUSE 1-68 MAR TSH construct. Passaging at this concentration of methotrexate was carried out for 45 days, until 91% viability was achieved. The pool production was 3.64 pg/cell/day (10.20 mg/L of culture).

The final step was to pass the cells in the same manner as described above into the medium but containing 2500 nM MTX. Passaging was carried out for 54 days until 91% viability was achieved. Pool production was 14.83 pg/cell/day (41.52 mg/L culture).

The resulting highly productive cell pool was cloned to obtain a homogeneous producer line, a descendant of a single cell. For this purpose, the cell culture was diluted to a concentration of 3 cells/ml in the base medium with the addition of 10% conditioned medium and 100 μl were pipetted into the wells of ten 96-well culture plates. On the same day, wells containing exactly one cell per well were found and marked in the plates using a microscope. The cells were grown in this form for 10 days, after which the contents of the previously selected wells with single clones were transferred to a 48-well plate for biomass growth and then to a 12-well plate. During this time, the relative growth rate of the clones was observed. Clones with the optimal growth rate and viability indicators were selected for product evaluation by the ELISA method. Of the 14 monoclones based on the production rates per cell in the plate, the 6 best were selected for expression analysis in flasks and for freezing the seeding pool of cells. In the next step, the two best clones based on production were selected for studying the stability of production, for which the lines were passaged for three weeks and then used again for thyrotropin production. The line with the minimal change in expression rates was used to obtain the main cell bank.

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taatgccaggcgggccatttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgat

gtactgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgt

tactatgggaacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggcca

tttaccgtaagttatgtaacgcctgcaggttaattaagaacatgtgagcaaaaggccagcaaaaggccag

gaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaat

cgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagct

ccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaag

cgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggc

tgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacc

cggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtagg

cggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgc

gctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctg

gtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatccttt

gatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatggctagt

taattaacatttaaatcagcggccgcaataaaatatctttattttcattacatctgtgtgttggtttttt

gtgtgaatcgtaactaacatacgctctccatcaaaacaaaacgaaacaaaacaaactagcaaaataggct

gtccccagtgcaagtgcaggtgccagaacatttctctatcgaaggatctgcgat</INSDSeq_sequen

ce>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (2)

1. Recombinant plasmid DNA pFUSE 1-68 MAR TSH SEQ ID NO: 1, encoding cDNA of the alpha and beta subunit genes of recombinant thyrotropin alpha and containing the nucleotide sequence of attachment to the nuclear matrix MAR1-68, the synthetic promoter hEF-HTLV, the nucleotide sequence of the cDNA of the beta subunit gene of human thyrotropin alpha (TSH beta), the nucleotide sequence of the internal ribosome entry region (IRES), the nucleotide sequence of the cDNA of the alpha subunit genes of human thyrotropin alpha (TSH alpha), the nucleotide sequence of the extended internal ribosome entry region, the nucleotide sequence of the cDNA of the human dehydrofolate reductase gene (hDHFR), located in a single reading frame with the nucleotide sequence of the 39 C-terminal amino acids mouse ornithine decarboxylase. 2. The cell line of Chinese hamster ovary cells rhTSHCHO - a producer of recombinant human thyrotropin alpha, created by transforming the CHO DG44 line by the nucleofection method of the recombinant plasmid DNA pFUSE 1-68 MAR TSH according to item 1.