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JP-7856785-B2 - Bacterial glutamine synthase as a selection marker in mammalian cells

JP7856785B2JP 7856785 B2JP7856785 B2JP 7856785B2JP-7856785-B2

Inventors

  • シュミット，モリッツ
  • ハインツェルマン，ダニエル
  • ロイス，フランツィスカ
  • フィッシャー，ジーモン
  • シュルツ，パトリック

Assignees

  • ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング

Dates

Publication Date
20260511
Application Date
20230322
Priority Date
20220323

Claims (20)

  1. A mammalian expression vector comprising a polynucleotide encoding bacterial glutamine synthase as a selection marker, wherein the bacterial glutamine synthase comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 .
  2. The mammalian expression vector according to claim 1, wherein the bacterial glutamine synthase is derived from bacteria of the Enterobacteriaceae order and the Morganellaceae family.
  3. The mammalian expression vector according to claim 2, wherein the bacterial glutamine synthase is derived from the genus Providencia or Photorhabdus.
  4. The mammalian expression vector according to claim 3, wherein the bacterial glutamine synthase is Providencia vermicola glutamine synthase or Photorhabdus luminescens glutamine synthase.
  5. The mammalian expression vector according to claim 1, wherein the bacterial glutamine synthase comprises an amino acid sequence having a sequence identity of at least 90% to the amino acid sequence of SEQ ID NO: 1 or at least 95% to the amino acid sequence of SEQ ID NO: 14, and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid.
  6. The mammalian expression vector according to claim 5, wherein the mutation at amino acid position E130 is a substitution with an aromatic amino acid or a hydrophobic amino acid.
  7. The mammalian expression vector according to claim 6, wherein the aromatic amino acid or hydrophobic amino acid is selected from the group consisting of Y, W, F, A, G, V, L, M, and I.
  8. The mammalian expression vector according to claim 1, wherein the bacterial glutamine synthase comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 14, and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid.
  9. The mammalian expression vector according to claim 1 , further comprising at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  10. The mammalian expression vector according to claim 1, further comprising an expression cassette containing at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  11. The mammalian expression vector according to claim 9 , wherein the protein of interest is a therapeutic protein.
  12. The mammalian expression vector according to claim 11, wherein the therapeutic protein is selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetic compounds.
  13. A nucleic acid comprising a polynucleotide encoding bacterial glutamine synthase, the amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1 or at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 14 , operably linked to a mammalian promoter.
  14. Furthermore, the nucleic acid according to claim 13, comprising at least one polynucleotide encoding a protein of interest and/or a non-coding RNA.
  15. The nucleic acid according to claim 13, wherein the bacterial glutamine synthase comprises an amino acid sequence having a sequence identity of at least 90% with respect to the amino acid sequence of SEQ ID NO: 1 or at least 95% with respect to the amino acid sequence of SEQ ID NO: 14 , and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid .
  16. A bacterial glutamine synthase comprising an amino acid sequence having at least 90 % sequence identity with the amino acid sequence of SEQ ID NO: 1 or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 , and a mutation selected from the group consisting of E130X, F226Y, R345A and combinations thereof, where X is any amino acid.
  17. A mammalian host cell comprising an expression vector according to any one of claims 1 to 12 , a nucleic acid according to any one of claims 13 to 15 , or a nucleic acid encoding bacterial glutamine synthase according to claim 16 .
  18. The mammalian host cell according to claim 17, wherein the mammalian host cell is (a) a rodent cell and/or (b) a glutamine synthase gene knockout cell.
  19. The mammalian host cell according to claim 18, wherein the rodent cell is a Chinese hamster ovary (CHO) cell.
  20. (a) Introducing into a mammalian host cell an expression vector according to any one of claims 1 to 12 or a nucleic acid according to any one of claims 13 to 15 , which comprises a polynucleotide encoding bacterial glutamine synthase and at least one polynucleotide encoding the protein of interest and/or non-coding RNA; and (b) culturing the mammalian host cell in a glutamine-free medium under conditions selected for bacterial glutamine synthase (wherein the at least one polynucleotide encoding the protein of interest and/or non-coding RNA is simultaneously incorporated into the host cell genome together with the polynucleotide encoding bacterial glutamine synthase). A method for preparing cells that stably express a protein of interest and/or non-coding RNA, including the above.

Description

Field of Invention The present invention relates to a mammalian expression vector comprising a polynucleotide encoding a bacterial glutamine synthase as a select marker having at least 85% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1, as well as a nucleic acid encoding the bacterial glutamine synthase and a mammalian host cell. In particular, the bacterial glutamine synthase is derived from bacteria of the order Enterobacteriales and the family Morganellaceae, preferably a glutamine synthase of the genus Providencia, and optionally further comprising mutations at positions E130 and/or F226 and/or R345. The present invention further relates to a method for preparing mammalian cells stably expressing a protein and/or non-coding RNA of interest, and a method for producing a protein in mammalian cells using the bacterial glutamine synthase. Background: Chinese hamster ovary (CHO) cells are one of the most frequently used mammalian cell lines for the production of therapeutic proteins, such as antibodies. A key aspect is the generation of productive and stable cell lines that express the protein of interest with high product titer and appropriate product quality in a short time. To produce stable and highly productive cell lines, a stable and heterogeneous cell pool consisting of various clones that need to be isolated and screened is generated, followed by a selection period to make the final productive cell line ready for storage in a cell bank. Metabolic selection systems, such as dihydrofolate reductase (DHFR) and glutamine synthase (GS) selection systems, are commonly used to improve this process and more efficiently produce stable cell lines. In cell lines lacking the dihydrofolate reductase gene, such as CHO-DG44 cells, selection is performed in the absence of hypoxanthine and thymidine in the culture medium. An amplification step involving the addition of gradually increasing concentrations of methotrexate (MTX) may be added. The glutamine synthase selection system is advantageous because it requires fewer gene copies for survival, and therefore selection is more rapid in highly productive cell pools. Glutamine synthase (EC 6.3.1.2, also known as γ-glutamyl ammonia ligase) catalyzes the ATP-dependent condensation of ammonia and glutamic acid to form glutamine. Glutamine synthases are classified into three subgroups: GSI, GSII, and GSIII. CHOGS is a class II enzyme, a subclass mainly expressed by eukaryotic cells, while bacterial GS proteins are typically members of the GSI class. Although GSI and GSII catalyze the same reaction, they are quite different overall, showing little to no sequence similarity except for residues that form part of the active site. For example, bacterial type I GS is a complex of 12 subunits (Eisenberg et al. (2000), Biochim. Biophys. Acta 1477, 122-145), and GSII has been reported to form a stack of two or three pentameric rings (Krajewski et al. (2008), J. Mol. Biol. 375, 217-228). Furthermore, bacterial glutamine synthases may exhibit only very slight sequence similarities to one another. For example, Bacillus coagulans (CN 12625930 A), Mycobacterium tuberculosis (International Publication No. 2006/000045), or Corynebacterium glutamicum (CN 1884501 A) have a low amino acid identity of approximately 50% or less compared to the glutamine synthase of Providencia vermicola (Zuo et al., Scientific Reports, 2018, 8(1): 1-8 and supplementary materials). Glutamine synthase is a ubiquitous enzyme essential for nitrogen metabolism. Therefore, glutamine synthase is used as a selective marker introduced via mammalian expression constructs. In cell lines that do not express sufficient levels of endogenous glutamine synthase, removal of glutamine supplementation from the cell culture medium increases the selective pressure on the cells. In cell lines with insufficient endogenous glutamine synthase levels, such as mouse myeloma cell lines, culturing in the absence of glutamine or without glutamine supplementation provides sufficient selective pressure to isolate stable recombinant cell lines. In cell lines with sufficient endogenous glutamine synthase, such as CHO cells, the addition of the glutamine synthase inhibitor methionine sulfoximine (MSX) or the creation of glutamine synthase knockout cells (GS-/- or GS-/+) is necessary to enable sufficient selective pressure to isolate producing cell lines in the absence of glutamine. The rigor of selection in CHO GS knockout cells has been significantly improved, and it has been reported that transfected GS gene expression under weak promoter control, with or without the use of MSX, improves the rigor of selection. In addition to the rigor of selection and productivity, the stability of protein production in highly productive clones has been shown to be a key feature. Furthermore, transfected glutamine synthase selection markers have a significant impact on the selection process, phenotypic stability, and productivity