Targeted integration of nucleic acids
Patent Information
- Application Number
- JP2024168021
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2018-12-21
- Filing Date
- 2024-09-27
- Publication Date
- 2025-01-23
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
的な薬剤)、例えば、化学療法剤または放射性薬剤などとモノクローナル抗体との複合化によって、毒性ペイロードを患部組織に特異的に送達するための薬剤が作製され、健康な組織への害が減少し得る。治療用抗体の「生物学的に機能的な断片」は、インタクトな抗体に起因する生物学的機能の一部または全部ではないにしても少なくとも1つを示し、その機能は標的抗原への少なくとも特異的な結合を含む。
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Figure 2025011135000001_ABST
Abstract
Claims
1. A host cell capable of expressing a polypeptide of interest, a) a targeted integration exogenous nucleic acid sequence of interest (SOI) encoding a first polypeptide of interest and a first selectable marker flanked by two recombination recognition sequences (RRS), wherein the targeted integration exogenous SOI is integrated into a targeted locus in the genome of the host cell; and b) a host cell comprising a random integration exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selectable marker, wherein said random integration SOI is integrated at least once into the genome of said host cell.
2. The host cell of claim 1 , wherein the first polypeptide of interest and the second polypeptide of interest are identical.
3. The host cell of claim 1 , wherein the first selection marker and the second selection marker are the same.
4. The host cell of claim 1, comprising from 1 to 10 randomly integrated exogenous nucleic acid SOIs.
5. The host cell of claim 1, wherein the targeted locus is at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.
6. The host cell of claim 1, further comprising a second targeted integration exogenous nucleic acid SOI encoding a second polypeptide of interest and a second selection marker integrated into a targeted locus in the genome of the host cell, wherein the first targeted integration exogenous nucleic acid SOI and the first selection marker are flanked by a first RRS and a third RRS, and the second targeted exogenous SOI and the second selection marker are flanked by a second RRS and a third RRS.
7. 6. The host cell according to any one of claims 1 to 5, wherein the polypeptide of interest is selected from the group consisting of a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv) and an Fc fusion protein.
8. The host cell according to any one of claims 1 to 5, wherein the host cell is a mammalian host cell.
9. The host cell of claim 8 , wherein the host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.
10. 10. The host cell of claim 9, wherein the host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.
11. The host cell of any one of claims 1 to 7, wherein the targeted integration of the SOI and selection marker is facilitated by an exogenous nuclease.
12. 12. The host cell of claim 11, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.
13. 1. A method for expressing a polypeptide of interest, comprising the steps of: a) providing a host cell comprising an exogenous nucleotide sequence integrated into a targeted locus in the genome of the host cell, said exogenous nucleotide sequence comprising two RRSs flanking a first selection marker; b) introducing into the cell provided in (a) a nucleic acid comprising two RRSs that match the two RRSs of the integrated exogenous nucleotide sequence and flank a first exogenous SOI encoding a first polypeptide of interest and a second selectable marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein said recombinase recognizes said RRS; d) selecting cells expressing the second selection marker; e) introducing, via random integration, a second exogenous SOI encoding a second polypeptide of interest and a third selection marker into the genome of the host cell; f) selecting cells expressing the third selection marker; and g) culturing said host cell under conditions sufficient to express said first polypeptide of interest and said second polypeptide of interest.
14. 14. The method of claim 13, further comprising recovering the first polypeptide of interest and the second polypeptide of interest from the host cell culture medium.
15. 14. The method of claim 13, wherein the first polypeptide of interest and the second polypeptide of interest are the same.
16. The method of claim 13, wherein the targeted locus is at least about 90% homologous to a sequence selected from SEQ ID NOs: 1-7.
17. 17. The method of any one of claims 13 to 16, wherein the first polypeptide of interest and the second polypeptide of interest are selected from the group consisting of a single chain antibody, an antibody light chain, an antibody heavy chain, a single chain Fv fragment (scFv) and an Fc fusion protein.
18. The method of any one of claims 13 to 17, wherein the host cell is a mammalian host cell.
19. 20. The method of claim 18, wherein the host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.
20. 20. The method of claim 19, wherein the host cell is a CHO host cell, a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S host cell, or a CHO K1M host cell.
21. The method of any one of claims 13 to 20, wherein the targeted integration of any of the SOIs is facilitated by an exogenous nuclease.
22. 22. The method of claim 21, wherein the exogenous nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), ZFN dimers, transcription activator-like effector nucleases (TALENs), TAL effector domain fusion proteins, RNA-guided DNA endonucleases, engineered meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonucleases.
23. The method of any one of claims 1 to 22, wherein expression of the SOI is controlled by a regulatable promoter.
24. 24. The method of claim 23, wherein the regulatable promoter is selected from the group consisting of SV40 and CMV promoters.