WO2023131330A1 - Vecteur d'expression et son utilisation - Google Patents

Vecteur d'expression et son utilisation Download PDF

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WO2023131330A1
WO2023131330A1 PCT/CN2023/071371 CN2023071371W WO2023131330A1 WO 2023131330 A1 WO2023131330 A1 WO 2023131330A1 CN 2023071371 W CN2023071371 W CN 2023071371W WO 2023131330 A1 WO2023131330 A1 WO 2023131330A1
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expression vector
screening
expression
marker
host cells
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PCT/CN2023/071371
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Chinese (zh)
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沈潇
李京浩
弗兰克·亚娜
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佛山汉腾生物科技有限公司
广州汉腾生物科技有限公司
佛山普津生物技术有限公司
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Publication of WO2023131330A1 publication Critical patent/WO2023131330A1/fr

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Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to an expression vector and a method for screening cell pools using it.
  • CHO cells Chinese hamster ovary (CHO) cells are the most commonly used expression hosts for the manufacture of complex biomolecules.
  • the two most common methods for expressing proteins in CHO cells are transient gene expression and stable gene expression.
  • Transient gene expression means that after the exogenous gene enters the recipient cell, it exists on the free carrier and does not integrate into the chromosome of the cell.
  • Transient gene expression is the method of choice for rapid production of small quantities of recombinant protein, usually in as little as 1-3 weeks.
  • transient gene expression is not practical when gram-scale protein production is required due to the large amounts of DNA and host cells required. Therefore, stable gene expression is often used to produce gram-scale recombinant proteins.
  • Stable gene expression refers to the integration of the transfected target gene into the chromosomal DNA to express the target protein.
  • stabilizing gene expression is time-consuming and laborious. It usually takes 3-9 months to generate CHO cell lines. Therefore, there is an urgent need for cell lines that are fast, stable, and high in producing target proteins.
  • the invention provides an expression vector, through which a cell pool can be rapidly screened, and a CHO clone can be obtained through further screening, so as to generate a stable CHO cell line.
  • a cell pool can be rapidly screened, and a CHO clone can be obtained through further screening, so as to generate a stable CHO cell line.
  • the expression level of the target protein is increased, and the rapidly produced CHO cell line is not only stable but also highly productive.
  • the present invention provides an expression vector, which includes a selection marker expression unit, at least one target gene expression unit, and inverted terminal repeats (ITRs) of the PiggyBac transposon.
  • ITRs inverted terminal repeats
  • the screening marker expression unit comprises a coding sequence of a screening marker, and a first regulatory sequence operably linked to the coding sequence of the screening marker, for example, a coding sequence of a promoter and a coding sequence of a terminator.
  • the selectable markers include, but are not limited to, Puromycin (Puro), Neomycin (Neo), Hygromycin B (Hygro B), and Blasticidin ( Blasticidin, Bsd), glutamine synthetase (Glutamine synthetase, GS) and dihydrofolate reductase (Dihydrofolate reductase, DHFR).
  • the screening marker is human GS.
  • the promoter of the expression unit of the selection marker can be a herpes simplex virus thymidine kinase (Thymidine kinasegene of Herpes simplex virus, HSV-tk) promoter or a simian vacuolar virus 40 (Simian virus40, SV40) promoter son.
  • the terminator of the selection marker expression unit can be a polyadenylation signal (PolyA), such as SV40 PolyA, bovine growth hormone (Bovine growth hormone, bGH) PolyA, HSV-tk PolyA or rabbit ⁇ - Globin (Rabbit beta-globin, RBG) PolyA, preferably SV40 PolyA.
  • PolyA polyadenylation signal
  • SV40 PolyA bovine growth hormone (Bovine growth hormone, bGH) PolyA
  • HSV-tk PolyA HSV-tk PolyA or rabbit ⁇ - Globin (Rabbit beta-globin, RBG) PolyA, preferably SV40 PolyA.
  • the screening marker expression unit comprises the coding sequence of HSV-tk promoter and the coding sequence of SV40 polyA.
  • the screening marker expression unit comprises the coding sequence of the HSV-tk promoter, the coding sequence of the human GS screening marker and the coding sequence of SV40 polyA.
  • the target gene expression unit comprises the target gene sequence and a second regulatory sequence operably linked to the target gene sequence, for example, the coding sequence of the target gene expression regulatory element and the coding sequence of the terminator.
  • the coding sequence regulatory sequence of the target gene expression regulatory element has a sequence selected from the sequences shown in SEQ ID NO.1-6.
  • the terminator of the target gene expression unit can be polyA, such as SV40 PolyA, bGH PolyA, tk PolyA, RBG PolyA, preferably bGH PolyA.
  • the target gene is one or more, such as 2, 3 or 4, and correspondingly, the expression vector has 2, 3 or 4 target gene expression units.
  • the target gene encodes the light chain and/or heavy chain of an anti-CD20 monoclonal antibody (Rituximab) or an anti-HER2 monoclonal antibody (Trastuzumab).
  • the expression vector has 1 or 2 target gene expression units.
  • the expression vector can be quickly screened to obtain a CHO cell pool, and further screened to obtain a CHO clone to generate a stable CHO cell line, and by optimizing the regulatory elements in the expression vector, the expression of the target protein is increased, and the rapid production
  • the CHO cell line is not only stable but also highly productive.
  • the present invention provides an expression system comprising the expression vector of the first aspect.
  • the expression system further comprises a helper plasmid encoding a PiggyBac transposase.
  • the present invention provides host cells transfected with the expression vector of the first aspect.
  • the host cell is transfected with the expression system of the second aspect.
  • the expression vector of the first aspect is stably integrated in the genome of the host cell.
  • the host cell does not contain a helper plasmid encoding a PiggyBac transposase.
  • the host cell is a CHO cell, such as a CHO-K1 cell or a CHOExpress TM cell.
  • the host cell is a stable CHO cell line.
  • the host cell has increased expression of the protein of interest.
  • the present invention provides a method for screening cells using the expression vector of the first aspect or the expression system of the second aspect, the method comprising the following steps:
  • step (b) a step of screening the host cells obtained in step (a) with a screening reagent directed against the screening marker in the expression vector.
  • the host cell is a CHO cell, such as a CHO-K1 cell or a CHOExpress TM cell.
  • the selection marker is puromycin, and 10 ⁇ g/ml of puromycin is used to select host cells transfected with the expression vector comprising a puromycin resistance selection marker .
  • the selection marker is puromycin, and increased concentrations of puromycin (10 ⁇ g/ml, 50 ⁇ g/ml, 250 ⁇ g/ml) are used to select host cells transfected with The expression vector for the puromycin resistance selection marker.
  • the selection marker is GS, and 25 ⁇ M methionine sulfoximine (MSX) is used to select host cells transfected with the expression vector comprising the GS selection marker .
  • MSX methionine sulfoximine
  • the selection marker is GS, and an increased concentration of MSX (25 ⁇ M, 50 ⁇ M) is used to select host cells transfected with the expression vector comprising the GS selection marker.
  • the selection marker is GS, and increased concentrations of MSX (25 ⁇ M, 50 ⁇ M, 100 ⁇ M) are used to select host cells transfected with the expression vector comprising the GS selection marker.
  • the screening marker is human GS.
  • the selection marker is DHFR, and 250 nM of methotrexate (methopterin, MTX) is used to select host cells transfected with the expression vector comprising the DHFR selection marker.
  • methotrexate methopterin, MTX
  • the selection marker is DHFR
  • increased concentrations of methotrexate 250 nM, 500 nM are used to select host cells transfected with the expression vector comprising the DHFR selection marker.
  • the screening reagent is puromycin, methionine iminosulfone or methotrexate.
  • the host cells are subjected to pressure selection with CHO cell culture medium containing puromycin, methionine sulfone imino, or methotrexate.
  • the medium is BalanCD CHO GROWTH A medium.
  • the screening pressure is removed after the viability rate recovers to above 90%.
  • the screening method of the present invention screens high-yield and stable CHO clones, and then produces stable CHO cell lines.
  • the present invention provides cells obtained by the screening method of the fourth aspect.
  • the present invention provides the expression vector of the first aspect, the expression system of the second aspect, the host cell of the third aspect, or the screening method of the fourth aspect, or the purpose of increasing the expression level of the cells of the fifth aspect protein application.
  • the expression vector provided in the present invention can be quickly screened to obtain a cell pool through the expression vector, and can be further screened to obtain a CHO clone to produce a stable CHO cell line, which greatly shortens the time for producing a stable CHO cell line.
  • the optimization of the regulatory elements in the medium increases the expression of the target protein, and the rapidly produced CHO cell line is not only stable but also highly productive.
  • Figure 1 shows a structural diagram of an expression vector comprising two target gene expression units.
  • Figure 2 shows the structural diagram of an expression vector comprising an expression unit of a target gene.
  • Figure 3 shows the selection strategy against the pool of cells using the puromycin resistance selection marker.
  • Figure 4 shows the screening strategy for cell pools using GS selection markers including rat GS as well as human GS.
  • Figure 5 shows the screening strategy against the cell pool using the DHFR selection marker.
  • Figure 6 shows the expression vector structure for expressing anti-CD20 mAb (Rituximab).
  • Figure 7 shows the cell viability (VIA) of the CHO-K1 cell pool.
  • Figure 8 shows the cell viability (VIA) of the CHOExpress TM cell pool.
  • Figure 9 shows the viable cell density (VCD) and cell viability (VIA) of the CHO-K1 cell pool with better recovery of cell viability.
  • Figure 10 shows the viable cell density (VCD) and cell viability (VIA) of the CHOExpress TM cell pool with better recovery of cell viability.
  • Figure 11 shows the growth curve of the cell pool at 37°C, where "KP10” refers to K1-PR10, “KP250” refers to K1-PR250, "EP10” refers to EXP-PR10, “EP250” refers to EXP-PR250, "KH25 “ refers to K1-HG25, “KH50” refers to K1-HG50, “KH100” refers to K1-HG100, “EH25” refers to EXP-HG25, “EH50” refers to EXP-HG50, and “EH100” refers to EXP-HG100.
  • Figure 12 shows the growth curve of the cell pool when the temperature was lowered to 33°C on the sixth day, where "KP10” refers to K1-PR10, “KP250” refers to K1-PR250, “EP10” refers to EXP-PR10, and “EP250” refers to EXP -PR250, "KH25” refers to K1-HG25, “KH50” refers to K1-HG50, “KH100” refers to K1-HG100, “EH25” refers to EXP-HG25, “EH50” refers to EXP-HG50, “EH100” refers to EXP-HG100 .
  • Figure 13 shows the results of Protein A HPLC detection expression level, where "KP10” refers to K1-PR10, “KP250” refers to K1-PR250, “EP10” refers to EXP-PR10, “EP250” refers to EXP-PR250, "KH25” refers to K1-HG25, “KH50” refers to K1-HG50, “KH100” refers to K1-HG100, “EH25” refers to EXP-HG25, “EH50” refers to EXP-HG50, and “EH100” refers to EXP-HG100.
  • Figure 14 shows the growth curve of the cells in Example 3.
  • FIG. 15 shows the expression detection results in Example 3.
  • FIG. 16 shows the expression detection results in Example 4.
  • the present invention can construct an expression vector comprising two target gene expression units (see FIG. 1 ), or construct an expression vector comprising one target gene expression unit (see FIG. 2 ).
  • the PiggyBac system that the present invention adopts comprises two vectors, and a vector is called helper plasmid, is responsible for coding transposase; Another vector is called transposon plasmid, comprises two terminal repeats (ITRs) and between the two The transposed region, promoter, expression cassette and gene of interest are cloned into this transposed region together.
  • helper plasmid and the transposon plasmid co-transfect the target cell, the transposase produced by the helper plasmid will recognize the two ITR elements of the transposon, and then insert the transposable region and the two ITR elements into the host genome .
  • Transposon insertions usually occur at host chromosomal loci that contain TTAA sequences, with TTAA repeats flanking the transposon.
  • PiggyBac is a class II transposon that moves by a "cut-and-paste" mechanism, transposing from one place to another without leaving behind the sequence itself. Since the helper plasmid is transiently transfected into the host cell, it is gradually lost. With the loss of the helper plasmid, the transposon becomes permanently integrated in the host genome. When these host cells are transfected again with the helper plasmid, the integrated transposons are again moved by the "cut-and-paste" mechanism.
  • the selection strategy for the pool of cells using the puromycin resistance selection marker is as follows (see Figure 3):
  • Puro strategy 1 Use 10 ⁇ g/ml puromycin for screening throughout the process;
  • Puro strategy 2 Screening with increased concentration of puromycin (10 ⁇ g/ml, 50 ⁇ g/ml, 250 ⁇ g/ml);
  • GS screening markers including rat GS and human GS.
  • GS strategy 1 Use 25 ⁇ M MSX for screening throughout the process
  • GS strategy 2 use MSX with varying concentrations (25 ⁇ M, 50 ⁇ M) for screening;
  • GS strategy 3 use MSX with varying concentrations (25 ⁇ M, 50 ⁇ M, 100 ⁇ M) for screening;
  • the screening strategy for cell pools using DHFR screening markers is as follows (see Figure 5):
  • DHFR strategy 1 use 250nM methotrexate (methopterin, MTX) for screening throughout;
  • DHFR strategy 2 Screening using varying concentrations of MTX (250 nM, 500 nM).
  • Example 1 Construction of expression vectors for expressing anti-CD20 mAb (Rituximab) and screening of cell lines
  • FIG. 6 the expression vector constructed for expressing anti-CD20 mAb (Rituximab) is shown.
  • the coding sequence of the target gene expression regulatory element is shown in SEQ ID NO.1
  • the PiggyBac transposon LTD sequence is shown in SEQ ID NO.7
  • the PiggyBac transposon RTD sequence is shown in SEQ ID NO.8, bGH
  • the polyA sequence is shown in SEQ ID NO.9
  • the HSV-tk promoter sequence is shown in SEQ ID NO.10
  • the SV40 polyA sequence is shown in SEQ ID NO.11.
  • PEI 25KD 37.5 ⁇ g
  • recombinant anti-CD20 monoclonal antibody plasmid 11.25 ⁇ g see the expression vector constructed in Figure 6 for expressing anti-CD20 mAb (Rituximab)
  • transposase plasmid 1.25 ⁇ g
  • HyCell Transfx containing CHO cells -C medium GE, SH30934.04
  • the density of CHO cells in the medium is 15 ⁇ 10 6 cell/ml.
  • BalanCD CHO GROWTH A medium containing screening reagents for pressurized screening, passaging once every three days, the passaging cell density is 0.3-0.5 ⁇ 10 6 cell/ml, remove after the viability rate recovers to more than 90% Screening pressure.
  • CHO-K1 purchased from ATCC
  • CHOExpress TM purchased from ExcellGene
  • four selection markers of puromycin, rat GS, human GS, and mouse DHFR were used to construct eight expression Anti-CD20 monoclonal antibody cells, and for these cells, different cell pool screening strategies were used, as shown in Table 1 below.
  • VAV cell viability
  • the cell pool using the puromycin resistance marker and human GS marker can recover within two weeks after transfection, while the cell pool using the DHFR marker recovers much slower.
  • the concentration of screening reagents in the screening process has a certain impact on the recovery of the cell pool.
  • VCD Viable cell density
  • VIA cell viability test results of the CHO-K1 cell pool with better recovery of cell viability (as shown in FIG. 9 ).
  • VCD Viable cell density
  • VIA cell viability
  • Example 2 Fed-batch culture evaluation of cell pools using human GS and the puromycin resistance selection marker
  • the culture conditions are: 37° C., 140 rpm, 5% CO 2 , and 85% humidity.
  • Another group was set up and the temperature was adjusted to 33°C when the culture reached the 6th day, and the other conditions were the same as those described above.
  • the growth curve of the cell pool at 37°C is shown in Figure 11 .
  • the growth curve of the cell pool under the condition of cooling down to 33° C. on the sixth day is shown in FIG. 12 .
  • Example 3 Monocloning of positive cloned cells and detection of expression levels of monoclonal cell lines
  • the cells were inoculated at a density of 0.3*10 ⁇ 6cells/ml in a 250ml shake flask with a culture volume of 50ml, 140rpm, 5% CO 2 , and cultured at 37°C.
  • the cell growth curve and expression level are shown in Figure 14 and Figure 15, respectively.

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Abstract

La présente invention concerne un vecteur d'expression. Le vecteur d'expression peut être utilisé pour un criblage rapide pour obtenir un groupe de cellules, et un criblage supplémentaire pour obtenir un clone CHO pour générer une ligne de cellule CHO stable. La quantité d'expression d'une protéine cible est améliorée par optimisation de l'élément de régulation et de commande dans le vecteur d'expression.
PCT/CN2023/071371 2022-01-10 2023-01-09 Vecteur d'expression et son utilisation WO2023131330A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090042297A1 (en) * 2007-06-01 2009-02-12 George Jr Alfred L Piggybac transposon-based vectors and methods of nucleic acid integration
CN106755096A (zh) * 2016-12-20 2017-05-31 上海药明生物技术有限公司 利用piggyBac转座子在CHO细胞中获得表达目标蛋白的稳定细胞群的方法
WO2020068631A1 (fr) * 2018-09-24 2020-04-02 Merck Sharp & Dohme Corp. Vecteurs d'expression pour systèmes d'expression eucaryotes
WO2020132382A1 (fr) * 2018-12-21 2020-06-25 Merck Sharp & Dohme Corp. Vecteurs d'expression pour systèmes d'expression eucaryotes
US20200318121A1 (en) * 2019-04-08 2020-10-08 Dna Twopointo Inc. Integration of nucleic acid constructs into eukaryotic cells with a transposase from oryzias
CN112513277A (zh) * 2019-04-08 2021-03-16 Dna2.0股份有限公司 使用来自螟蛾属的转座酶将核酸构建体转座入真核基因组
WO2021164704A2 (fr) * 2020-02-19 2021-08-26 Wuxi Biologics (Shanghai) Co., Ltd. Système d'expression amélioré et son procédé d'utilisation

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090042297A1 (en) * 2007-06-01 2009-02-12 George Jr Alfred L Piggybac transposon-based vectors and methods of nucleic acid integration
CN106755096A (zh) * 2016-12-20 2017-05-31 上海药明生物技术有限公司 利用piggyBac转座子在CHO细胞中获得表达目标蛋白的稳定细胞群的方法
WO2020068631A1 (fr) * 2018-09-24 2020-04-02 Merck Sharp & Dohme Corp. Vecteurs d'expression pour systèmes d'expression eucaryotes
WO2020132382A1 (fr) * 2018-12-21 2020-06-25 Merck Sharp & Dohme Corp. Vecteurs d'expression pour systèmes d'expression eucaryotes
US20200318121A1 (en) * 2019-04-08 2020-10-08 Dna Twopointo Inc. Integration of nucleic acid constructs into eukaryotic cells with a transposase from oryzias
CN112424362A (zh) * 2019-04-08 2021-02-26 Dna2.0股份有限公司 使用来自青鳉属的转座酶将核酸构建体整合入真核细胞
CN112513277A (zh) * 2019-04-08 2021-03-16 Dna2.0股份有限公司 使用来自螟蛾属的转座酶将核酸构建体转座入真核基因组
WO2021164704A2 (fr) * 2020-02-19 2021-08-26 Wuxi Biologics (Shanghai) Co., Ltd. Système d'expression amélioré et son procédé d'utilisation

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