Host cells with improved protein expression efficiency and uses thereof

Abstract

To provide new types of host cells that have improved protein (e.g., antibody) production efficiencies.SOLUTION: A host cell for protein expression has a lower expression level of a gene as compared to a wild-type cell, and the gene is selected from HDAC8, Dab2, Caspase3, Sys1, Ergic3, Grasp, Trim 23, or a combination thereof. The host cells are CHO cells. The lower expression level of the gene results from RNA interference, which may be achieved by transfecting a vector that contains an shRNA targeting the gene.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to host cells for protein production, in particular engineered host cells such as CHO cells that are capable of producing proteins at higher levels compared to wild-type cells. [Background technology]

[0002] Protein drugs are usually produced by expression in suitable host cells. Chinese hamster ovary (CHO) cells are the most widely used host cells for protein drug production. Optimization of host cells (e.g., by genetic modification of the host cells) or optimization of downstream processes are being explored to increase the efficiency of protein drug production. Currently, many strategies are available to improve protein expression and / or secretion, for example, by using chemical reagents or by genetic modification of cells.

[0003] Genetic modifications typically target genes involved in transcription, post-transcriptional regulation, translation, and post-translational events. For example, post-transcriptional regulatory elements (PTREs) have been targeted for manipulation to improve protein expression (see Mariati et al., "Post-transcriptional regulatory elements for enhancing transient gene expression levels in mammalian cells," Methods Mol Biol., 2012, 801:125-35). Summary of the Invention [Problem to be solved by the invention]

[0004] Although conventional techniques for modifying protein-expressing host cells are useful, there remains a need for other methods for improving protein expression in host cells. [Means for solving the problem]

[0005] Embodiments of the present invention relate to a new type of host cell with improved efficiency in protein (e.g., antibody) production. The new type of host cell has been genetically engineered to modify one or more genes that have unexpectedly been found to affect protein expression or secretion. Such genes are distinct from previously known genes, such as post-transcriptional regulatory elements, that have been targeted for manipulation to improve protein expression. The genes manipulated in the present invention are not related to transcription, post-transcriptional regulation, translation, or post-translational events. Therefore, it was unexpected that suppressing the expression of these genes can result in improved protein expression / secretion.

[0006] According to an embodiment of the present invention, the genetic engineering of host cells can comprise the knockdown of one or more genes selected from HDAC8, Dab2, Caspase3, Sys1, Ergic3, Grasp and Trim23.Gene knockdown can be achieved by any suitable genetic engineering technique known in the art, such as RNA interference with target genes.The RNAi targeting these genes can be carried out by transfecting cells with suitable constructs to knockdown these target genes, thereby producing engineered host cells.

[0007] In a preferred embodiment, the host cell is a CHO cell, and the target gene can be the HDAC8, DAB2, or Caspase 3 gene, or a combination thereof. Inhibition or suppression of one or more of these genes by short hairpin RNA (shRNA) or siRNA produces host cells that can support improved protein expression and / or secretion. For example, shRNA inhibition of Caspase 3 can lead to reduced apoptosis of host cells. Thus, inhibition of the Caspase 3 gene by siRNA or shRNA, either short-term or long-term, can increase the production of proteins (such as antibodies).

[0008] According to some embodiments of the present invention, siRNA or shRNA inhibition of HDAC8, DAB2, or Caspase 3 can result in stable cell lines. These stable cell lines can be selected after evaluating their transfection efficiency, increased antibody expression, lactate metabolism, growth rate, adaptability to new media, and / or stability over long periods of time (e.g., 60 or more generations). In addition to being able to produce / secrete larger amounts of protein (e.g., antibodies), these stable cells also have the characteristics of greater stability and adaptability to new media. Therefore, they are suitable for downstream process development.

[0009] One aspect of the present invention relates to a host cell for protein expression. According to one embodiment of the present invention, the host cell comprises a lower expression level of HDAC8, Dab2, Caspase3, Sys1, and / or Trim23 genes compared to wild-type cells. For example, the engineered host cell may have an expression level that is 15% or more lower, which is manifested as gene knockdown. That is, the knockdown cell may express a particular gene at a level of 85% or less compared to wild-type cells.

[0010] According to a preferred embodiment of the present invention, the gene with lower expression level is HDAC8, Dab2, Caspase3, or a combination thereof. According to some embodiments of the gene, the host cell is a CHO cell. According to some embodiments of the present invention, the lower expression level of post-transcriptional regulatory genes or apoptosis genes is due to RNA interference.

[0011] Other aspects of the present invention will become apparent from the following description, drawings, and appended claims. [Brief explanation of the drawings]

[0012] [Figure 1]Figure 1 shows protein production levels after knockdown of various genes in 1C9 cells. 1C9 is a low IgG-producing cell line (1.28 mg / L on day 6 of batch culture). 1C9 cells were used to determine whether IgG secretion could be increased by siRNA inhibition of various genes.

[0013] [Figure 2] Candidate genes for knockdown selected from analysis of high and low producing cells using gene arrays from NimbleGene and Agilent are shown.

[0014] [Figure 3] Protein (Avastin and Herceptin) expression levels in various cell lines with target gene knockdown are shown.

[0015] [Figure 4] Suitable non-lentiviral vectors (plasmids) for shRNA construction are shown.

[0016] [Figure 5] 1 shows lentivirus-derived plasmids for shRNA construction.

[0017] [Figure 6] An example of the sequence format of shRNA is shown.

[0018] [Figure 7] Selection of cell pools using puromycin at various concentrations is shown.

[0019] [Figure 8] Protein expression levels in various transfectant cells selected with puromycin as described in FIG. 7 are shown.

[0020] [Figure 9] FIG. 1 shows a schematic illustrating the procedure for isolating single clones of engineered cells.

[0021] [Figure 10] Protein expression levels (transient expression) in the top 5 single clones with Caspase3 knockdown are shown.

[0022] [Figure 11A] The results of an analysis of the top five single clones in terms of their characteristics (population doubling time, lactate level, and Caspase 3 gene expression level) in long-term culture (up to 6 weeks) are shown.

[0023] [Figure 11B] Caspase 3 knockdown levels in the top five single clones at different time points during long-term culture are shown.

[0024] [Figure 12] The long-term stability of the top five single clones is shown, with doubling time and percentage of viable cells plotted as a function of time (up to 100 generations).

[0025] [Figure 13] Protein expression levels using the top three single clones as a function of time are shown (weeks 0, 3 and 6).

[0026] [Figure 14A] The characteristics of second generation cells derived from the top three single clones are shown.

[0027] [Figure 14B] 1 shows the transfection rate of CHO cells according to an embodiment of the present invention.

[0028] [Figure 14C] Caspase 3 expression levels in second-generation cells are shown.

[0029] [Figure 15] Protein expression levels in second generation cells compared to first generation and parental cells are shown.

[0030] [Figure 16] 1 shows the glycan profile of Herceptin produced in CHO cells of the present invention, showing that the major glycans include G0F, G1Fa, G1Fb, and G2F, similar to those of commercially available Herceptin. DETAILED DESCRIPTION OF THE INVENTION

[0031] Embodiments of the present invention relate to the development of new cell lines for protein production. These new host cells have improved efficiency in the production and / or secretion of proteins (such as antibodies). Because the genes engineered in these cells are not directly linked to transcription, post-transcriptional regulation, translation, or post-translational events, it was unexpected that suppressing the expression of these genes could result in improved protein expression / secretion.

[0032] By analyzing the CHO cell genome and transcriptome, the inventors of the present invention found that suppression of one or more of the HDAC8, Dab2, Caspase3, Sys1, Ergic3, Grasp, and Trim23 genes can result in CHO cells with improved protein expression and / or secretion. HDAC8 is involved in regulating chromosome structure and organization during cell division. Dab2 is an adaptor protein that functions as a clathrin-associated sorting protein (CLASP) required for clathrin-mediated endocytosis of selected cargo proteins. Caspase3 is involved in the caspase activation cascade involved in the execution of apoptosis. At the onset of apoptosis, Caspase3 proteolytically cleaves poly(ADP-ribose) polymerase (PARP). Sys1 is a Golgi-localized integral membrane protein homolog and is involved in Golgi transport. Trim23 (tripartite motif-containing 23) plays a role in the formation of intracellular transport vesicles and their movement from one compartment to another. Ergic3 encodes a cycling membrane protein, an endoplasmic reticulum-Golgi intermediate compartment (ERGIC) protein that interacts with other members of this protein family to increase metabolic turnover. Grasp encodes a protein that functions as a molecular scaffold, linking receptors, including group 1 metabotropic glutamate receptors, to neuronal proteins. None of these genes is directly involved in regulating transcription or translation. Therefore, the fact that suppression of these genes can lead to increased protein expression and / or secretion is unexpected.

[0033] According to an embodiment of the present invention, the CHO chromosome and transcriptome were analyzed to select target genes for manipulation. In brief, gene chips provided by the CHO consortium were analyzed. In one example, a total of four CHO cell lines containing no transgenes were analyzed. One line was a CHO-S producing cell line, and the other three lines were suspension CHO cell lines domesticated in the laboratory.

[0034] Furthermore, three sets of CHO cells with either high- or low-yield characteristics were analyzed. Genes expressed at low levels in high-yield CHO cells but at high levels in low-yield CHO cells may not lead to high levels of protein expression. Therefore, such genes could be candidate targets for RNAi intervention to improve protein expression in host cells. To investigate whether such genes actually affect protein expression / secretion efficiency, the expression levels of these target genes were reduced by RNA interference. Various RNAi constructs were prepared for interference with these genes. These constructs could be transiently transfected into CHO cells to screen for their effects on protein expression and cell growth and stability.

[0035] As shown in Figure 1, knockdown of HDAC8, Trim23, Sys1, Ergic3, Dab2, and Grasp genes resulted in improved protein expression and / or secretion in engineered cells. Furthermore, we found that the combination of HDAC8 and Dab2 knockdown was most effective in improving protein expression. Specifically, knockdown of both HDAC8 and Dab2 genes resulted in cells capable of expressing 1.65- to 1.8-fold more protein.

[0036] Using a similar approach, we analyzed two additional CHO cell gene sequences, one from Agilent (Santa Clara, CA) and one from Roche NimbleGen (Madison, WI). These genes, highly expressed in low-yielding cell lines, were identified as targets for interference. From these analyses, two additional genes, BAX and Caspase 3, were identified as knockdown candidates, as shown in Figure 2.

[0037] Based on these target genes, various approaches to RNA interference can be used to suppress their function. For example, shRNA plasmids containing target sequences (HDAC8+Dab2, Caspase3, BAX, and Caspase3+BAX) can be constructed. In embodiments of the present invention, any suitable plasmid or vector can be used. For example, lentiviral plasmids or pcDNA3.1(+) vectors are often used for shRNA. Commercially available kits, such as the BLOCK-iT™ lentiviral RNAi expression system from Thermo Fisher Scientific (Waltham, Massachusetts), can be used.

[0038] These plasmids are amplified in bacteria. The shRNA-containing plasmids are then transfected into a cell line of interest (e.g., DXB11-S1). The transfection efficiency of the transfectants can be assessed (based on antibiotic resistance in the plasmid, e.g., by determining a death curve using 0.5–10 μg / ml puromycin) and the long-term effects of gene silencing can be analyzed. Gene silencing of target genes in these cell lines can be analyzed using real-time PCR. Finally, these cells are analyzed with biochemical, cellular, or molecular biological assays to investigate the function of these genes.

[0039] In addition to interfering with these genes individually, combinatorial knockdown of these target genes was also investigated. As shown in Figure 3, combined knockdown of HDAC8 and Dab2 increased protein expression levels by 1.28- to 1.88-fold in most cell lines (1C9, 1G9, 1G7, 1E3, and 3C8). The production improvement was more pronounced in low-producing cells (1C9, 1G9, and 1G7) compared to high-producing cells (1E3, 3C8, and 3G7). These improvements were observed for various antibodies (Avastin and Herceptin) expressed in these cells, demonstrating that the production improvement is not limited to specific proteins but can be applied to general protein production.

[0040] The simultaneous knockdown of two target genes can produce synergistic effects.For example, the knockdown of HDAC8 and Dab2 respectively produces 1.28-fold and 1.32-fold increase in Avastin in 1C9 cells, while the knockdown of HDAC8 and Dab2 combined produces 1.65-fold increase in expression in the same cells.Similarly, the knockdown of HDAC8 and Dab2 respectively produces 0.78-fold and 1.32-fold increase in Avastin in 1G9 cells, while the knockdown of HDAC8 and Dab2 combined produces 1.88-fold increase in expression in the same cells, showing synergistic effects.

[0041] A variety of shRNA vectors are available that allow for transient and stable transfection and stable delivery of shRNA expression cassettes into host cells. According to embodiments of the present invention, transfection of shRNA constructs into CHO cells can use any suitable vector, including commercially available vectors such as pcDNA3.1(+) (Figure 4, available from Thermo Fisher Scientific, Waltham, Massachusetts) and pGFP-C-ShLenti (Figure 5, available from OriGene, Rockville, Maryland). Other suitable vectors known in the art can also be used.

[0042] Although lentiviral vectors can provide a convenient method for delivering and integrating shRNA constructs into the cellular genome, it may be preferable to produce pharmaceutical proteins without the use of lentiviral elements. The following example demonstrates the use of pcDNA3.1(+) from Thermo Fisher (Waltham, MA).

[0043] Using the pcDNA3.1(+) (Figure 4) plasmid as an example, a stem-loop sequence / framework with the structure shown in Figure 6 can be inserted into the plasmid. The oligos in Figure 6 show typical stem-loop structure constructs. Using these plasmids, several constructs containing the sequence of the target gene were prepared. Successful construction of these plasmids can be confirmed by restriction enzyme digestion to generate fragments of the correct size.

[0044] These constructs were used to transfect CHO cells, such as DXB11 cells. The transfected cells were then evaluated for target gene inhibition. As shown in Figure 7, various transfected cell lines (DXB11 sh-HDAC8+Dab2, DXB11 sh-Caspase3, DXB11 sh-BAX-pool, and DXB11 sh-BAX+Caspase3) may be selected based on selectable markers (e.g., puromycin resistance) and then screened for better target gene inhibition. Briefly, these transfected cell lines were screened with various concentrations of antibiotics (e.g., puromycin), and target gene inhibition was assessed by real-time PCR. Based on these screens, the best candidate host cells were identified. These best cell lines include, for example, DXB11 sh-HDAC8+Dab2 (HD50P) selected with 50 μg / ml puromycin, DXB11 sh-Caspase3 (C10P) selected with 10 μg / ml puromycin, DXB11 sh-BAX (B7.5P) selected with 7.5 μg / ml puromycin, and DXB11 sh-BAX+Caspase3 (BC10P) selected with 10 μg / ml puromycin.

[0045] The best candidate host cells were further evaluated for their ability to support improved protein production. These cells were tested for the production of various proteins, such as SEAP (secreted alkaline phosphatase), Herceptin, and Avastin. As shown in Figure 8, most of these cell lines produced more protein under various culture conditions.

[0046] To obtain stable cell lines, these cells may be serially diluted and selected for single clones. Using DXB11 sh-Caspase3 (C10P) cells selected with 10 μg / ml puromycin as an example, single clones are isolated using the procedure shown in Figure 9 using ClonePix or other appropriate equipment / protocol. Figure 9 illustrates one protocol for the isolation of single clones. Briefly, after cell transfection, the cells are expanded and screened, then subcloned. The cell line can be adapted to suspension culture in serum-free, chemically defined medium. Stable pools of transfectants are then generated and characterized, followed by the generation of high-producing, stable single clones. Those skilled in the art will understand that this is merely exemplary and that other procedures for achieving single clone isolation may also be used.

[0047] As shown in Figure 9, the suppression of gene expression in these cells can be confirmed using real-time PCR. From these analyses, cells with different target gene (e.g., caspase 3) knockdown percentages can be obtained. These cells are then seeded into 6-well plates at appropriate concentrations (e.g., 3 x 10 cells in each 5 ml well). 5 The cells were cultured at a concentration of 10 ...

[0048] The effect of target gene knockdown in these cells on protein production can then be investigated using transient transfection of an expression vector carrying a protein gene (e.g., IgG). Once it has been confirmed that these cells support high levels of protein expression, stable cell pools can be selected by adding a selection drug (e.g., geneticin or puromycin) to the culture medium. The appropriate drug concentration to use is first titrated, and then the cells are grown at the selected drug concentration so that only transfectants carrying the selected drug resistance marker are viable and able to proliferate at a reasonable rate.

[0049] After stable pools of cells are selected, these cell pools can be further diluted and their stability tested. For example, these stable cell pools can be subjected to limiting dilution to select subclones whose stability can be assessed.

[0050] Finally, if desired, single clones of stable transfectants can be isolated to establish a research cell bank (RCB), from which master cell banks (MCBs) or working cell banks (WCBs) can be obtained and cryopreserved. Specifically, single clones are evaluated based on their characteristics (e.g., clonal stability and protein production efficiency), and optimal clones are selected for creation of the RCB. The RCB cells can be further tested and characterized before cryopreservation as an MCB.

[0051] As an example, we investigated the efficiency of these cells in supporting protein expression using the top five single clones of DXB11-sh-Caspase 3 transfectants. As shown in Figure 10, all five clones (CI-1B, CI-1H, CII-1H, CII-4G, and CII-3B) showed improved protein expression after transient transfection of vectors containing Herceptin or Avastin compared to the parental DXB11-J1.0 cells. The improvement ranged from 1.5-fold to 2.4-fold. These results indicate that suppression of Caspase 3 can lead to higher protein-producing host cells. This finding was unexpected, as inhibition of Caspase-1 was found to result in improved protein folding but not enhanced protein production or accumulation in a baculovirus-insect cell (sf9) expression system (X Zhang et al., BMC Biotechnol. 2018 May 2;18(1):24; doi:10.1186 / s12896-018-0434-1).

[0052] These top five clones were further investigated for their long-term behavior. As shown in Figure 11A, from week 0 to week 6, the cell densities of these five clones did not change significantly, regardless of seeding density (D0, D4, and D5, 0.3–25 × 10). 6 The long-term stability of cell numbers indicates that these cells have long-term viability, which may be due to the suppression of apoptosis.

[0053] The population doubling times (PDT) of these cells ranged from 16 to 19 hours and did not change significantly over time. These population doubling times are comparable to those of untransfected CHO cells under the same conditions. Again, these results demonstrate that caspase 3-inhibited cells have virtually the same biological properties as parental CHO cells.

[0054] In fed-batch processes, prolonged cultivation can lead to significant accumulation of lactic acid and ammonia in the medium. High levels of lactic acid are detrimental to cell growth and production quality. CHO cells also exhibit deregulated glucose metabolism, which is associated with high lactic acid production, which can cause medium acidification or undesirable changes in osmolality. Therefore, lactic acid production can be used as an indicator of cell health.

[0055] As shown in Figure 11A, the lactate levels in these cell cultures did not change significantly over a 6-week period. The lactate levels and lactate / cell counts were relatively low. The low lactate levels of these top clones suggest that these cells can efficiently utilize the energy source (glucose).

[0056] Figure 11B shows the expression levels of the Caspase 3 gene. Caspase 3 expression was low in all five clones and did not change significantly over a 6-week period. These results indicate that the suppression of the Caspase 3 gene was relatively stable.

[0057] As shown in Figure 12, these cells maintained a nearly 100% viability for a long period of time (more than 100 generations). Furthermore, the population doubling time (PDT) of these cells was relatively constant. All of these results indicate that these transfectant cells are highly stable for a long period of time (e.g., more than 100 generations).

[0058] Not only are these cells stable over time, but their ability to support enhanced protein expression is also very stable. As shown in Figure 13, the expression levels of both Avastin and Herceptin remained at essentially the same levels, except for the CII-4G clone, which showed some decrease in expression levels at week 6.

[0059] To further investigate the long-term health of these cells, a second generation of these cells was generated by serial dilution and single clone selection as described above. These second generation cells were also evaluated for various properties. [Table 1]

[0060] Table 1 shows the results of analyses of four second-generation cell lines derived from the first-generation cell line CI-1B (CI-1B-D3, CI-1B-E2, CI-1B-F6, and CI-1B-G5) and two second-generation cell lines derived from the first-generation cell line CII-4G (CII-4G-F6 and CII-4G-G6). The population doubling times (PDTs) of these second-generation cells were similar to those of the first-generation cells, indicating a slightly lower proliferation rate for the second-generation cells. However, the difference was minimal. Furthermore, lactate levels and lactate levels per cell were slightly higher in the second-generation cells.

[0061] Figure 14A shows the characteristics of three exemplary second-generation clones, C1-1B-D3, CII-4G-G5, and CII-4G-G6. The long-term stability of these second-generation clones is evidenced by their nearly 100% viability after 9 weeks of culture. Furthermore, these cells maintain consistent transfection efficiency over time. As shown in Figure 14B, CHO-C (i.e., CII-4G-G6) and C1-1B-G5 cells maintained similar transfection rates (approximately 15%) from 0 to 9 weeks.

[0062] Caspase 3 gene expression levels in these second-generation cells are more altered than in the first-generation cells, as assessed by real-time PCR (Figure 14C). For example, CI-1B-D3 and CI-1B-E2 cells show excellent suppression of the Caspase 3 gene, whereas CI-1B-F6 and CII-4G-F6 show little change from the first-generation cells. Interestingly, CII-4G-G6 cells show significantly improved suppression of the Caspase 3 gene, whereas CI-1B-F6 cells have lost the ability to suppress Caspase 3 expression.

[0063] Figure 15 shows results from the evaluation of second-generation cells for supporting enhanced expression of antibodies (Avastin and Herceptin). As shown, these second-generation cells exhibit 1.21- to 2.4-fold higher expression levels of these antibodies compared to those of control cells (DXB-11).

[0064] The above results clearly demonstrate that knockdown of caspase 3, as well as Hasp8, Dab2, Sys1, and Trim23, can produce CHO cells with improved production capacities. These improved capacities were found in various CHO cell lines, including CHO-DXB11, CHO-S, CHO-K1, and CHOC cells. Furthermore, these cells express various proteins, including antibodies against Her2, mesothelin (MSLN), T cell immunoglobulin, and mucin domain-containing 3 (Tim3). In general, these cells can produce proteins (antibodies) at levels of approximately 200 mg / L or higher.

[0065] Proteins expressed in these cells have typical characteristics, including post-translational modifications. For example, Herceptin expressed in DXB11 and CHOC cells was compared to commercially available Herceptin and found to have a similar molecular weight (approximately 145 kDa). RP-HPLC (reduced and non-reduced) showed similar banding patterns for Herceptin produced in these cells compared to commercially available Herceptin / trastuzumab. Figure 16 shows the N-linked glycan profile (after PNGase F release) of Herceptin produced in CHOC cells analyzed on an ACQUITY UPLC BEH Glycan column (1.7 μm, Waters Corp., Milford, MA, USA) and eluted with a gradient of acetonitrile and 50 mM ammonium formate. Glycan analysis revealed that the protein primarily contained G0F, G1Fa, G1Fb, and G2F glycans.

[0066] The above description clearly illustrates an embodiment of the present invention. It has been unexpectedly found that genes not directly related to protein production affect protein expression and / or secretion. According to an embodiment of the present invention, these genes are selected as targets for RNAi. RNAi targeting these genes is carried out by manipulating host cells (e.g., CHO cells) by transfecting cells with appropriate constructs to knock down these target genes.

[0067] Embodiments of the present invention demonstrate that inhibition of selected target genes (e.g., HDAC8, Dab2, and Caspase 3 genes) with siRNA and shRNA, either by short-term or long-term inhibition, can produce cells capable of supporting enhanced protein expression and / or secretion. These results demonstrate that selected gene-inhibited host cells have great potential as new hosts for protein drug production. While Caspase 3 is used in the specific examples to illustrate embodiments of the present invention, other genes (particularly HDAC8, Dab2, and HDAC8+Dab2) can also be targeted for knockdown to improve protein production and / or secretion.

[0068] Various procedural methods are known in the art. The following description provides exemplary procedures and various examples to illustrate the embodiments of the present invention. Those skilled in the art will understand that these specific examples are merely illustrative, and other variations and modifications are possible without departing from the scope of the present invention. For example, the following uses HDAC8, Dab2, BAX, and Caspase3 as examples to describe the embodiments of the present invention. However, other genes can be used without departing from the scope of the present invention.

[0069] Cell culture and media The Chinese hamster ovary (CHO) cell line DXB11 was obtained from Dr. Lawrence Chasin of Columbia University. Cell culture was performed in an incubator at 37°C, 95% humidity, and 5% CO2. The cell culture medium contained Hyclone and mixed medium (50% CDFortiCHO and 50% ActiCHO). Cell counting and viability analysis were performed after trypan blue staining using an automated cell counter TC10 (Bio-Rad, USA).

[0070] The transfection construct contains a puromycin resistance gene. Stable pools were selected based on puromycin resistance. Single clones no longer require selection.

[0071] Vector structure In this example, HDAC8, Dab2, BAX, and Caspasae3 were selected as target genes for RNA interference. The specific sequence fragments from these genes selected for RNA interference are shown in Table 2. [Table 2]

[0072] These target sequences were cloned into the pcDNA3.1(+) vector (Figure 5) to generate shRNA plasmids used to suppress these genes and generate engineered host cells.

[0073] The plasmid structure was as follows: separate primers were designed to contain an MfeI restriction site at the 5' end and a KpnI restriction site at the 3' end. PCR was performed using pGFP-C-Dab2 and pGFP-C-BAX as templates to amplify the desired fragment (corresponding to the desired RNA sequence) containing the U6 promoter and puromycin selection gene. GFP-C-Dab2 and p-GFP-C-BAX are HuSH shRNA plasmids constructed by OriGene by cloning a polynucleotide corresponding to the desired RNA sequence into the pGFP-C-shLenti vector (Figure 6).

[0074] Similar primers were separately designed, each with an EcoRI restriction site at the 5' end and an XmaI restriction site at the 3' end. Similarly, PCR was performed using pGFP-HDAC8 and pGFP-C-Caspase3 as templates to amplify the desired fragment (corresponding to the desired RNA sequence) containing the U6 promoter and puromycin selection gene. Table 3 shows the various primer sequences. [Table 3]

[0075] The PCR fragments were temporarily cloned into the pJET1.2 vector (Thermo Fisher) and sequence verified. The HDAC8, Dab2, BAX, and Casepase3 fragments were excised from the temporary pJET1.2 vector using the restriction enzymes MfeI / KpnI and EcoRI / XmaI, respectively.

[0076] The pcDNA3.1(+) vector is digested with restriction enzymes EcoRI and XmaI. Then, the HDAC8 and Caspase3 fragments are cloned into the vector, respectively, to obtain the pcDNA3.1(+)-HDAC8 and pcDNA3.1(+)-Caspase3 vectors.

[0077] The pcDNA3.1(+)-HDAC8 vector is digested with restriction enzymes MfeI and KpnI, and the Dab2 fragment is assembled into the digested vector to obtain the pcDNA3.1(+)-Dab2-HDAC8 vector.

[0078] Additionally, the pcDNA3.1(+) and pcDNA3.1(+)-Caspase3 vectors were cut using the restriction enzymes MfeI and KpnI. The BAX fragment was then ligated into the cut vectors to obtain pcDNA3.1(+)-BAX and pcDNA3.1(+)-BAX-Caspase3. After construction of these vectors, proper construction was confirmed by restriction enzyme digestion to ensure that the appropriate fragment (i.e., the appropriate size) was assembled into the vector.

[0079] Transfection of CHO cells DXB11 cells were cultured in 6-well plates, with each well containing 3 × 10 cells in 3 mL of Hyclone™ HyCell™ CHO medium (GE Healthcare) containing 8 mM GlutaMAX™ (Thermo Fischer). 6 It has cells.

[0080] Transfection of a vector (e.g., pcDNA3.1) containing an shRNA construct can be performed using any suitable reagent known in the art, such as the lipophilic agent FreeStyle MAX (Thermo Fischer). For example, the RNAi / shRNA vector and transfection reagent FreestyleMAX™ (Thermo Fischer) were separately added to OptiPRO™ SFM (Thermo Fischer) to prepare a vector solution as shown in Table 4. These solutions were left for 5 minutes before being added to the transfection reagent and mixed well. The resulting solution was left for 20 minutes before being transfected into cells. Cells were then evaluated 3 days after transfection. [Table 4]

[0081] Protein expression For protein / antibody production in test CHO cells, antibody / protein expression constructs can be obtained from commercial sources or prepared based on procedures known in the art and transfected into test CHO cells for transient expression of antibodies or proteins. The transfected CHO cells were cultured for an appropriate period (e.g., 3 days) to produce antibodies.

[0082] Protein expression levels can be assessed using any suitable method. For example, the GreatEscAPe™ Chemiluminescence Kit was obtained from Clontech. Dilute 5X dilution buffer 1:5 with ddH2O to prepare 1X dilution buffer.

[0083] To assess protein expression levels, transfer 25 μl of cell media from transfected or mock-transfected cells into a 96-well microtiter plate. If desired, the plate can be sealed and frozen at -20°C for later analysis. Add 75 μl of 1X dilution buffer to each sample in the 96-well microtiter plate. Seal the plate with adhesive aluminum foil or a regular 96-well lid and incubate the diluted samples at 65°C for 30 minutes using a heat block or water bath.

[0084] Cool the samples on ice for 2-3 minutes, then allow them to return to room temperature. Add 100 μl of SEAP substrate solution to each sample. Incubate at room temperature for 30 minutes before reading. Detect and record the chemiluminescent signal using a 96-well plate reader luminometer (e.g., CLARIOstar®).

[0085] Knockdown analysis using real-time PCR The expression level of the target gene can be evaluated by QPCR. Briefly, RNA from cells was extracted using RNA purification reagent (from Qiagen) and quantified using a NanoDrop 2000. QPCR reactions were performed using the following conditions (Table 5). [Table 5] Use a StepOne real-time PCR machine to perform QPCR with the following protocol (Table 6). [Table 6] To assess the expression levels of apoptotic genes (Caspase3 and BAX) and other genes (such as HDAC8 and DAB2), the following primers were used (Table 7). [Table 7]

[0086] While embodiments of the present invention have been described with a limited number of examples, those skilled in the art will recognize that other modifications and variations are possible without departing from the scope of the present invention. Accordingly, the scope of protection of the present invention should be limited only by the appended claims.

Claims

1. A host cell for protein expression, said host cell comprising a lower expression level of a gene compared to a wild-type cell, said gene being Caspase 3; The host cell is a stable cell line with a 100% survival rate for more than 60 generations, The host cell is produced by transfecting Chinese hamster ovary (CHO) cells with short hairpin RNA (shRNA) targeting the gene; the lower expression level of the gene is 85% or less compared to a control; the control is the expression level in wild-type cells; The host cell is a Chinese hamster ovary (CHO) cell. host cell.

2. Transfecting the host cell of claim 1 with an expression vector encoding the protein; and culturing the transfected host cells. A method for producing a protein using the host cell of claim 1.

3. the protein is an antibody; The method of claim 2.

4. the antibody is an antibody against Her2, an antibody against mesothelin (MSLN), or an antibody against T-cell immunoglobulin and mucin domain-containing-3 (Tim3); The method of claim 3.

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