293T cell strain for expressing Claudin18.2 and preparation method thereof

Abstract

The invention provides a 293T cell strain for expressing Claudin18.2 and a preparation method of the 293T cell strain. The preparation method comprises the following steps: packaging lentivirus by using a lentiviral vector for expressing Claudin18.2, and transfecting cells to obtain a virus solution; infecting 293T cells with the virus liquid according to the MOI of 0.1-1, culturing, and screening monoclone. The lentiviral vector for expressing the Claudin18.2 comprises an expression box for expressing the Claudin18.2, and a promoter of the expression box is CMV (Cytomegalovirus) and does not comprise a co-expression gene. The stably transfected cell strain obtained by the invention does not need to depend on drug screening, and can still stably express the Claudin18.2 gene and keep a good cell state after passage for more than 10 times. The cell strain can be used as a Claudin18.2 targeted monoclonal antibody, a bispecific antibody, an antibody coupling drug and an in-vitro killing experiment positive target cell of a chimeric antigen receptor T cell, an effective cell model is constructed for in-vitro functional verification of a targeted drug, and a reliable basis is provided for evaluating the killing efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to an expression vector and a cell line for expressing foreign proteins / polypeptides in mammalian cells. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Claudin18.2 is a tight junction transmembrane protein and a member of the Claudin family. Under normal physiological conditions, it is mainly expressed in differentiated epithelial cells of the gastric mucosa at a low level and with limited distribution. Under pathological conditions, such as in digestive system tumors such as gastric cancer, esophageal cancer, and pancreatic cancer, its expression level is greatly increased. Claudin18.2 is involved in maintaining the integrity of tight junctions between tumor cells. This abnormally high expression may be closely related to tumor growth, invasion, and metastasis. On the one hand, it may participate in the remodeling of connections between tumor cells, creating favorable conditions for the growth and proliferation of tumor cells; on the other hand, during the invasion and metastasis of tumor cells, Claudin18.2 may help tumor cells break through the barrier of normal tissues and spread to surrounding tissues and distant organs by affecting the adhesion properties between cells. This tumor-specific expression makes Claudin18.2 a highly potential tumor marker and therapeutic target. Monoclonal antibodies, bispecific antibodies, antibody-drug conjugates, and chimeric antigen receptor T cell immunotherapy targeting Claudin18.2 have been developed one after another. Compared with small molecule (chemical) drugs (chemotherapy), the above drugs / methods have great advantages in that they can recognize cancer cell surface markers with high specificity, and side effects are usually fewer or milder, resulting in a better quality of life.

[0004] The first thing that needs to be completed in the efficacy study of targeted drugs is in vitro research, and suitable target cells are required at this stage. In existing studies, some gastric cancer or pancreatic cancer cell lines are used to detect the killing effect. However, gastric cancer and pancreatic cancer cell lines accumulate a large number of complex changes such as gene mutations and chromosomal abnormalities in the process of tumor development. These factors will increase the difficulty of studying the function of Claudin18.2, and the heterogeneity of such cancer cell lines may cause deviations in experimental results, thereby affecting the accuracy and reliability of the study. In contrast, the 293T cell line is derived from human embryonic kidney, with a clear and single genetic background, and no complex tumor-related genetic variations and signal pathway disorders, which can effectively reduce the interference with the study of Claudin18.2 function. 293T cells have high transfection efficiency and their culture conditions are easy to control. As a widely used universal cell model, they are conducive to variable control in immunotherapy research. When Claudin18.2 is overexpressed in 293T cells, the protein can be analyzed more purely without worrying about other tumor-specific factors interfering with the action of Claudin18.2.

[0005] At present, the methods for preparing 293T cell lines that stably express Claudin18.2 mostly use lentiviral vectors containing resistance, such as lentiviral vectors containing puromycin. This screening method is relatively efficient and can quickly obtain cell lines that stably express the target gene. However, there are still many problems with resistant vectors when constructing overexpression stable strains, such as potential cytotoxicity, the possibility of introducing additional gene expression changes, and the screening efficiency may be affected by multiple factors. The above problems may have a multi-faceted impact on 293T cells. Therefore, it is of great significance to develop a method for preparing a 293T stable cell line that overexpresses Claudin18.2 without expressing a resistance gene. Summary of the invention

[0006] In order to continuously obtain a 293T stable cell line overexpressing Claudin18.2, the present invention provides a method for preparing a 293T cell line expressing Claudin18.2.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A lentiviral expression vector carrying the Claudin18.2 gene comprises an expression cassette for expressing Claudin18.2 and does not co-express a selection marker gene, wherein the promoter of the expression cassette is CMV.

[0009] The above-mentioned lentiviral expression vector also includes a replicon (Ori), a long terminal repeat sequence (LTRs) for integrating the target gene into the host genome, and may also include expression regulatory elements such as enhancers, cPPT, WPRE, PolyA signals, etc.

[0010] Preferably, the backbone of the vector is a pHR plasmid.

[0011] The selection marker gene includes but is not limited to antibiotic resistance genes, fluorescent protein genes, and synthetic genes of nutritional deficiency.

[0012] The above-mentioned lentiviral expression vector is constructed by inserting the Claudin18.2 encoding gene into the pHR-CMV-GFP vector between the BamH1 and Not1 restriction sites, and is referred to as pHR-CMV-Claudin18.2.

[0013] The above-mentioned lentiviral expression vector can be co-packaged with a lentiviral packaging plasmid and a lentiviral envelope plasmid to form a lentivirus, and infect mammalian cells to produce a lentivirus; and further used to produce a cell line stably expressing Claudin18.2.

[0014] A method for preparing a 293T cell line stably expressing Claudin18.2 (293T-Claudin18.2), comprising the following steps: (1) The above-mentioned lentiviral expression vector carrying Claudin18.2, pCMV plasmid, and pMD.2G plasmid were co-transfected into 293T cells to obtain virus solution; (2) 293T cells were cultured at a rate of (1-4) × 10 4 / cm 2 Initial density culture was performed for 24 h until the confluence reached 20-30%; (3) Infect the 293T cells in step (2) with the virus solution at an MOI of 0.1-1 and culture them. The confluence is not less than 90% and the cell viability is not less than 95% 48 hours after virus infection. (4) The cells in step (3) are subjected to monoclonal screening to obtain a 293T cell line that stably expresses Claudin18.2.

[0015] Preferably, in step (1), the specific steps are: (a) 293T cells were cultured at a rate of (1-1.25)×10 5 / cm 2 Initial density culture was performed for 24 h until the confluence was no less than 80%; (b) The lentiviral expression vector, pCMV plasmid, and pMD.2G plasmid were mixed in a mass ratio of (2.5-3):(2.5-3):(0.4-0.5) and the amount of the lentiviral expression vector added was (0.25-0.5) μg / cm 2 , then add PEI transfection reagent to obtain transfection solution; (c) Add the transfection solution to the cells in step (a) and harvest the supernatant containing the virus after 48 h of culture.

[0016] In step (b), the amount of PEI transfection reagent added is lentiviral expression vector: PEI = 1 μg: 3 μg.

[0017] Preferably, in step (2), the initial density is 2×10 4 cm 2 ; In step (3), the MOI is 0.3-1; more preferably, the MOI is 0.3.

[0018] A 293T cell line expressing Claudin18.2 obtained by the above method.

[0019] The present invention has the following advantages: The method for constructing Claudin18.2 293T cells provided by the present invention uses a high-quality lentivirus packaging method to obtain a high titer of lentivirus liquid without the need for concentration; by optimizing the optimal cell plating number, the optimal MOI value of virus infection, the monoclonal screening method, etc., the cell proliferation rate is improved; the entire method greatly reduces the workload of cell culture, saving time and cost; The 293T cell strain expressing Claudin18.2 obtained by the present invention does not need to rely on screening drug intervention, and can still stably express the Claudin18.2 gene and maintain a good cell state after continuous passage for more than 10 times. The cell strain can be used as a positive target cell for in vitro killing experiments of monoclonal antibodies, bispecific antibodies, antibody-drug conjugates and chimeric antigen receptor T cells targeting Claudin18.2, construct an effective cell model for in vitro functional verification of targeted drugs, and provide a reliable basis for evaluating the killing efficacy of Claudin18.2 targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the map of the Claudin18.2 lentiviral vector; Figure 2 This is the titer result of Claudin18.2 lentivirus; Figure 3 The cell infection efficiency of 293T cells infected with Claudin18.2 lentivirus; Figure 4is the cell viability of 293T cells infected with Claudin18.2 lentivirus; Figure 5 To compare the proliferation of different 293T-Claudin18.2 stably transfected cell lines; Figure 6 This is the result of 293T-Claudin18.2 monoclonal identification; Figure 7 This is the result of continuous passage identification of 293T-Claudin18.2 monoclone; Figure 8 The 293T-Claudin18.2 stable transfected strain was used as the target cell killing result of the CAR-T killing experiment. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with examples and drawings, but the present invention is not limited to the following examples; methods not particularly described in detail are all conventional experimental methods and can be obtained from the prior art.

[0022] Example 1 Construction of Claudin18.2 Lentiviral Vector Commercially synthesized DNA containing BamH1+Claudin18.2 genes (NCBI Reference Sequence: NM_001002026.2, GeneID: 51208 )+Not1 fragment pUC57-Amp plasmid, double digested with BamH1-HF+Not1-HF, and the 793bp target fragment CLDN18.2 (sticky end) was recovered by gel. pHR-CMV-GFP (9717bp) vector was double digested with BamH1-HF+Not1-HF, and the 8997bp vector backbone fragment was recovered by gel. The target fragment and the vector backbone fragment were connected with T4DNA ligase at a molar ratio of 1:3. The ligation product was transformed with Trans5a competence. The monoclonal colony was picked from the plate, identified, the plasmid was extracted, and the correct sequence was the Claudin18.2 lentiviral vector plasmid pHR-CMV-Claudin18.2. The vector map is shown in Figure 1 .

[0023] Example 2 Packaging of Claudin18.2 Lentivirus 1. Lentivirus packaging (1) 24 h before transfection, adherent 293T cells were plated in DMEM (containing 10% FBS and 1% Glutamax) complete medium, and the cell seeding density was (1.14×10 5 ) / cm 2Bottom area; 2 hours before transfection, observe that the cell confluence reaches 80-90%, remove the original culture medium in the biosafety cabinet, and slowly add 37°C preheated DMEM complete culture medium along the wall. The culture medium added at this time is 9 / 10 of the original volume; (2) Dilute the DNA plasmid with serum-free DMEM medium. The mass ratio of the plasmids used is: viral packaging plasmid (pCMV): viral envelope plasmid (pMD.2G): target DNA plasmid = 2.66:0.45:3. The mass of the target DNA plasmid is 0.313 µg / cm 2 The bottom area and culture medium volume were 1 / 20 of the final volume in the container; all plasmids used were extracted with an endotoxin removal kit, with an A260 / A280 range of 1.8-2.0; Dilute PEI to 1 / 20 of the total volume of the lentiviral packaging system with serum-free DMEM medium; Add the above PEI reagent dilution to the above DNA dilution to make the mass ratio of PEI to target DNA 3:1, immediately mix and vortex for 15s-20s, and let stand at room temperature for 15min; (3) Gently add the PEI / DNA mixture along the wall to the cell suspension in step (1) and gently shake to mix. Place the cells in a 37°C, 5% CO2 incubator; 8 hours after transfection, remove the original culture medium and slowly add 37°C preheated DMEM complete culture medium along the wall; 48 hours after transfection, collect the viral supernatant, centrifuge at 2000 rpm for 5 minutes to remove debris, and filter with a 0.45μm filter into a new centrifuge tube to obtain the viral liquid.

[0024] 2. Lentivirus titer detection Take a small amount of the above virus liquid and measure the titer according to the following method: (1) Take K562 cells in good condition and count them using Count Star counter. Take 1×10 5 K562 cells were added to 24-well plates and 1640 complete culture medium was added to 500 µL per well; (2) Add the virus solution into the K562 cells plated in (1) at volumes of 1 µL, 3 µL, 10 µL, and 30 µL respectively; (3) The K562 cells with virus solution were cultured in a 37°C, 5% CO2 incubator; after 48 hours, the cells were collected and placed in a 1.5 mL EP tube, centrifuged at 500 g for 3 min, resuspended in 200 µL PBS, and Claudin18.2 antibody (Dima Biotechnology, Catalog No.: DME100179) was added at a final concentration of 2 µg / mL, and incubated on ice for 30 min; after the antibody incubation, the cells were washed twice with 1 mL PBS, and the secondary antibody was diluted at a ratio of 1:500, and 100 µL of secondary antibody (Dima Biotechnology, Catalog No.: DME300002) was added and incubated on ice for 30 min; after washing twice with 1 mL PBS, the EP tube was placed in a centrifuge and centrifuged at 300 g for 5 min, resuspended in 300 µL PBS, and the lentivirus titer was detected by flow cytometry; The results are as follows Figure 2 As shown, the virus titer was 5.95×10 6 TU / mL.

[0025] Example 3 Construction of 293T Stable Transfectant Expressing Claudin18.2 1. Optimization of viral infection conditions 24 h before virus infection of target cells, cells cultured in DMEM complete medium (DMEM + 10% FBS) were revived and plated (6-well plate) with 2 × 10 cells per well. 5 / 9.6cm², 24h after plating, the confluence of 293T cells reached 20-30%, and virus solution was added according to the virus infection MOI value of 0.1, 0.3, 0.6, 1. 48h after virus infection of 293T cells, the cell confluence was about 95%, the cells were digested and stained with Claudin18.2 antibody, and flow cytometry was used to detect the virus infection efficiency and cell viability.

[0026] Virus infection efficiency and cell viability were Figure 3 and 4 As shown, the infection efficiency of the virus infection MOI value = 0.3 group was 66.1%, the cell viability was 98.7%, and the cell state was good. The cells of the MOI value = 0.3 group were selected to plate 96-well plates, and 1 cell / well / 100μL DMEM complete medium was used to screen single clones.

[0027] 2. Viral Transfection 24 h before virus infection of target cells, cells cultured in DMEM complete medium (DMEM + 10% FBS) were revived and 293T cells of generation P3 were plated in 96-well plates. The number of cells plated was 2 × 10 5 / 9.6cm², 24 hours after plating, the confluence of 293T cells reached 20-30%, and the virus solution was added according to the virus infection MOI of 0.3. After plating, the cells were placed in an incubator for three days to prevent the dispersion of monoclones and the formation of polyclones. 7 days after plating, the wells were observed under a microscope to see whether monoclones were formed. The wells with monoclonal formation were screened and the cells were digested and transferred to 48-well plates. The cells were digested and transferred to 24-well plates after 48 hours of cell culture, and to 6-well plates after 48 hours of cell culture. After culturing for 24-72 hours, the cells in the wells with a confluence of about 95% were digested and stained with Claudin18.2 antibody. The monoclones with high positive rates were screened by flow cytometry. The results are as follows Figure 4 shown.

[0028] The successfully identified monoclonal clones were continuously amplified 10-15 times at a frequency of subculturing every 2 days. Cells were cryopreserved and sampled for each generation to detect the positive rate of target cells and cell proliferation times (cell proliferation times = number of harvested cells / number of initial cultured cells). Figure 5 The results of the target cell positive rate experiment are as follows Figure 6 According to the results, cell lines that can be continuously passaged for more than 10 times, have high cell viability, good condition and maintain stable expression of the Claudin18.2 gene were screened.

[0029] Comparative Example 1 Construction of a Puromycin-resistant 293T Stable Transgenic Line Expressing Claudin18.2 1. Lentiviral vector plasmid construction The pUC57-Amp plasmid containing BamH1+Claudin18.2 gene (GeneID: 51208)+Not1 fragment was synthesized commercially. The target fragment CLDN18.2 (containing sticky ends) of 793bp was recovered by gel digestion using BamH1-HF+Not1-HF. The pHR-CMV-PuroR (10864bp) vector was double-digested with BamH1-HF+Not1-HF, and the vector backbone fragment of 10144bp was recovered by gel digestion. The target fragment and the vector backbone fragment were connected with T4 DNA ligase at a molar ratio of 1:3. The ligation product was transformed with Trans5a competence. The monoclonal colony was picked on the plate, identified, the plasmid was extracted, and the correct sequence was the Claudin18.2 lentiviral vector plasmid pHR-CMV- Claudin18.2 -Puro R .

[0030] 2. Lentivirus packaging (1) 24 h before transfection, adherent 293T cells were plated in DMEM (containing 10% FBS and 1% Glutamax) complete medium, and the cell seeding density was (1.14×10 5 ) / cm2 Bottom area; 2 hours before transfection, observe that the cell confluence reaches 80-90%, remove the original culture medium in the biosafety cabinet, and slowly add 37°C preheated DMEM complete culture medium along the wall. The culture medium added at this time is 9 / 10 of the original volume; (2) Dilute the DNA plasmid with serum-free DMEM medium. The ratio of the plasmids used is: viral packaging plasmid (pCMV): viral capsid plasmid (pMD.2G): target DNA plasmid = 2.66:0.45:3. The mass of the target DNA plasmid is 0.313 µg / cm 2 The bottom area and the culture medium volume are 1 / 20 of the final volume in the container; the plasmids used are extracted with an endotoxin removal kit, and the A260 / A280 range is 1.8-2.0; PEI is diluted to 1 / 20 of the total volume with serum-free DMEM medium; Add the above PEI reagent dilution to the above DNA dilution to make the mass ratio of PEI to target DNA 3:1, immediately mix and vortex for 15s-20s, and let stand at room temperature for 15-20 min; (3) Gently add the PEI / DNA mixture along the wall to the cell suspension in step (1) and gently shake to mix. Place the cells in a 37°C, 5% CO2 incubator; 8 hours after transfection, remove the original culture medium and slowly add 37°C preheated DMEM complete culture medium along the wall; 48 hours after transfection, collect the viral supernatant, centrifuge at 2000 rpm for 5 minutes to remove debris, and filter with a 0.45μm filter into a new centrifuge tube to obtain the viral liquid; 3. Viral Transfection 24 h before virus infection of target cells, cells cultured in DMEM complete medium (DMEM + 10% FBS) were revived and 293T cells of generation P3 were plated in 6-well plates. The number of cells plated was 2 × 10 5 / 9.6cm², 24h after plating, the confluence of 293T cells reached 20-30%, and the virus solution was added according to the virus infection MOI value of 0.3. After 48h of virus infection of 293T target cells, 2µg / mL of puromycin was added for screening for 48-72h; the surviving cells after screening were cultured in DMEM complete medium containing 2µg / mL of puromycin, and monoclonal screening was performed. The obtained cells were stained with Claudin18.2 antibody, and the positive rate of target cells was 100%.

[0031] The selected monoclonal clones were subcultured 10 times and the cell proliferation times were recorded. The proliferation times of the cell line and the cells obtained in Example 3 were compared. Figure 6 As shown, the proliferation capacity of cells without resistance is much higher than that of resistant 293T stable transfectants.

[0032] Application Example 1 Application of 293T-Claudin18.2 Stable Transformant The 293T cell line (293T-CLDN18.2) stably expressing Claudin18.2 obtained in Example 3 was used as a positive target cell for the Claudin18.2 CAR-T cell in vitro killing experiment, and the 293T cell line was used as a negative target cell for the killing experiment, and an in vitro killing experiment was performed using an RTCA instrument. The specific implementation method is as follows: (1) 24 h before the experiment, target cells 293T cells and 293T-CLDN18.2 were inoculated into RTCA-specific 16-well plates. The number of cells inoculated was 1×10 4 / 100µL / well; at the beginning of the experiment, the DMEM medium was replaced with AIM+5%CTS medium (Gibco), 100µL per well; (2) Prepare Claudin18.2 CAR-T cells and T cells according to the effector-target ratio of 3:1 and 1:1, dilute the effector cells in sequence, resuspend them and add them to the RTCA plate, 100 µL cell suspension / well; RTCA detects the killing effect.

[0033] The experimental results showed that Claudin18.2 CAR-T can specifically kill Claudin18.2 positive target cells 293T-CLDN18.2. Figure 8 This shows that the 293T-Claudin18.2 prepared by the present invention can construct an effective cell model for in vitro functional verification of CAR-T cells. At the same time, this cell line can also effectively bind to the Claudin18.2 antibody and can also be used to detect the biological activity of the Claudin18.2 antibody.

[0034] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A lentiviral expression vector carrying the Claudin18.2 gene, comprising an expression cassette for expressing Claudin18.2, characterized in that: The promoter of the expression cassette is CMV, and no selection marker gene is co-expressed.

2. The lentiviral vector according to claim 1, characterized in that The backbone of the vector is a pHR plasmid; The selection marker gene is selected from at least one of an antibiotic resistance gene, a fluorescent protein gene and a nutrient deficiency synthetic gene; The lentiviral expression vector also includes a replicon, a long terminal repeat sequence for integrating the target gene into the host genome, an enhancer, a cPPT, a WPRE and a PolyA signal.

3. The lentiviral vector according to claim 1, characterized in that The lentiviral vector is constructed by inserting the Claudin18.2 encoding gene into the pHR-CMV-GFP vector between the BamH1 and Not1 restriction sites.

4. Use of the lentiviral vector according to any one of claims 1 to 3 in producing a lentivirus expressing Claudin18.2 and a cell line stably expressing Claudin18.

2.

5. A method for preparing a 293T cell line expressing Claudin18.2, characterized in that: The following steps are involved: (1) The above-mentioned lentiviral expression vector carrying Claudin18.2, pCMV plasmid, and pMD.2G plasmid were co-transfected into 293T cells to obtain virus solution; (2) 293T cells were cultured at a rate of (1-4) × 10 4 / cm 2 Initial density culture was performed for 24 h until the confluence reached 20-30%; (3) Infect the 293T cells in step (2) with the virus solution at an MOI of 0.1-1 and culture them. The confluence is not less than 90% and the cell viability is not less than 95% 48 hours after virus infection. (4) The cells in step (3) are subjected to monoclonal screening to obtain a 293T cell line that stably expresses Claudin18.

2.

6. The preparation method according to claim 5, characterized in that: In step (1), the specific steps are: (a) 293T cells were cultured at a rate of (1-1.25)×10 5 / cm 2 Initial density culture was performed for 24 h until the confluence was no less than 80%; (b) The lentiviral expression vector, pCMV plasmid, and pMD.2G plasmid were mixed in a mass ratio of (2.5-3):(2.5-3):(0.4-0.5) and the amount of the lentiviral expression vector added was (0.25-0.5) μg / cm 2 , then add PEI transfection reagent to obtain transfection solution; (c) Add the transfection solution to the cells in step (a) and harvest the supernatant containing the virus after 48 h of culture.

7. The preparation method according to claim 6, characterized in that: In step (b), the amount of PEI transfection reagent added is: lentiviral expression vector: PEI = 1 μg: 3 μg.

8. The preparation method according to claim 5, characterized in that: In step (2), the initial density is 2×10 4 cm 2 ; In step (3), the MOI is 0.3-1; more preferably, the MOI is 0.

3.

9. A 293T cell line expressing Claudin18.2 obtained by the preparation method according to any one of claims 5 to 8.

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