Preparation method of adenovirus p53 loaded DC cell vaccine
Through peripheral blood collection, cell isolation and adenovirus transfection on DC cells, the problem of difficulty in overexpressing Ad-p53 protein in DC cells was solved, and efficient anti-tumor T cell activation and tumor killing effects were achieved.
Patent Information
- Application Number
- CN202411631162.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to integrate the P53 gene into DC cells, resulting in DC cells being unable to effectively overexpress the Ad-p53 protein, which in turn affects the anti-tumor effect.
By collecting peripheral blood, isolating and purifying DC cells, and using adenovirus as a transfection tool, Ad-P53 virus transfection is carried out according to specific infection plural and infection methods, the expression of Ad-p53 protein in DC cells is improved.
High purity and efficient Ad-p53 protein overexpression of DC cells were achieved, significantly improving the activation ability and tumor killing efficiency of anti-tumor T cells.
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Figure CN119454942A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a DC cell vaccine loaded with adenovirus p53. Background Art
[0002] The p53 gene is recognized as the "gene guardian". Among more than 100 human tumors, more than 60% of tumors are related to p53 gene mutations. The exogenous tumor suppressor gene (p53 gene) is introduced into the tumor through adenovirus vectors, and the growth of tumor tissue is inhibited through a variety of anti-tumor mechanisms such as apoptosis, bystander effects, and inhibition of multidrug resistance gene (MDR) expression. At present, adenovirus-mediated p53 gene therapy has been used clinically to treat various malignant tumors, including: head and neck tumors, lung cancer, liver cancer, breast cancer, brain tumors, ovarian cancer, bladder cancer, prostate cancer, etc.
[0003] Dendritic cells (DC) are the most powerful professional antigen-presenting cells (APC) in the body. They can effectively take up, process and present antigens. Immature DCs have strong migration and antigen uptake abilities. Mature DCs can effectively activate naive T cells and produce effector cells with anti-tumor activity. Loading DCs with adenovirus p53 (Ad-P53) can effectively activate anti-tumor T cells in the human body. However, conventional methods make it difficult to integrate the P53 gene into DCs. Therefore, it is urgent to develop a culture method with high purity of DCs that can overexpress Ad-p53 protein. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing adenovirus p53-loaded DC cell vaccine. The DC cells finally prepared by the method can make the DC cells overexpress Ad-p53 protein, and have broad application prospects.
[0005] The objective of the present invention is achieved through the following technical solutions:
[0006] The present invention provides a method for preparing a DC cell vaccine loaded with adenovirus p53, comprising the following steps:
[0007] (1) peripheral blood was collected and centrifuged to separate the plasma layer and the cell layer without the plasma layer, and the plasma layer was centrifuged again to collect the supernatant of the plasma layer as autologous plasma for later use, and the cell layer without the plasma layer was mixed with lymphocyte separation solution at a ratio of 2:1 and added to a centrifuge tube containing lymphocyte separation solution for centrifugation, and then the buffy coat layer was aspirated to collect PBMC, and the PBMC was centrifuged and washed again;
[0008] (2) using CD14 magnetic beads to incubate and sort PBMCs, collect CD14+ cells and CD14- cells, and culture CD14+ adherent cells;
[0009] (3) discarding the supernatant of the CD14+ cells that have completed the adherent culture in step (2), adding DC complete culture medium and the autologous plasma prepared in step (1), and continuing the culture to obtain DC cells;
[0010] (4) The DC cells prepared in step (3) were centrifuged, resuspended, and placed in serum-free XVIVO-15 medium containing 1 mg / mL Perminede, transfected with Ad-P53 virus, and then continued to be cultured;
[0011] (5) Resuspending the DC cells cultured in step (4) in physiological saline to obtain the DC cell vaccine.
[0012] Furthermore, in step (1), the centrifugation parameters for collecting peripheral blood and performing centrifugal treatment are 2300 rpm for 15 min, and the centrifugation parameters for centrifuging the plasma layer again are 2700 rpm for 10 min. The plasma layer also includes a step of being bathed in 56°C water for 30 min before being centrifuged again.
[0013] Furthermore, in step (1), the specific separation method of PBMC is Ficoll density gradient centrifugation, the lymphocyte separation fluid is Ficoll, the centrifugation parameters when added to the centrifuge tube containing the lymphocyte separation fluid are 2000rpm, centrifugation for 20min, and the centrifugation parameters when performing the centrifugal washing treatment again are 1500rpm, centrifugation for 5min.
[0014] Furthermore, in step (2), the incubation is performed using 80 μL buffer + 20 μL CD14 magnetic beads at 2-8°C in the dark for 30 minutes, wherein 1E7 PBMCs are incubated with every 5 μL magnetic beads.
[0015] Furthermore, in step (3), the DC complete culture medium contains 50 ng / ml IL-4 and 100 ng / ml GM-CSF, the amount of autologous plasma added is 2%, and the continued culture is carried out at 37°C, 5% CO 2 Continue culturing in the incubator.
[0016] Furthermore, in step (4), the centrifugation parameter is 1800 rpm for 5 min, and the serum-free XVIVO-15 culture medium containing 1 mg / mL Permine contains L-Gln and Phenol Red.
[0017] Furthermore, in step (4), the transfection is carried out at a rate of 1×10 6 DC cells were infected at a multiplicity of infection of MOI = 25000:1, and virus Ad-P53 was added in proportion, then incubated at room temperature for 120 minutes, and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded and DC complete culture medium containing 5% autologous plasma was added.
[0018] Furthermore, in step (5), the resuspension is to resuspend the transfected DC cells at 5×10 6 The cells were resuspended at a density of 1.54 × 10 / mL in 2 mL of 0.9% saline.
[0019] The present invention also provides a DC cell vaccine loaded with adenovirus p53 prepared by the preparation method.
[0020] The present invention also provides an application of the adenovirus p53-loaded DC cell vaccine in the preparation of anti-tumor products.
[0021] Beneficial effects:
[0022] Conventional methods of infecting DC cells cannot effectively increase the expression of Ad-P53 protein in DC cells, but the present invention can increase the infection rate of P53 through DC cell purification and specific infection multiplicity and infection method; and the use of adenovirus as a transfection tool can increase the virus infection rate, and the use of P53 protein to replace the currently available common tumor-associated antigen (TAA) technology as the main component of the vaccine has a more obvious antigen presentation effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0024] Figure 1 The figure is a specific preparation flow chart of the adenovirus p53-loaded DC cell vaccine of the present invention.
[0025] Figure 2 This is a flow cytometry analysis of P53 infected DC cells.
[0026] Figure 3 This is a diagram of in vitro killing of T cells activated by P53-infected DC cells. DETAILED DESCRIPTION
[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0028] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.
[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0032] The test methods used in the following implementations, unless otherwise specified, are all commonly used experimental methods in this field;
[0033] Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.
[0034] The specific preparation process of the adenovirus p53-loaded DC cell vaccine of the present invention is as follows: Figure 1 shown.
[0035] Example 1
[0036] The specific preparation method of the adenovirus p53 (Ad-P53) loaded DC cell vaccine in this embodiment includes the following steps:
[0037] Step 1: Peripheral blood collection and peripheral blood mononuclear cell (PBMC) isolation
[0038] 1.1 Sample centrifugation: Collect 50 mL of peripheral blood from the tumor patient using a collection tube. Use a 10 mL disposable sterile pipette to transfer all the peripheral blood in the collection tube to a new 50 mL disposable sterile centrifuge tube. Centrifuge at 2300 rpm for 15 min (9 for ascending and 7 for descending) at room temperature.
[0039] 1.2 Plasma separation: Use a 10mL disposable sterile pipette to carefully aspirate the upper plasma layer, add it to a new disposable 50mL sterile centrifuge tube, and then place it in a 56℃ water bath for 30 minutes. After inactivation, cool it in a 4℃ refrigerator overnight, centrifuge it at 2700rpm for 10 minutes at room temperature, use a 10mL disposable sterile pipette to collect the plasma supernatant into a new 50mL disposable sterile centrifuge tube, use it as autologous plasma for later use, and store it in a 4℃ refrigerator.
[0040] 1.3PBMC separation: Ficoll density gradient centrifugation was used for separation. The blood cell layer after plasma removal was diluted to 30mL with Ficoll in a volume ratio of 2:1 using a 10mL disposable sterile pipette and physiological saline. Then, 15mL of lymphocyte separation solution was added to a 50mL centrifuge tube using a 10mL disposable sterile pipette. The lymphocyte separation solution was purchased from Tianjin Meide Pacific Technology Co., Ltd. Then, a new 10mL disposable sterile pipette was used to mix the diluted blood cells and add them to the centrifuge tube (note that it should be added very slowly at the beginning, and the blood cells should be added carefully above the liquid surface of Ficoll, and the contact surface between the separation solution and the cell suspension should not be damaged). Centrifuge at room temperature, 2000rpm for 20min (0 rise, 0 fall);
[0041] 1.4 PBMC collection: After centrifugation, use a 10 mL disposable sterile pipette to remove the top layer of liquid layer by layer, then carefully aspirate the buffy coat layer to collect PBMCs, and transfer to a new 50 mL disposable sterile centrifuge tube. After sampling and counting, centrifuge at room temperature at 1500 rpm for 5 minutes (9 for ascending and 7 for descending), and wash for the second time.
[0042] Step 2: PBMC isolation
[0043] 2.1 CD14+ magnetic bead sorting and cell adhesion: prepare MACS Running Buffer buffer to balance room temperature, use two 200μL disposable sterile pipette tips to add 80μL buffer + 20μL CD14 magnetic beads respectively, incubate for 30min at 2-8℃ in the dark, shake gently every 5min, mix once (every 5μL magnetic beads are incubated with 1E7 PBMCs, the amount of magnetic beads is increased or decreased according to the specific number of cells, and the volume ratio of magnetic bead incubation buffer: magnetic beads is 4:1) Place the LS sorting column on the sorting rack, place a 15mL disposable sterile centrifuge tube under the sorting column, use a 1000μL boxed sterile pipette tip to absorb 3mL of pre-cooled buffer to balance the sorting column, balance 1-2 times, and discard the buffer collected during balance; after the CD14 magnetic beads are incubated, resuspend them with 3mL of buffer (mix gently and do not blow), and add them to the sorting column (0-1E8 total If the number of cells is greater than 1E8, use 3 mL. If the number of cells is greater than 1E8, resuspend in 6 mL and divide evenly into two columns).
[0044] 2.2 Collection of CD14+ cells and CD14- cells: The filtered cells are CD14- cells. The CD14+ cells in the sorting column were collected and centrifuged at 1800rpm for 5min (9 up and 9 down). After centrifugation, a 10mL pipette was used to discard the supernatant. A new 10mL pipette was used to add 2mL CIK complete medium 1. CIK complete medium is X-VIVO medium containing 50ng / ml CD3 monoclonal antibody and 100ng / l IFN-γ. CD3 monoclonal antibody was purchased from Zhuhai Beso Cell Science and Technology Co., Ltd., and IFN-γ was purchased from Novus Biologicals. After the cells were mixed, 20μL was taken to count and calculate the viability. CD14- cells were used for CIK culture.
[0045] 2.3CD14+ cell adhesion: The collected CD14+ cells were aspirated and the supernatant was discarded. The serum-free X-VIVO medium containing L-Gln and Phenol Red was used to adjust the cell concentration to 1*10 6 / mL, and placed in a 37℃ carbon dioxide incubator to adhere to the wall for 30 minutes.
[0046] Step 3: DC cell activation
[0047] 3.1 After the cells are attached to the wall, use a new 10mL disposable sterile pipette to discard the supernatant and observe under a microscope whether there are any cells that have not attached to the wall. If there are, use a 1000μL disposable pipette tip to add 1mL of X-VIVO serum-free medium for washing (note that the pipette should not touch the bottle wall or blow during the whole process) until there are no obvious T cells remaining by naked eye observation.
[0048] 3.2 Use a 1000 μL pipette tip to add DC complete medium, which consists of serum-free X-VIVO medium containing 50 ng / ml IL-4 and 100 ng / ml GM-CSF and 2% autologous plasma. Shake gently and place at 37°C, 5% CO 2 Continue to culture in an incubator;
[0049] Step 4: Ad-P53 transfected DC cells. Ad-P53 was obtained from Shenzhen SaiBianuo Biotechnology Co., Ltd.
[0050] 4.1 Take the DC cells cultured to D5, collect all the cell suspension in a 50 mL centrifuge tube, take a sample for live cell counting, and centrifuge at 1800 rpm for 5 min.
[0051] 4.2 After counting, resuspend the cells in 1 mL of serum-free XVIVO-15 medium containing 1 mg / mL Perminede. The serum-free X-VIVO medium contains L-Gln and Phenol Red. 6 DC cells were infected at a multiplicity of infection of MOI = 25000:1, and Ad-P53 virus was added in proportion. After incubation at room temperature for 120 minutes, the cells were centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was discarded and DC complete culture medium containing 5% autologous plasma was added to continue the culture.
[0052] 4.3 After 48 hours of continuous culture, discard the supernatant, rinse the non-adherent cells with PBS, digest the DC cells with mild digestive enzymes, and take 1×10 6 The expression of P53 was detected by flow cytometry in each cell, and the specific method is shown in Table 1;
[0053] Table 1 Different types of DC cells
[0054]
[0055] (1) Number the samples to be tested, with No. 0 being the negative control tube and No. 1 being the sample tube, and they are numbered in sequence according to the number of samples to be tested;
[0056] (2) After the sample is centrifuged, the supernatant is discarded, and 200 μL PBS is added to tube 0 to resuspend it in preparation for loading on the machine. At the same time, FVS780 antibody is diluted with PBS at a ratio of 1:1000, that is, 1 μL antibody is added with 1 mL PBS;
[0057] (3) After the test sample is centrifuged, the supernatant is discarded and 200 μL of diluted FVS620 antibody is added and incubated at 4°C in the dark for 30 min;
[0058] (4) After incubation in tube 1, add 1 mL of PBS to resuspend, centrifuge at 300 g for 5 min, discard the supernatant, add 2 μL of anti-P53 according to the amount of antibody in the above table, mix well with 200 μL of PBS, resuspend the cells, and incubate at 4°C in the dark for 30 min;
[0059] (5) Resuspend tube 1 with 1 mL of PBS, centrifuge at 1500 g for 3 min, discard the supernatant, resuspend with 200 μL of PBS and prepare for flow cytometry;
[0060] (6) Flow cytometry showed that the expression of P53 on the surface of mature DC cells infected with P53 was approximately 11.94%. Figure 2 As shown;
[0061] Step 5 DC vaccine preparation
[0062] After DC cells were harvested and centrifuged, the supernatant was discarded and the cells were centrifuged at 1×10 6 The cells with a density of 100 μg / mL were resuspended in 2 mL of 0.9% saline and mixed evenly, and 200 μL of the cell suspension was taken for endotoxin detection.
[0063] Step 6: Co-culture of P53-loaded DCs and T cells
[0064] 5.1 Resuspend the CD14- cells sorted in step 2.2 above in T cell complete medium, which is X-VIVO medium containing 50 ng / ml CD3 monoclonal antibody and 100 ng / l IFN-γ. CD3 monoclonal antibody was purchased from Zhuhai Beso Cell Science and Technology Co., Ltd., and IFN-γ was purchased from Novus Biologicals. Transfer the cell suspension to the corresponding culture container and place it at 37°C, 5% CO 2 Continue to culture in an incubator;
[0065] 5.2 DC cells cultured to D7 were co-cultured with T cells cultured to D7. After the co-cultured DCT cells were cultured to D9, an in vitro killing experiment was performed on HK1 tumor cells at an effector-target ratio of 10:1. It was finally found that T cells activated by DC cells loaded with P53 adenovirus had a higher killing efficiency against HK1 tumor cells infected with P53 in vitro than T cells activated by DC cells not loaded with P53. Figure 3 shown.
[0066] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A method for preparing adenovirus p53-loaded DC cell vaccine, characterized in that: The following steps are involved: (1) Collect peripheral blood and centrifuge to separate blood cells and autologous plasma, collect the upper plasma layer, inactivate the plasma layer and centrifuge again, collect the inactivated plasma supernatant as autologous plasma for later use, dilute the cells removed from the plasma layer with physiological saline, mix with lymphocyte separation solution at a ratio of 2:1, add to the centrifuge tube containing lymphocyte separation solution and centrifuge, then aspirate the buffy coat layer to collect PBMCs, and centrifuge and wash again; (2) Using CD14 magnetic beads to incubate and sort PBMCs, collect CD14+ cells and CD14- cells, and culture CD14+ adherent cells; (3) discarding the supernatant of the CD14+ cells that have completed the adherent culture in step (2), adding DC complete culture medium and the autologous plasma prepared in step (1), and continuing the culture to obtain DC cells; (4) The DC cells prepared in step (3) were centrifuged at 1500 rpm for 5 min, resuspended and placed in serum-free XVIVO-15 medium containing 1 mg / mL Perminede, transfected with Ad-P53 virus, and then continued to be cultured; (5) Resuspending the DC cells cultured in step (4) in physiological saline to obtain the DC cell vaccine.
2. The method for preparing adenovirus p53-loaded DC cell vaccine according to claim 1, characterized in that: In step (1), the centrifugation parameters for collecting peripheral blood and performing centrifugal treatment are 2300 rpm for 15 min, and the centrifugation parameters for centrifuging the plasma layer again are 2700 rpm for 10 min. The plasma layer also includes a step of being water bathed at 56°C for 30 min before being centrifuged again.
3. The method for preparing adenovirus p53-loaded DC cell vaccine according to claim 1, characterized in that: In step (1), the specific separation method of PBMC is Ficoll density gradient centrifugation, the lymphocyte separation fluid is Ficoll, the centrifugation parameters when added to the centrifuge tube containing the lymphocyte separation fluid are 2000 rpm and centrifugation for 20 minutes, and the centrifugation parameters when performing the centrifugal washing treatment again are 1500 rpm and centrifugation for 5 minutes.
4. The method for preparing adenovirus p53-loaded DC cell vaccine according to claim 1, characterized in that: In step (2), the incubation is performed using 80 μL buffer + 20 μL CD14 magnetic beads at 2-8°C in the dark for 30 minutes, wherein 1E7 PBMCs are incubated with every 5 μL magnetic beads.
5. The method for preparing adenovirus p53-loaded DC cell vaccine according to claim 1, characterized in that: In step (3), the components of the DC complete culture medium are 50 ng / ml IL-4 and 100 ng / ml GM-CSF, the amount of autologous plasma added is 2%, and the continued culture is continued in an incubator at 37° C. and 5% CO 2 .
6. The method for preparing adenovirus p53-loaded DC cell vaccine according to claim 1, characterized in that: In step (4), the centrifugation parameters are 1800 rpm for 5 min, and the serum-free XVIVO-15 culture medium containing 1 mg / mL Permine contains L-Gln and Phenol Red.
7. The method for preparing adenovirus p53-loaded DC cell vaccine according to claim 1, characterized in that: In step (4), the transfection was carried out at 1×10 6 DC cells were infected at a multiplicity of infection of MOI = 25000:1, and virus Ad-P53 was added in proportion, then incubated at room temperature for 120 min, and centrifuged at 1500 rpm for 5 min. After centrifugation, the supernatant was discarded and DC complete culture medium containing 5% autologous plasma was added.
8. The method for preparing adenovirus p53-loaded DC cell vaccine according to claim 1, characterized in that: In step (5), the resuspension is to resuspend the transfected DC cells at 5×10 6 The cells were resuspended at a density of 1.54 × 10 / mL in 2 mL of 0.9% saline.
9. An adenovirus p53-loaded DC cell vaccine prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the adenovirus p53-loaded DC cell vaccine as claimed in claim 9 in the preparation of anti-tumor products.