CN114028552A - Monocyte-loaded LMP1 protein vaccine composition and preparation method and application thereof - Google Patents
Monocyte-loaded LMP1 protein vaccine composition and preparation method and application thereof Download PDFInfo
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- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses a monocyte load LMP1 protein vaccine composition, a preparation method and an application thereof, wherein the vaccine composition consists of LMP1 protein solution with certain concentration and monocytes, and is prepared by the following method: preparing a LMP1 protein solution with a certain concentration; separating peripheral blood or bone marrow to obtain mononuclear cells; and mixing the LMP1 protein solution with a certain concentration obtained from S1 with a certain amount of monocytes obtained from S2 to prepare the vaccine composition of which the monocytes load LMP1 protein. The monocyte-loaded LMP1 protein vaccine composition, the preparation method and the application thereof disclosed by the invention can solve the problem of complicated preparation process of antigen vaccines in the prior art, greatly reduce the preparation cost of the vaccines, achieve the obvious tumor-resistant effect and provide an early-stage technical basis for wider clinical tests.
Description
Technical Field
The invention relates to the technical field of immunotherapy, in particular to a monocyte-loaded LMP1 protein vaccine composition, and a preparation method and application thereof.
Background
Nasopharyngeal carcinoma refers to a cancer species occurring on nasopharyngeal mucosa epithelia, about 80% of nasopharyngeal carcinomas occur in China all over the world according to statistics, the nasopharyngeal carcinoma is secret, most of nasopharyngeal carcinomas are in the middle and late stages, and the survival rate is less than 50% after the prognosis is poor for 5 years. EB virus DNA was first discovered in a nasopharyngeal carcinoma patient specimen in 1970, and EB virus (EBV) infection was identified as a major causative factor of nasopharyngeal carcinoma in 2005. EB virus can express multiple virus proteins after entering nasopharyngeal epithelial cells. The research proves that the LMP1 protein is the only protein which can transform normal epithelial cells into tumor cells in a plurality of proteins expressed by EB virus. LMP1 can promote generation and development of nasopharyngeal carcinoma by activating nuclear factor-kB cell inflammation signal to cause tissue chronic inflammation and further cancerate, can also up-regulate EGFR to promote cell proliferation, and can assist tumor cell in resisting tumor immune monitoring and promoting angiogenesis. According to the research, LMP1 protein epitope is found on the surface of cancer cells of nasopharyngeal carcinoma patients. Therefore, the specific immunoreaction of the body aiming at the EB virus oncogenic protein LMP1 is induced by an immunization means, and EB positive nasopharyngeal carcinoma cells can be effectively eliminated theoretically so as to inhibit the occurrence and development of tumors. Therefore, the development of an antigen vaccine composition of LMP1 protein is necessary, but the preparation process of the antigen vaccine in the prior art is complicated.
Disclosure of Invention
Aiming at the technical problems in the related art, the invention provides a vaccine composition with LMP1 protein loaded on monocytes, and a preparation method and application thereof, which can overcome the defects in the prior art.
In recent years, research shows that monocytes can take up antigens and directly process the antigens into an antigen peptide form to activate dendritic cells in vivo to induce specific immune response, so that the LMP1 antigen loaded by the monocytes is worthy of being developed as a tumor therapeutic vaccine.
In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:
a vaccine composition of monocyte-loaded LMP1 protein, which consists of a certain concentration of LMP1 protein solution and monocytes, and is prepared by the following method:
s1, preparing LMP1 protein solution with a certain concentration;
s2 separating peripheral blood or bone marrow to obtain mononuclear cells;
s3, mixing the LMP1 protein solution with a certain concentration obtained from S1 with a certain amount of monocytes obtained from S2 to prepare the vaccine composition of which the monocytes load LMP1 protein.
Preferably, the concentration of the LMP1 protein solution is 1 mg/mL.
Preferably, the mononuclear cells are prepared by adopting a lymph separation liquid Ficoll density gradient centrifugation method.
According to another aspect of the present invention, there is provided a method for preparing a monocyte-loaded LMP1 protein vaccine composition, the method comprising the steps of:
s1, preparing LMP1 protein solution with a certain concentration;
s2 separating peripheral blood or bone marrow to obtain mononuclear cells;
s3, mixing the LMP1 protein solution with a certain concentration obtained from S1 with a certain amount of monocytes obtained from S2 to prepare the vaccine composition of which the monocytes load LMP1 protein.
Further, the LMP1 protein solution with a certain concentration prepared in S1 is LMP1 protein solution with a certain concentration prepared from LMP1 protein by PBS.
Further, the obtaining of mononuclear cells from bone marrow isolation in S2 includes the steps of:
s21 taking mouse femur, cutting off two ends of femur, repeatedly washing femoral bone cavity with PBS to obtain bone marrow eluate, filtering with 40 μ M screen, and collecting into centrifuge tube;
s22 centrifuging the centrifuge tube in S21 to obtain a bone marrow cell sediment; adding PBS to resuspend the bone marrow cells; adding the bone marrow cell suspension into a centrifuge tube containing Ficoll lymph separation liquid;
s23 centrifuging the centrifuge tube in S2, sucking the second layer of annular milky white monocyte layer into another centrifuge tube by a suction tube, supplementing PBS and centrifuging;
s24 discarding the supernatant, adding PBS for resuspension, centrifuging again, discarding the supernatant to obtain monocyte mass, resuspending with appropriate amount of RPMI1640 medium containing 10% inactivated FBS, and counting.
Further, mixing the LMP1 protein solution with the monocytes in S3 comprises the steps of:
s31 LMP1 protein solution was dissolved in RPMI1640 medium containing 10% inactivated FBS to a concentration of 1 mg/mL;
s32 mononuclear cells are added into the LMP1 protein solution, the consistency of the density of the mononuclear cells is ensured, and the mononuclear cells are all 2 multiplied by 107Individual cells/mL;
s33 placing the cell suspension of step S32 into a constant temperature incubator at 37 ℃ and 5% CO2Incubating for 2h, and oscillating for 1 time every half hour;
after the S34 incubation is finished, centrifuging the cell suspension, removing the supernatant, adding a proper amount of PBS for resuspension, centrifuging again, and removing the supernatant to obtain a monocyte pellet loaded with LMP1 protein solution antigen;
s35 monocyte loaded with LMP1 protein solution antigen was resuspended in RPMI1640 medium to a final cell concentration of 5X 107Individual cells/mL to obtain the vaccine composition.
The invention also provides application of the vaccine composition of the monocyte loaded LMP1 protein in preparation of a product for treating LMP1 positive tumors.
Further, the vaccine composition is injected intravenously.
The invention has the beneficial effects that: the invention provides a novel LMP1 protein solution antigen vaccine composition and a preparation method thereof, which reduces the operation steps of conventional monocyte separation and differentiation to generate DC through simplified monocyte separation operation, and greatly reduces the vaccine preparation cost; the separated monocyte is directly used for loading LMP1 protein solution antigen, so that the remarkable anti-tumor effect can be achieved, and the early-stage technical basis is provided for wider clinical tests.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a graph showing the measurement of the phagocytosis of OVA-FITC by mouse bone marrow-derived mononuclear cells according to an embodiment of the present invention, including a flow chart and a statistical bar chart;
FIG. 2 shows the result of detecting peripheral blood antibodies at day 14 after the monocyte LMP1 protein solution tumor vaccine immunized the A2 receptor mouse according to the embodiment of the present invention;
FIG. 3 is the result of detecting peripheral blood specific T cells at day 14 after immunization of A2 recipient mice with the monocyte LMP1 protein solution tumor vaccine according to the embodiment of the present invention, including flow-type representative graph and statistical data graph;
FIG. 4 shows the tumor growth curve (preventive model) of LMP1 protein solution monocyte vaccine against LMP1 positive TC-1 cell line tumor-bearing mice injected on days-21, -14 and-7 and inoculated with tumor cells on day 0 according to the embodiment of the present invention;
FIG. 5 shows the tumor growth curve (therapeutic model) of LMP1 protein solution monocyte vaccine against LMP1 positive TC-1 cell line tumor-bearing mice, inoculated with tumor cells on day 0, and injected with vaccine on days 7 and 14, according to the embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
Specifically, the raw material manufacturers involved in the examples of the present invention are shown in table 1:
table 1: raw material table
Example 1
Isolation of monocytes comprising the steps of:
(1) the mice were sacrificed by cervical dislocation, 2 femurs were taken, both ends of the femurs were cut off, and appropriate amount of PBS was sucked in by a syringe to repeatedly wash the femoral bone cavity to obtain bone marrow eluate, which was filtered by a 40 μ M mesh screen and then collected into a PE centrifuge tube.
(2) Centrifuging at 500g for 5min at 300-; adding 4mL of PBS to resuspend the bone marrow cells; at the same time, 3mL of Ficoll lymph separation medium was added to another PE centrifuge tube. The bone marrow cell suspension was gently added to the PE centrifuge tube containing the Ficoll lymph separation medium, taking care not to disrupt the interface between the cell suspension and the lymph separation medium.
(3) Centrifuging a PE centrifuge tube for 30min at 400g, wherein the centrifuge tube is divided into four layers from top to bottom due to different densities: the first layer is a plasma layer, the second layer is an annular milky white monocyte layer, the third layer is a transparent separation liquid layer, and the fourth layer is a red blood cell layer. Carefully sucking the second annular milky white monocyte layer into another 50ml centrifuge tube by using a pipette, supplementing PBS, and centrifuging for 5-10 min at 400 g.
(4) Discarding the supernatant, adding PBS for resuspension, centrifuging for 5-10 min at 400g, discarding the supernatant to obtain monocyte mass, resuspending in a proper amount of RPMI1640 medium containing 10% inactivated FBS, and counting.
Example 2
The mononuclear cells obtained in example 1 are loaded with LMP1 protein solution or OVA-FITC antigen, and the method comprises the following steps:
(1) LMP1 protein solution or OVA-FITC was dissolved in RPMI1640 medium containing 10% inactivated FBS at a concentration of 1 mg/mL.
(2) Adding the mononuclear cells into the LMP1 protein solution to ensure that the mononuclear cells have consistent density which is 2 multiplied by 107Individual cells/mL.
(3) Putting the cell suspension obtained in the step (2) into a constant-temperature incubator at 37 ℃ and 5% CO2Incubate for 2h, shake 1 time every half hour.
(4) And after the incubation is finished, centrifuging the cell suspension for 5-10 min at 400g, removing the supernatant, adding a proper amount of PBS (phosphate buffer solution) for heavy suspension, centrifuging for 5-10 min at 400g, and removing the supernatant to obtain the LMP1 protein solution antigen-loaded monocyte pellet.
(5) The LMP1 protein solution antigen-loaded monocytes obtained in step (4) were resuspended in RPMI1640 medium to a final cell concentration of 5X 107Each cell/mL, and the mixed solution is the vaccine composition loaded with LMP1 protein. The loading of OVA-FITC on monocytes was measured by flow cytometry, and the results are shown in FIG. 1.
Example 3
The vaccine composition obtained in example 2 was used to immunize a mouse model, comprising the following steps:
(1) the monocyte suspension loaded with LMP1 protein solution was injected into A2 mice in a volume of 200. mu.L from the tail vein, and each mouse was inoculated with a cell amount of 1X 107Control mice were injected with an equal volume of PBS. The inoculation was continued 3 times, 1 time every 7 days.
(2) The growth status of the tumor of the mice was continuously followed and measured starting on day 7 after the tumor inoculation, and the serum antibody production was analyzed on day 14 after the first inoculation, and the detection results are shown in fig. 2, and fig. 3 is the detection results of peripheral blood specific T cells on day 14 after the monocyte LMP1 protein solution tumor vaccine immunized a2 receptor mice.
(3) Preventive models: each mouse was inoculated with tumor cells 14 days from the first vaccination. The tumor cell inoculation amount of each mouse is 1 × 106The inoculation mode is subcutaneous injection, and the tumor growth curve of the mice is shown in figure 4.
(4) Therapeutic models: the tumor cell inoculation amount of each mouse is 1 × 106The inoculation mode is subcutaneous injection. The LMP1 protein solution monocyte vaccine was inoculated at days 7 and 14 after tumor inoculation, respectively, and the tumor growth curves of the mice are shown in fig. 5.
The vaccine composition has strong specificity and high-efficiency resistance effect, and can be applied to preparation of products for treating solid tumors.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A vaccine composition of a monocyte-loaded LMP1 protein, which is characterized by consisting of a certain concentration of LMP1 protein solution and monocytes, and is prepared by the following method:
s1, preparing LMP1 protein solution with a certain concentration;
s2 separating peripheral blood or bone marrow to obtain mononuclear cells;
s3, mixing the LMP1 protein solution with a certain concentration obtained from S1 with a certain amount of monocytes obtained from S2 to prepare the vaccine composition of which the monocytes load LMP1 protein.
2. The vaccine composition of claim 1, wherein the concentration of the LMP1 protein solution is 1 mg/mL.
3. The vaccine composition of claim 1, wherein said monocytes are prepared using a lymph separator Ficoll density gradient centrifugation method.
4. A preparation method of a vaccine composition of which the monocytes are loaded with LMP1 protein is characterized by comprising the following steps:
s1, preparing LMP1 protein solution with a certain concentration;
s2 separating peripheral blood or bone marrow to obtain mononuclear cells;
s3, mixing the LMP1 protein solution with a certain concentration obtained from S1 with a certain amount of monocytes obtained from S2 to prepare the vaccine composition of which the monocytes load LMP1 protein.
5. The method according to claim 4, wherein the step of preparing the LMP1 protein solution at a certain concentration in S1 is to prepare the LMP1 protein into LMP1 protein solution at a certain concentration by PBS.
6. The method according to claim 4, wherein the isolation of mononuclear cells from bone marrow in S2 comprises the steps of:
s21 taking mouse femur, cutting off two ends of femur, repeatedly washing femoral bone cavity with PBS to obtain bone marrow eluate, filtering with 40 μ M screen, and collecting into centrifuge tube;
s22 centrifuging the centrifuge tube in S21 to obtain a bone marrow cell sediment; adding PBS to resuspend the bone marrow cells; adding the bone marrow cell suspension into a centrifuge tube containing Ficoll lymph separation liquid;
s23 centrifuging the centrifuge tube in S2, sucking the second layer of annular milky white monocyte layer into another centrifuge tube by a suction tube, supplementing PBS and centrifuging;
s24 discarding the supernatant, adding PBS for resuspension, centrifuging again, discarding the supernatant to obtain monocyte mass, resuspending with appropriate amount of RPMI1640 medium containing 10% inactivated FBS, and counting.
7. The method of claim 4, wherein the step of mixing the LMP1 protein solution with the mononuclear cells in S3 comprises the steps of:
s31 LMP1 protein solution was dissolved in RPMI1640 medium containing 10% inactivated FBS to a concentration of 1 mg/mL;
s32 mononuclear cells are added into the LMP1 protein solution, the consistency of the density of the mononuclear cells is ensured, and the mononuclear cells are all 2 multiplied by 107Individual cells/mL;
s33 placing the cell suspension of step S32 into a constant temperature incubator at 37 ℃ and 5% CO2Incubating for 2h, and oscillating for 1 time every half hour;
after the S34 incubation is finished, centrifuging the cell suspension, removing the supernatant, adding a proper amount of PBS for resuspension, centrifuging again, and removing the supernatant to obtain a monocyte pellet loaded with LMP1 protein solution antigen;
s35 monocyte loaded with LMP1 protein solution antigen was resuspended in RPMI1640 medium to a final cell concentration of 5X 107Individual cells/mL to obtain the vaccine composition.
8. Use of a monocyte-loaded LMP1 protein vaccine composition of claim 1 in the preparation of a product for treating LMP1 positive tumors.
9. The use according to claim 8, wherein the vaccine composition is administered intravenously.
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