DC vaccine for improving tumor prevention and / or treatment effect, preparation method and application thereof

Abstract

The invention provides a DC vaccine for improving the tumor prevention and / or treatment effect and a preparation method and application thereof, and relates to the technical field of vaccine preparation. According to the DC vaccine for improving the tumor prevention and / or treatment effect, cholesterol of a DC cell membrane is deprived through a reagent capable of regulating and controlling the cholesterol content of the cell membrane, so that the antigen presentation efficiency of the DC vaccine carrying the polypeptide antigen is enhanced, and the formation efficiency of immune synapses is improved as a starting point; initial T cells of an organism are domesticated to be differentiated into antigen-specific effector T cells and antigen-specific memory T cells, effective prevention and treatment of tumors are achieved by enhancing infiltration and killing of the effector T cells in the tumors, and the curative effect of the DC vaccine is improved. According to the application, the cell membrane surface hardness of the antigen presenting cell is effectively regulated and controlled by adopting a specific reagent, the vaccine-induced cellular immune effect is enhanced from another perspective, and a new thought is provided for the development of anti-tumor vaccines.

Description

Technical Field

[0001] The present application relates to the field of DC vaccine preparation, and particularly to a DC vaccine for improving the efficacy of preventing and / or treating tumors, a preparation method thereof, and an application thereof. Background Art

[0002] Traditional means for cancer treatment, such as surgery, chemotherapy, and radiotherapy, cannot effectively improve the survival rate of patients with advanced malignant tumors. The main reasons include tumor recurrence and metastasis, drug resistance, and the generation of side effects in the body. With the discovery of CTLA-4 and PD-1 immune checkpoints, and the breakthrough progress made by immune checkpoint inhibitors in various clinical solid tumors, immunotherapy has become the most promising anti-tumor treatment strategy in recent years. Currently, strategies that are developing rapidly and applied to clinical tumor immunotherapy include anti-tumor personalized vaccine strategies. For example, the mRNA-4157 vaccine jointly developed by Moderna and Merck, which encodes up to 34 tumor-specific mutant antigens, combined with pembrolizumab of PD-1 can be effectively used to treat stage III / IV melanoma with a high risk of recurrence; at the same time, the mRNA vaccine BNT122 combined with chemotherapy and immunotherapy (up to 20 neoantigens per patient) can induce the generation of specific T cells against pancreatic cancer neoantigens and achieve a recurrence-free survival period of up to 18 months. In addition, the mRNA vaccine CARVac expressing the CLDN6 antigen combined with the CAR-T therapy targeting the CLDN6 antigen can effectively treat patients with CLDN6-positive recurrent / refractory solid tumors and extend their survival period. The above results prove the effectiveness and importance of anti-tumor personalized vaccines in assisting other clinical anti-tumor therapies. Therefore, developing an efficient anti-tumor personalized vaccine strategy can provide a new treatment method for clinical treatment of malignant tumors.

[0003] The main target for a vaccine to exert its efficacy is the antigen-presenting cells in the body's lymph nodes. Existing vaccines mainly promote the activation of antigen-presenting cells and release a large amount of pro-inflammatory cytokines to guide the development of natural CD8 + T cells into antigen-specific memory T cells; however, due to the inherent barrier of the lymph nodes, it limits the efficient infiltration of existing vaccines into the lymph nodes to play a role; although the DC vaccine that has been loaded with antigens and activated by in vitro reinfusion has shown high efficacy in tumor treatment, the heterogeneity existing in DC cells themselves and the MHC restriction among different populations have brought difficulties to the widespread clinical popularization of DC vaccines. This patent intends to start from the perspective of metabolism, regulate cholesterol metabolism, effectively control the surface softness and hardness of the cell membrane of antigen-presenting cells, thereby further enhancing the efficacy of DC vaccines, and providing new ideas for the development of anti-tumor vaccines.

[0004] Content of the Application

[0005] The present application provides a DC vaccine for improving the efficacy of preventing and / or treating tumors, a preparation method thereof, and an application thereof, so as to solve the following technical problem: how to improve the efficacy of the DC vaccine.

[0006] In a first aspect, an embodiment of the present application provides an application of a reagent capable of regulating the cholesterol content of cell membranes in improving the efficacy of DC vaccines for preventing and / or treating tumors.

[0007] Optionally, the reagent capable of regulating cell membrane cholesterol improves the domestication ability of the DC vaccine on the body's immune system by regulating the cholesterol content on the surface of DC cells, and enhances the anti-tumor efficacy of the DC vaccine.

[0008] Optionally, the reagent capable of regulating cell membrane cholesterol includes at least one of methyl-β-cyclodextrin and related small molecule drugs, polypeptides, proteins, or bioenzymes having functions similar to those of methyl-β-cyclodextrin.

[0009] In a second aspect, an embodiment of the present application provides a preparation method of a DC vaccine for improving the efficacy of preventing and / or treating tumors, including the following steps:

[0010] Obtain mature DC cells loaded with antigens;

[0011] Co-incubate the mature DC cells with a reagent capable of regulating the cholesterol of DC cell membranes to obtain a DC vaccine;

[0012] The reagent capable of regulating the cholesterol of DC cell membranes includes at least one of methyl-β-cyclodextrin and related small molecule drugs, polypeptides, proteins, or bioenzymes having functions similar to those of methyl-β-cyclodextrin.

[0013] Optionally, the obtaining of the mature DC cells loaded with antigens specifically includes: co-culturing immature DC cells with the antigen to be loaded for 12 h to 36 h to obtain mature DC cells loaded with antigens.

[0014] Optionally, the ratio of the immature DC cells to the antigen to be loaded is 10 6 cells corresponding to 1 to 10 μg of polypeptide for incubation; and / or, the antigen concentration is 1 to 10 μg / mL.

[0015] Optionally, the ratio of the mature DC cells loaded with antigens to the reagent capable of regulating the cholesterol of DC cell membranes is 10 6 cells corresponding to 0.5 μmol of the total amount of the reagent for incubation.

[0016] In a third aspect, an embodiment of the present application provides a DC vaccine prepared by the preparation method described in the second aspect.

[0017] Fourthly, the embodiments of the present application provide an application of the DC vaccine described in the third aspect in improving the efficacy of preventing and / or treating tumors.

[0018] Optionally, the DC vaccine is co-incubated with specific CD8 + T cells and / or specific CD69 + T cells to determine the proliferation ratio of CD8 + T cells and / or the activation ratio of CD69 + T cells.

[0019] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0020] The embodiments of the present application provide an application of a reagent capable of regulating the cholesterol content of cell membranes in improving the efficacy of DC vaccines for preventing and / or treating tumors. By depriving the cholesterol of DC cell membranes with a reagent capable of regulating the cholesterol content of cell membranes, the antigen presentation efficiency of DC vaccines carrying polypeptide antigens is enhanced. Starting from improving the formation efficiency of immune synapses, naive T cells in the body are domesticated to differentiate into antigen-specific effector T cells and antigen-specific memory T cells. By enhancing the infiltration and killing of effector T cells in tumors, effective prevention and treatment of tumors are achieved, and the efficacy of DC vaccines is improved.

[0021] The present application effectively regulates the surface softness and hardness of the cell membranes of antigen-presenting cells by using specific reagents, enhancing the cellular immune response induced by vaccines from another perspective, and providing new ideas for the development of anti-tumor vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present application and, together with the specification, are used to explain the principles of the present application.

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is an image in Experimental Example 1 of the present application that proves the deprivation of cholesterol in DC cell membranes by methyl-β-cyclodextrin through a confocal microscope: where Figure 1 A is the DC vaccine, Figure 1 B is the DC-Cho vaccine; Figure 1C is the DC-MeβCD vaccine; where the red is the PKH26 dye (MCE), the blue is the Flipin3 dye (Thermofisher, tracing the cholesterol content in the cell membrane), and the image scale bar is 50μm;

[0025] Figure 2 It is the ratio of the proliferation and maturation of OT-1 T cells counted by flow cytometry in Experimental Example 2 of this application; where Figure 2 A is the result of flow cytometry detection after mature DC cells loaded with antigen were incubated with different concentrations of methyl-β-cyclodextrin and then incubated for 48 hours at a ratio of DC:T cells of 1:5; Figure 2 B is the result of flow cytometry detection after mature DC cells loaded with antigen were incubated with different concentrations of methyl-β-cyclodextrin and then incubated for 48 hours at a ratio of DC:T cells of 1:1;

[0026] Figure 3 It is to verify the effect of preventing tumor growth after immunization at the tail root of the DC vaccine loaded with OVA antigen that deprives the cell membrane of cholesterol in Experimental Example 3 of this application; Figure 3 A is the experimental flow chart of DC vaccine immunization, tumor inoculation and statistics;

[0027] Figure 3 B is the statistical chart of tumor growth curve; Figure 3 C is the physical picture of tumor growth points in different groups; Figure 3 D is the statistical chart of tumor weights in different groups;

[0028] Figure 4 It is to analyze the infiltration of related immune cells in the tumor microenvironment of different groups by flow cytometry in Experimental Example 4 of this application; Figure 4 A is the flow cytometry gating diagram of different immune cell subsets in the tumor microenvironment; Figure 4 B is to count the infiltration of CD8+ T cells in different treatment groups; Figure 4 C is to count IFN-γ + CD8 + + T cell infiltration; Figure 4 D is to count TNF-α + CD8 + + T cell infiltration; Figure 4 E is to count the infiltration of tissue-resident effector memory T cells (CD44 + , CD62L - , CD8 + ) in different treatment groups;

[0029] Figure 5It is the proportion of antigen-specific effector T cells in the lymph node microenvironment of mice in different treatment groups inoculated with tumors analyzed by flow cytometry in Experimental Example 5 of this application; Figure 5 A is a flow cytometry gating map of different immune cell subsets in the lymph node microenvironment; Figure 5 B is a proportion map of typical antigen-specific effector T cells in different treatment groups; Figure 5 C is to count the proportion of antigen-specific effector T cells (CD8 + CD103 + ) in different treatment groups;

[0030] Figure 6 It is the number of T cells reactive to OVA antigen in the spleens of different treatment groups verified by ELISPOT in Experimental Example 6 of this application; Figure 6 A is a white light image of Elispot after co-incubating single cell suspensions of spleens from different treatment groups with OVA; Figure 6 B is the statistical result of the number of spots in Elispot after co-incubating single cell suspensions of spleens from different treatment groups with OVA;

[0031] Figure 7 is the chemical structural formula of methyl-β-cyclodextrin;

[0032] Figure 8 is the flow chart of the preparation method of the DC vaccine provided in some embodiments of this application. Detailed implementation manners

[0033] To make the objectives, technical solutions and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some, but not all, of the embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts shall fall within the scope of protection of this application.

[0034] The various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of this application; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and individual values within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, which applies regardless of the range; additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0035] In this text, terms such as "comprising" mean "including but not limited to". Relative terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "a plurality" means two or more; "at least one kind", "at least one of the following items" or similar expressions refer to any combination of these items, including any combination of single items or plural items; for example, "at least one of a, b, or c", or, "at least one of a, b, and c" can both represent: a, b, c, a-b (that is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple respectively. "Parts representation" such as parts by weight, parts by mass, etc. represents the proportional relationship between each component. In the proportional relationships involved in this text, the parameters that need to be described by proportion should be understood as the antecedents of the proportion formula in the order of description, and the proportional numbers should be understood as the consequents of the proportion formula. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substance A, substance B, and substance C should correspond one by one with the proportional numbers in the proportion formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0036] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in this text can be obtained through market purchase or can be prepared by existing methods.

[0037] In a first aspect, the embodiments of the present application provide an application of a reagent capable of regulating the cholesterol content in the cell membrane in improving the efficacy of DC vaccines in preventing and / or treating tumors.

[0038] In the above embodiment, a reagent capable of regulating the cholesterol content in the cell membrane is used to regulate the cholesterol content on the DC cell membrane, so as to regulate the surface softness and hardness of the cell membrane of antigen-presenting cells, thereby enhancing the antigen presentation efficiency of DC vaccines carrying polypeptide antigens. Starting from improving the formation efficiency of immune synapses, the body's naive T cells are domesticated to differentiate into antigen-specific effector T cells and antigen-specific memory T cells. By enhancing the infiltration and killing of effector T cells in tumors, effective prevention and treatment of tumors are achieved, and the efficacy of DC vaccines is improved.

[0039] As an alternative embodiment, the reagent for regulating cell membrane cholesterol improves the ability of the DC vaccine to domesticate the body's immune system by regulating the cholesterol content on the surface of DCs, and enhances the efficacy of the DC vaccine in preventing and treating tumors.

[0040] As an alternative embodiment, the reagent for regulating cell membrane cholesterol includes at least one of methyl-β-cyclodextrin and related small molecule drugs, polypeptides, proteins or bioenzymes having a similar function to methyl-β-cyclodextrin.

[0041] In the above embodiment, methyl-β-cyclodextrin, as a small molecule compound that can regulate the cholesterol content in the DC cell membrane, has a structural formula as Figure 7 shown. The principle of action of methyl-β-cyclodextrin is that methyl-β-cyclodextrin can combine with cholesterol in the DC cell membrane, causing cholesterol to be removed from the cell membrane, thereby regulating the cholesterol content on the cell membrane. Other related small molecule drugs, polypeptides, proteins or bioenzymes that achieve the regulation of the cholesterol content on the cell membrane by the same mechanism also fall within the protection scope of this application.

[0042] In a second aspect, an embodiment of the present application provides a method for preparing a DC vaccine for improving the efficacy of preventing and / or treating tumors, including the following steps:

[0043] Obtain mature DC cells loaded with antigens;

[0044] Co-incubate the mature DC cells with a reagent for regulating DC cell membrane cholesterol to obtain a DC vaccine;

[0045] The reagent for regulating DC cell membrane cholesterol includes at least one of methyl-β-cyclodextrin and related small molecule drugs, polypeptides, proteins or bioenzymes having a similar function to methyl-β-cyclodextrin.

[0046] In the above embodiment, antigens are first loaded onto DC cells, and the antigens include, but are not limited to, antigen polypeptides for treating and / or preventing specific tumors, protein antigens, lipid antigens, carbohydrate antigens, DNA-encoded antigens, and mRNA-encoded antigens. Then, the obtained mature DC cells are co-incubated with a reagent for regulating DC cell membrane cholesterol, so that the cholesterol content on the DC cell membrane is reduced, to regulate the surface softness of the cell membrane of antigen-presenting cells (DC cells), improve the antigen presentation efficiency, and starting from improving the formation efficiency of immune synapses, domesticate naive T cells in the body to differentiate into antigen-specific effector T cells and antigen-specific memory T cells, and finally obtain a DC vaccine with improved efficacy in preventing and treating tumors by enhancing the infiltration and killing of effector T cells in tumors.

[0047] As an alternative embodiment, the method for obtaining mature DC cells loaded with antigens specifically includes: co-culturing immature DC cells with the antigens to be loaded for 12 h to 36 h to obtain mature DC cells loaded with antigens.

[0048] In the above embodiment, the immature DC cells are co-cultured with the antigens to be loaded for 12 h to 36 h to achieve successful antigen loading. Exemplarily, the co-culture time can be 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h or 36 h.

[0049] As an alternative embodiment, the antigen can be OVA antigen polypeptide, virus-related antigen polypeptide or related antigen peptide specifically expressed by tumor cells.

[0050] As an alternative embodiment, the immature DC cells are formed by co-incubating and inducing differentiation of bone marrow-derived hematopoietic stem cells with cell stimulating factors.

[0051] In the above embodiment, DC cells (dendritic cells) are lymphoid or myeloid cells originating from the bone marrow and residing in peripheral and lymphoid tissues. Cell stimulating factors are used to induce the gradual differentiation and proliferation of DC precursor cells in the direction of DC to obtain immature DC cells.

[0052] As an alternative embodiment, the cell stimulating factor can be granulocyte-macrophage colony-stimulating factor GM-CSF, and the concentration can be 15 ng / mL to 25 ng / mL, that is, the concentration of the cell stimulating factor in each milliliter of complete medium is 15 ng to 25 ng.

[0053] In the above embodiment, the reason for controlling the concentration of the cell stimulating factor to be 15 ng / mL to 25 ng / mL is that within this concentration range, it can effectively mediate the differentiation of bone marrow myeloid cells into DC cells and will not cause a large number of deaths of DC cells. Exemplarily, the concentration of the cell stimulating factor can be 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL or 25 ng / mL.

[0054] As an alternative embodiment, the immature DC cells and the antigens to be loaded are incubated in RPMI 1640 complete medium for 12 h to 36 h, then the medium is replaced, and the cells are stimulated with RPMI 1640 complete medium containing 1 μg / mL of LPS for 12 h to 36 h, and then the medium is replaced again to obtain mature DC cells loaded with antigens.

[0055] As an alternative embodiment, the ratio of the immature DC cells to the antigen to be loaded is 10 6 cells corresponding to incubation with 1 μg to 10 μg of polypeptide.

[0056] In the above embodiment, controlling the ratio of the immature DC cells to the antigen to be loaded to be 10 6 cells corresponding to incubation with 1 μg to 10 μg of polypeptide is due to the fact that a lower antigen concentration cannot efficiently mediate the antigen presentation of cells and subsequent T cell domestication characteristics, while a higher antigen concentration has exceeded the upper limit that antigen-presenting cells can present. Antigens within this concentration range can effectively promote the antigen presentation function of antigen-presenting cells and mediate the formation of memory T cells.

[0057] As an alternative embodiment, the antigen concentration is 1 μg / mL to 10 μg / mL.

[0058] In the above embodiment, controlling the antigen concentration to be 1 μg / mL to 10 μg / mL is because a lower antigen concentration cannot efficiently mediate the antigen presentation of cells and subsequent T cell domestication characteristics, while a higher antigen concentration has exceeded the upper limit that antigen-presenting cells can present. Therefore, antigens within this concentration range can effectively promote the antigen presentation function of antigen-presenting cells and mediate the formation of memory T cells. Exemplarily, the antigen concentration can be 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL.

[0059] As an alternative embodiment, the ratio of the mature DC cells loaded with antigen to the reagent that can regulate DC cell membrane cholesterol is 10 6 cells corresponding to incubation with a total of 0.5 μmol of reagent.

[0060] In the above embodiment, controlling the ratio of the mature DC cells loaded with antigen to the reagent that can regulate DC cell membrane cholesterol to be 10 6 cells corresponding to incubation with a total of 0.5 μmol of reagent is because a lower amount of reagent cannot effectively deprive cholesterol from the DC cell membrane, while a higher amount of reagent will cause a large number of DC cells to die.

[0061] In a third aspect, based on a general inventive concept, the embodiments of the present application provide a DC vaccine prepared by the preparation method described in the second aspect.

[0062] The DC vaccine is achieved based on the above-mentioned method for preparing a DC vaccine for improving the efficacy of preventing and / or treating tumors. The specific steps of this preparation method can be referred to the above-mentioned embodiments. Since this DC vaccine adopts some or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, and will not be elaborated here one by one.

[0063] Fourthly, the embodiments of the present application provide an application of the DC vaccine described in the third aspect in improving the efficacy of preventing and / or treating tumors.

[0064] In the above-mentioned embodiment, the DC vaccine described in the present application can efficiently present antigens, promote the proliferation and activation of effector T cells that recognize antigens; subcutaneous injection of the DC vaccine can significantly prevent or treat the growth of tumors expressing related antigens. Its main principle is to play a role by enhancing the infiltration and activation of antigen-specific effector T cells and the formation of tissue memory T cells, and subcutaneous injection can also enhance the response rate of tumors to immunotherapy, thereby providing a new strategy for anti-tumor immunity and enhancing the response rate of immunotherapy.

[0065] As an alternative embodiment, the DC vaccine is co-incubated with specific CD8 + T cells and / or specific CD69 + T cells to determine the proliferation ratio of CD8 + T cells and / or the activation ratio of CD69 + T cells.

[0066] In the above-mentioned embodiment, the above-mentioned method can be used to verify that the DC vaccine described in the present application can enhance the antigen presentation effect. The specific operation process includes the following content:

[0067] Taking the DC vaccine loaded with OVA antigen as an example, verify that it can induce the proliferation of OT-1 CD8 + T cells and the activation of CD69 + T cells:

[0068] Based on CD8 + T cell magnetic beads (MojoSort TM Mouse CD8a Selection Kit, Biolegend) to sort CD8 in the spleen of OT-1 specific TCR T cell mice (C57BL / 6-Tg(TcraTcrb)1100Mjb / J from Jackson labratory) +T cells, OT-1T cells were stained with 5 μM CFSE fluorescent dye (Biyuntian) in serum-free 1640 medium for 15 minutes, terminated with 1640 medium containing 10% FBS, and washed twice with PBS; the DC vaccine loaded with OVA antigen obtained in the third aspect of the present invention was mixed with OT-1 specific CD8 T cells containing cell proliferation dye CFSE. + T cells were incubated in 1640 complete medium at a cell number ratio of 1:1 to 1:5 for 48 hours, and then flow cytometry was used to determine the proportion of T cell proliferation to reflect the strength of DC vaccine antigen presentation ability.

[0069] CD8-based + T cell magnetic bead sorting OT-1 specific TCR T cells CD8 in mouse spleen + The DC vaccine loaded with OVA antigen obtained in the third aspect of the present invention and OT-1 specific CD8 + T cells were co-cultured in 1640 complete medium at a cell number ratio of 1:1 to 1:5 for 48 hours, and flow cytometry and flow fluorescent antibodies that can bind to mouse CD69 molecules were used to detect CD69. + The proportion of T cell activation reflects the strength of DC vaccine antigen presentation ability.

[0070] As an optional embodiment, the method for using the DC vaccine to enhance the anti-tumor effect of the DC vaccine is vaccine immunization, and the effect is verified by mouse tail immunization injection, which specifically includes:

[0071] 6-8 week old C57BL / 6 female mice were selected and divided into an experimental group and a control group. The mice in the experimental group were treated as follows: the DC vaccine described in the third aspect of the present invention was diluted in PBS solution at a concentration of 1-8×10 6 The number of mice per day was immunized at the base of the tail for 2 to 3 times, with an interval of 1 to 2 weeks between each immunization. The order of each immunization was intraperitoneal injection of TNF-α cytokine, DC vaccine at the base of the tail on the second day, and PD-1 antibody intraperitoneal injection on the third day; 2×10 6 Tumor cells from C57BL / 6 mice of any cancer type expressing OVA antigen. The mice in the control group were treated in the same way as the experimental group, except that the DC vaccine described in the third aspect was replaced with mature DC cells loaded with antigens that had not been treated with reagents. The tumor growth conditions of the mice in the experimental group and the control group were counted respectively, and the domestication of the body's immune system and the immune memory characteristics were compared to evaluate the efficacy of the vaccine.

[0072] As an alternative embodiment, the immunization injection method further includes subcutaneous injection, intraperitoneal injection, intramuscular injection or intraperitoneal injection.

[0073] The relevant tumors in the anti-tumor immunity of the DC vaccine mentioned above are not limited to cancer types and include 33 cancer types commonly classified clinically at present.

[0074] The following further elaborates the present application in conjunction with specific embodiments. For the experimental methods without specific conditions noted in the following embodiments, they are usually determined according to national standards / industry standards; if there are no corresponding national standards / industry standards, they are carried out according to general international standards, conventional conditions or the conditions recommended by the manufacturer.

[0075] Example 1

[0076] This example provides a DC vaccine for improving the efficacy of preventing and / or treating tumors, and the preparation method includes the following steps:

[0077] 1) Obtaining immature DC cells derived from C57BL / 6 mice: Take a 6-8-week-old C57BL / 6 mouse and euthanize it by cervical dislocation. Immerse it in 75% alcohol for 3-5 minutes. Use surgically sterile instruments after high-pressure sterilization to dissect the tibia and femur in a sterile operating table. After cutting open the bone marrow cavity with ophthalmic scissors, repeatedly rinse with sterile PBS (1 mL syringe) until all bone marrow cells are flushed out. Filter the collected bone marrow cell suspension through a 70 μm filter into a 15 mL centrifuge tube, centrifuge at 500 g for 5 minutes at 4 °C, discard the supernatant, add 1 mL ACK to resuspend the cells, lyse at room temperature for 1 minute, and then add 10 mL PBS to terminate the reaction. Centrifuge at 500 g for 5 minutes at 4 °C, discard the supernatant, wash once with PBS, and plate the cell pellet with the prepared DC medium. Perform semi-medium change on the cells on the 3rd and 5th days of culture, and the immature DC cells are obtained on the 6th day of culture. The DC medium described in this method includes RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum + 1% double antibody + 20 ng / mL GM-CSF.

[0078] 2) Obtaining mature DC cells loaded with OVA antigen: Incubate the immature DC cells prepared in step 1) with a concentration of 10 μg / mL OVA polypeptide antigen in 1640 complete medium for 24 hours, then replace the medium, add 1640 complete medium containing 1 μg / mL LPS to stimulate for 24 hours, and after replacing the 1640 complete medium, obtain mature DC cells loaded with antigen. The sequence of the OVA polypeptide in this method is SIINFEKL, synthesized by Hefei Guotai Biotech; the 1640 complete medium is provided by Gibco and includes 1% penicillin / streptomycin double antibody and 10% inactivated Sijiqing fetal bovine serum.

[0079] 3) Obtaining DC vaccine: The antigen-loaded mature DC cells described in step 2) are co-incubated with RPMI 1640 complete medium containing 5 mM methyl-β-cyclodextrin for 2 hours, and then replaced with normal RPMI 1640 complete medium to obtain mature DC cells loaded with OVA antigen with cholesterol stripped from the cell membrane, namely the DC vaccine for enhancing the efficacy of tumor prevention and / or treatment.

[0080] Examples 2 to 3

[0081] Examples 2 and 3 both provide a DC vaccine for enhancing the efficacy of tumor prevention and / or treatment. The preparation method is the same as that in Example 1, except that the dosage of methyl-β-cyclodextrin in Example 2 is 1 mM, and the dosage of methyl-β-cyclodextrin in Example 3 is 2 mM.

[0082] Comparative Example 1

[0083] This comparative example provides a DC vaccine. The preparation method is the same as steps 1) and 2) in Example 1, and the obtained antigen-loaded mature DC cells are the DC vaccine provided by this comparative example.

[0084] Example 4

[0085] This example provides a method for a DC vaccine to enhance the efficacy of tumor prevention and / or treatment, which is realized by using the DC vaccine prepared in any one of Examples 1 to 3, and includes the following steps:

[0086] Select 6-8-week-old female C57BL / 6 mice, and immunize the tail root with the DC vaccine in PBS solution at a quantity of 1 - 8×10 6 cells / mouse, and immunize a total of 2 - 3 times, with an interval of 1 - 2 weeks between each immunization. The order of each immunization is to intraperitoneally inject TNF-α cytokine first, immunize the tail root with the DC vaccine on the next day, and intraperitoneally inject PD-1 antibody on the third day. The role of TNF-α is to promote the maturation of lymph node DC cells and avoid the formation of immune tolerance DC cells; the reason for injecting PD-1 antibody is to shield the negative regulatory effect of the PD-L1 / PD-1 signaling axis that appears during the immunization process of the DC vaccine in the lymph nodes.

[0087] Experimental Example 1

[0088] This experimental example is to verify that methyl-β-cyclodextrin can effectively strip the cholesterol component from the DC cell membrane, specifically including:

[0089] 1) Obtaining mature DC cells BMDCs: Take a 6-week-old C57BL / 6 mouse and sacrifice it by cervical dislocation. Immerse it in 75% alcohol for 3 - 5 min. Use surgically sterile instruments after autoclaving to dissect the tibia and femur in a sterile operating table. After cutting open the bone marrow cavity with ophthalmic scissors, repeatedly rinse it with sterile PBS (1 mL syringe) until all bone marrow cells are flushed out. Filter the collected bone marrow cell suspension through a 70 μm filter into a 15 mL centrifuge tube, centrifuge at 500 g for 5 min at 4 °C, discard the supernatant, add 1 mL ACK to resuspend the cells, lyse at room temperature for 1 min, and then add 10 mL PBS to terminate the reaction. Centrifuge at 500 g for 5 min at 4 °C, discard the supernatant, wash once with PBS, and plate the cell pellet with the prepared DC medium for culture. The DC medium contains RPMI 1640 medium with 10% heat-inactivated fetal bovine serum + 1% double antibody + 20 ng / mL GM-CSF. Perform half-medium change on the cells on the 3rd and 5th days of culture. On the 6th day of culture, add 10 μg / mL OVA antigen and incubate for 24 h; on the 7th day, add 1 μg / mL LPS and incubate for 18 h to stimulate them to become mature DCs. Collect the mature DC cells carrying OVA antigen, centrifuge at 1000 g for 5 min, retain the cell pellet, wash it twice with PBS and then count to obtain the mature DC cells BMDCs loaded with OVA antigen.

[0090] 2) Divide the mature DC cells BMDCs loaded with OVA antigen into three equal parts. The first part is added with 5 mM cholesterol Cholesterol and denoted as DC-Cho vaccine. The second part is added with 5 mM MeβCD and denoted as DC-MeβCD vaccine. The third part is the control group, added with an equal amount of normal saline and denoted as DC vaccine. Incubate the three groups of DC cells at 37 °C for 2 h, and then co-incubate the three different DC vaccines with 1640 complete medium containing 10 μg / mL Flipin-3 dye (provided by Thermofisher, used to trace the cell membrane cholesterol content) and 10 μg / mL PKH26 dye (provided by MCE, used for labeling and tracking of DC cells in vitro) for 30 minutes. Then wash twice with PBS, and capture the fluorescence intensities of Flipin-3 and PKH26 on the cell membranes of the three types of DCs through a confocal microscope. The obtained images are as Figure 1 shown. Among them, the excitation wavelength used by the confocal microscope is 405 nm, and the receiving band is 440 - 460 nm.

[0091] As can be seen from Figure 1 the images, from right to left are the first, second, and third parts of the mature DC cells loaded with OVA antigen. Among them, the red is the fluorescence of the PKH26 dye, and the blue is the fluorescence of the Flipin3 dye. From Figure 1It can be seen that after co-culturing with methyl-β-cyclodextrin, cholesterol on the cell membrane surface of DC cells is stripped under the action of methyl-β-cyclodextrin. At the same time Figure 1 The results also show that by co-incubating cholesterol with DC cells, the content of cholesterol on the cell membrane surface can also be efficiently increased.

[0092] Experimental Example 2

[0093] This experimental example is used to verify the effect of DC vaccines with stripped cell membrane cholesterol on antigen-specific T cells, specifically including:

[0094] 1) Obtain OT-I antigen-specific T cells stained with CFSE proliferation dye: Based on CD8 + T cell magnetic beads (MojoSort TM Mouse CD8a Selection Kit, Biolegend) were used to sort CD8 + T cells in the spleen of 6-8-week-old OT-1 specific TCR T cell mice (C57BL / 6-Tg(TcraTcrb)1100Mjb / J from Jackson labratory). The OT-1 T cells were stained with 5 μM CFSE fluorescent dye (Beyotime) in serum-free 1640 medium for 15 minutes. The staining was terminated with 1640 medium containing 10% FBS, and the stained cells were washed twice with PBS.

[0095] 2) The DC vaccines obtained in Examples 1-2 and Comparative Example 1 were co-incubated with CFSE-stained OT-1 specific CD8 + T cells at a cell number ratio of 1:5 in 1640 complete medium for 48 hours. Flow cytometry was used to reflect the strength of the antigen presentation ability of the DC vaccine through the proportion of T cell proliferation. The results are as Figure 2 shown in A; at the same time, the DC vaccines obtained in Examples 1-3 and Comparative Example 1 were co-incubated with OT-1 specific CD8 positive T cells at a cell number ratio of 1:1 in 1640 complete medium for 48 hours. Flow cytometry was used to reflect the strength of the antigen presentation ability of the DC vaccine through the proportion of T cell activation (the expression of CD69 is a marker of T cell activation). The relevant experimental results are as Figure 2 shown in B.

[0096] 3) From Figure 2As can be seen from A, compared with Comparative Example 1, the DC vaccines provided in Example 1 and Example 2 were co-incubated with methyl-β-cyclodextrin to regulate the cholesterol content in the cell membrane, which was helpful for the proliferation of antigen-specific T cells. Moreover, when the incubation concentration of methyl-β-cyclodextrin added in Example 1 with DC mature cells was 5 mM, it could best promote the proliferation of antigen-specific T cells. In addition, from Figure 2 As can be seen from B, compared with Comparative Example 1, the DC vaccines provided in Examples 1 to 3 were co-incubated with methyl-β-cyclodextrin to regulate the cholesterol content in the cell membrane, which could promote the activation of antigen-specific T cells. And when the incubation concentration of methyl-β-cyclodextrin with DC in Example 2 was 5 mM, the activation promotion effect was the best.

[0097] Experimental Example 3

[0098] This experimental example was used to compare the efficacy of DC-Cho group vaccine, DC-MeβCD group vaccine and DC vaccine in improving the prevention of tumor growth, specifically including:

[0099] 1) Obtain the three groups of vaccines obtained in step 2) of Experimental Example 1: DC-Cho group vaccine, DC-MeβCD group vaccine and DC vaccine. Immunize 6-8-week-old C57 mice through the tail root with the three groups of vaccines, twice in total. Before each vaccination, intraperitoneally inject the same dose of TNF-α cytokine. And after each immunization, intraperitoneally inject 150 μg of PD-1 antibody, twice in total, with a one-week interval between the two injections. The relevant experimental flow chart is as Figure 3 shown in A;

[0100] 2) On the 7th day after the second vaccination, all mice were inoculated with tumors, that is, subcutaneously inject B16 cells expressing OVA antigen, and the injection dose was 2×10 6 cells / mouse. On the 6th day after tumor inoculation, the tumor sizes of different groups were counted, and the counting was carried out until the 22nd day after tumor inoculation. The statistical results of the sizes of different tumors in different treatment groups are as Figure 3 shown in B, the physical pictures of the tumor sizes in different treatment groups on the 22nd day are as Figure 3 shown in C, and the statistical results of the tumor weights in different treatment groups on the 22nd day are as Figure 3 shown in D.

[0101] 3) From Figure 3The results showed that the DC vaccine loaded solely with OVA antigen only had a certain degree of effect in inhibiting tumor growth; while the DC-Cho group vaccine with excessive cholesterol loading reversed the effect of the ordinary DC vaccine in inhibiting tumor growth, and the DC-MeβCD group vaccine that had its cell membrane cholesterol depleted more efficiently inhibited tumor growth, showing significant differences compared with the control group and the DC vaccine loaded solely with OVA antigen.

[0102] Experimental Example 4

[0103] This experimental example was used to compare the effects of the DC-Cho group vaccine, the DC-MeβCD group vaccine, and the DC vaccine on effector T cells in the tumor microenvironment, specifically including:

[0104] 1) The tumors of different treatment groups in Experimental Example 3 were respectively enzymolyzed to obtain single-cell suspensions of the tumor microenvironment, and in the process of enzymolysis, hyaluronidase and collagenase IV were selected.

[0105] 2) Fascia and impurities were sieved out through a 70-μm filter, and then the single-cell tumor suspension was labeled with relevant flow antibodies. The selected flow antibodies were as shown in Figure 4 the flow cytometry gating diagram in Figure A, which also included the ZoomNIR dye for staining dead and live cells. All relevant flow antibodies were purchased from Biolegend Biotechnology Co., Ltd.

[0106] 3) Since the mechanism by which the DC vaccine exerts its anti-tumor immune effect mainly lies in inducing cellular immune responses, in this experimental example, it was necessary to count the proportion of infiltrating CD8 Figure 4 T cells in the tumor microenvironment as shown in + B, the proportion of infiltrating and activated IFN-γ Figure 4 CD8 + T cells in the tumor microenvironment as shown in + C, the proportion of infiltrating and activated TNF-α Figure 4 CD8 + T cells in the tumor microenvironment as shown in + D, and the proportion of tissue-resident memory T cells as shown in Figure 4 E.

[0107] 4) As Figure 4 known, the DC vaccine loaded solely with OVA antigen only had a certain degree of effect in promoting cellular immunotherapy, while the DC vaccine with cholesterol added (DC-Cho group vaccine) masked this effect, being similar to the infiltration of effector T cells in the PBS group; while the DC vaccine with cholesterol depleted (DC-MeβCD group vaccine) highly promoted the infiltration of effector T cells and the formation of activated T cells and memory T cells, showing significant differences compared with other treatment groups, and its results were consistent with those of Experimental Example 3.

[0108] Experimental Example 5

[0109] This experimental example was used to compare the effects of DC-Cho group vaccine, DC-MeβCD group vaccine, and DC vaccine on memory T cells in the lymph node microenvironment, specifically including:

[0110] 1) Collect the lymph nodes of mice in different treatment groups in Experimental Example 3, obtain a single-cell suspension of lymph nodes through grinding and filtration, and then label the single-cell suspension of lymph nodes with relevant flow antibodies. The selected flow antibodies are as shown in the flow cytometry gating diagram in Figure A, which also includes the ZoomNIR dye for staining dead and live cells. All relevant flow antibodies were purchased from Biolegend Biotechnology Co., Ltd. Figure 5 As shown in the flow cytometry gating diagram in Figure A, which also includes the ZoomNIR dye for staining dead and live cells. All relevant flow antibodies were purchased from Biolegend Biotechnology Co., Ltd.

[0111] 2) Perform flow cytometry analysis on the single-cell suspension of lymph nodes of mice in different treatment groups, and use relevant antigen-specific effector T cells as the evaluation index for the efficacy of DC vaccine; the typical antigen-specific effector T cell ratios in the lymph nodes of mice in different groups are as shown in Figure B, and the statistical results of antigen-specific effector T cells in the lymph nodes of mice in different groups are as shown in Figure C. Figure 5 As shown in Figure B, and the statistical results of antigen-specific effector T cells in the lymph nodes of mice in different groups are as shown in Figure C. Figure 5 As shown in Figure C.

[0112] 3) From the results of Figure 5 it can be seen that the cholesterol-deprived DC vaccine efficiently promoted the formation of antigen-specific effector T cells in the lymph nodes, showing significant differences compared with other treatment groups, and the results were consistent with those of Experimental Example 3.

[0113] Experimental Example 6

[0114] This experimental example was used to compare the effects of DC-Cho group vaccine, DC-MeβCD group vaccine, and DC vaccine on memory T cells in the lymph node microenvironment, specifically including:

[0115] 1) Collect the spleens of mice in different treatment groups in Experimental Example 3, obtain a single-cell suspension of the spleen through grinding and filtration, and analyze the T cells with effector OVA antigen reactivity in the single-cell suspension of the spleen of mice in different groups using an IFN-γ Elispot detection kit; among them, the actual Elispot diagram results of reactive T cells in the spleens of different mice in different groups are as shown in Figure A, and the statistical results of the number of spots on the Elispot are as shown in Figure B. Figure 6 As shown in Figure A, and the statistical results of the number of spots on the Elispot are as shown in Figure B. Figure 6 As shown in Figure B.

[0116] 2) From the results of Figure 6As can be seen from the results, the cholesterol-depleted DC vaccine efficiently promoted the formation of antigen-reactive T cells in the spleen, showing a significant difference compared with other treatment groups, and the results were consistent with those of Experimental Example 3.

[0117] In summary, regulating the cholesterol content on the DC cell membrane provided in the embodiments of the present application is beneficial to improving the effect of treating and / or preventing tumors, specifically manifested as follows:

[0118] 1) The DC vaccine with cholesterol-depleted cell membrane (DC-MeβCD group vaccine) can efficiently present antigens and promote the proliferation and activation of antigen-specific T cells;

[0119] 2) The DC vaccine with cholesterol-depleted cell membrane (DC-MeβCD group vaccine) can efficiently enhance the anti-tumor immune ability of the body, and its anti-tumor mechanism is related to its induction of highly efficient antigen memory T cells and effector T cells;

[0120] 3) Subcutaneous injection of antigen-loaded mature DC cells with cholesterol-depleted cell membrane (DC-MeβCD group vaccine) can significantly prevent the growth of tumors expressing related antigens by enhancing the infiltration and activation of antigen-specific effector T cells and the formation of tissue memory T cells, thereby efficiently enhancing its anti-tumor immune effect.

[0121] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features claimed in the present application.

Claims

1. Application of agents capable of regulating cell membrane cholesterol content in improving the efficacy of DC vaccines in preventing and / or treating tumors.

2. According to the use according to claim 1, the agent capable of regulating cell membrane cholesterol improves the ability of the DC vaccine to tame the body's immune system and enhances the anti-tumor efficacy of the DC vaccine by regulating the cholesterol content on the surface of DC cells.

3. According to the use of claim 1 or 2, the reagent capable of regulating cell membrane cholesterol comprises methyl β-cyclodextrin and at least one of related small molecule drugs, polypeptides, proteins or biological enzymes having similar functions to methyl β-cyclodextrin.

4. A method for preparing a DC vaccine for improving the efficacy of preventing and / or treating tumors, comprising the following steps: Obtaining antigen-loaded mature DC cells; The mature DC cells are co-incubated with a reagent capable of regulating DC cell membrane cholesterol to obtain a DC vaccine; The reagent capable of regulating DC cell membrane cholesterol includes methyl β-cyclodextrin and at least one of related small molecule drugs, polypeptides, proteins or biological enzymes having functions similar to methyl β-cyclodextrin.

5. The preparation method according to claim 4, wherein obtaining mature DC cells loaded with antigens comprises: The immature DC cells are co-cultured with the antigen to be loaded for 12 hours to 36 hours to obtain mature DC cells loaded with the antigen.

6. The preparation method according to claim 5, wherein the ratio of immature DC cells to the antigen to be loaded is 10 6 1 to 10 μg of polypeptide is incubated with each cell; and / or, the concentration of the antigen is 1 μg / mL to 10 μg / mL.

7. The preparation method according to claim 4, wherein the ratio of the antigen-loaded mature DC cells to the agent capable of regulating DC cell membrane cholesterol is 10 6 Each cell was incubated with 0.5 μmol of reagent.

8. A DC vaccine for improving the efficacy of preventing and / or treating tumors, obtained by the preparation method according to any one of claims 4 to 7.

9. Use of the DC vaccine according to claim 8 in improving the efficacy of preventing and / or treating tumors.

10. The use according to claim 9, wherein the DC vaccine is combined with a specific CD8 + T cells and / or specific CD69 + T cells were co-incubated to determine CD8 + T cell proliferation ratio and / or CD69 + The proportion of T cell activation.

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