Nano vaccine as well as preparation method and application thereof

Abstract

The invention discloses a nano vaccine as well as a preparation method and application thereof. The nano vaccine provided by the invention is prepared by fusing a liposome of which the surface is coupled with an inhibitor of an inhibitory immune checkpoint and a cell membrane of a tumor antigen sensitized dendritic cell cultured by utilizing a tumor cell lysate and keyhole limpet hemocyanin. The obtained nano vaccine is uniform in particle size, the outer layer of the nano vaccine is wrapped by a cell membrane, and the nano vaccine has proper surface potential, size and structural flexibility and is beneficial to aggregation in tumors. After the nano vaccine reaches tumor tissues, antigens can be efficiently and directly presented to T lymphocytes through cell membranes of tumor antigen sensitized dendritic cells, a costimulatory signal necessary for T cell activation is fully provided, meanwhile, T cell depletion can be reversed through an immune checkpoint inhibitor, the function of the inhibited T cells is re-excited, and the T cells are activated. The cytotoxic function and the multiplication capacity of the T cells are effectively and specifically recovered through a double-line strategy.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor immunotherapy, and more specifically, relates to a nano vaccine and a preparation method and application thereof. Background Art

[0002] Pancreatic cancer is a type of digestive system malignancy that occurs in the exocrine pancreatic glands and has an extremely poor prognosis. The five-year survival rate is only about 10%, and it is known as the "king of cancer." In recent years, immune checkpoints represented by programmed death receptor-1 (PD-1) and its ligands PD-L1 and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) have made breakthrough progress in the treatment of various tumors, making immunotherapy show broad prospects in the clinical treatment of tumors. However, limited by the immunosuppressive tumor microenvironment, the objective response rate (ORR) of immunotherapy or combined therapy in pancreatic cancer is not ideal. Among them, the immune microenvironment of pancreatic cancer is an important factor causing its insensitivity to immunotherapy and related poor prognosis.

[0003] T cell exhaustion is a state of T cell dysfunction that occurs during cancer, manifested by the persistent high expression of a series of inhibitory immune checkpoint molecules and progressive defects in effector function. For example, high expression of TIM-3 indicates that T cells are in a state of severe exhaustion. However, exhausted T cells (exhausted T cells, Tex) are not irreversible. The use of monoclonal antibodies to inhibit negative immune regulatory receptors can reverse the effector anergy of tumor infiltrating lymphocytes (Tumor Infiltrating Lymphocytes, TILs) to a certain extent and restore the proliferation and effector ability of TILs. At present, the most popular inhibitory target for clinical research to reverse Tex is the PD-1 / PD-L1 signaling pathway. Unfortunately, many tumor types do not respond to this type of treatment, or develop drug resistance after an initial response to treatment. For example, due to the low immunogenicity and lack of co-stimulatory signals in the pancreatic cancer immune microenvironment, anti-PD-1 antibodies cannot achieve the same therapeutic effect as other "hot" tumors. Clinical research data show that it is still difficult to reverse the phenomenon of T cell exhaustion in pancreatic cancer by immune checkpoint inhibition alone.

[0004] TIM-3 in various tumor CD8 + Sustained expression on TILs is associated with an exhausted state, which is also present in CD4 + TIM-3 is highly expressed on Treg cells. + CD8 +TILs are a group of T cells with the most severe functional disorders. As an emerging immune-oncology target, TIM-3 has a good translational prospect in combination therapy and is crucial in overcoming immunotherapy resistance. However, the common TIM-3 inhibitors are monoclonal antibodies. The abnormally dense extracellular matrix of pancreatic cancer hinders the infiltration of most drugs and immune cells, so that the monoclonal antibodies of TIM-3 cannot effectively reach the tumor site and obtain the local dose used.

[0005] Major histocompatibility complex class I molecules (MHC-I) play an important role in tumor immune surveillance and immunotherapy. Tumors may evade immune control by losing the MHC-I antigen presentation machinery (APM), which not only impairs the ability of natural immune responses to control tumors, but also hinders the ability to reactivate anti-tumor CD8 + Immunotherapy that works with T cells. Tumor cells in pancreatic cancer tissues express low levels of MHC-I, and their low immunogenicity results in insufficient presentation efficiency and co-stimulatory signals by antigen presenting cells (APCs), thereby weakening T cell activation.

[0006] Dendritic cells (DCs) are the strongest APCs in the body, and the immune response mediated by them is an important bridge between innate immunity and adaptive immunity. Given that DCs are the only innate immune cells that can activate resting T cells, DC cell therapy has become a powerful weapon against tumors. For example, the DC vaccine sensitized with tumor cell lysates prepared by researchers reduced the metastasis rate of liver cancer patients and prolonged their survival in clinical trials (Sun T, Yan W, Yang C, et al. Clinical research on dendritic cell vaccines to prevent postoperative recurrence and metastasis of liver cancer [J]. Genetics and Molecular Research, 2015, 14 (4): 16222-32.). However, liver cancer is an invasive "hot" tumor, and immune cells can enter liver cancer tissues. However, the extracellular matrix of pancreatic cancer is abnormally dense, and the cell-level size (~18 μm) is difficult to pass through the matrix barrier of pancreatic cancer, making the results of DC cell therapy in pancreatic cancer clinical trials unsatisfactory. Summary of the invention

[0007] The present invention provides a nano vaccine and a preparation method thereof to address the problems of low immunogenicity of pancreatic cancer, insufficient immune response caused by T cell exhaustion, and the effect of pancreatic cancer matrix barrier on drug delivery efficiency.

[0008] The first object of the present invention is to provide a nano vaccine.

[0009] The second object of the present invention is to provide a method for preparing the nanovaccine.

[0010] The third object of the present invention is to provide the use of the nanovaccine in the preparation of a preparation for improving the exhausted state of T cells in tumors.

[0011] The fourth object of the present invention is to provide the use of the nano vaccine in the preparation of cancer immunotherapy drugs.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The present invention is to prepare a nano vaccine with uniform particle size and outer layer wrapped with cell membrane, and excellent targeting and antigen presentation by mixing liposomes with inhibitors of inhibitory immune checkpoints on the surface (coupled by pH-sensitive chemical bonds) with cell membranes of tumor antigen-sensitized dendritic cells obtained by culturing tumor cell lysates and keyhole limpet hemocyanin through FNC technology. The nano vaccine prepared by the liposomes with TIM-3 monoclonal antibodies (aTIM-3 antibodies) coupled to the surface and cell membranes of tumor antigen-sensitized dendritic cells can target T cells with high expression of TIM-3, and the pMHC and B7-1 / 2 molecules on the surface of the nano vaccine are respectively combined with TCR-CD3 and CD28 molecules on the surface of the T cells to directly present tumor antigens; at the same time, based on the slightly acidic conditions of the tumor, the pH-sensitive chemical bonds are broken to release aTIM-3 antibodies, which reverses T cell exhaustion from two aspects and restores the cytotoxicity and proliferation ability of exhausted T cells.

[0014] That is, the present invention provides a strategy for reversing T cell exhaustion through aTIM-3 antibodies and enhancing the activity and proliferation of in situ T cells through the cell membrane of tumor antigen-sensitized dendritic cells, thereby fundamentally changing the state of T cell response anergy in pancreatic cancer. When the nano vaccine of the present invention enters the blood circulation, its suitable surface potential, size and structural flexibility help liposomes to aggregate in the tumor. After reaching the tumor tissue, on the one hand, the cell membrane of tumor antigen-sensitized dendritic cells efficiently and directly presents antigens to T lymphocytes, fully providing the necessary costimulatory signals for T cell activation; on the other hand, immune checkpoint inhibitors (aTIM-3 antibodies) are introduced into the nano vaccine through pH-sensitive chemical bonds, and the release on demand reverses T cell exhaustion and re-stimulates the suppressed T cell function. This two-pronged strategy can effectively and specifically restore the cytotoxic function and proliferation ability of in situ T cells, fundamentally solving the problem of small number and poor quality of pancreatic cancer T cells. Therefore, the present invention requests protection of the nano vaccine and its preparation method.

[0015] The method for preparing the nano vaccine of the present invention comprises the following steps:

[0016] S1. preparing liposomes loaded with inhibitors of inhibitory immune checkpoints;

[0017] S2. Tumor antigen-sensitized dendritic cells were obtained by culturing tumor cell lysate and keyhole limpet hemocyanin;

[0018] S3. The liposomes obtained in S1 and the cell membranes of the dendritic cells obtained in S2 are mixed by FNC technology, and the obtained mixture is then extruded by an extruder to obtain the nanovaccine.

[0019] Specifically, the inhibitor described in S1 is an inhibitor of the inhibitory immune checkpoint TIM-3.

[0020] More specifically, the TIM-3 inhibitor is an anti-TIM-3 monoclonal antibody.

[0021] Specifically, the inhibitor described in S1 is coupled to the liposome surface via a pH-sensitive chemical bond.

[0022] Optionally, the pH sensitive chemical bond is an imide bond, an acetal bond or a benzoic acid imide bond.

[0023] In a specific embodiment of the present invention, the pH sensitive chemical bond is an imide bond.

[0024] Optionally, the imide bond is obtained by a ring-opening reaction of distearoylphosphatidylethanolamine-polyethylene glycol-amino and 2,3-dimethylmaleic anhydride.

[0025] When using tumor cell lysate to cultivate tumor antigen-sensitized dendritic cells, which tumor cell lysate to use is selected according to the tumor to be treated. As in a specific embodiment of the present invention, the present invention utilizes the lysate of pancreatic cancer cells (Panc02-OVA) to cultivate tumor antigen-sensitized dendritic cells, and then utilizes the cell membrane of dendritic cells to prepare a nano vaccine that can improve the T cell exhaustion state in pancreatic cancer.

[0026] Specifically, when tumor cell lysate and keyhole limpet hemocyanin are used to culture tumor antigen-sensitized dendritic cells in S2, the final concentration of the tumor cell lysate in the liquid culture medium used is 50-200 μg / mL, and the final concentration of keyhole limpet hemocyanin is 20-50 μg / mL.

[0027] More specifically, the final concentration of the tumor cell lysate is 200 μg / mL, and the final concentration of the tumor cell lysate is 50 μg / mL.

[0028] Alternatively, the cell membrane described in S3 is obtained by hypotonic lysis of cultured dendritic cells followed by differential centrifugation.

[0029] Specifically, the liposomes described in S3 are mixed with cell membranes by FNC technology at a mass ratio of lipid to membrane protein of 2.5 to 1.5:1.

[0030] Specifically, the mixture obtained by mixing by FNC technology was coextruded through polycarbonate films of 400 nm, 200 nm and 100 nm in sequence.

[0031] Specifically, each polycarbonate film was extruded 14 to 16 times.

[0032] More specifically, each polycarbonate film was extruded 15 times.

[0033] Specifically, when mixed by FNC technology, the flow rate of the liposome and cell membrane mixture is 2 to 3 mL / min, and the flow rate of the buffer solution is 8 to 10 mL / min.

[0034] Optionally, the buffer is phosphate buffer, purified water or physiological saline.

[0035] In a specific embodiment of the present invention, the buffer is a phosphate buffer.

[0036] The present invention also claims protection for the nano vaccine prepared by the above method.

[0037] The present invention also claims to protect the use of the nano vaccine in preparing a preparation for improving the exhausted state of T cells in tumors.

[0038] The present invention also claims to protect the use of the nano vaccine in preparing cancer immunotherapy drugs.

[0039] Specifically, the tumor / cancer is pancreatic cancer.

[0040] The present invention has the following beneficial effects:

[0041] The present invention prepares a nano vaccine with uniform particle size and cell membrane wrapped in the outer layer, which has excellent targeting and antigen presentation effects, by mixing liposomes with inhibitors of inhibitory immune checkpoints on the surface (coupled by pH-sensitive chemical bonds) with cell membranes of tumor antigen-sensitized dendritic cells obtained by culturing tumor cell lysates and keyhole limpet hemocyanin through FNC technology and then extruding through an extruder. The nano vaccine of the present invention has a suitable surface potential, size and structural flexibility, which helps it to aggregate in the tumor. After reaching the tumor tissue, it can efficiently and directly present antigens to T lymphocytes through the cell membranes of tumor antigen-sensitized dendritic cells, fully providing the necessary costimulatory signals for T cell activation, and can reverse T cell exhaustion through immune checkpoint inhibitors, re-stimulate the suppressed T cell function, and effectively and specifically restore the cytotoxic function and proliferation ability of in situ T cells through a two-pronged strategy.

[0042] The invention is beneficial to the immunotherapy of tumors and the development of related immunotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the H NMR spectrum of the pH-sensitive DSPE-PEG-CDM prepared.

[0044] Figure 2 These are the electron microscopy observation results of the prepared nanovaccine MLP-aTIM-3.

[0045] Figure 3 The electron microscopy observation results of the nanovaccine prepared using only the extrusion method.

[0046] Figure 4 To investigate the antibody release of the prepared nanovaccine MLP-aTIM-3 under different pH conditions.

[0047] Figure 5 These are the SDS-PAGE test results of DC cell membrane extracts, cell membrane liposomes MLP and nanovaccine MLP-aTIM-3.

[0048] Figure 6 After culturing immature DC cells with different stimuli, the maturation of DC cells was detected by multi-color flow cytometry.

[0049] Figure 7 The results show the detection results of TIM-3 expression levels in peripheral blood and tumor T cells of pancreatic cancer-bearing mice.

[0050] Figure 8 These are the test results of the nanovaccine MLP-aTIM-3 targeting tumor-exhausted T cells.

[0051] Fig. 9 This is the result of detecting the expression level of IFN-γ in exhausted T cells.

[0052] Fig.10 The results of the proliferation assay of exhausted T cells. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0054] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0055] Example 1 Preparation of Nano-Vaccine

[0056] 1. Synthesis of pH-sensitive lipid material DSPE-PEG-CDM

[0057] The nano vaccine described in this embodiment is prepared by coupling liposomes to aTIM-3 antibody (anti-TIM-3 monoclonal antibody, clone number RMT3-23, manufacturer Bioxcell, catalog number BE0115) and cell membranes of dendritic cells sensitized with pancreatic cancer antigens. The aTIM-3 antibody is coupled to the liposome via a pH-sensitive chemical bond (imide bond), which is obtained by a ring-opening reaction of DSPE-PEG-NH2 (distearoylphosphatidylethanolamine-polyethylene glycol-amino) and CDM (2,3-dimethylmaleic anhydride).

[0058] The preparation method of pH-sensitive lipid material DSPE-PEG-CDM is as follows:

[0059] 1.35 g of DSPE-PEG-NH2 was dissolved in 10 mL of freshly distilled chloroform and 0.25 g of CDM was added. The mixture was stirred at room temperature for 48 h under nitrogen protection. The resulting solution was dialyzed in methanol for 24 h using a dialysis bag with a molecular cutoff of 5000 Da, and then precipitated in ten times the volume of ether. The precipitate was collected by centrifugation, washed with ether and vacuum dried. The precipitate obtained by vacuum drying was analyzed by nuclear magnetic resonance. The results are as follows: Figure 1 As shown. Figure 1 As shown in the H NMR spectrum, the pH-sensitive DSPE-PEG-CDM was successfully prepared in the present invention.

[0060] The synthetic route of the DSPE-PEG-CDM is as follows:

[0061]

[0062] 2. Preparation of liposomes by thin film hydration method and coupling with aTIM-3 antibody

[0063] In this embodiment, liposomes were synthesized by a thin film hydration method, and the preparation method was as follows: 9 mg HSPC (hydrogenated soybean phosphatidylcholine), 3.88 mg PE (phosphatidylethanolamine), 2.18 mg CHO (cholesterol) and 3.15 mg of the prepared DSPE-PEG-CDM were added to 10 mL of chloroform, and the organic solvent was removed by rotary evaporation at 40° C., so that the liposome membrane material formed a thin film at the bottom of the eggplant-shaped bottle; the remaining chloroform was placed under vacuum until it was completely removed, 10 mL of PBS buffer was added to the bottle, and then ultrasonicated with an ultrasonic probe for 10 minutes to obtain liposomes.

[0064] The prepared liposomes were dispersed in PBS buffer, and NHS (N-hydroxysuccinimide) and EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) were added in a molar amount of 1.5 times that of DSPE-PEG-CDM in the obtained liposomes, respectively. After activating the carboxyl group for 4 hours, aTIM-3 antibody was added in a molar amount of 0.2 times that of DSPE-PEG-CDM in the obtained liposomes, and the mixture was incubated overnight at 4°C. The mixture was washed with PBS buffer and concentrated by ultrafiltration for later use to obtain aTIM-3-coupled liposomes, referred to as LP-aTIM-3.

[0065] 3. Extract and activate dendritic cells (DC)

[0066] Mouse bone marrow was extracted and dissociated into monodispersed cells, and stem cells were separated and cultured with complete cell culture medium containing 20 ng / mL GM-CSF (granulocyte-macrophage colony-stimulating factor) and 10 ng / mL IL-4 cytokines to promote stem cell differentiation into DC cells; on the fifth day, some cells were taken for purity identification (cells were labeled with anti-CD11c antibody and CD11c expression was detected by flow cytometry); immature DC cells (iDCs) were cultured with complete cell culture medium containing GM-CSF and IL-4 cytokines, and lysate of mouse pancreatic cancer cells (Panc02-OVA) overexpressing egg white protein (final concentration of 200 μg / mL) and 50 μg / mL KLH (keyhole limpet hemocyanin) were added to incubate iDCs cells for 48 hours. After the incubation, the expression of MHC, CD80 and CD86 on the surface of DC cells was detected by multicolor flow cytometry to detect the maturation of DC cells, and mature DC cells (mDCs) were taken for subsequent experiments.

[0067] 4. Extraction of mDCs cell membrane and fusion with liposomes

[0068] The mDCs cell membrane was extracted by hypotonic lysis followed by differential centrifugation. The mDCs incubated with tumor cell lysate and KLH were lysed in a hypotonic buffer (0.25×PBS) at 4°C for 30 minutes, and then centrifuged at 15,000×g for 30 minutes to collect the cell membranes. The protein concentration was determined by the BCA method. The extracted cell membranes (i.e., the cell membranes of tumor antigen-sensitized dendritic cells) were mixed with the pre-prepared liposome LP-aTIM-3 in an aqueous solution at a mass ratio of lipid to membrane protein of 2:1, and then the membranes were centrifuged by Flash. NanoComplexation (FNC) technology was used for mixing. The solution entered through pipes 1 and 2 at a flow rate of 2.5 mL / min, and the buffer (phosphate buffer) entered through pipes 3 and 4 at a flow rate of 10 mL / min. After FNC treatment, the mixture was co-extruded through 400 nm, 200 nm and 100 nm polycarbonate membranes in sequence using an extruder (each membrane was extruded 15 times), and the cell membrane was embedded in a lipid bilayer with good fluidity, thereby obtaining a nanovaccine with uniform particle size and complete coating, which is called nanovaccine MLP-aTIM-3.

[0069] 5. Electron microscopic observation of nanovaccine MLP-aTIM-3

[0070] The electron microscopic observation results of the nanovaccine MLP-aTIM-3 prepared in this example are as follows Figure 2 As shown. Figure 2 It can be seen that the particle size of the prepared nano vaccine is about 110nm, and it can be seen that its outer layer has a cell membrane coating with a thickness of about 10.7nm. If the cell membrane is mixed with the liposome MLP-aTIM-3, and the mixture is co-extruded through 400nm, 200nm and 100nm polycarbonate membranes in sequence (each membrane is extruded 15 times), the electron microscopy observation results of the prepared nano vaccine are as follows Figure 3 As shown. Figure 3 It can be seen that the fusion rate between cell membrane and liposome is low and uncontrollable.

[0071] 6. Antibody release and potential detection of nanovaccine MLP-aTIM-3

[0072] The prepared nanovaccine MLP-aTIM-3 was used to test its antibody release under different pH conditions (pH 7.4 / pH 6.5), and its potential under different pH conditions was also tested.

[0073] The process is as follows: take 5 mL of the quantified MLP-aTIM-3 solution into a dialysis bag, and then soak it in 50 mL of PBS buffer containing different pH values; stir slowly in a constant temperature shaker at 37°C, take 1 mL of buffer every 10 hours, detect the aTIM-3 antibody in the buffer by enzyme-linked immunosorbent assay, and calculate the cumulative release percentage of the antibody at the specific time point.

[0074] Antibody release of nanovaccine MLP-aTIM-3 under different pH conditions Figure 4 As shown. Figure 4 It can be seen that the nano vaccine MLP-aTIM-3 prepared by the present invention has a higher release rate under slightly acidic conditions (pH 6.5), indicating that it can adapt to the slightly acidic environment of the tumor, release a large amount of aTIM-3 antibodies in the tumor microenvironment and function. In addition, from the potential test results, it can be seen that the potential of MLP-aTIM-3 under neutral conditions (pH 7.4) is -9.4mV, and the potential under slightly acidic conditions (pH 6.5) is +6.3mV. The potential between -10mV and +10mV is suitable for intravenous injection.

[0075] 7. SDS-PAGE detection

[0076] Referring to the same method as above, the present invention also prepares cell membrane liposome MLP, which differs from the nano vaccine MLP-aTIM-3 in that it is not coupled with aTIM-3, and the coupling of aTIM-3 is detected by SDS-PAGE.

[0077] DC cell membrane extracts, cell membrane liposomes MLP and nanovaccine MLP-aTIM-3 with the same protein content were mixed with protein loading buffer at a volume ratio of 5:1, and denatured in a 95°C water bath for 5 min; the above samples containing 10 μg of protein were added to the polyacrylamide gel (SDS-PAGE) loading wells and run at 90 mV for 3 h; the SDS-PAGE gel was incubated with Coomassie brilliant blue dye for 2 h, placed on a horizontal shaker and washed with decolorizing solution until the protein bands were clear, and photographed and recorded using a digital camera.

[0078] The SDS-PAGE results of DC cell membrane extracts, cell membrane liposomes MLP and nanovaccine MLP-aTIM-3 are shown in Figure 5 As shown. Figure 5It can be seen that the nano vaccine MLP-aTIM-3 has a similar band composition to the DC cell membrane extract and the cell membrane liposome MLP. In addition, two bands can be found in the nano vaccine MLP-aTIM-3, with sizes between 40 and 55 kDa and 15 and 25 kDa, respectively, which are consistent with the size of the heavy chain and light chain that constitute the aTIM-3 antibody. The above results show that the DC cell membrane extracted by the present invention is successfully embedded in the liposome, and the aTIM-3 antibody is successfully coupled to the liposome.

[0079] 8. Others

[0080] In the process of preparing the nano vaccine, the present invention found that the effect of using tumor cell lysate to stimulate DC cell maturation was not good, so it was considered to add new stimulants to promote DC cell maturation. It was found that the lysate of mouse pancreatic cancer cell Panc02-OVA and KLH combined to stimulate DC cells can significantly increase the DC cell maturation rate.

[0081] iDCs were seeded in a 24-well plate at a density of 2×106 cells per well, and equal amounts of mouse pancreatic cancer cell Panc02-OVA lysate (Lysate), KLH, and a complex of Lysate and KLH were added and cultured for 2 days. iDCs treated with equal amounts of PBS were used as blank controls. After the culture, the cells were washed twice with PBS, collected in a centrifuge tube, and fluorescently conjugated antibodies CD80 and CD86 were added to stain the cell surface (protected from light, 4°C, 30 min), followed by centrifugation to remove excess fluorescently conjugated antibodies, resuspended with 400 μL flow cytometry buffer, and the expression levels of CD80 and CD86 on the DC cell surface were detected using a multicolor flow cytometry method.

[0082] After culturing immature DC cells with different stimuli, the maturation of DC cells was detected by multicolor flow cytometry. Figure 6 As shown. Figure 6 It can be seen that the use of tumor cell lysate or KLH alone cannot effectively promote DC cell maturation, while the combination of tumor cell lysate and KLH has a synergistic effect, which can effectively promote DC cell maturation and significantly improve the DC cell maturation rate, making the maturation rate as high as more than 91%.

[0083] Example 2 TIM-3 expression level in pancreatic cancer-bearing mice and detection of targeted nanovaccine

[0084] 1. Detection of TIM-3 expression levels in peripheral blood and tumor T cells of pancreatic cancer-bearing mice

[0085] Six-week-old female C57BL / 6J mice were anesthetized and the abdominal area was depilated. 20 μL of 1×10 5The matrix gel of pancreatic cancer cells was injected into the pancreatic tail of mice to establish an orthotopic pancreatic cancer model (pancreatic cancer-bearing mice). Peripheral blood and tumor tissues were obtained from pancreatic cancer-bearing mice; the peripheral blood was diluted with the same volume of PBS, separated using Ficoll separation solution, centrifuged at 400g for 30 minutes, and the peripheral blood mononuclear cell layer was aspirated into a new centrifuge tube and washed twice with PBS; the tumor tissue was cut into small pieces with a scalpel and ophthalmic shears, resuspended with PBS and filtered with a 70μm cell sieve to obtain a single cell suspension of tumor tissue; the single cell suspension separated from peripheral blood and tumor tissue was incubated with fluorescent-conjugated antibodies CD3, CD8, and TIM-3 antibodies at 4°C in the dark for 30 minutes, washed and resuspended, and detected using a flow cytometer.

[0086] The results of the detection of TIM-3 expression levels in peripheral blood and tumor T cells of pancreatic cancer-bearing mice are as follows Figure 7 As shown. Figure 7 It can be seen that tumor infiltrates CD3 + CD8 + T cells highly express the immune checkpoint TIM-3, while peripheral blood T cells hardly express TIM-3, indicating that TIM-3 is a potential T cell target for pancreatic cancer.

[0087] 2. Detection of the targeting effect of nanovaccine MLP-aTIM-3 on tumor-exhausted T cells

[0088] Based on the above results, the present invention fluorescently labeled the prepared nanovaccine MLP-aTIM-3 (purple), and used the fluorescently labeled nanovaccine MLP-aTIM-3 to detect the targeting effect of the nanovaccine on tumor-depleted T cells.

[0089] 100 μL of fluorescently labeled (purple) nanovaccine MLP-aTIM-3 was injected into pancreatic cancer-bearing mice via the tail vein. After 2 hours, the mice were euthanized, and the pancreatic cancer tissue of the mice was removed. It was cut into small pieces with a scalpel and ophthalmic shears and resuspended with PBS. After resuspension, it was filtered with a 70 μm cell sieve to obtain a single cell suspension of pancreatic cancer tumor tissue in the mice. The single cell suspension was stained with 0.5 μM Calcein-AM for 15 minutes (green, indicating living cells), and then stained with fluorescent conjugated antibodies such as anti-CD8 antibody (blue) and anti-TIM-3 antibody (red) at room temperature for 1 hour; the excess dye was removed by centrifugation, and 10 μL of the cell suspension was placed on a slide and observed using a laser confocal microscope.

[0090] The results of the nanovaccine MLP-aTIM-3 targeting tumor-depleting T cells are shown in Figure 8 As shown. Figure 8 It can be seen that CD8 +The T cells have nanovaccine MLP-aTIM-3 on their surface, while CD8 + The nanovaccine MLP-aTIM-3 was not detected on the surface of T cells. This indicates that the nanovaccine MLP-aTIM-3 prepared by the present invention can specifically target tumor CD8 + TIM-3 + T cells.

[0091] Example 3 Effect of Nanovaccine MLP-aTIM-3 on T Cell Exhaustion

[0092] The present invention detects the effect of the prepared nano vaccine MLP-aTIM-3 on T cell exhaustion by inducing exhausted T cells.

[0093] 1. Detection of IFN-γ expression level in exhausted T cells

[0094] The spleen cells of OT-I transgenic mice were inoculated in a 24-well plate at a density of 2×106 cells per well and 1 mL of complete medium containing 100 U / mL recombinant IL-2 factor was added. The cells were induced and cultured with 50 ng / mL phorbol ester and 1 μg / mL ionomycin for 12 days to induce exhausted T cells. The induced exhausted T cells were incubated with TIM-3-coupled liposomes LP-aTIM-3, cell membrane fusion liposomes MLP and nanovaccine MLP-aTIM-3 for 48 hours, respectively, and PBS was used as a blank control. Before flow cytometry analysis, the cells were first incubated with 10 μM protein transport inhibitor Brefeldin A for 4 hours, and then fluorescent-coupled antibodies anti-CD3 antibody and anti-CD8 antibody were added and incubated at 4°C in the dark for 30 minutes. After washing, the cells were fixed and permeated, and incubated with anti-IFN-γ antibody at room temperature for 30 minutes. After washing and resuspending with PBS, the cells were detected by flow cytometry.

[0095] The results of IFN-γ expression level detection in exhausted T cells are as follows Fig. 9 As shown. Fig. 9 It can be seen that the nanovaccine MLP-aTIM-3 prepared by the present invention can promote the secretion of proinflammatory cytokine IFN-γ by exhausted T cells, indicating that the effector ability of exhausted T cells is enhanced.

[0096] 2. Proliferation detection of exhausted T cells

[0097] The spleen cells of OT-I transgenic mice were labeled with 0.5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE), seeded in a 24-well plate at a density of 2×106 cells per well, and 1 mL of complete culture medium containing 100 U / mL recombinant IL-2 factor was added and induced with 50 ng / mL phorbol ester and 1 μg / mL ionomycin for 12 days to induce exhausted T cells; they were incubated with LP-aTIM-3, MLP, and MLP-aTIM-3 for 5 days, respectively, and PBS was used as a blank control; CFSE-labeled T cells were collected by centrifugation, incubated with fluorescent-conjugated antibodies, anti-CD3 antibodies, and anti-CD8 antibodies at 4°C in the dark for 30 min, washed and resuspended, and detected by flow cytometry.

[0098] The results of the proliferation assay of exhausted T cells are as follows Fig.10 As shown. Fig.10 It can be seen that the CFSE fluorescence signal of 75.57% T cells in the MLP-aTIM-3 group was weakened, which was caused by the division and proliferation of T cells. That is, the nanovaccine prepared by the present invention can promote the proliferation of exhausted T cells and increase the number of T cells in tumors.

[0099] The above results indicate that the nanovaccine MLP-aTIM-3 of the present invention can promote the secretion of proinflammatory cytokine IFN-γ and proliferation of exhausted T cells, which is beneficial to the immunotherapy of pancreatic cancer.

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a nano vaccine, characterized in that: The following steps are involved: S1. preparing liposomes loaded with inhibitors of inhibitory immune checkpoints; S2. Tumor antigen-sensitized dendritic cells were obtained by culturing tumor cell lysate and keyhole limpet hemocyanin; S3. The liposomes obtained in S1 and the cell membranes of the dendritic cells obtained in S2 are mixed by FNC technology, and the obtained mixture is then extruded by an extruder to obtain the nanovaccine.

2. The preparation method according to claim 1, characterized in that: The inhibitor described in S1 is an inhibitor of the inhibitory immune checkpoint TIM-3.

3. The preparation method according to claim 1, characterized in that: In the liquid culture medium of the culture in S2, the final concentration of the tumor cell lysate is 50 to 200 μg / mL, and the final concentration of keyhole limpet hemocyanin is 20 to 50 μg / mL.

4. The preparation method according to claim 1, characterized in that: The liposomes and cell membranes described in S3 were mixed by FNC technology at a mass ratio of lipid to membrane protein of 2.5 to 1.5:

1.

5. The preparation method according to claim 4, characterized in that: The mixture obtained by mixing by FNC technology was coextruded through polycarbonate films of 400 nm, 200 nm and 100 nm in sequence.

6. The preparation method according to claim 4, characterized in that: When mixed by FNC technology, the flow rate of the liposome and cell membrane mixture is 2 to 3 mL / min, and the flow rate of the buffer is 8 to 10 mL / min.

7. The preparation method according to claim 6, characterized in that: The buffer is phosphate buffer, purified water or physiological saline.

8. The nano vaccine prepared by the method according to any one of claims 1 to 7.

9. Use of the nanovaccine according to claim 8 in the preparation of a preparation for improving the exhausted state of T cells in tumors.

10. Use of the nano vaccine according to claim 8 in the preparation of cancer immunotherapy drugs.

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