Anti-pancreatic cancer dendritic cell vaccine as well as preparation method and application thereof

Abstract

The invention provides an anti-pancreatic cancer dendritic cell vaccine as well as a preparation method and application thereof, and belongs to the technical field of tumor vaccines. The anti-pancreatic cancer dendritic cell vaccine comprises dendritic cells loaded with at least one of the following antigens: induced pluripotent stem cell lysate and induced pluripotent stem cell exosome. The vaccine can obviously stimulate proliferation of T cells and can specifically induce proliferation of CD8 + T lymphocytes, so that the vaccine has obvious killing activity on three pancreatic cancer tumor cell lines. Meanwhile, the vaccine shows a good anti-pancreatic cancer effect in preventive inoculation in a human immune reconstruction mouse pancreatic cancer tumor model.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor vaccines, and in particular relates to an anti-pancreatic cancer dendritic cell vaccine and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy is considered to be the fourth tumor treatment method after surgery, radiotherapy and chemotherapy. In 2013, Science magazine named it one of the most valuable scientific breakthroughs of the year. The strategies of tumor immunotherapy can be divided into four categories: adoptive immune cell therapy, immune checkpoint monoclonal antibody therapy, tumor vaccine therapy and nonspecific immune stimulation therapy (cytokine therapy). Tumor vaccines, as the name suggests, are vaccines that can induce immunity to specific tumors. Tumor vaccines are usually tumor cells or fragments or fragments containing tumor-specific antigens (TSA) or tumor-associated antigens (TAA). After entering the human body, these cell fragments can activate the body's own immunity, induce specific immune responses, overcome the immunosuppressive state, and increase resistance to specific tumors. It is an active immunotherapy method. According to their different uses, they are generally divided into two categories: one is preventive tumor vaccines, and vaccination with such vaccines can reduce or eliminate the chance of tumor occurrence, such as the famous HPV vaccine; the other is therapeutic tumor vaccines, which are mainly based on tumor antigens and are used for adjuvant therapy after chemotherapy or surgical resection, such as DC vaccines (such as Provenge <sipuleucel-t>).

[0003] Dendritic cells (DC cells) are the most powerful professional antigen-presenting cells (APCs) known in the body and are also the "starters" of the body's immune response. Only DC cells can activate naive T cells, and even a small number can trigger a strong T cell response. Compared with other antigen-presenting cells, DC cells have a stronger presentation ability. Therefore, dendritic cell vaccines have become an important means of tumor immune cell therapy. Although multiple clinical trials have confirmed the safety and immunogenicity of dendritic cell vaccines, the clinical response effect is still not ideal. The main reasons for this result are insufficient dendritic cell antigen presentation, low migration ability, and insufficient immune activation to counteract the immunosuppressive environment in the body.

[0004] Pancreatic cancer is a highly malignant tumor with the characteristics of dangerous onset and strong invasiveness. It can be treated surgically in the early stage of the disease, but requires systematic chemotherapy in the middle and late stages. Currently, there are not many drugs related to targeted therapy. There are no reports on anti-pancreatic cancer dendritic cell vaccines. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an anti-pancreatic cancer dendritic cell vaccine that can stimulate CD8 + It promotes T cell proliferation, has a strong killing effect on a variety of pancreatic cancer cells, and has an anti-pancreatic cancer effect in vivo.

[0006] The present invention provides an anti-pancreatic cancer dendritic cell vaccine, comprising dendritic cells loaded with at least one of the following antigens: induced pluripotent stem cell lysate and induced pluripotent stem cell exosomes.

[0007] Preferably, the mass ratio of the dendritic cells to the antigen is 1×10 6 : 15~25μg.

[0008] Preferably, the mass ratio of the dendritic cells to the antigen is 1×10 6 : 20μg.

[0009] Preferably, the method for preparing the induced pluripotent stem cell lysate comprises repeatedly freezing and thawing induced pluripotent stem cells in the logarithmic growth phase, collecting the freeze-thaw solution, removing cell debris and impurity particles, and obtaining the induced pluripotent stem cell lysate.

[0010] Preferably, the method for preparing induced pluripotent stem cell exosomes comprises culturing induced pluripotent stem cells to a logarithmic growth phase, collecting the culture supernatant, removing cells, cell debris, impurity particles and water, and obtaining induced pluripotent stem cell exosomes.

[0011] The present invention provides a method for preparing the anti-pancreatic cancer dendritic cell vaccine, comprising the following steps:

[0012] CD34 + Hematopoietic stem cells were induced and cultured twice to obtain immature dendritic cells;

[0013] The immature dendritic cells and antigens are inoculated into a third induction culture medium for induction culture to obtain an anti-pancreatic cancer dendritic cell vaccine.

[0014] Preferably, the concentration of the antigen is 10-160 μg / mL.

[0015] Preferably, the concentration of the antigen is 15-25 μg / mL.

[0016] Preferably, the induction culture time is 2 to 3 days.

[0017] The present invention provides the anti-pancreatic cancer dendritic cell vaccine or the use of the anti-pancreatic cancer dendritic cell vaccine obtained by the preparation method in preparing a drug for preventing and / or treating pancreatic cancer.

[0018] The present invention provides an anti-pancreatic cancer dendritic cell vaccine, comprising dendritic cells loaded with at least one of the following antigens: induced pluripotent stem cell lysate and induced pluripotent stem cell exosomes. Under in vitro conditions, the anti-pancreatic cancer dendritic cell vaccine can promote the proliferation of T lymphocytes and can effectively stimulate the proliferation of CD8+T cells. The results of the pancreatic cancer cell killing experiment under in vitro conditions showed that the cytotoxic T cells activated by the anti-pancreatic cancer dendritic cell vaccine had good killing activity against three types of pancreatic cancer cells, and the killing activity was dependent on the effect-target ratio. In addition, the preventive vaccination experiment in the pancreatic cancer tumor model of human immune reconstructed mice evaluated the tumor volume and tumor mass respectively, and found that the anti-pancreatic cancer dendritic cell vaccine can effectively inhibit the growth of pancreatic cancer tumors after administration. It can be seen that the anti-pancreatic cancer dendritic cell vaccine provided by the present invention has a good effect of inhibiting the growth of pancreatic cancer, providing a new means for the clinical treatment and drug development of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 DC cells loaded with different antigens stimulate the proliferation activity of T lymphocytes;

[0020] Figure 2 Inducing T cell subsets CD8 by DC cells loaded with different antigens + With CD4 + Proportional changes;

[0021] Figure 3 The killing activity of CTL activated by DC cells loaded with different concentrations of iPSCs Lysate and PANC-1 Lysate antigens on PANC-1 pancreatic cancer cells; A is the killing activity of CTL activated by DC cells loaded with different concentrations of iPSCs Lysate antigens on PANC-1 pancreatic cancer cells, and B is the killing activity of CTL activated by DC cells loaded with different concentrations of PANC-1 Lysate antigens on PANC-1 pancreatic cancer cells;

[0022] Figure 4 The figures show the killing activity of T cells activated by DC cells loaded with different antigens against pancreatic cancer cell lines; A is the result of PANC-1, B is the result of BXPC-3, and C is the result of PaCa-2;

[0023] Figure 5 This is the animal weight growth curve;

[0024] Figure 6 is the tumor growth curve;

[0025] Figure 7 The results are compared based on tumor weight. DETAILED DESCRIPTION

[0026] The present invention provides an anti-pancreatic cancer dendritic cell vaccine, comprising dendritic cells loaded with at least one of the following antigens: induced pluripotent stem cell lysate and induced pluripotent stem cell exosomes.

[0027] In the present invention, the method for preparing the induced pluripotent stem cell lysate preferably freezes and thaws the induced pluripotent stem cells in the logarithmic growth phase repeatedly, collects the freeze-thaw solution, removes cell debris and impurity particles, and obtains the induced pluripotent stem cell lysate. The culture medium of the induced pluripotent stem cells is preferably a complete stem cell culture medium. The repeated freezing and thawing method suspends the induced pluripotent stem cells in PBS and alternately treats them at -20 to -80°C and 36 to 38°C. The treatment time is preferably 4 to 6 minutes, more preferably 5 minutes. The number of repeated freezing and thawing is preferably 4 to 6 times, more preferably 5 times. The method for removing cell debris is preferably centrifugation. The speed of the centrifugation is preferably 2000g. The time of the centrifugation is preferably 10 minutes. The method for removing impurity particles is preferably carried out by membrane filtration. The pore size of the filter membrane is preferably 0.22μm. After the induced pluripotent stem cell lysate is prepared, the protein concentration is preferably measured for quantitative addition.

[0028] In the present invention, the method for preparing induced pluripotent stem cell exosomes preferably cultured induced pluripotent stem cells to the logarithmic growth phase, collected the culture supernatant, removed cells, cell debris, impurity particles and water, and obtained induced pluripotent stem cell exosomes. The method for removing cells is preferably centrifugation at 300×g for 10 min. The method for removing cell debris is preferably centrifugation at 2000×g for 10 min. The method for removing impurity particles is preferably membrane filtration. The pore size of the filter membrane is preferably 0.22 μm, which is conducive to removing impurity particles larger than 220 nm. The method for removing water is preferably ultracentrifugation at 100000×g for 90 min, and the solid phase is collected.

[0029] In the present invention, the mass ratio of the number of dendritic cells to the antigen is preferably 1×10 6 : 15 to 25 μg, more preferably 1×10 6 : 20 μg. The method of loading the antigen on the dendritic cells is preferably that the dendritic cells use the antigen phagocytic activity to achieve the loading of the antigen.

[0030] The present invention provides a method for preparing the anti-pancreatic cancer dendritic cell vaccine, comprising the following steps:

[0031] CD34 + Hematopoietic stem cells were induced and cultured twice to obtain immature dendritic cells;

[0032] The immature dendritic cells and antigens are inoculated into a third induction culture medium for induction culture to obtain an anti-pancreatic cancer dendritic cell vaccine.

[0033] In the present invention, the CD34 + The hematopoietic stem cells are preferably isolated from mononuclear cells of umbilical cord blood. Before being cultured, they are preferably subjected to expansion culture. The expansion culture medium is preferably based on a serum-free medium and further comprises the following components: 50-100 ng / mL granulocyte-macrophage colony stimulating factor (GM-CSF), 10-50 ng / mL stem cell growth factor (SCF), 50-100 ng / mL FMS-like tyrosine kinase 3 (FLT-3), 50-100 ng / mL thrombopoietin (TPO), 10-50 ng / mL IFNα, 10-50 ng / mL IL-7, 1-5 μm aromatic hydrocarbon receptor inhibitor SR1 and 1-10 μg / mL heparin, more preferably 60-80 ng / mL granulocyte-macrophage colony stimulating factor, 20-40 ng / mL stem cell growth factor, 60-80 ng / mL FMS-like tyrosine kinase 3, 60-90 ng / mL thrombopoietin, 20-40 ng / mL IFNα, 20-40 ng / mL IL-7, 2-4 μm aryl hydrocarbon receptor inhibitor SR1 and 3-7 μg / mL heparin, most preferably 75 ng / mL granulocyte-macrophage colony stimulating factor, 30 ng / mL stem cell growth factor, 75 ng / mL FMS-like tyrosine kinase 3, 75 ng / mL thrombopoietin, 30 ng / mL IFNα, 30 ng / mL IL-7, 5 μm aryl hydrocarbon receptor inhibitor SR1 and 5 μg / mL heparin. The expansion culture time is preferably 7-10 days, with shaking every 12 hours. The CD34 + The initial culture density of hematopoietic stem cells is preferably (1-3)×10 5 / ml, more preferably 2×10 5 / ml. + Hematopoietic stem cells are expanded and cultured, with the expansion multiples ranging from 30 to 100 times.

[0034] In the present invention, the two induction cultures are preferably carried out using a first induction culture medium and a second induction culture medium in sequence. The first induction culture medium is based on a serum-free culture medium;

[0035] The third induction culture medium is based on a serum-free culture medium and further comprises the following components: 10-50 ng / mL tumor necrosis factor α and 100-500 ng / mL lipopolysaccharide.

[0036] The first induction culture medium is preferably based on a serum-free medium, and preferably further comprises the following components: 50-100 ng / mL granulocyte-macrophage colony stimulating factor and 10-50 ng / mL stem cell growth factor; more preferably comprises the following components: 60-80 ng / mL granulocyte-macrophage colony stimulating factor and 20-40 ng / mL stem cell growth factor; most preferably 70 ng / mL granulocyte-macrophage colony stimulating factor and 30 ng / mL stem cell growth factor. The second induction culture medium is based on a serum-free medium, and further comprises the following components: 50-100 ng / mL granulocyte-macrophage colony stimulating factor and 10-50 ng / mL interleukin-4, more preferably 60-80 ng / mL granulocyte-macrophage colony stimulating factor and 20-40 ng / mL interleukin-4, most preferably 75 ng / mL granulocyte-macrophage colony stimulating factor and 30 ng / mL interleukin-4. The third induction culture medium is preferably based on a serum-free medium, and further comprises the following components: 10-50 ng / mL tumor necrosis factor α and 100-500 ng / mL lipopolysaccharide, more preferably 20-40 ng / mL tumor necrosis factor α and 200-400 ng / mL lipopolysaccharide, and most preferably 30 ng / mL tumor necrosis factor α and 300 ng / mL lipopolysaccharide. The serum-free medium preferably comprises one of the following: TheraPEAK TM X-VIVO 15(LONZA), CTS TM AIM V TM SFM(RUO) and KBM 581. The culture conditions are preferably such that when the density of adherent cells reaches 80% to 90% in the first induction culture medium, the suspension cells continue to be cultured, while the adherent cells are transferred to the second induction culture medium for culture for 1 to 5 days, and the immature dendritic cells are harvested.

[0037] In the present invention, the concentration of the antigen is preferably 10 to 160 μg / mL, more preferably 15 to 25 μg / mL, and most preferably 20 μg / mL. The seeding density of immature dendritic cells in the third induction culture medium is preferably (2 to 5)×10 5 / mL, more preferably 3×10 5 The induction culture time is preferably 2 to 3 days.

[0038] The present invention provides the anti-pancreatic cancer dendritic cell vaccine or the use of the anti-pancreatic cancer dendritic cell vaccine obtained by the preparation method in preparing a drug for preventing and / or treating pancreatic cancer.

[0039] In the present invention, the anti-pancreatic cancer dendritic cell vaccine has a good growth inhibition effect on pancreatic cancer of different cell types, such as pancreatic cancer ductal cells PANC-1, human in situ pancreatic cancer cells BXPC-3 and MIAPaCa-2 pancreatic cancer cells. Experiments have shown that antigens derived from induced pluripotent stem cells are more widely applicable to the treatment of pancreatic cancer than antigens derived from pancreatic cancer ductal cells PANC-1. The anti-pancreatic cancer dendritic cell vaccine achieves tumor inhibition by promoting the proliferation of T lymphocytes, especially CD8+T cells in the body.

[0040] In the present invention, the drug further comprises a pharmaceutically acceptable excipient. The type of the excipient is selected according to the dosage form of the drug. The dosage form of the drug is preferably an injection. The effective cell concentration of the anti-pancreatic cancer dendritic cell vaccine in the injection is preferably 3×10 6 / mL~10×10 6 / mL, more preferably 5×10 6 / mL~8×10 6 / mL, and the most preferred is 6×10 6 / mL. The drug is used for subcutaneous injection or lymph node injection. The dosage for each injection is 1mL.

[0041] The anti-pancreatic cancer dendritic cell vaccine and its preparation method and application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Expansion Media Configuration: Choose TheraPEAK TM X-VIVO 15 (LONZA: 04-418Q) serum-free medium is the basal medium and also contains GM-CSF (75 ng / mL), SCF (30 ng / mL), FLT-3 (75 ng / mL), TPO (75 ng / mL), IFNα (30 ng / mL), IL-7 (30 ng / mL), SR1 (5 μm) and Heparin (5 μg / mL).

[0043] Configuration of DC Induction Medium I: Select TheraPEAK TM X-VIVO 15 serum-free medium is the basal medium and also contains GM-CSF (75 ng / mL) and SCF (30 ng / mL).

[0044] Configuration of DC Induction Medium II: Select TheraPEAK TM X-VIVO 15 serum-free medium was used as the basal medium and also contained GM-CSF (75 ng / mL) and IL-4 (30 ng / mL).

[0045] Configuration of DC Induction Medium III: Select TheraPEAK TM X-VIVO 15 serum-free medium is a basal medium, and further comprises TNF-α (30 ng / mL), LPS (300 ng / mL) and tumor cell lysate antigen. The preparation method of tumor cell lysate antigen is described in Example 6 of the specification of the patent publication number CN 118064365A.

[0046] Example 1

[0047] A method for preparing dendritic cell vaccine loaded with iPSC cell lysate antigen

[0048] Step 1 Isolation of cord blood mononuclear cells:

[0049] 1) Mononuclear cells were extracted by Ficoll density gradient centrifugation. The umbilical cord blood without plasma was diluted twice with physiological saline. The suspension was blown evenly with a pipette and carefully aspirated and slowly added to the top of the Ficoll separation solution. Centrifuge was used at 2000 rpm, 2 for 20 minutes, and the speed was increased by 2 and decreased by 2 for 20 minutes.

[0050] 2) After centrifugation, the components of the cord blood will be stratified due to density differences. Use a pipette to gently aspirate the middle white cell layer. Add physiological saline to wash and centrifuge at 1600 rpm for 5 minutes. Repeat the wash twice. Add red blood cell lysis buffer, lyse at 4℃ in the dark for 5 minutes, and centrifuge at 1600 rpm for 5 minutes. Discard the supernatant to obtain cord blood mononuclear cells.

[0051] Step 2 Magnetic bead separation of CD34 hematopoietic stem cells

[0052] Isolation of CD34 hematopoietic stem cells using Miltenyi CD34 microbead isolation kit

[0053] 1) Prepare buffer: Weigh 0.5 g of BSA (bovine serum albumin) and 0.074 g of EDTA using an electronic balance, dissolve them in 100 mL of PBS to obtain a buffer containing 0.5% BSA and 2 mM EDTA, and filter through a 0.2 μm bacterial filter for later use.

[0054] 2) Count the mononuclear cells washed in step 1 and filter them with a 30μm cell strainer to avoid clogging the MS sorting column. After centrifugation, discard the supernatant and add 300μL of buffer to resuspend the cells. (General concentration is 300μL / 10 8 cells); add 100μL FcR (generally 100μL / 10 8 cells) and 100 μL CD34 magnetic bead reagent (usually 100 μL / 10 8 After fully mixing, incubate at 4-8°C for 30 min.

[0055] 3) After incubation, add 6 mL of buffer to wash the cells and centrifuge (300 g / min, 10 min), discard the supernatant, add 500 μL of buffer to reselect the cells (500 μL / 10 8 cells); fix the MS sorting column on the magnetic bead sorter to collect non-target cells; rinse the sorting column with 500μL buffer, introduce the cell suspension into the sorting column, repeat the collected negative liquid through the column twice, and rinse the column three times with 500μL buffer (each time rinsing, wait until there is no liquid left from the previous rinse before adding new rinse solution); remove the sorting column from the magnetic field, add 1mL buffer, and use the piston to quickly pump the cells into a 15mL centrifuge tube, which are CD34 positive cells (CD34+ hematopoietic stem cells), wash the cells and count them.

[0056] 4) Resuspend the cell pellet with an appropriate amount of PBS according to the cell quantity and add it to the flow cytometry tube. Each group is divided into a blank control group and a CD34 antibody-added group. After adding the antibody, incubate in the dark for 15 minutes, wash and centrifuge at 1500 rpm / min for 5 minutes, resuspend the cell pellet with an appropriate amount of PBS, and detect the cell CD34 positivity rate on the flow cytometry tube.

[0057] Step 3: Induction of differentiation of DC cells

[0058] 1) Adjust the cell density of the sorted CD34+ hematopoietic stem cells to an appropriate level (the appropriate density is (1-3)×10 5 / ml), add expansion medium to resuspend and transfer to culture flask for culture, gently shake every 12 hours. The expansion culture time is 7 to 10 days, and the cell expansion multiple is 30 to 100 times.

[0059] 2) Continue to culture CD34 hematopoietic stem cells under the conditions of DC induction medium I. The cells gradually change from a suspended state to an adherent state. When the density of adherent cells reaches 80% to 90%, transfer the suspended cells to a new culture bottle and continue to culture under the conditions of DC induction medium I. The adherent cells are replaced with DC induction medium II and cultured for 5 days to harvest immature DC cells.

[0060] Step 4 Preparation of DC vaccine loaded with iPSC cell lysate antigen or exosomes

[0061] 1) Preparation of iPSC Lysate: Take 1 tube of iPSC cells (purchased from Saibei Biotechnology) from liquid nitrogen and quickly put it into a 37°C water bath for recovery. After recovery until only a small piece of ice crystal remains, transfer the cell suspension to a 15mL centrifuge tube containing 4mL of PGM1 human pluripotent stem cell culture medium equilibrated to room temperature. Gently pipette twice to mix, centrifuge at 22g for 5min, and discard the supernatant. Add appropriate amount of Resuspend the cells in human pluripotent stem cell recovery medium, gently pipette twice to mix, and inoculate into the T75 culture flask with the bottom laid the day before (before inoculation, carefully suck out the flask with a Pasteur pipette). Human pluripotent stem cell working solution was used as the bottom layer. The cells were observed to be clumps of 2-10 cells under a microscope. Shake horizontally in a cross shape and place at room temperature for 10 to 15 minutes. After most of the clones were observed to be attached to the bottom of the bottle under a microscope, they were placed in a 37°C constant temperature cell culture incubator with 5% CO2 and cultured for 24 hours. The next day, the recovery medium in the bottle was aspirated and 20 mL of complete stem cell culture medium equilibrated to room temperature was added. The iPSCs in the logarithmic growth phase were digested with DissoEasy human pluripotent stem cell digestion solution. Resuspend the cells with PBS and centrifuge at 300×g for 5 minutes to wash the remaining culture medium twice (to remove foreign proteins). Resuspend the cells with 1 mL of PBS and place them in a 1.8 mL cryopreservation tube. The cell density was 1×10 7 The cryovial was placed in liquid nitrogen for 5 min, taken out and thawed in a 37°C water bath, and the cells were frozen and thawed 5 times. The freeze-thaw solution was centrifuged at 2000g for 10 min at 4°C to remove cell debris, and the supernatant was filtered through a 0.22μm filter membrane. After the protein concentration was determined using a BCA kit, it was aliquoted and stored in a -80°C refrigerator.

[0062] 2) Add DC induction medium III to the harvested immature DC cells, and add iPSC cell lysate antigen (10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL) to induce culture for 2-3 days, and harvest the DC cell vaccine loaded with iPSC cell lysate antigen.

[0063] Example 2

[0064] A method for preparing dendritic cell vaccine loaded with iPSC cell exosomes

[0065] 1) Prepare immature DC cells according to the method of steps 1 to 3 in Example 1.

[0066] 2) Preparation of iPSCs EXOs:

[0067] When iPSC cells are cultured normally to the logarithmic growth phase, the culture supernatant of the cells is collected to extract exosomes. The whole process is carried out at 4°C. Collect all the supernatant liquid in the culture bottle into a 50mL centrifuge tube, centrifuge at 300×g for 10min to remove dead cells, then centrifuge at 2000×g for 10min to remove cells and cell debris, and then centrifuge at 10000×g for 30min, take the supernatant and filter it through a 0.22μm filter membrane to remove particles larger than 220nm; ultracentrifuge at 100000×g for 90min; remove the supernatant, and the obtained precipitate is the exosome. Resuspend the precipitate with 100μL PBS buffer and determine the concentration by BCA method, and store in a refrigerator at 4°C.

[0068] 3) Add DC induction medium III to the harvested immature DC cells, and add iPSC cell exosomes (10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL) to induce culture for 2 to 3 days, and harvest the DC cell vaccine loaded with iPSC cell lysate antigen.

[0069] Comparative Example 1

[0070] Preparation method of DC vaccine loaded with antigen from human pancreatic cancer cell line PANC-1 cell lysate

[0071] 1) Prepare immature DC cells according to the method of steps 1-3 in Example 1.

[0072] 2) Preparation of human pancreatic ductal carcinoma PANC-1 cell lysate (PANC-1Lysate) antigen: PANC-1 cells frozen in liquid nitrogen were taken out and quickly placed in a 37°C water bath for recovery. The recovered cell suspension was then quickly transferred to a 50 mL centrifuge tube containing physiological saline, centrifuged at 1100 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in DMEM / high glucose medium containing a mixture of 10% FBS and 1% penicillin-streptomycin, and cultured in a culture bottle after adjusting the appropriate cell density. When the cells reached 90% confluence, they were subcultured. When the subcultured cells entered the logarithmic growth phase, the supernatant in the bottle was poured out, and the bottom of the bottle was rinsed twice with blank DMEM / high glucose medium. After washing the residual FBS, an appropriate amount of blank DMEM / high glucose medium was poured into each bottle, and the cells were placed in an incubator for continued culture for 48 h. After the PANC-1 cells were digested with 0.25% trypsin-EDTA, they were counted, prepared into a cell suspension, and placed in a cryopreservation tube, 1 mL per tube. Place the cryovial in liquid nitrogen for 5 minutes, take it out and thaw it at 37°C. Repeat the freezing and thawing of the cells 5 times. Finally, centrifuge the freeze-thaw solution at 2000×g for 10 minutes at 4°C. Filter the supernatant through a 0.22μm filter membrane to remove cell debris. The resulting liquid is the PANC-1 cell lysate antigen.

[0073] 3) Add DC induction medium III to the harvested immature DC cells, and add iPSC cell lysate antigen (10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL) to induce culture for 2-3 days, and harvest DC cell vaccine loaded with human pancreatic cancer cell line PANC-1 cell lysate antigen.

[0074] Comparative Example 2

[0075] Preparation method of DC vaccine loaded with exosomes of human pancreatic cancer cell line PANC-1

[0076] 1) Prepare immature DC cells according to the method of steps 1-3 in Example 1.

[0077] 2) Preparation of human pancreatic ductal carcinoma PANC-1 cell exosomes (PANC-1EXO)

[0078] PANC-1 cells frozen in liquid nitrogen were taken out and quickly placed in a 37°C water bath for recovery. The recovered cell suspension was quickly transferred to a 50mL centrifuge tube filled with physiological saline, and the supernatant was discarded after centrifugation at 1100 rpm for 5 minutes. The cells were resuspended in DMEM / high glucose medium containing a mixture of 10% FBS and 1% penicillin-streptomycin, and the appropriate cell density was adjusted and cultured in a culture flask. When the cells reached 90% confluence, they were subcultured. When the subcultured cells entered the logarithmic growth phase, the supernatant in the bottle was poured out, and the bottom of the bottle was rinsed twice with blank DMEM / high glucose medium. After washing the residual FBS, an appropriate amount of blank DMEM / high glucose medium was poured into each bottle, and the culture was continued in the incubator for 48 hours. On the third day, DMEM / high glucose medium without FBS was added. After one day of culture, the culture supernatant of the cells was collected to extract exosomes. The whole process was carried out at 4°C. Collect all the supernatant liquids from the six culture flasks into a 50 mL centrifuge tube, centrifuge at 300 × g for 10 min to remove dead cells, then centrifuge at 2000 × g / min for 10 min to remove cells and cell debris, centrifuge at 10000 × g for 30 min, filter the supernatant through a 0.22 μm filter membrane to remove particles larger than 220 nm, ultracentrifuge at 100000 × g for 90 min, remove the supernatant, and the obtained precipitate is the exosome. Resuspend the precipitate with 100 μL of PBS buffer and store the extracted exosomes at 4 °C.

[0079] 3) DC induction medium III was added to the harvested immature DC cells, and iPSC exosomes (10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL) were added for induction culture for 2-3 days, and DC cell vaccines loaded with exosomes from human pancreatic cancer cell line PANC-1 cells were harvested.

[0080] Example 3

[0081] DC vaccine promotes T lymphocyte proliferation in vitro

[0082] 1) On the fifth day of DC cell culture, place the cells in five 25 cm 2 iPSCs EXO, PANC-1EXO, iPSCs Lysate, PANC-1Lysate antigen (20 μg / mL) and an equal volume of blank culture medium were added to the culture flask. After 24 hours, 100 ng / mL of LPS and 50 ng / mL of TNF-α were added to promote maturation and culture for 24 hours. The mature DC vaccine was harvested on the 7th day.

[0083] 2) Inoculate 2×10 cells per well in a 96-well plate. 5 T lymphocytes were inoculated with the four DC vaccines harvested above at a ratio of 1:5, 1:10, and 1:20, and DC without antigen was used as a blank control, that is, 4×10 4 , 2×10 4 , 1×10 4 Each well of DC vaccine was set, and 3 blank DC vaccine culture wells, blank T lymphocyte culture wells and blank culture medium background wells with different effect-target ratios were set. Each group was set with 3 duplicate wells, and the total volume of each well was 200 μL. The culture system was X-VIVO15 culture medium containing 5% inactivated autologous plasma and IL-2 (200 IU / mL). The cells were cultured in a 37°C incubator for 2 days, and 20 μL CCK8 was added at 48 hours for incubation for 4 hours. The absorbance OD value was detected at 450 nm with an enzyme marker, and a cell proliferation curve was drawn. The stimulation index was calculated according to formula I.

[0084] Stimulation index (SI, %) = (A value of experimental well - A value of blank DC group) / (A value of blank lymphocyte group - A value of background group) × 100% Formula I

[0085] The results are as follows Figure 1 As shown, compared with the blank DC control group, DC cells loaded with four antigens can significantly promote the proliferation of T lymphocytes.

[0086] Example 4

[0087] DC vaccines induce T cell subsets CD8 + With CD4 + Proportional Change

[0088] When DC cells were cultured to the 5th day, they were loaded with iPSCs EXO, PANC-1EXO, iPSCs Lysate, and PANC-1Lysate antigens (20 μg / mL), and a blank DC group without antigen loading was set up. After 2 days of maturation promotion with maturation-promoting factors, T lymphocytes were added at a ratio of 1:10 between the number of DCs and T cells, and after 7 days of co-incubation, the suspended cells were taken for flow cytometry detection of the expression of CD3, CD8, and CD4.

[0089] The results are as follows Figure 2 As shown in the figure, compared with the PBS control group and the blank DC control group, the DC cells loaded with the four antigens can significantly stimulate the proliferation of CD8+T cells, and the difference is statistically significant. DC cells loaded with iPSC cell lysate antigens and exosomes can significantly promote the proliferation of CD8+T cells compared with the corresponding DC cells loaded with pancreatic cancer tumor cell lysate antigens and exosomes. + T cell proliferation, the difference was statistically significant.

[0090] Example 5

[0091] Killing experiment of CTL activated by DC vaccine on pancreatic cancer cell line

[0092] 1) Preparation of CTL activated by DC vaccine:

[0093] The DC vaccines loaded with different antigens prepared in Examples 1 to 2 and Comparative Examples 1 to 2 were co-incubated with T lymphocytes at a ratio of 1:10. The culture medium was X-VIVO medium containing 5% autologous plasma and 1000 IU / mL IL-2. The culture density was 2×10 6 / mL. Incubate for 4 days, and replenish the fluid at an appropriate time. The suspended cells harvested after 4 days are the corresponding CTL cells.

[0094] 2) Pancreatic cancer cell line killing experiment

[0095] CTL activated by DC cells loaded with different concentrations of iPSCs Lysate and PANC-1 Lysate antigens were plated with PANC-1 pancreatic cancer cells at effector-target ratios of 5:1, 10:1, 20:1, and 40:1 for killing experiments. LDH kit was used to detect killing activity.

[0096] The results are as follows Figure 3 As shown, under different effector-target ratios, the killing effect of CTL activated by DC cells loaded with lysate antigen at a concentration of 20 μg / mL was the best.

[0097] Example 6

[0098] Killing experiment of CTL activated by DC vaccine on different types of pancreatic cancer cell lines

[0099] CTL activated by DC cells loaded with iPSCs EXO, PANC-1EXO, iPSCs Lysate, PANC-1Lysate antigen (20μg / mL) and without antigen were plated with PANC-1, BXPC-3 and MIA PaCa-2 pancreatic cancer cells at effector-target ratios of 5:1, 10:1, 20:1, and 40:1 for killing experiments. LDH kit was used to detect killing activity.

[0100] like Figure 4 As shown, at different effector-target ratios, compared with the PBS control group and the blank DC control group, T cells activated by DC cells loaded with the four antigens had significant killing activity against the three pancreatic cancer cell lines, while T cells activated by DC cells loaded with PANC-1 pancreatic cancer cell lysate antigen and PANC-1 exosome antigen only had significant killing activity against PANC-1 pancreatic cancer cells.

[0101] Example 7

[0102] Study on the preventive vaccination of DC vaccine loaded with iPSC lysate antigen in pancreatic cancer tumor model of human immune reconstitution mice

[0103] Human pancreatic cancer PANC1 mouse transplant tumor model, the selected animals are huHSC-(M-NSG) mice, female, 16-18 weeks old. The mice were placed in a sterile isolator for one week to adapt to the environment, randomly divided into groups, weighed, and the experimental animals were divided into 3 groups: solvent control group, blank UCB-DC group, and UCB-DC vaccine group, with 7 animals in each group. Then the first solvent control, blank UCB-DC and UCB-DC vaccine were vaccinated (D1), with a dose of 1×10 6 / 50μL, the solvent control group was infused with the same amount of PBS, and each mouse was intradermally injected with 50μL on the back. Two days later (D4), the second solvent control, blank UCB-DC and dendritic vaccine (UCB-DC vaccine) prepared in Example 1 were infused (different from the first location), with a dose of 1×10 6 / 50μL, the control group was infused with an equal amount of PBS, and 50μL was injected intradermally in the back of each mouse. On D7, PANC1 tumor tissue was removed under sterile conditions, cut into tumor blocks of 2.0mm×2.0mm×2.0mm, and inoculated subcutaneously in the left axillary back of the experimental animal. During the experiment, the clinical status of the mice was observed twice a week, the mice were weighed, and the tumor volume was measured using a vernier caliper. The experiment was terminated on D26, and the remaining animals were killed by cervical dislocation, and the mouse tumors were removed and weighed.

[0104] At D26, compared with the solvent control (PBS) group, the average weight of tumor-bearing mice in the UCB-DC vaccine group was higher than that in the solvent control (PBS) group and the blank UCB-DC group, and the average weight of tumor-bearing mice in the blank UCB-DC group was lower than that in the solvent control (PBS) group, but the above differences were not statistically significant ( Figure 5 ).

[0105] Except for one animal in the solvent control (PBS) group, the tumors grew well. Compared with the solvent control (PBS) group, the tumor growth inhibition rates (calculated based on tumor volume) of the blank UCB-DC group and the UCB-DC vaccine group at D26 were 5.83% and 40.46%, respectively, both less than 60%. The tumor growth inhibition rates of the 7 animals in the UCB-DC vaccine group were 47.18%, 35.24%, 60.04%, 40.46%, 63.47%, 9.43% and 27.38%, respectively. The tumor growth inhibition rates of two animals were greater than 60% ( Figure 6 ).

[0106] Calculated by tumor weight, compared with the solvent control (PBS) group, the tumor growth inhibition rates (calculated based on tumor weight) of the blank UCB-DC group and the UCB-DC vaccine group at D26 were 6.69% and 31.31%, respectively, both less than 60%. The tumor growth inhibition rates of the 7 animals in the UCB-DC vaccine group were 43.62%, 31.91%, 51.06%, 28.72%, 46.81%, -10.64% and 27.66%, respectively. Figure 7 ).

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-pancreatic cancer dendritic cell vaccine, characterized in that: The method comprises dendritic cells loaded with at least one of the following antigens: induced pluripotent stem cell lysate and induced pluripotent stem cell exosomes.

2. The anti-pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The mass ratio of the dendritic cells to the antigen is 1×10 6 : 15~25μg.

3. The anti-pancreatic cancer dendritic cell vaccine according to claim 1, characterized in that: The mass ratio of the dendritic cells to the antigen is 1×10 6 : 20μg.

4. The anti-pancreatic cancer dendritic cell vaccine according to any one of claims 1 to 3, characterized in that: The method for preparing the induced pluripotent stem cell lysate comprises repeatedly freezing and thawing the induced pluripotent stem cells in the logarithmic growth phase, collecting the freeze-thaw solution, removing cell debris and impurity particles, and obtaining the induced pluripotent stem cell lysate.

5. The anti-pancreatic cancer dendritic cell vaccine according to any one of claims 1 to 3, characterized in that: The method for preparing induced pluripotent stem cell exosomes comprises culturing induced pluripotent stem cells to a logarithmic growth phase, collecting the culture supernatant, removing cells, cell debris, impurity particles and water, and obtaining induced pluripotent stem cell exosomes.

6. The method for preparing the anti-pancreatic cancer dendritic cell vaccine according to any one of claims 1 to 5, characterized in that: The following steps are involved: CD34 + Hematopoietic stem cells were induced and cultured twice to obtain immature dendritic cells; The immature dendritic cells and antigens are inoculated into a third induction culture medium for induction culture to obtain an anti-pancreatic cancer dendritic cell vaccine.

7. The preparation method according to claim 6, characterized in that: The concentration of the antigen is 10-160 μg / mL.

8. The preparation method according to claim 6, characterized in that: The concentration of the antigen is 15-25 μg / mL.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The induction culture time is 2 to 3 days.

10. Use of the anti-pancreatic cancer dendritic cell vaccine according to any one of claims 1 to 5 or the anti-pancreatic cancer dendritic cell vaccine obtained by the preparation method according to any one of claims 6 to 9 in the preparation of a medicament for preventing and / or treating pancreatic cancer.

Images