Dendritic cell vaccine for multiple myeloma as well as preparation method and application of dendritic cell vaccine

Abstract

The invention relates to the technical field of biological medicines, in particular to a dendritic cell vaccine for multiple myeloma as well as a preparation method and application of the dendritic cell vaccine, and the dendritic cell vaccine is obtained by co-culturing dendritic cell vaccines MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1. S whole cell line lysates and dendritic cells. According to the present invention, the whole cell line lysates of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1. S are adopted as the antigen of the DC vaccine, and the mixed lysates contain rich tumor antigens, can activate the immune system, and can generate the cytotoxic T cells aiming at the multiple myeloma antigens so as to specifically kill the tumor cells. The DC vaccine prepared by the invention not only stimulates T cells to generate more IFN-gamma, but also induces to generate stronger tumor killing T cells, and provides a new thought for research and development of multiple myeloma DC vaccines.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a dendritic cell vaccine for multiple myeloma and a preparation method and application thereof. BACKGROUND

[0002] Multiple myeloma (MM) is a malignant plasma cell disease, which is a malignant tumor disease caused by abnormal proliferation of bone marrow plasma cells accompanied by excessive production of monoclonal immunoglobulin or light chain (M protein), which can cause bone destruction and bone marrow failure. Amyloidosis is a kind of plasma cell disease, and the most common one is light chain amyloidosis, which accounts for more than 80% of amyloidosis. It is a disease caused by the deposition of amyloid protein formed by the incorrect folding of monoclonal immunoglobulin light chains in tissues and organs, causing tissue structure damage, organ dysfunction and progressive progression. At present, the pathogenesis of MM combined with light chain amyloidosis is not clear, about 10% of MM patients will have light chain amyloidosis at the time of diagnosis, and about 10%-15% of MM patients may develop light chain amyloidosis.

[0003] MM has insidious onset and lacks specific initial symptoms, which can delay diagnosis and treatment, increase the risk of complications, and even shorten the progression-free survival and overall survival of patients. In the prior art, with the use of proteasome inhibitors, immunomodulators, monoclonal antibodies and epigenetic drugs, the treatment efficacy and survival of plasma cell diseases represented by MM have been significantly improved. However, overall, plasma cell diseases are still incurable diseases, and the goal of treatment is still to control disease progression, improve quality of life and prolong survival. The vast majority of patients will eventually still face disease recurrence or progression. The more times of recurrence in clinic, the more difficult the treatment is, and the available therapies are limited, and the outcome is usually poor, and the progression-free survival and survival time after recurrence of patients are shorter.

[0004] At present, for newly diagnosed MM patients, autologous stem cell transplantation after induction therapy is the preferred treatment method. The induction therapy recommends a three-drug combination regimen of proteasome inhibitors combined with immunomodulators and dexamethasone. Proteasome inhibitors can be selected from bortezomib, ixazomib or carfilzomib. Immunomodulators can be selected from lenalidomide, pomalidomide and thalidomide. For patients who are not suitable for autologous stem cell transplantation, such as induction therapy regimen, it is recommended to continue using 8-12 courses to the maximum effect and then enter the maintenance treatment, or continue the original regimen. However, the treatment regimen for relapsed MM is first recommended to enter a suitable clinical trial.

[0005] In recent years, immunotherapy has ushered in a new era of MM treatment. Monoclonal antibodies, bispecific antibodies, immune checkpoint inhibitors, vaccines, and adoptive T cell therapy have been actively studied, and some have achieved remarkable clinical success in MM patients. Monoclonal antibodies targeting CD38, Daratumumab or Isatuximab, Elotuzumab targeting CS1, antibody-drug conjugate Belantamab mafodotin targeting B-cell maturation antigen (BCMA), and Selinexor targeting CRM1 are approved drugs for the treatment of MM. In April 2021, the first CAR-T cell therapy targeting BCMA in MM patients was approved by the US FDA for the treatment of relapsed MM patients. Although great progress has been made in cancer immunotherapy, almost all patients will eventually relapse, and there is still an urgent need for new treatment options.

[0006] Using dendritic cells (DCs) as a platform for cancer vaccine development is a safe and promising direction for development. DCs are the main antigen-presenting cells present in all tissues, which present various antigens to naive T cells through major histocompatibility complex molecules, causing the activation and expansion of naive T cells. DCs are indispensable for a full immune response because they connect the innate and adaptive immune systems to respond to different pathogens, viruses, bacteria, and even tumor cells.

[0007] Currently, there are studies trying to use DC vaccine in the treatment of MM, but there is no successful product on the market. The DC-based strategies implemented in MM patient clinical trials mainly include loading ID protein, myeloma-associated antigen mRNA (such as MAGE3, BCMA and Survivin) or whole myeloma cells with monocyte-derived DCs (MoDCs). The initial clinical trial used ID protein as a MM-specific tumor-associated antigen (TAA) to induce specific immune response in patients with advanced MM, but the effect was limited and most patients had disease progression. Hobo et al. demonstrated the safety of MM TAAs MAGE3, Survivin and BCMA in a phase I clinical trial. The results showed that TAA mRNA-loaded DC vaccine had good tolerance and TAA-specific CTL response was found in two MM patients. However, there is a potential limitation of using a single TAA in DC immunotherapy, which is difficult to observe long-term clinical response. With the development of research, it is found that MoDCs can be loaded with total MM antigens to break through the limitation of immune escape. Rosenblatt et al. fused MoDCs with patient-derived MM tumor cells and observed a certain T cell expansion response. In addition to using DC and MM tumor cell fusion, there are also studies using tumor cell apoptotic bodies, tumor cell lysates or total tumor RNA to load DCs. In vitro analysis shows that both of these two schemes can induce MM-specific immune response. In summary, DC-based immunotherapy is very promising in MM, and the key point is to select the appropriate antigen to load DCs, and the DC vaccine targeting tumor whole cell lysate has potential development value.

[0008] Through data research, it is found that multiple myeloma cell lines MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S express abundant tumor-associated antigens of multiple myeloma, such as BCMA, CD38, CD138, etc. The use of tumor whole cell lysate-loaded dendritic cell vaccine to treat multiple myeloma patients avoids tumor immune escape of single antigen and does not need to worry about the patient's HLA-restricted universal antigen. Therefore, it is feasible to use MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole multiple myeloma cell lysates as antigens for DC loading to treat multiple myeloma patients. SUMMARY

[0009] In view of the deficiencies in the prior art, the present application aims to provide a dendritic cell vaccine using multiple myeloma cell lines MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S as antigens, a preparation method and application thereof. The present application separates the patient's own CD14+ Monocytes are induced by various cytokines and specific agonists, and are added to multiple myeloma cell line lysates to become mature dendritic cells loaded with multiple myeloma related antigens. The antigen-loaded mature dendritic cells are returned to the human body, which can activate the immune system, stimulate innate immunity and acquired immunity, produce cytotoxic T cells to kill tumor cells; the dendritic cell vaccine preparation cycle is about 1 week, the time is short, the cost is low, and there is a large amount of operation space. In order to prepare a dendritic cell vaccine for treating multiple myeloma, the specific technical solutions of the present application are as follows:

[0010] The first aspect of the present application provides a dendritic cell vaccine for multiple myeloma, which comprises at least one of multiple myeloma cell lines MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates and a vaccine co-cultured with dendritic cells.

[0011] The second aspect of the present application provides the use of a dendritic cell vaccine for preparing a drug for treating multiple myeloma.

[0012] The third aspect of the present application provides a preparation method of a dendritic cell vaccine for multiple myeloma, which comprises the following steps:

[0013] S1. Preparation of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell line lysates: collect multiple myeloma MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell lines, centrifuge to remove supernatant, resuspend and freeze-thaw multiple times, centrifuge to take supernatant, filter and sterilize, and obtain;

[0014] S2. Induction of immature dendritic cells: transfer CD14+ cell liquid to a six-well plate, 2mL per well, cell density is 2×10 5 -2×10 6 Add recombinant human GM-CSF and recombinant human IL-4 to the six-well plate.

[0015] S3. Induction of mature dendritic cells: co-culture MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates with imDC described in S2, and add recombinant human TNF-α, LPS and Poly to the culture medium to obtain mature dendritic cells, which are dendritic cell vaccines.

[0016] As a preferred embodiment, the preparation method comprises the following steps:

[0017] S1. Preparation of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell line lysates: Collect multiple myeloma MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell lines, centrifuge after counting, remove supernatant, resuspend in a cryotube with RPMI-1640 / 10% FBS, lyse and break the cells by repeated freeze-thaw method, then centrifuge the cells to take the supernatant, and filter to remove bacteria. The prepared MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell lysates are stored in liquid nitrogen for later use.

[0018] S2. Induction of immature dendritic cells (imDC): Take CD14 + Transfer the cell solution to a six-well plate, 2 mL per well, with a cell density of 2 x 10 5 -2 x 10 6 IU / mL-4000 IU / mL, and the final concentration of recombinant human IL-4 is 200 IU / mL-1000 IU / mL;

[0019] S3. Induction of mature dendritic cells (mDC): Co-culture MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates with imDC described in step (2), and add recombinant human TNF-α, LPS and Poly(I:C) to the culture medium, wherein the final concentration of recombinant human TNF-α is 200 IU / mL-2000 IU / mL, the final concentration of LPS is 2 ng / mL-2 μg / mL, and the final concentration of Poly(I:C) is 2 ng / mL-2 μg / mL.

[0020] The amount of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates used is: co-culture according to the ratio of DC cells to MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates, DC:MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S = 1:1, 1:5 and 1:10.

[0021] Further, the specific procedure of S1 is as follows:

[0022] S11. Collect multiple myeloma cell lines MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S, count the total cell number with 0.4% trypan blue, centrifuge at 700g for 5 min at room temperature, discard the supernatant;

[0023] S12. Resuspend the cell pellet in a cryotube with an appropriate amount of RPMI / 10% FBS according to the cell number counted in the previous step, at a density of (5-10) x 10 6 / mL;

[0024] S13. Freeze the cells and the cryotube together in liquid nitrogen until completely frozen;

[0025] S14. Thaw the cells and the cryotube together in a 37°C water bath until completely thawed;

[0026] S15. Repeat steps (3) and (4) 4 times, for a total of 5 times;

[0027] S16. After complete thawing, remove all the cell suspensions, centrifuge at 1000-3000 rpm for 5-10 min, and remove the supernatant

[0028] S17. Filter to remove bacteria to obtain MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell lysates, and store in liquid nitrogen for use.

[0029] Further, S2 is divided into:

[0030] S21. Isolate peripheral blood mononuclear cells (PBMCs) from the peripheral blood of a patient;

[0031] S22. Further isolate CD14 + mononuclear cells from the PBMCs;

[0032] S23. Induce CD14 + mononuclear cells to produce immature dendritic cells.

[0033] Further, the specific process of S21 is:

[0034] S211. Collect the peripheral blood of a multiple myeloma patient, and add 4.5 mL of Paque Plus solution to each of two centrifuge tubes;

[0035] S212. Slowly adhere the blood sample to the upper layer of the Ficoll solution, 10 mL per tube, the upper layer is the blood layer, and the lower layer is the Ficoll layer, centrifuge at 800g for 20 min at room temperature. The Ficoll density gradient centrifugation method is the most commonly used method for separating and purifying PBMCs;

[0036] S213. Take out the centrifugal tube, and the sample is divided into four layers from top to bottom, which are plasma layer, mononuclear cell layer, Ficoll solution layer, and red blood cell and granulocyte layer;

[0037] S214. Suck the plasma layer, and transfer the mononuclear cell layer to a new 15 mL centrifugal tube. Add PBS / 1% FBS solution and mix by blowing, and the total amount is 14 mL. Centrifuge at room temperature at 800 g for 5 min;

[0038] S215. Discard the supernatant, add 14 mL of PBS / 1% FBS solution and mix by blowing, and centrifuge at room temperature at 700 g for 5 min;

[0039] S216. Discard the supernatant, add 14 mL of PBS / 1% FBS solution and mix by blowing, and centrifuge at room temperature at 700 g for 5 min;

[0040] S217. Discard the supernatant, add 10 mL of RPMI / 10% FBS solution and mix by blowing, resuspend the cells, and count the cell suspension with 0.4% trypan blue to obtain the total cell amount;

[0041] S218. Centrifuge at room temperature at 700 g for 5 min, discard the supernatant, resuspend the cells with appropriate amount of PBS / 1% FBS, and obtain the PBMC suspension for subsequent experiments.

[0042] Further, the specific process of S22 is as follows:

[0043] S221. Transfer the PBMC suspension in the previous step to a 5 mL flow tube;

[0044] S222. Add selection cocktail solution to the tube, and the final concentration of the selection cocktail solution is 100 μL / mL. After mixing by blowing, stand, incubate at room temperature for 10 min;

[0045] S223. Take out the RapidSphere TM solution from the 4°C refrigerator, and vortex for 30 s to mix the magnetic bead particles;

[0046] S224. Add RapidSphere TM solution to the tube, and the final concentration of the RapidSphere TM solution is 100 μL / mL. After mixing by blowing, stand, incubate at room temperature for 3 min;

[0047] S225. Add PBS / 2% FBS solution containing 1 mM EDTA to the tube to make the total volume in the tube 2.5 mL, and mix by blowing;

[0048] S226. Insert the flow tube vertically into the EasySep TM magnet, do not cover, room temperature, stand for 3 min;

[0049] S227. Prepare a new 15 mL centrifuge tube, invert the magnet and flow tube together, collect the cell solution flowing out of the flow tube, keep the magnet inverted for 3 s, do not shake or suck the liquid on the wall of the tube;

[0050] S228. Place the magnet upright, remove the flow tube;

[0051] S229. Repeat steps (5), (6) and (7) once;

[0052] S2210. Blow the cell suspension in the obtained 15 mL centrifuge tube to mix, centrifuge at 700 g for 5 min at room temperature;

[0053] S2211. Discard the supernatant, resuspend the cells with RPMI / 10% FBS, count with 0.4% trypan blue to obtain the cell density, and the obtained cell suspension is CD14 + monocyte suspension.

[0054] Further, the specific process of S23 is as follows:

[0055] S231. In the clean bench, transfer the CD14 + monocyte suspension to a six-well plate, 2 mL per well, with a cell density of 2 x 10 5 -2 x 10 6 6 / mL, add recombinant human GM-CSF and recombinant human IL-4 to the six-well plate, wherein the final concentration of recombinant human GM-CSF is 400 IU / mL-4000 IU / mL, and the final concentration of recombinant human IL-4 is 200 IU / mL-1000 IU / mL;

[0056] S232. Place the six-well plate on the clean bench table, gently shake it left and right to disperse the cells evenly, then put the cell culture plate back into the 37℃ cell culture incubator, and culture for 3 days in a 5% CO2 environment;

[0057] S233. Take the six-well plate out of the incubator and place it on the clean bench table, add 2 mL of RPMI1640 / 10% FBS containing recombinant human GM-CSF with a final concentration of 400 IU / mL-4000 IU / mL and recombinant human IL-4 with a final concentration of 200-1000 IU / mL to the six-well plate;

[0058] S234. Place in a 37℃ cell culture incubator with 5% CO2 for 2 days to obtain immature dendritic cells.

[0059] Further, the specific process of S3 is as follows:

[0060] S31. The MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates prepared in step (1) are co-cultured with the immature dendritic cells prepared in step (3) according to the ratio of DC cells to MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates, i.e. DC:MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S = 1:1, 1:5 and 1:10;

[0061] S32. After 6 hours of co-culture, recombinant human TNF-α, LPS and Poly(I:C) are added to the culture medium to induce maturation of the DCs, wherein the final concentration of recombinant human TNF-α is 200 IU / mL-2000 IU / mL, the final concentration of LPS is 2 ng / mL-2 μg / mL, and the final concentration of Poly(I:C) is 2 ng / mL-2 μg / mL.

[0062] S33. After 2 days of addition of the reagent, mature dendritic cells are obtained. The mature dendritic cells grow adherently, and the surface protrusions are increased and elongated in a long strip radial manner. The harvested mature DCs are the multiple myeloma dendritic cell vaccine.

[0063] Compared with the prior art, the application has the following beneficial effects:

[0064] 1. In the application, the DC vaccine is prepared using MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates as antigens, which can stimulate the body to produce more extensive and powerful T cell immune responses against tumor cells. The MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates contain a large number of antigens in multiple myeloma tumor cells, solving the problem of single antigen loading in the prior art DC vaccine, and effectively reducing the immune escape of tumor cells. These antigens can be presented to the immune system by dendritic cells, effectively activating immune cells.

[0065] 2. The application stimulates a broad immune response by using whole cell lysates in the DC vaccine, including T cell immune responses against multiple tumor antigens, enhances antigen presentation and T cell activation, and thus promotes the exertion of immune response. In addition, using whole tumor cell line lysates as antigens can not be limited by the MHC type of the patient, allowing more patients to benefit, and enhancing the universality of the vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1is an optical schematic diagram of mature dendritic cells in the present application;

[0067] Figure 2 is a schematic diagram for detecting the expression of IL-12p70 of immature dendritic cells and mature dendritic cells by ELISA detection;

[0068] Figure 3 is a schematic diagram of the detection results of the secretion of IFN-γ in vitro;

[0069] Figure 4 is a schematic diagram of the results of detecting lactate dehydrogenase (LDH) in vitro; DETAILED DESCRIPTION

[0070] In order to facilitate understanding of the technical means, creative features, purposes and effects of the present application, the present application will be further described below in combination with specific embodiments.

[0071] The following will be described in combination with the accompanying Figures 1-4 The present application will be further described in detail.

[0072] The embodiments of the present application disclose a dendritic cell vaccine for multiple myeloma and a preparation method and application thereof.

[0073] Embodiment 1

[0074] A dendritic cell vaccine for multiple myeloma, and a preparation method thereof, comprises the following steps:

[0075] S1. Preparation of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell lines lysates: collect multiple myeloma MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell lines, centrifuge after counting, remove supernatant, resuspend in a cryotube with RPMI-1640 / 10% FBS, lyse and break the cells by repeated freeze-thaw method, then centrifuge the cells to take the supernatant, and filter and sterilize. The prepared MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell lysates are stored in liquid nitrogen for standby.

[0076] S2. Induction of immature dendritic cells (imDC): take CD14 + Transfer the cell solution to a six-well plate, 2 mL per well, with a cell density of 2×10 5 -2×10 6 IU / mL-4000 IU / mL, and the final concentration of recombinant human IL-4 is 200 IU / mL-1000 IU / mL;

[0077] S3. Induction of mature dendritic cells (mDC): take MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysate and co-culture with imDC described in step (2), add recombinant human TNF-α, LPS and Poly (I:C) to the culture medium, wherein the final concentration of recombinant human TNF-α is 200 IU / mL-2000 IU / mL, the final concentration of LPS is 2 ng / mL-2 μg / mL, and the final concentration of Poly (I:C) is 2 ng / mL-2 μg / mL.

[0078] The amount of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysate used: co-culture according to the ratio of DC cells to MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysate, DC:MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S = 1:1, 1:5 and 1:10.

[0079] The specific procedure of S1 is as follows:

[0080] S11. Collect multiple myeloma cell lines MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S, count with 0.4% trypan blue, get total cell number, centrifuge at 700g for 5 min at room temperature, discard the supernatant;

[0081] S12. According to the amount of counted cells in the previous step, resuspend the cell pellet in a cryotube with an appropriate amount of RPMI / 10% FBS, with a density of (5-10) x 10 6 / mL;

[0082] S13. Freeze the cells and cryotubes together in liquid nitrogen until completely frozen;

[0083] S14. Thaw the cells and cryotubes together in a 37°C water bath until completely thawed;

[0084] S15. Repeat steps (3) and (4) 4 times, a total of 5 times;

[0085] S16. After complete thawing, take out all the cell suspensions, centrifuge at 1000-3000 rpm for 5-10 min, take the supernatant

[0086] S17. Filter sterilization to obtain MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S cell lysate, store in liquid nitrogen for use.

[0087] The specific procedure of S2 is as follows:

[0088] S21. Isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood of the patient;

[0089] S211. Collect peripheral blood from multiple myeloma patients and add 4.5 mL of Paque Plus solution;

[0090] S212. Draw a blood sample and slowly pipette it onto the top layer of the Ficoll solution, 10 mL per tube. The top layer is the blood layer, and the bottom layer is the Ficoll layer. Centrifuge at 800g for 20 minutes at room temperature. Ficoll density gradient centrifugation is the most commonly used method for isolating and purifying PBMCs.

[0091] S213. Remove the centrifuge tube and separate the sample into four layers from top to bottom: plasma layer, mononuclear cell layer, Ficoll solution layer, and red blood cell and granulocyte layer.

[0092] S214. Aspirate the plasma layer and transfer the mononuclear cell layer to a new 15 mL centrifuge tube. Add PBS / 1% FBS solution and mix thoroughly by pipetting, totaling 14 mL. Centrifuge at 800 g for 5 minutes at room temperature.

[0093] S215. Discard the supernatant, add 14 mL of PBS / 1% FBS solution, mix thoroughly by pipetting, and centrifuge at 700 g for 5 min at room temperature.

[0094] S216. Discard the supernatant, add 14 mL of PBS / 1% FBS solution, mix thoroughly by pipetting, and centrifuge at 700 g for 5 min at room temperature.

[0095] S217. Discard the supernatant, add 10 mL of RPMI / 10% FBS solution and mix thoroughly by pipetting to resuspend the cells. Count the cell suspension using 0.4% trypan blue to determine the total cell count.

[0096] S218. Centrifuge at 700 g for 5 min at room temperature, discard the supernatant, and add an appropriate amount of PBS / 1% FBS to resuspend the cells. The resulting cell suspension is the PBMC suspension, which is used for subsequent experiments.

[0097] S22. Further isolation of CD14 from PBMC + Monocytes;

[0098] S221. Transfer the PBMC suspension from the previous step to a 5 mL flow cytometry tube;

[0099] S222. Add selection cocktail solution to the tube to a final concentration of 100 μL / mL, pipette to mix, and incubate at room temperature for 10 minutes.

[0100] S223. Take out the RapidSphere TM solution, vortex for 30 s to mix the magnetic bead particles evenly;

[0101] S224. Add the RapidSphere TM solution into the tube, vortex to mix the magnetic bead particles evenly; TM solution to a final concentration of 100 μL / mL, vortex to mix, and then incubate at room temperature for 3 min;

[0102] S225. Add the PBS / 2% FBS solution containing 1 mM EDTA into the tube to make the total volume in the tube 2.5 mL, and vortex to mix;

[0103] S226. Insert the flow tube into the EasySep TM magnet vertically, do not cover the cap, and incubate at room temperature for 3 min;

[0104] S227. Prepare a new 15 mL centrifuge tube, invert the magnet and the flow tube together, collect the cell solution flowing out of the flow tube, keep the magnet inverted for 3 s, and do not shake or suck the liquid on the tube wall;

[0105] S228. Place the magnet upright, and take out the flow tube;

[0106] S229. Repeat steps (5), (6) and (7) once;

[0107] S2210. Vortex the cell suspension in the obtained 15 mL centrifuge tube, centrifuge at 700 g for 5 min at room temperature;

[0108] S2211. Discard the supernatant, resuspend the cells with RPMI / 10% FBS, count the cells with 0.4% trypan blue, obtain the cell density, and the obtained cell suspension is the CD14 + mononuclear cell suspension.

[0109] S23. CD14 + Monocytes are induced to produce immature dendritic cells;

[0110] S231. In the clean bench, transfer the CD14 + mononuclear cell suspension into a six-well plate, 2 mL per well, the cell density is 2 x 10 5 -2 x 10 6 -2 x 10

[0111] S232. Place the six-well plate on the clean bench table, gently shake it left and right to disperse the cells evenly, then put the cell culture plate back into the 37℃ cell incubator and culture for 3 days in the condition of 5% CO2;

[0112] S233. Take the six-well plate out of the incubator and place it on the clean bench table, add 2 mL of RPMI1640 / 10% FBS containing recombinant human GM-CSF with a final concentration of 400 IU / mL-4000 IU / mL and recombinant human IL-4 with a final concentration of 200-1000 IU / mL into the six-well plate;

[0113] S234. Place it in the cell incubator at 37℃, 5% CO2 for 2 days to obtain immature dendritic cells.

[0114] The specific procedure of S3 is as follows:

[0115] S31. Co-culture the MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates prepared in step (1) with the immature dendritic cells prepared in step (3) according to the ratio of DC cells to MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates, according to the ratio of DC:MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S = 1:1, 1:5 and 1:10;

[0116] S32. After 6h of co-culture, add recombinant human TNF-α, LPS and Poly(I:C) to the culture medium to induce DC maturation, wherein the final concentration of recombinant human TNF-α is 200 IU / mL-2000 IU / mL, the final concentration of LPS is 2 ng / mL-2 μg / mL, and the final concentration of Poly(I:C) is 2 ng / mL-2 μg / mL.

[0117] S33. After adding the reagent for 2 days, mature dendritic cells are obtained. Mature dendritic cells grow adherently, with increased and elongated surface protrusions in long strip radial pattern. The harvested mature DCs are multiple myeloma dendritic cell vaccines.

[0118] Figure 1The images of mature dendritic cells were observed under an optical microscope with a 10x objective lens. As can be seen from the images, the dendritic cells grow in high density, the cell protrusions are connected with each other, and interweave into a network structure, increasing the contact and interaction between cells, promoting information transmission and signal exchange between immune cells. In order to continue to verify that the dendritic cells have been successfully induced to mature, the culture supernatant of immature dendritic cells and mature dendritic cells was taken respectively, and the content of IL-12p70 in the supernatant was detected by ELISA. The detection results are shown in Figure 2 Figure 2, it is found that the immature dendritic cells hardly secrete IL-12p70, while the induced mature dendritic cells secrete a large amount of IL-12p70.

[0119] Example 2

[0120] Verification of the function of the DC cells prepared in Example 1:

[0121] S1. Preparation of T cells:

[0122] (1) After counting the obtained PBMC, resuspend and mix evenly with PBS / 1% FBS, and transfer to a 5 mL sterile flow tube;

[0123] (2) According to the instructions of CD3 + T sorting kit (STEMCELL, Cat# 17951), add an appropriate amount of selection cocktail solution to the tube, and the final concentration of the selection cocktail solution is 100 μL / mL. After mixing evenly by blowing, stand, incubate at room temperature for 5 min;

[0124] (3) Take the RapidSphere TM solution from the 4°C refrigerator, vortex for 30 s to mix the magnetic bead particles evenly;

[0125] (4) According to the instructions of the kit, add an appropriate amount of RapidSphere TM solution to the tube, and the final concentration of the RapidSphere TM solution is 100 μL / mL. After mixing evenly by blowing, stand, incubate at room temperature for 3 min;

[0126] (5) Add PBS / 2% FBS solution containing 1 mM EDTA to the tube to make the total volume in the tube 2.5 mL, and mix evenly by blowing;

[0127] (6) Insert the flow tube vertically into the EasySep TM magnet without covering the lid, stand at room temperature for 3 min;

[0128] (7) Prepare a new 15 mL centrifuge tube, invert the magnet and flow tube together, collect the cell fluid flowing out of the flow tube, and keep the magnet inverted for 3 seconds. Do not shake or absorb the liquid on the tube wall;

[0129] (8) Place the magnet upright and remove the flow tube;

[0130] (9) Repeat steps (5), (6) and (7) once;

[0131] (10) The cell suspension in the 15 mL centrifuge tube was mixed by pipetting and centrifuged at 700 g for 5 min at room temperature;

[0132] (11) Discard the supernatant, add RPMI / 10% FBS to resuspend the cells, and count the cells with 0.4% trypan blue to obtain the cell density. The resulting cell suspension is the CD3 + T cell suspension.

[0133] S2. DC activated in vitro CD3 + T cells:

[0134] The experiment was divided into two groups: negative control group (DC cells without antigen loading, unLoad-DC) and experimental group (DC cells loaded with MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S lysate, Load-DC), with 3 replicates in each group. + The density of T cells was adjusted to an appropriate level, generally to a DC density of 2×10 5 / mL, CD3 + T cell density 2×10 6 / mL.

[0135] According to DC cells: CD3 + T cells = 1:10 ratio, first add 100 μL of CD3 + T cells were seeded in 96-well plates, and 100 μL of DC cells were added to the CD3 + At the same time, 10-100 U / mL of IL-2 was added to the T cell wells, which was marked as Day 0. During the co-culture period, half of the co-cultured cells were replaced with RPMI / 10% FBS containing 10-100 U / mL of IL-2 every 2-3 days. + Add 1 / 10 of the number of DC cells to the number of T cells. Day 14, according to CD3 + The number of T cells was 1 / 10 of the number of DC cells. On Day 21, CD3 + T cells, and obtain CTL cells.

[0136] S3. Detection of CD3 in vitro+ IFN-γ secreted by T cells:

[0137] To detect the secretion of IFN-γ in vitro, each group of CTL cells obtained by the above process was co-cultured with the NCI-H929 tumor cell line, mixed in a U-bottom 96-well plate at an effector-target ratio of 20:1, and cultured for 72 h. Then, the supernatant of the co-cultured cells was collected, and the content of IFN-γ in the culture supernatant was detected using an IFN-γ enzyme-linked immunoassay kit according to the instruction procedure.

[0138] The detection results are shown in Table 1. Figure 3 As shown in Table 1, the T lymphocytes stimulated by the dendritic cell vaccine (loaded with tumor antigens) (Load-DC group) can produce a large amount of interferon γ, reaching about 1272 pg / ml, which is significantly higher than that of the T lymphocytes stimulated by the dendritic cells without loading tumor antigens (unLoad-DC group).

[0139] The experimental results show that the dendritic cell vaccine prepared in the present application can activate T cells and induce T cells to produce a large amount of INF-γ.

[0140] S4. Detection of CD3 + Detection of the killing activity of T cells on multiple myeloma cells:

[0141] Lactate dehydrogenase (LDH) is an enzyme stably present in the cytoplasm of cells, which only exists in cells under normal conditions. When cells are stimulated to death, the plasma membrane is ruptured, and LDH is rapidly released to the outside of the cells (in the experiment, it is released to the cell culture solution), and thus it is one of the most widely used markers in cytotoxicity studies.

[0142] To detect the secretion of LDH in vitro, each group of CTL cells obtained by the above process was co-cultured with the NCI-H929 tumor cell line, mixed in a U-bottom 96-well plate at an effector-target ratio of 10:1, and cultured for 72 h. Then, the supernatant of the co-cultured cells was collected, and the content of LDH in the culture supernatant was detected using an LDH detection kit according to the instruction procedure, so as to determine the death of the target cells.

[0143] The detection results are shown in Table 2. Figure 4 As shown in Table 2, the T lymphocytes stimulated by the dendritic cell vaccine (loaded with tumor antigens) (Load-DC group) can produce a large amount of interferon γ, reaching about 1272 pg / ml, which is significantly higher than that of the T lymphocytes stimulated by the dendritic cells without loading tumor antigens (unLoad-DC group).

[0144] The experimental results show that the dendritic cell vaccine prepared in the application can activate T cells, activate T lymphocyte immune function, and play a role in killing tumors.

[0145] The above are preferred embodiments of the application, and are not intended to limit the protection scope of the application, so: any equivalent changes made in the structure, shape, principle of the application should be covered in the protection of the application.

Claims

1. A dendritic cell vaccine for multiple myeloma, characterized in that: The dendritic cell vaccine comprises a vaccine obtained by co-culturing dendritic cells with at least one of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell line lysates.

2. A use of the dendritic cell vaccine according to claim 1, characterized in that: Dendritic cell vaccines are used to prepare drugs for the treatment of multiple myeloma.

3. A drug for treating multiple myeloma, characterized in that: The invention comprises the dendritic cell vaccine according to claim 1 and / or pharmaceutically acceptable excipients.

4. A method for preparing a dendritic cell vaccine for multiple myeloma according to claim 1, characterized in that: The steps include: Preparation of S1.MOLP-8, RPMI-8226, NCI-H929, U266B1, and MM1.S cell line lysates: Multiple myeloma MOLP-8, RPMI-8226, NCI-H929, U266B1, and MM1.S cell lines were collected, centrifuged, and the supernatant was removed. The cells were resuspended and freeze-thawed multiple times. The supernatant was collected after centrifugation and sterilized by filtration. S2. Induction of immature dendritic cells: CD14 + The cell suspension was transferred to a six-well plate, 2 mL per well, and recombinant human GM-CSF and recombinant human IL-4 were added to the six-well plate; S3. Induction of mature dendritic cells: Whole-cell lysates of MOLP-8, RPMI-8226, NCI-H929, U266B1, and MM1.S were co-cultured with the imDCs described in S2. Recombinant human TNF-α, LPS, and Poly I:C were added to the culture medium to obtain mature dendritic cells, i.e., the dendritic cell vaccine.

5. The preparation method according to claim 4, characterized in that The S2 CD14 + The cell density of the cell suspension was 2×10 5 -2×10 6 pieces / mL.

6. The preparation method according to claim 4, characterized in that The final concentration of recombinant human GM-CSF in S2 is 400 IU / mL-4000 IU / mL, and the final concentration of recombinant human IL-4 is 200 IU / mL-1000 IU / mL.

7. The preparation method according to claim 4, characterized in that The final concentration of the recombinant human TNF-α is 200 IU / mL-2000 IU / mL, the final concentration of LPS is 2 ng / mL-2 μg / mL, and the final concentration of Poly I:C is 2 ng / mL-2 μg / mL.

8. The preparation method according to claim 4, characterized in that The amount of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S whole cell lysates is as follows: DC cells are co-cultured according to the ratio of MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S corresponding to the whole cell lysates, DC:MOLP-8, RPMI-8226, NCI-H929, U266B1 and MM1.S = 1:1, 1:5 and 1:

10.

9. The preparation method according to claim 4, characterized in that The S2 specifically includes the following steps: S21. Isolate peripheral blood mononuclear cells from peripheral blood; S22. Further separation of PBMC to obtain CD14 + Monocytes; S23.CD14 + Monocytes induce the generation of immature dendritic cells.

10. The preparation method according to claim 4, characterized in that The mature dendritic cells grow by adhering to the wall, and the protrusions on the surface increase in number and become longer, forming long radial strips.

Images