Dendritic cell vaccine for clear cell renal cell carcinoma and use thereof and preparation method therefor

Abstract

The present invention relates to the technical field of cellular immunology. Provided are a dendritic cell vaccine for clear cell renal cell carcinoma and the use thereof and a preparation method therefor. The dendritic cell vaccine comprises a vaccine obtained by means of co-culturing an OS-RC-2 whole cell lysate with a dendritic cell. The dendritic cell vaccine is used in the preparation of a drug for treating clear cell renal cell carcinoma. The method for preparing the dendritic cell vaccine comprises: the induction of an immature dendritic cell (imDC): transferring a CD14+ cell solution into a well plate, adding human recombinant GM-CSF into a six-well plate, and adding human recombinant IL-4 to induce and prepare the immature dendritic cell; and the induction of a mature dendritic cell: co-culturing an OS-RC-2 whole cell lysate with the imDC of the above-described step, adding TNF-α, LPS and Poly(I:C) to stimulate the maturation of the immature dendritic cell, thereby preparing a dendritic cell vaccine. The use of the OS-RC-2 whole cell lysate as an antigen to prepare a DC vaccine can effectively activate T cells and other immune cells, and enhance antigen presentation and T-cell activation, thereby promoting immune-mediated killing of tumors.

Description

A dendritic cell vaccine for clear cell renal cell carcinoma and its application and preparation method Technical Field

[0001] The present invention relates to the technical field of cellular immunology, and in particular to a dendritic cell vaccine for clear cell renal cell carcinoma and its application and preparation method. Background Art

[0002] Renal cell carcinoma (RCC) is a common and fatal urinary tract disease. It is a malignant tumor that originates from the renal tubular epithelium and accounts for 80% to 90% of renal malignancies. Surgical cure rates exceed 90% for patients with early-stage or localized lesions, while the five-year survival rate for patients with locally advanced RCC is 40% to 60%. Advanced metastatic RCC is incurable. The efficacy and survival benefit of targeted therapy vary significantly, with severe adverse reactions and high resistance. Immune checkpoint inhibitors may achieve long-term survival, but 75% of patients do not respond. Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of renal cancer, accounting for approximately 60% to 85% of renal cancers, according to the 2022 guidelines for the diagnosis and treatment of renal cancer issued by the National Health Commission. ccRCC has the worst prognosis of all RCC pathological types and the highest potential for recurrence and metastasis. To address these unmet clinical needs, there is an urgent need for safe, convenient, and durable treatments for advanced ccRCC.

[0003] International studies have examined the major circulating lymphocyte subsets, circulating DCs, and the number of NK cells within tumors in multiple renal cancer patients, finding a significant decrease in all of them. Immunohistochemical identification (anti-S100 monoclonal antibody) of dendritic cell (DC) infiltration in RCC also showed a significant decrease in number compared to normal tissue. Not only are DC cells in tumor tissues of patients with renal cancer reduced in number, but their function is also severely impaired. Immunohistochemical double staining combined with optical microscopy evaluated multiple primary RCC tissues and found a negative correlation between DC cells (CD54+) and the expression of the activation co-stimulatory molecule CD86 (B7.2) on their cell surface. This indicates that the antigen presentation ability of DC cells in patients with renal cancer is impaired, and they cannot effectively perform their antigen presentation and T cell activation functions. Furthermore, the number of T cells that continuously kill tumors has also decreased significantly. Stem-like CD8+ T cells (TCF1+CD8+) in renal cell carcinoma tissue are primarily found in areas where DCs accumulate, while the distribution of non-stem-like CD8+ T cells (TCF1-CD8+) is unrelated to DC accumulation. In patients with advanced renal cell carcinoma, the density of DCs and CD8+ T cells, as well as the areas where TCF1 and CD8 are double positive, are significantly decreased, suggesting that tumor suppression / destruction of immune cell accumulation within the tumor leads to tumor immune escape.

[0004] To compensate for the reduced number and impaired function of DCs in renal cancer patients, mature DCs must be generated in vitro to activate the body's immune response and achieve the goal of treating renal cancer. It is well known that DCs are currently the most powerful professional antigen-presenting cells in the body. Their surface expression of MHC class I and II molecules, as well as multiple costimulatory molecules such as CD80 and CD86, effectively stimulates the proliferation and activation of naive T cells. Therefore, DCs loaded with highly immunogenic tumor antigens offer unique advantages. Clinical studies of DC vaccines conducted internationally have primarily used antigens including tumor lysates, tumor-associated antigens (TAAs) (G250, the immunohistochemical marker for renal cancer, CA-IX, survivin, MUC-1, and telomerase), and neoantigens. One clinical study used DCs loaded with the tumor-associated antigens MUC1 and survivin in combination with CIK cells to treat advanced RCC, achieving an objective response rate (ORR) of 39% and a disease control rate (DCR) of 75%, with no significant clinical side effects. Another study used tumor tissue lysate-loaded DCs in combination with IL-2 and IFN-α to treat metastatic RCC, achieving an ORR of 50% and a DCR of 75%, with no adverse clinical events reported. However, clinical guidelines for the diagnosis and treatment of renal cell carcinoma recommend the use of the targeted single-agent sunitinib for the treatment of advanced RCC. Sunitinib has achieved an ORR of 35.7% and a DCR of 75.1% in patients with advanced RCC; axitinib combined with pembrolizumab has achieved an ORR of 59.3% and a DCR of 83.3%; and ipilimumab combined with nivolumab has achieved an ORR of 59.3% and a DCR of 83.3%. These findings suggest that while clinical studies of DC vaccines for advanced RCC have demonstrated good safety, their efficacy has been suboptimal. Therefore, the search for new targeted antigen-loaded DCs is needed to treat patients with advanced RCC.

[0005] Renal cell carcinoma (RCC) cell lines contain numerous tumor-associated antigens. For example, data analysis has revealed that the ccRCC cell line OS-RC-2 expresses renal cell tumor-associated antigens G250, Survivin, MUC-1, and MAGE-3. Therefore, using whole-cell tumor lysate-loaded dendritic cell vaccines to treat renal cell cancer patients avoids the potential for single-antigen tumor immune escape and eliminates the concern of HLA-restricted universal antigens. Therefore, using OS-RC-2 whole-cell renal cell line tumor lysate as DC-loaded antigens for renal cell cancer treatment is feasible.

[0006] Therefore, there are areas for improvement. The present invention provides a dendritic cell vaccine and its application and preparation method.

[0007] Summary of the Invention

[0008] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a dendritic cell vaccine with OS-RC-2 clear cell renal cell carcinoma as an antigen and its application and preparation method. The present invention stimulates the patient's own dendritic cells in vitro, loads the cell line lysate of clear cell renal cell carcinoma, and becomes mature dendritic cells under the induction of multiple cytokines and specific agonists, and then returns them to the human body to activate the immune system, stimulate natural immunity and acquired immunity, and produce cytotoxic T cells to kill cancer cells; compared with chemotherapy, it is safe and has no side effects; and the preparation cycle of the dendritic cell vaccine is about 1 week, which is short in time and low in cost. In order to achieve the above-mentioned purpose of the preparation method of the dendritic cell vaccine for clear cell renal cell carcinoma, specifically, the technical solution of the present invention is as follows:

[0009] A dendritic cell vaccine comprises a vaccine prepared by co-culturing OS-RC-2 whole cell lysate and dendritic cells.

[0010] An application of a dendritic cell vaccine, wherein the dendritic cell vaccine is used to prepare a drug for treating clear cell renal cell carcinoma.

[0011] A method for preparing a dendritic cell vaccine for clear cell renal cell carcinoma, the preparation method being as follows:

[0012] (1) Induction of immature dendritic cells: CD14+ cell suspension was transferred to a well plate, 2x10 per well. 5 -2x10 6 / mL, add human recombinant GM-CSF to a six-well plate at a concentration of 400IU / mL-4000IU / mL, and add human recombinant IL-4 at a final concentration of 200-1000IU / mL to induce the preparation of immature dendritic cells;

[0013] (2) Induction of mature dendritic cells: OS-RC-2 whole cell lysate was co-cultured with the imDCs described in step (1), and TNF-α at a final concentration of 200 IU / mL-2000 IU / mL, LPS at a final concentration of 2 ng / mL-2 μg / mL, and Poly(I:C) at a final concentration of 2 ng / mL-2 μg / mL were added to stimulate the maturation of immature dendritic cells to prepare a dendritic cell vaccine;

[0014] In step (2), the amount of OS-RC-2 whole cell lysate is used according to the ratio of DC cells to OS-RC-2 whole cell lysate, DC:OS-RC-2 = 1:1, 1:3, 1:6 and 1:9 for co-culture.

[0015] In practice, step (1) is divided into:

[0016] Further separation and acquisition of CD14 from monocytes + Monocytes;

[0017] CD14 + Monocytes induce the generation of immature dendritic cells.

[0018] The source of monocytes is: PBMCs are separated from the patient's peripheral blood mononuclear cells, and monocytes are separated from PBMCs.

[0019] Furthermore, CD14 was further isolated from monocytes. + The specific process of monocytes is:

[0020] (1) Transfer the mononuclear cell suspension from step 1 to a 5 mL flow cytometry tube;

[0021] (2) Add an appropriate amount of selection cocktail solution to the flow tube to a final concentration of 100 μL / mL, pipette thoroughly to mix, and incubate at room temperature for 10 min;

[0022] (3) Prepare CD14 + Magnetic beads, RapidSphere TM The solution was vortexed for 30 seconds to allow CD14 + The magnetic beads are evenly dispersed;

[0023] (4) Add appropriate amount of RapidSphere to the flow tube TM The solution was adjusted to a final concentration of 100 μL / mL, pipetted thoroughly to mix, and incubated at room temperature for 3 min;

[0024] (5) Add an appropriate amount of PBS / 2% FBS with 1 mM EDTA solution to the flow cytometry tube to a total volume of 2.5 mL and pipette thoroughly to mix.

[0025] (6) Insert the flow tube vertically into EasySep TM magnet, incubate at room temperature for 3 min;

[0026] (7) Invert the magnet and collect the cell fluid flowing out of the flow tube into a 15 mL centrifuge tube. Keep the magnet inverted for 3 seconds. Do not shake or absorb the liquid on the tube wall.

[0027] (8) Place the magnet upright and remove the flow tube;

[0028] (9) Repeat steps (7) and (8) twice;

[0029] (10) Add 2 mL of RPMI / 10% FBS to the flow cytometry tube to resuspend the cells, count the cells using Trypan Blue, and prepare CD14+ monocytes.

[0030] Furthermore, CD14 + The specific process of inducing immature dendritic cells from monocytes is as follows:

[0031] (1) Use a pipette to draw CD14 in a clean bench + The mononuclear cell suspension was transferred to a six-well plate, with 2 x 10 cells per well. 5 -2x10 6 / mL, continue to add human recombinant GM-CSF to the six-well plate at a concentration of 400IU / mL-4000IU / mL, and the final concentration of human recombinant IL-4 is 200-1000IU / mL;

[0032] (2) Place the six-well plate on a clean bench and gently shake it three times to evenly disperse the cells. Place the plate in a cell culture incubator at 37°C and 5% CO2 for three days.

[0033] (3) Remove the six-well plate from the incubator and continue to add 2 mL RPMI1640 / 10% FBS, 1 μL human recombinant GM-CSF (final concentration of 400 IU / mL-4000 IU / mL), and 1 μL human recombinant IL-4 (final concentration of 200-1000 IU / mL) to the six-well plate in the clean bench;

[0034] (4) The cells were placed in a cell culture incubator at 37° C. and 5% CO 2 for 2 days to prepare immature dendritic cells.

[0035] In practice, step (2) is divided into:

[0036] Whole cell lysates of the clear cell renal cell carcinoma cell line OS-RC-2;

[0037] OS-RC-2 whole cell lysate is co-cultured with immature dendritic cells to stimulate the immature dendritic cells to become mature dendritic cells, thereby preparing a dendritic cell vaccine.

[0038] Furthermore, the specific process for whole cell lysis of the clear cell renal cell carcinoma cell line OS-RC-2 is as follows:

[0039] (1) Pre-set the water bath temperature to 37°C;

[0040] (2) Clear cell renal cell carcinoma OS-RC-2 cells were collected by centrifugation at 700 g for 5 min at room temperature;

[0041] (3) Remove the supernatant and resuspend in RPMI / 10% FBS;

[0042] (4) Count the cells after staining with trypan blue;

[0043] (5) Centrifuge at 700 g for 5 min at room temperature, apply the brake gently, and remove the supernatant.

[0044] (6) Resuspend the cells in RPMI / 10% FBS in a 1 mL cryovial at a density of (5-10) × 10 6 / mL;

[0045] (7) Freeze the cells in liquid nitrogen for 20 seconds;

[0046] (8) Immediately thaw the cells completely and quickly in a 37°C water bath;

[0047] (9) Repeat steps (7) and (8) 4 times, for a total of 5 times;

[0048] (10) The prepared OS-RC-2 cell lysate was stored in liquid nitrogen until use.

[0049] Furthermore, the OS-RC-2 whole cell lysate prepared in step 3 and the immature dendritic cells prepared in step 2 were co-cultured at a ratio of DC:OS-RC-2 = 1:1, 1:3, 1:6 and 1:9, according to the DC cell-to-OS-RC-2 whole cell lysate;

[0050] Furthermore, the specific process of stimulating immature dendritic cells to become mature dendritic cells is:

[0051] After 6 hours of culture, immature dendritic cells were stimulated with 2 μL of TNF-α at a final concentration of 200 IU / mL-2000 IU / mL, 2 μL of LPS at a final concentration of 2 ng / mL-2 μg / mL, and 1 μL of Poly(I:C) at a final concentration of 2 ng / mL-2 μg / mL to mature the immature dendritic cells for 2 days.

[0052] When mature dendritic cells grow attached to the wall, the number of surface protrusions increases and becomes longer, forming long radial strips with obvious dendritic morphology, which means they are mature dendritic cells.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] In the present invention, a DC vaccine prepared using OS-RC-2 whole-cell lysate as an antigen can stimulate the body to produce a broader and more powerful T cell immune response against renal cell carcinoma. The OS-RC-2 whole-cell lysate contains a large number of antigens from this renal cell carcinoma cell line, solving the problem of existing DC vaccines with a single antigen load and reducing the occurrence of immune escape. These antigens can be presented to the immune system by dendritic cells, effectively activating dendritic cells and other immune cells. By using whole-cell lysate in DC vaccines, a broad immune response is stimulated, including T cell immune responses against multiple tumor antigens, enhancing antigen presentation and T cell activation, thereby promoting the immune response. In addition, the use of whole tumor cell lysate as an antigen is not limited by the patient's MHC type, and can benefit more patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG1 is an optical schematic diagram of mature dendritic cells in the present invention;

[0056] FIG2 is a schematic diagram of flow cytometry analysis of immature dendritic cells and mature dendritic cells;

[0057] FIG3 is a schematic diagram of detecting IL-12p70 expression in immature dendritic cells and mature dendritic cells using ELISA;

[0058] FIG4 is a schematic diagram showing the killing rate of T cells stimulated by dendritic cell vaccines against clear cell renal cell carcinoma cells derived from corresponding patients;

[0059] FIG5 is a schematic diagram showing the results of in vitro detection of interferon-γ secretion. DETAILED DESCRIPTION

[0060] The present invention will be further described in detail below with reference to specific embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0061] ccRCC, also known as clear cell renal cell carcinoma (CCRCC), is a common malignant tumor of the kidney. ccRCC patients are individuals diagnosed with CRCC. Treatment options for ccRCC include surgical resection, radiotherapy, targeted therapy, and immunotherapy. Surgical resection is the mainstay of treatment for early-stage tumors, while targeted therapy (such as tyrosine kinase inhibitors and vascular endothelial growth factor (VEGF) inhibitors) or immunotherapy (such as checkpoint inhibitors) are often used for advanced or metastatic ccRCC.

[0062] OS-RC-2 whole cell lysate-loaded DC vaccine specifically amplifies patient-derived T cells, which can target and kill patient-derived ccRCC tumor cells. Therefore, it is feasible to prepare a DC vaccine for ccRCC using OS-RC-2 whole cell lysate as an antigen. The present invention first proposes a dendritic cell vaccine for the preparation of a drug for treating clear cell renal cell carcinoma. The dendritic cell vaccine includes a vaccine co-cultured with OS-RC-2 whole cell lysate and dendritic cells. Secondly, a method for preparing a dendritic cell vaccine is proposed. The prepared vaccine is used to treat clear cell renal cell carcinoma, and the steps are as follows:

[0063] (1) Induction of immature dendritic cells: CD14+ cell suspension was transferred to a well plate, 2x10 per well. 5 -2x10 6 / mL, add human recombinant GM-CSF to a six-well plate at a concentration of 400IU / mL-4000IU / mL, and add human recombinant IL-4 at a final concentration of 200-1000IU / mL to induce the preparation of immature dendritic cells;

[0064] (2) Induction of mature dendritic cells: OS-RC-2 whole cell lysate was co-cultured with the imDCs described in step (1), and TNF-α at a final concentration of 200 IU / mL-2000 IU / mL, LPS at a final concentration of 2 ng / mL-2 μg / mL, and Poly(I:C) at a final concentration of 2 ng / mL-2 μg / mL were added to stimulate the maturation of immature dendritic cells;

[0065] The amount of the OS-RC-2 whole cell lysate was as follows: DC cells were co-cultured with the corresponding OS-RC-2 whole cell lysate at a ratio of DC:OS-RC-2 = 1:1, 1:3, 1:6 and 1:9.

[0066] In practice, step (1) is divided into:

[0067] Further separation and acquisition of CD14 from monocytes + Monocytes;

[0068] CD14 + Monocytes induce the generation of immature dendritic cells.

[0069] The source of monocytes is: PBMCs are separated from the patient's peripheral blood mononuclear cells, and monocytes are separated from PBMCs.

[0070] When the patient's peripheral blood mononuclear cells are separated for PBMC, based on the differences in the density of each cell component in the peripheral blood (peripheral blood mainly contains platelets, mononuclear cells, granulocytes, red blood cells and other cells, the platelet density is 1.030-1.035kg / m 3 , the mononuclear cell density is 1.075-1.090kg / m 3 The granulocyte density is 1.092 kg / m 3 , the red blood cell density is 1.093 kg / m 3 ), added to peripheral blood samples Paque Plus (GE Healthcare) solution. Density is 1.075-1.089 kg / m 3 , perform density gradient centrifugation to separate different cell components, and quickly separate mononuclear cells from human peripheral blood. The specific process is as follows:

[0071] (1) Peripheral blood was collected from the vein of ccRCC patients, and 4.5 mL of ... Paque Plus solution;

[0072] (2) Use a pipette to draw up the blood sample and slowly inject it along the wall of the centrifuge tube. The upper layer of Paque Plus solution (10 mL per tube) was centrifuged at 800 g for 20 min at room temperature.

[0073] (3) Take out the centrifuge tube and separate the sample into four layers, from top to bottom: plasma, monocytes, Paque Plus solution, red blood cells and granulocytes;

[0074] (4) Carefully transfer the mononuclear cells to a 15 mL centrifuge tube. Add PBS / 1% FBS solution to make up to 14 mL and pipette to mix. Centrifuge at 800 g for 5 min at room temperature.

[0075] (5) Remove the supernatant, gently flick the bottom of the tube to loosen the cells, add 14 mL of PBS / 1% FBS solution to resuspend the cells, pipette to mix, and centrifuge at 700 g for 5 min at room temperature;

[0076] (6) Remove the supernatant, gently tap the bottom of the tube to loosen the cells, add 14 mL of RPMI / 10% FBS solution to resuspend the cells, pipette to mix, and centrifuge at 400 g for 5 min at room temperature;

[0077] (7) Remove the supernatant, gently tap the bottom of the tube to loosen the cells, add 10 mL of RPMI / 10% FBS solution to resuspend the cells, and mix thoroughly by pipetting;

[0078] (8) Pipette 10 μL of cell fluid into a new 1.5 mL centrifuge tube, add 90 μL of RPMI / 10% FBS solution to dilute 10-fold, then pipette another 10 μL of diluted cell fluid, add 10 μL of Trypan Blue to stain, and then add to a hemocytometer for counting under an inverted microscope;

[0079] (9) Centrifuge at 700 g for 5 min at room temperature, remove the supernatant, and add appropriate amount of PBS / 1% FBS for subsequent steps.

[0080] Through the above processes (1)-(9), PBMC can be quickly isolated from human peripheral blood in step 1. It should be noted that FBS solution generally plays a cell protection role for PBMC, provides cell nutrition and proliferation, and reduces red blood cell contamination. FBS solution contains rich nutrients, growth factors and cytokines, which can provide a suitable environment to protect PBMC from damage; adding FBS solution when resuspending cells can reduce the stress and damage of cells during centrifugation and sedimentation, and improve cell survival rate; adding an appropriate amount of FBS solution when resuspending cells can help reduce red blood cell contamination and improve the purity of PBMC. In processes (4)-(7), the concentration of PBS / 1% FBS solution added in processes (4) and (5) is the same, the content of PBS / 1% FBS solution is different, and the centrifugation time is the same based on the different total amount of solution in the centrifuge tube. The difference between processes (5) and (6) is that the concentration of FBS solution is different, the total amount of solution in the centrifuge tube is different, and the content of FBS solution in process (7) is reduced. By adjusting the process parameters such as concentration, content and centrifugation time, the removal effect of the supernatant during PBMC separation is improved, thereby improving the purity and quality of monocytes separated from PBMC.

[0081] In order to further separate the obtained PBMCs into monocytes, immature dendritic cells, including S1 and S2, were prepared.

[0082] S1 is to further separate and obtain CD14 from PBMC + The purpose of monocytes is to obtain a higher purity monocyte subpopulation, which can obtain a purer monocyte population, facilitating subsequent further separation and purification to obtain higher purity immature dendritic cells. The specific process is:

[0083] (1) Transfer the mononuclear cell suspension from step 1 to a 5 mL flow cytometry tube;

[0084] (2) Add an appropriate amount of selection cocktail solution to the flow tube to a final concentration of 100 μL / mL, pipette thoroughly to mix, and incubate at room temperature for 10 min;

[0085] (3) Prepare CD14+ Magnetic beads, RapidSphere TM The solution was vortexed for 30 seconds to allow CD14 + The magnetic beads are evenly dispersed;

[0086] (4) Add appropriate amount of RapidSphere to the flow tube TM The solution was adjusted to a final concentration of 100 μL / mL, pipetted thoroughly to mix, and incubated at room temperature for 3 min;

[0087] (5) Add an appropriate amount of PBS / 2% FBS with 1 mM EDTA solution to the flow cytometry tube to a total volume of 2.5 mL and pipette thoroughly to mix.

[0088] (6) Insert the flow tube vertically into EasySep TM magnet, incubate at room temperature for 3 min;

[0089] (7) Invert the magnet and collect the cell fluid flowing out of the flow tube into a 15 mL centrifuge tube. Keep the magnet inverted for 3 seconds. Do not shake or absorb the liquid on the tube wall.

[0090] (8) Place the magnet upright and remove the flow tube;

[0091] (9) Repeat steps (7) and (8) twice;

[0092] (10) Add 2 mL RPMI / 10% FBS to the flow cytometry tube to resuspend the cells, count the cells with Trypan Blue, and prepare CD14 + Monocytes.

[0093] Through processes (1)-(10), using CD14 with a surface marker that specifically binds to CD14 + Magnetic beads, monocytes and CD14 + After mixing the magnetic beads, stir them thoroughly to make CD14 + Magnetic beads bind to the CD14 surface marker, and the cells bound to the beads are adsorbed to the magnetic separation device (EasySep TM The non-bound cells are retained in the suspension by combining processes (7)-(8) to remove the non-bound cells in the suspension. Under the action of magnetic field separation, the bound CD14 + Monocytes are released into flow cytometry tubes and collected.

[0094] It should be noted that CD14 + The isolation and extraction process of monocytes includes but is not limited to the CD14 + Magnetic bead sorting, CD14 +Negative selection, Miltenyi immunomagnetic cell sorting (MACS), cell attachment and other methods. The principle is based on the specific binding characteristics of antigen and antibody, CD14 + The magnetic bead separation kit can specifically recognize and bind CD14 in PBMCs + Monocytes are indirectly coupled to magnetic beads through biotin or dextran, and CD14 + Purpose of monocyte separation. This example uses EasySep TM CD14 positive selection kit.

[0095] S2 is CD14 + Monocytes are induced to produce immature dendritic cells. The specific process is as follows:

[0096] (1) Use a pipette to draw CD14 in a clean bench + The mononuclear cell suspension was transferred to a six-well plate, with 2 x 10 cells per well. 5 -2x10 6 / mL, continue to add human recombinant GM-CSF to the six-well plate at a concentration of 400IU / mL-4000IU / mL, among which 1000IU / mL-2000IU / mL has the best effect, and the final concentration of human recombinant IL-4 is 200-1000IU / mL, among which 500IU / mL-1000IU / mL has the best effect;

[0097] (2) Place the six-well plate on a clean bench and gently shake it three times to evenly disperse the cells. Place the plate in a cell culture incubator at 37°C and 5% CO2 for three days.

[0098] (3) Remove the six-well plate from the incubator and continue to add 2 mL RPMI1640 / 10% FBS, 1 μL human recombinant GM-CSF to the six-well plate in the clean bench to a final concentration of 1000 IU / mL-2000 IU / mL, and 1 μL human recombinant IL-4 to a final concentration of 500 IU / mL-1000 IU / mL;

[0099] (4) The cells were placed in a cell culture incubator at 37° C. and 5% CO 2 for 2 days to prepare immature dendritic cells.

[0100] Through the above processes (1)-(4), human recombinant GM-CSF and human recombinant IL-4 were used as cytokines to induce CD14 + Monocytes were differentiated into immature dendritic cells and the cells were incubated at 37°C with 5% CO2 to promote the generation and development of immature dendritic cells.

[0101] It should be noted that in vitro, granulocyte-macrophage colony stimulating factor (GM-CSF) can promote the survival of immature dendritic cells (imDC) and induce the proliferation of imDC. Interleukin-4 (IL-4) can inhibit the excessive growth of macrophages, reduce the expression of CD14 molecules on the cell surface, and induce CD14 + Monocytes differentiate into imDCs. In the above induction process, the total amount of human recombinant GM-CSF and human recombinant IL-4 is not added directly into the six-well plate at one time, but added twice. + After monocytes were cultured in a cell culture incubator for 3 days, adding new human recombinant GM-CSF and human recombinant IL-4 and continuing to culture them could continuously stimulate CD14 + Monocytes develop in the direction of dendritic cell differentiation, enhance the production and differentiation of dendritic cells, help increase the yield of dendritic cells, and increase the number of immature dendritic cells during the culture process. Moreover, by intermittently adding these two factors, it can further promote the transformation of immature dendritic cells into mature dendritic cells and enhance their antigen presentation and immune stimulation capabilities.

[0102] In practice, step (2) is divided into:

[0103] Whole cell lysates of the clear cell renal cell carcinoma cell line OS-RC-2;

[0104] OS-RC-2 whole cell lysate is co-cultured with immature dendritic cells to stimulate the immature dendritic cells to become mature dendritic cells, thereby preparing a dendritic cell vaccine.

[0105] Among them, the clear cell renal cell carcinoma cell line OS-RC-2 whole cell lysis, the lysis method specifically uses a mechanical lysis method of repeated freezing and thawing to lyse the cell line. The principle is that the formation of ice particles in the cells and the increase in the salt concentration of the remaining cell fluid cause swelling, which breaks the cell structure, but retains the immunogenicity of the cells. Freezing is usually performed in liquid nitrogen or on ice at -20°C, and thawing can be performed by heat shock in a water bath at 37°C, 50°C, 65°C or 100°C, which is milder than chemical lysis. The specific process of whole cell lysis of the clear cell renal cell carcinoma cell line OS-RC-2 is as follows:

[0106] (1) Pre-set the water bath temperature to 37°C.

[0107] (2) Clear cell renal cell carcinoma OS-RC-2 cells were collected by centrifugation at 700 g for 5 min at room temperature.

[0108] (3) Remove the supernatant and resuspend in RPMI / 10% FBS.

[0109] (4) Count the cells after staining with trypan blue.

[0110] (5) Centrifuge at 700g for 5 min at room temperature, brake gently, and remove the supernatant.

[0111] (6) Resuspend the cells in RPMI / 10% FBS in a 1 mL cryovial at a density of (5-10) × 10 6 / mL.

[0112] (7) Freeze the cells in liquid nitrogen for 20 seconds.

[0113] (8) Immediately thaw the cells completely and quickly in a 37°C water bath.

[0114] (9) Repeat steps (7) and (8) 4 times, for a total of 5 times.

[0115] (10) The cells were centrifuged at 700 g for 5 min at room temperature and the supernatant was collected.

[0116] (11) The prepared OS-RC-2 cell lysate supernatant was stored in liquid nitrogen until use.

[0117] Through the above processes (1)-(11), the cells are frozen at low temperatures (liquid nitrogen) and then thawed at room temperature, repeatedly to achieve cell wall disruption. Due to freezing, on the one hand, the hydrophobic bond structure of the cell membrane is broken, thereby increasing the hydrophilicity of the cell. On the other hand, the intracellular water crystallizes, forming ice crystals, causing the cells to swell and rupture. In this way, the supernatant of the cell lysate is obtained for use as the antigen protein.

[0118] Afterwards, the OS-RC-2 whole cell lysate prepared above was co-cultured with the immature dendritic cells prepared in the above step (1) according to the ratio of DC cells to OS-RC-2 whole cell lysate (DC:OS-RC-2=1:1, 1:3, 1:6 and 1:9). The immature dendritic cells of the control group were co-cultured with the culture medium of the cell lysate. After 6 hours, the immature dendritic cells were stimulated to become mature dendritic cells, which were subsequently used to prepare the tumor antigen-loaded Load group and the unloaded control group. The specific stimulation process was as follows:

[0119] After 6 hours of co-culture, 2 μL of TNF-α (i.e., tumor necrosis factor-α, final concentration of 200 IU / mL-2000 IU / mL, of which 1000 IU / mL-2000 IU / mL has the best effect), 2 μL of LPS (i.e., lipopolysaccharide, final concentration of 2 ng / mL-2 μg / mL, of which 1 μg / mL-2 μg / mL has the best effect), and 1 μL of Poly (I:C) (i.e., artificially synthesized oligonucleotide immune adjuvant, 2 ng / mL-2 μg / mL, of which 1 μg / mL-2 μg / mL has the best effect) were added to stimulate dendritic cell maturation after 2 days.

[0120] During the process of stimulating dendritic cell maturation, first, immature dendritic cells undergo phenotypic changes after these stimuli. They express more co-stimulatory molecules on their surface, such as CD80 (B7-1), CD86 (B7-2), and CD83. The increased expression of these molecules enhances the interaction between dendritic cells and other immune cells, promoting the initiation of immune responses. Second, their antigen presentation ability gradually increases, allowing them to more effectively capture, process, and present antigens to T cells, thereby activating T cell immune responses. Third, mature dendritic cells secrete cytokines, such as interferon-γ (IFN-γ), interleukin-12 (IL-12), and interleukin-6 (IL-6). The secretion of these cytokines promotes the development and regulation of immune responses. Fourth, their migration ability is enhanced. Mature dendritic cells possess a stronger ability to migrate from tissues to immune-related sites such as lymph nodes, where they interact with T cells and trigger immune responses.

[0121] Observe the morphology of mature dendritic cells and assess their maturity based on their morphology. Specifically, place a culture dish of mature dendritic cells under an optical microscope (10x objective lens). If mature dendritic cells are observed adhering to the wall, with increased and elongated surface protrusions forming long, radial strips and a distinct dendritic morphology, they are considered mature. As shown in Figure 1, at high density, multiple cells interconnect, forming a network-like structure. This increases intercellular contact and interaction, promoting information transfer and signal exchange between immune cells. Furthermore, the dendritic cell network provides a larger surface area, enhancing antigen capture and processing capabilities.

[0122] It should be noted that the formation of the reticular structure of mature dendritic cells is affected by a variety of factors. Different process conditions result in different morphologies and structures of the reticular structure. It is precisely because the immature dendritic cells obtained in step (1) have already shown a tendency to transform into mature dendritic cells under the action of human recombinant GM-CSF and human recombinant IL-4 that these immature dendritic cells can quickly become mature dendritic cells under the stimulation of TNF-α, LPS, and Poly (I: C) in the subsequent step (2).

[0123] Dendritic cells play a crucial role in immune responses, but their functions and properties differ between their immature and mature states. Immature dendritic cells are typically located in tissues and have high antigen uptake and processing capabilities, but are less capable of antigen presentation and T cell activation. Mature dendritic cells, on the other hand, have enhanced antigen presentation and T cell stimulation capabilities and are typically found in lymphoid tissues such as lymph nodes.

[0124] In order to more clearly evaluate the maturity of dendritic cells, their functional status can be evaluated and distinguished by detecting the surface molecular markers of immature dendritic cells and mature dendritic cells obtained in step (2). Some commonly used markers are used to distinguish immature and mature dendritic cells, such as:

[0125] Markers of immature dendritic cells: CD11c (a dendritic cell marker), CD80, CD86, CD1a, CD14 (expressed on certain types of dendritic cells), etc.

[0126] Markers of mature dendritic cells: CD83, CD40, CD80, CD86, HLA-DR (MHC class molecule), CD1a (expressed on certain types of dendritic cells), etc.

[0127] Flow cytometry was used to examine the surface markers CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC on immature and mature dendritic cells. The results are shown in Figure 2 . Iso represents the flow cytometric profile of the corresponding antibody isotype control, imDC represents the flow cytometric profile of immature dendritic cell surface markers, and mDC represents the flow cytometric profile of mature dendritic cell surface markers. In Figure 2 , the abscissa represents the relative intensity of the fluorescence or scattered light signal, and the ordinate represents the relative cell number. As shown in Figure 2 , mature dendritic cells (mDCs) exhibited higher expression of CD11c, CD14, CD40, CD80, CD83, CD86, HLA-DR, and HLA-ABC than imDCs, confirming that the dendritic cells had been induced to mature.

[0128] To further verify that the dendritic cells had been induced to mature, culture supernatants from immature dendritic cells and mature dendritic cells induced in step 4 were collected and assayed for IL-12p70 expression by ELISA. The results, shown in Figure 3, indicate that immature dendritic cells secrete almost no IL-12p70, whereas IL-12p70 expression and secretion are enhanced after induction into mature dendritic cells.

[0129] Based on the above, mature dendritic cells loaded with tumor lysates from renal cancer cell lines were obtained, i.e., vaccines, and tested. The specific process is as follows:

[0130] A. Processing of patient-derived tumor tissue to prepare target cells

[0131] Tumor tissue samples (0.5 g sample, for example) from patients undergoing renal cancer surgery were transferred to a 50 ml centrifuge tube. The sample was washed 3-5 times with sterile saline containing 1% penicillin-streptomycin-amphotericin B solution, using 15-20 ml of wash solution each time. Using sterile scissors, the tissue was cut into approximately 1 mm³ pieces on ice. The sheared tissue was collected in 5 ml of HBSS buffer and placed in a 15 ml centrifuge tube. The sheared tissue was centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. 3 ml of tissue digestion buffer was added to the pellet, and the pellet was shaken at 37°C for 1 hour. After digestion, 5 ml of HBSS was added to terminate the digestion, and the pellet was centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. If red blood cells were present in the pellet, 2 ml of red blood cell lysis buffer was added to resuspend the pellet. After standing at room temperature for 2-5 minutes, the pellet was diluted with 4 ml of HBSS buffer, centrifuged at 1200 rpm for 3 minutes, and the supernatant was discarded. The resulting single cells were counted and frozen for use as target cells.

[0132] When testing the cytotoxicity of T cells stimulated by dendritic cell vaccines against ccRCC cells, the following steps are generally followed:

[0133] Prepare target cells: Target cells prepared after processing patient-derived tumor tissue need to be revived one day in advance to restore the cell state.

[0134] Dendritic cells stimulate T cell-specific proliferation: Antigen-loaded dendritic cells are co-cultured with T cells to activate the T cells and induce a cytotoxic response against ccRCC target cells. This typically involves incubating dendritic cells with T cells so that the dendritic cells present ccRCC antigens and activate the T cells.

[0135] Prepare effector and target cell co-culture: Co-culture T cells stimulated by dendritic cells with patient-derived ccRCC tumor cells to allow T cells to kill ccRCC target cells.

[0136] Cytotoxicity assay: Use appropriate cytotoxicity assays to assess the cytotoxicity of T cells against ccRCC cells. Commonly used methods include CCK8, flow cytometry, fluorescence microscopy, or radiolabeling.

[0137] Therefore, the present invention also requires T lymphocyte preparation, in vitro stimulation and induction of CTL cells, and corresponding detection.

[0138] B. T lymphocyte preparation

[0139] (1) Single cells isolated from PBMCs of ccRCC patients prepared in process A were placed in a 37°C, 5% CO2 incubator for 2 h, and the suspended cells were collected and prepared into 1 mL cell suspension;

[0140] (2) Add the cell suspension to a nylon wool column incubated at 37°C, lay the column flat, add 200 μL of pre-warmed RPMI 1640 containing 10% FBS to seal the column, and incubate at 37°C for 2 h;

[0141] (3) Wash the nylon wool column with RPMI 1640 containing 10% FBS at a flow rate of approximately 1 mL / min, and collect the initial 10 mL of cell suspension, which is enriched in T cells and NK cells;

[0142] (4) Centrifuge at 700g for 5 minutes at room temperature to collect the bottom cells. Count and adjust the cell concentration to 1×10 7 / mL, placed in RPMI1640 complete medium containing 80IU / mL IL-2 for later use. Alternatively, magnetic bead separation can be used to separate CD3 + T lymphocytes were separated by magnetic beads. The cells were first incubated with monoclonal antibodies against surface antigens for 12 minutes, 7 Each cell was treated with 50 μL of anti-CD3 monoclonal antibody. After washing, the cells were incubated with 100 μL of biotinylated goat anti-mouse secondary antibody for 10 minutes. After washing, 25 μL of FITC-labeled streptavidin was added and the reaction continued for 8 minutes. After washing, biotinylated magnetic particles (100 μL of magnetic particles in the case of anti-CD3 monoclonal antibody) were added and the reaction continued for 8 minutes. After each reaction step, the cells were washed with 1 mL of PBS containing 1% bovine serum albumin and centrifuged at 2000 rpm for 10 minutes. T lymphocytes were isolated by immunomagnetic separation using a magnetic cell separator (MACS).

[0143] C. In vitro stimulation and induction of CTL cells

[0144] Resuspend the tumor cell lysate (OS-RC-2 whole cell lysate) in step 4 with RPMI complete medium, and adjust the density of dendritic cell vaccine (load group) loaded with tumor antigen and dendritic cell vaccine (unload group) not loaded with tumor antigen to 2×10 5 The T lymphocyte suspension separated in step B was adjusted to a density of 2×10 6 100 μL of dendritic cells in the Load group and the Unload group were co-cultured with 100 μL of T lymphocytes in 96-well plates.

[0145] The above experimental groups were all added with the same auxiliary cytokines, IL-2 at 50 U / mL, IL-12 at 150 U / mL, and TNF-α at 100 U / mL. After culturing in a 37°C, 5% CO2 constant temperature and humidity incubator for one week, IL-2 was added at a final concentration of 30 U / mL, and each group was further added with 2×10 5 The cells were stimulated twice and three times with the corresponding dendritic cell vaccines and cultured for three weeks. The cells were collected on the 21st day to obtain CTL cells.

[0146] IFN-γ is an important cytokine secreted by activated CTL cells. It plays a crucial regulatory and effector role in immune responses. IFN-γ secretion can be used as an indicator of CTL activation and cytotoxicity. IFN-γ secretion testing can reveal the following information:

[0147] CTL activation: The secretion of IFN-γ indicates that CTL cells have been activated and indicates that they have mounted an immune response against tumor cells.

[0148] CTL cell function: IFN-γ secretion can be used as an indicator to evaluate the function and effector capacity of CTL cells. It reflects the ability of CTL cells to kill tumor cells and the cellular immune activity.

[0149] Immune monitoring: IFN-γ secretion detection can be used to monitor the immunotherapy effect of CTL cells. During immunotherapy, monitoring IFN-γ secretion can assess CTL cell activity and therapeutic response.

[0150] To test the cytotoxicity of T cells stimulated by dendritic cell vaccines against clear cell renal cell carcinoma cells derived from the corresponding patients, the cells obtained in step C were centrifuged and suspended in RPMI1640 complete medium. The cell concentrations were adjusted and divided into three experimental groups with different effector-target ratios of 5:1, 10:1, and 20:1. The number of T cells per well in each group was maintained at (4-10) x 10 5Added to 96-well culture plates as effector cells;

[0151] The corresponding number of clear cell renal cell carcinoma cells from the same patient was added to each well as target cells, with a final volume of 200 μL. A cell-free blank culture medium control group was also set up, with 5 replicates per well.

[0152] After 24 hours, the free effector cells in each well were aspirated, and the cells were washed twice with PBS. 100 μL of 20 μL CCK8 reagent was added to each well and cultured for another 2 hours. The absorbance (OD) at 450 nm was measured with a microplate reader, and the specific lymphocyte killing rate (%) was calculated.

[0153] The test results are shown in Figure 4. In vitro, T lymphocytes stimulated by dendritic cell vaccines (loaded with tumor antigens) (Load-DC group) were compared with T lymphocytes stimulated by dendritic cells not loaded with tumor antigens (Unload-DC group). By testing the killing activity of the collected cells against ccRCC cells, it was found that among the three experimental groups, the Load-DC group had a significant ccRCC cell-killing effect compared with the Unload-DC group, inhibiting the proliferation of ccRCC cells.

[0154] To detect the secretion of interferon-γ in vitro, the CTL cells of each group obtained in process C above were mixed with the ccRCC tumor cells of the corresponding patients at an effector-target ratio of 20:1 in a U-bottom 96-well plate and cultured for 72 h. The IFN-γ content in the culture supernatant was then detected using an interferon-γ enzyme-linked immunosorbent assay kit according to the instructions.

[0155] The test results are shown in Figure 5. Compared with T lymphocytes stimulated by dendritic cells without tumor antigen loading (Unload-DC group), T lymphocytes stimulated by dendritic cells vaccine (loaded with tumor antigen) (Load-DC group) can produce a large amount of interferon γ, reaching about 1700 pg / ml, which is significantly higher than that of the Unload-DC group.

[0156] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A dendritic cell vaccine, characterized in that: The dendritic cell vaccine comprises a vaccine formed by co-culturing OS-RC-2 whole cell lysate and dendritic cells.

2. An application of a dendritic cell vaccine, characterized in that: The dendritic cell vaccine according to claim 1 is used to prepare a medicament for treating clear cell renal cell carcinoma.

3. A method for preparing a dendritic cell vaccine for clear cell renal cell carcinoma, characterized in that: The preparation method is as follows: (1) Induction of immature dendritic cells: CD14+ cell suspension was transferred to a well plate, 2 x 10 cells per well. 5 -2x10 6 / mL, add human recombinant GM-CSF to a six-well plate at a concentration of 400IU / mL-4000IU / mL, and add human recombinant IL-4 at a final concentration of 200-1000IU / mL to induce the preparation of immature dendritic cells; (2) Induction of mature dendritic cells: Take OS-RC-2 whole cell lysate and co-culture it with the imDC described in step (1), add TNF-α with a final concentration of 200 IU / mL-2000 IU / mL, LPS with a final concentration of 2 ng / mL-2 μg / mL, and Poly(I:C) with a final concentration of 2 ng / mL-2 μg / mL to stimulate the maturation of immature dendritic cells; The amount of OS-RC-2 whole cell lysate used: DC cells correspond to OS-RC-2 whole cell lysate, and the co-culture is carried out at a ratio of DC:OS-RC-2=1:1, 1:3, 1:6 and 1:9.

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