A method for preparing dendritic cell exosome liver cancer vaccine

Abstract

The present invention discloses a method for preparing dendritic cell exosomes as a liver cancer vaccine, wherein exosomes are extracted from the culture supernatant of mature dendritic cells (DC) to prepare an exosome vaccine for liver cancer immunotherapy. The DC exosome vaccine of the present invention is loaded with a liver cancer-specific short peptide hAFP 158‑166 、hGPC3 144‑152 , HMGN1 functional domain (N1ND) that enhances the immunogenicity of dendritic cells, is a non-cellular liver cancer vaccine. The DEX vaccine combines the dual advantages of DC vaccines and exosomes, can be used for liver cancer immunotherapy, and provides a way to improve dendritic cell-based immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and medicine, and specifically relates to a method for preparing dendritic cell exosomes as liver cancer vaccines. Background Art

[0002] Liver malignancies remain a global health threat, with the number of cases expected to exceed one million in 2025. The clinical treatment of liver cancer is not ideal due to its multiple drug resistance mechanisms, complex physiological barriers and immune microenvironment. The occurrence, development, metastasis and recurrence of liver cancer are closely related to the body's immune system. Immunotherapy can activate anti-tumor immune responses by enhancing the body's immune function, thereby inhibiting tumor progression, recurrence and metastasis, and has become a hot topic in clinical tumor research. Liver cancer vaccines can activate anti-tumor immune responses through antigens and effectively inhibit tumor recurrence and metastasis, including cell vaccines, peptide vaccines, dendritic cell vaccines (DC), DNA vaccines, etc. Generally, liver cancer vaccines are insufficient to induce effective immune responses because patients with hepatocellular carcinoma have a long-term chronic liver disease background and their immune function is suppressed. Dendritic cells are used to load tumor antigens and prepare cellular vaccines due to their powerful antigen presentation function. They have been proven to effectively induce antigen-specific immune responses, such as the first DC vaccine approved for prostate cancer. However, after in vitro culture and proliferation, DC cells are rejected by the body and the operation is cumbersome, time-consuming and expensive. In addition, it is impossible to obtain DC vaccines with strong specific immunogenicity, which seriously limits the development and application of DC clinical vaccines. To address this problem, exosome vaccines have been developed, which can co-deliver adjuvants and multi-epitope antigens to lymphoid organs and antigen-presenting cells to a certain extent. Through exosome vaccines, the intracellular release of vaccines and the cross-expression of antigens can be fine-tuned. Exosomes (DEX) secreted by dendritic cells have been shown to carry most of the DC cell surface molecules, especially the tissue compatibility complex (MHC-I and MHC-II) related to immune activation and the costimulatory factors CD80, CD86, CD54, etc. Due to these characteristics, DEX is considered to be a non-cellular biological nano-vaccine that can be used to replace dendritic cells to activate immune responses. DEX vaccines loaded with specific antigen peptides are one of the emerging immunotherapy strategies. As a non-cellular vaccine, DEX vaccine has a clear composition, stable structure, high safety, can be frozen for a long time, and can be mass-produced according to GMP. It overcomes many shortcomings of DC cell vaccines and has more advantages in tumor immunotherapy. However, its side effects and the complexity of its preparation method make it worrying in terms of preparation and application.

[0003] In recent years, liver cancer vaccines have received much attention and have become one of the research hotspots in liver cancer immunotherapy. Liver cancer vaccines, which are constructed based on liver cancer cells, liver cancer antigens, dendritic cells and nucleic acids, stimulate the body's immune system and induce the body to produce an immune response against liver cancer-specific antigens, thereby killing tumor cells expressing liver cancer antigens and producing anti-tumor effects. Clinical trials have found that liver cancer vaccines can reduce the recurrence and metastasis of liver cancer, improve the quality of life of patients, and prolong survival time. They have become an important part of the comprehensive treatment of liver cancer. This article summarizes the research and latest research results of several liver cancer vaccines that have been studied more:

[0004] Liver cancer vaccines mainly include DC vaccines, gene-modified vaccines, high-intensity focused ultrasound (HIFU) solidified vaccines, chemically modified liver cancer cell vaccines, human cell fusion liver cancer vaccines, and cytokine-microparticle liver cancer vaccines. There are many reports on DC vaccines and gene-modified vaccines, but there is currently no liver cancer-specific tumor vaccine that is simple to prepare and has good effects.

[0005] The DC exosome vaccine in the present invention is loaded with liver cancer-specific polypeptides and immune adjuvants, and is a new non-cellular tumor vaccine. The DEX vaccine combines the dual advantages of DC vaccine and exosomes. It is very effective as a liver cancer vaccine and can be used for liver cancer immunotherapy. Summary of the invention

[0006] Based on this, the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a method for preparing dendritic cell exosomes as a liver cancer vaccine.

[0007] To achieve the above object, the technical solution adopted by the present invention includes the following aspects:

[0008] The object of the present invention is achieved as follows: a dendritic cell exosome vaccine, wherein the exosome vaccine is obtained by extracting exosomes from dendritic cells, loading liver cancer specific antigens and immune adjuvants.

[0009] To this end, the present invention provides a dendritic cell exosome vaccine, which includes exosomes derived from dendritic cells, liver cancer-specific antigens and an immune adjuvant.

[0010] Wherein, the liver cancer specific antigen is selected from: hAFP 158-166 Short peptide, its amino acid sequence is: FMNKFIYEI, SEQ ID NO.1; hGPC3 144-152 A short peptide, whose amino acid sequence is: FVGEFFTDV, SEQ ID NO.2;

[0011] Wherein, the immune adjuvant is selected from: N1ND, whose amino acid sequence is:

[0012] MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQ T, SEQ ID NO.3;

[0013] The dendritic cell exosome vaccine of the present invention is connected in the following manner: DEX carries DC major histocompatibility complex (MHC I / II), and MHC I / II can bind to hAFP 158-166 、hGPC3 144-152 Antigenic peptide fragments combine to form MHC I / II-antigenic peptide complexes, which are recognized by T cell receptors (TCR) and presented on the cell surface for T cell recognition and immune response. The highly specific binding of MHC I / II-antigenic peptide complexes to TCR is essential for adaptive immunity. The amino acid sequence of N1ND is connected to the FAM fluorescent group on the left end and the cholesterol polyethylene glycol active ester (Chol-PEG2000) that can be inserted into the plasma membrane on the right end, which can increase drug solubility and stability, reduce the immunogenicity of peptides, and inhibit the nonspecific binding of charged molecules on the modified surface. It is used to modify peptides and other small molecule materials.

[0014] The final structure of the dendritic cell exosome vaccine of the present invention is as follows:

[0015]

[0016] Another object of the present invention is to provide a method for preparing a liver cancer vaccine, comprising the following steps:

[0017] (1) Cultivation of primary dendritic cells: Dendritic cell precursor mononuclear cells obtained from human peripheral blood mononuclear cells (PBMC) were plated in a culture dish and cultured in RPIM 1640 incomplete medium. After the cells adhered to the wall, the medium was changed to serum-free X-VIVO medium and 1000 U / mL rmIL-4 and 1000 U / mL rmGM-CSF induction factors were added to induce the mononuclear cells to differentiate into dendritic cells. Half of the medium was changed every other day.

[0018] (2) Stimulating dendritic cell maturation: On the 5th day, TNF-α was added to the cells obtained in step (1) for stimulation and culture for at least 24 hours, and the mature dendritic cells and supernatant were collected; the concentration of TNF-α was 1000 U / mL.

[0019] (3) Extracting exosome vaccine: extracting exosomes from the cell culture supernatant obtained in step (2),

[0020] (4) Peptide-modified dendritic cell exosomes: hepatocellular carcinoma-specific peptide hAFP 158-166 、hGPC3 144-152 The HMGN1 functional domain N1ND that enhances the immunogenicity of dendritic cells was co-incubated at 0-4°C for 12-24 hours, filtered and sterilized using a vacuum filter to obtain a specific polypeptide-modified liver cancer exosome vaccine.

[0021] Wherein, in step (1), 1000U / mL rmIL-4 and 1000U / mL rmGM-CSF induction factors are added to the cell culture plate to induce the mononuclear cells to differentiate into dendritic cells, and the medium is replaced by half every other day to maintain the concentration of the induction factors in the cell growth environment unchanged; the dendritic cells are used on the 10th day or after further maturation.

[0022] Wherein, in step (1), all culture media contain 100 U / mL penicillin and 100 U / mL streptomycin.

[0023] Wherein, in step (2), the concentration of TNF-α for stimulating dendritic cell maturation is 1000 U / mL.

[0024] Wherein, in step (4), 0.05-0.1 mg / mL of liver cancer specific antigen short peptide hAFP 158-166 、hGPC3 144-152 , and the HMGN1 functional domain N1ND that enhances the immunogenicity of dendritic cells were incubated with dendritic cell exosomes at 0-4°C for 12-24 hours, and then filtered and sterilized to obtain a polypeptide-modified liver cancer exosome vaccine.

[0025] Most preferably, the method for preparing the liver cancer vaccine comprises the following steps:

[0026] The preparation method of the present invention comprises the following steps:

[0027] (1) Cultivation of primary dendritic cells:

[0028] Mononuclear cells were isolated from peripheral blood, 3×10 6 cells / mL cell density was moderate; cultured with RPMI 1640 medium for 2 hours, cells adhered to the wall; after 2 hours, the medium was gently aspirated, and the cells were gently washed once with PBS to remove the suspended cells; the non-adherent lymphocytes in the supernatant were collected, centrifuged at 500×g for 5 minutes, and frozen. The collected lymphocytes can be used for subsequent experiments; the cells were cultured with X-VIVO complete medium, and 1000U / mL rmIL-4 and 1000U / mL rmGM-CSF induction factors were added. Half of the medium was replaced on the second day.

[0029] (2) Stimulate dendritic cell maturation:

[0030] On the 5th day, the medium was completely replaced and TNF-α, which stimulates the maturation of dendritic cells, was added to the X-VIVO medium. On the 8th day, the supernatant secreted by mature dendritic cells could be harvested, and the medium was replaced by half every two days, and the supernatant was collected.

[0031] (3) Extraction of exosomes

[0032] The collected dendritic cell supernatant was centrifuged at room temperature, 500×g for 5 minutes to remove residual cells and debris; the supernatant was transferred to a new centrifuge tube; the supernatant was centrifuged at 4°C, 2000×g for 10 minutes, and large cell secretions and residual debris were removed; the supernatant was transferred to an ultracentrifuge tube with a pipette, and a 40Ti ultracentrifuge was selected. The supernatant was removed and resuspended with 10mL pre-cooled PBS, and a 40Ti ultracentrifuge was selected, and then the supernatant was removed and resuspended with 100μL pre-cooled PBS to obtain exosomes.

[0033] (4) Peptide-modified dendritic cell exosomes

[0034] The exosomes obtained after separation and purification were surface modified with liver cancer specific peptides and immune adjuvants, and 0.05-0.1 mg / mL of liver cancer specific antigen peptide hAFP was added. 158-166 、hGPC3 144-152 , and the HMGN1 functional domain N1ND that enhances the immunogenicity of dendritic cells were incubated with dendritic cell exosomes at 0-4°C for 12-24 hours, and then filtered and sterilized to obtain a polypeptide-modified liver cancer exosome vaccine.

[0035] In the Chinese population, HLA-A2 subtypes account for 53.5% of all HLA-A phenotypes, and among the HLA-A2 subtypes, the most important subtype is HLA-A*0201. Therefore, we selected the HLA-A*0201 restricted epitope as the screening condition. The results showed that the loading of hGPC3 144-152 , hAFP 158-166DCs with epitope antigen peptides can increase the proportion of CD8+T cells in T lymphocyte subsets. High mobility nucleosome binding protein 1 (HMGN1) can help improve DC maturation and activation of exogenous antigens, and continuously induce Th1 immune responses. Therefore, in different tumor models, HMGN1 has been used as a DNA vaccine or in combination with chemotherapeutic agents and activators (or checkpoint inhibitors) to promote the generation of anti-tumor immune responses. However, since HMGN1 needs to be administered intratumorally, the effects of DNA vaccines and combination therapies are very limited, which is not clinically applicable to most tumors. Therefore, HMGN1 needs to be effectively and systematically delivered to cells to release the full therapeutic potential of HMGN1 as an immune adjuvant. The present invention uses dendritic cell exosomes to load the functional N-terminal domain (N1ND) of HMGN1, which enhances the immunostimulatory activity of DC and lymph node migration, thereby accelerating the generation of protective T cell memory responses, which is the reason for activating DC's sustained anti-tumor immunity.

[0036] Explanation of terms and terms:

[0037] Dendritic cells (DCs): The most powerful antigen-presenting cells, named for the many dendritic or pseudopodia-like protrusions they extend when mature. They are the only cells found so far that can activate the proliferation of naive T cells and establish primary immune responses, and they play a key role in initiating anti-tumor immune responses.

[0038] DC vaccine: The patient's own monocytes are cultured in vitro to induce the generation of DCs, which are then loaded with corresponding tumor antigens to make DCs loaded with tumor antigens. These DC cells are then injected into the body to stimulate the proliferation of tumor-killing lymphocytes in the body, exerting long-term tumor surveillance and tumor-killing effects, thereby achieving the purpose of eliminating the tumor.

[0039] Dendritic cell-secreted exosomes (DEX): DC-derived exosomes that carry a large number of characteristic proteins of parental cells, including co-stimulatory molecules, major histocompatibility complex (MHC) / antigenic peptides, and surface expression of interleukin (IL)-15 receptor α (IL-15Rα).

[0040] DEX vaccine: A bio-nano vaccine that can be used to replace DC to activate immune response. It has strong immune properties, such as antigen presentation ability and immune stimulation function. At the same time, exosomes, as non-cellular carriers, have advantages that cell carriers do not have, such as lower cost, more conducive to industrial large-scale production, more stable environment in the body and no problems such as cell division and viral infection. DEX contains all the necessary immune stimulatory elements, such as MHC I (class 1 major histocompatibility complex), MHC II (class 2 major histocompatibility complex), and high levels of co-stimulatory molecules. It may be used as a cell-free carrier to overcome the limitations of DC cell-based tumor vaccines.

[0041] Liver cancer specific antigen: liver cancer specific antigen short peptides including hAFP 158-166 、hGPC3 144-152 . GPC3: (Glypican-3, phosphatidylinositol proteoglycan-3) is specifically and highly expressed in liver cancer tissues, has obvious sensitivity and specificity for liver cancer diagnosis, and can be used as an effective target for liver cancer treatment; AFP: Alpha-fetoprotein, which is closely related to the occurrence and development of liver cancer and various tumors. It is a highly specific and sensitive tumor marker for primary liver cancer and is used for the diagnosis of primary liver cancer and monitoring of therapeutic effects.

[0042] Immune adjuvant: also known as non-specific immunoproliferative agent, refers to auxiliary substances that are injected into the body together with antigens or in advance and can enhance the body's immune response to antigens or change the type of immune response.

[0043] Serum-free X-VIVO medium: does not contain any exogenous growth factors, artificial cell proliferation stimulating factors or additives of unknown ingredients, and does not contain any protein kinase C stimulating factors. It is particularly suitable for the study of the second signaling system of activated human and mouse lymphocytes, and supports the proliferation of CD3+ cells isolated and purified from peripheral blood and humans, as well as the proliferation of dendritic cells, monocytes, lymphocytes, and human lymphocyte cell lines.

[0044] HMGN1: High mobility group nucleosome binding protein 1, an immune adjuvant that can help improve DC maturation and activation of exogenous antigens and continuously induce Th1 immune response.

[0045] The present invention utilizes dendritic cell exosomes as carriers to load liver cancer-specific antigen peptides, thereby achieving efficient lymph node enrichment and significant immune activation of the liver cancer vaccine, achieving effective antigen presentation to activate immunity, clear tumors, and prevent tumor metastasis and recurrence.

[0046] The preparation method of the present invention is simple. After the polypeptide and dendritic cell exosomes are co-incubated for 12-24 hours under 0-4°C conditions, the polypeptide and exosomes are combined, which simplifies the vaccine preparation method, reduces the vaccine preparation cost, and reduces toxic side effects.

[0047] In summary, the beneficial effects of the present invention are:

[0048] The present invention has been experimentally confirmed that exosomes can effectively enrich surface antigens CD80, CD86, CD54 and the expression of antigen presenting molecules MHC, thereby promoting the proliferation and activation of T cells, regulating the tumor microenvironment, and effectively inhibiting tumor growth and metastasis.

[0049] The dendritic cell exosome vaccine of the present invention is loaded with liver cancer-specific polypeptides and is a non-cellular liver cancer-specific vaccine. The DEX vaccine combines the strengths of DC vaccines and the unique advantages of exosomes. It is very effective as a tumor vaccine and can be used for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the morphology of mature dendritic cells after stimulation with TNF-α;

[0051] Figure 2 The extracted dendritic cell exosomes were identified by transmission electron microscopy;

[0052] Figure 3 The particle size and particle concentration of exosomes detected by nanoflow cytometry:

[0053] Figure 3 A. Nano-flow cytometry detection of changes in the number of exosomes in dendritic cells;

[0054] Figure 3 B Scattering signal diagram of exosomes;

[0055] Figure 3 C Exosome particle size distribution;

[0056] Figure 3 D Exosome particle size;

[0057] Figure 4 Immune co-stimulatory molecules on the surface of dendritic cell exosomes detected by Amnis imaging flow cytometry:

[0058] Figure 4 A. Flow cytometry was used to detect the expression level of CD86 on the surface of DEX;

[0059] Figure 4 B. Flow cytometry was used to detect the expression level of CD80 on the surface of DEX;

[0060] Figure 4 C Flow cytometry was used to detect the expression level of MHC-II (HLA-DR) on the surface of DEX;

[0061] Figure 4D Flow cytometry was used to detect the expression level of MHC-I (HLA-ABC) on the surface of DEX;

[0062] Figure 4 E. Flow cytometry was used to detect the expression level of CD54 on the surface of DEX;

[0063] Figure 5 Amnis imaging flow cytometry for DEX and hAFP 158-166 、hGPC3 144-152 , N1ND three short peptide binding efficiency:

[0064] Figure 5 A Flow Cytometry Analysis of DEX and hAFP 158-166 Short peptide binding efficiency;

[0065] Figure 5 B Flow cytometry detection of DEX and hGPC3 144-152 Short peptide binding efficiency;

[0066] Figure 5 C flow analysis technology detects the binding efficiency of DEX and N1ND connected with FAM fluorescent group;

[0067] Figure 5 DEX and hAFP 158-166 、hGPC3 144-152 Quantitative analysis of short peptide binding efficiency;

[0068] Figure 6 The expression of IFN-γ was detected by flow cytometry:

[0069] A. Flow cytometry was used to detect the expression of intracellular cytokine IFN-γ in autologous T cells (NC);

[0070] B Flow cytometry was used to detect the expression of intracellular cytokine IFN-γ in T cells after the DEX group (DEX) and autologous T cells were co-incubated for 12 hours;

[0071] C Flow Cytometry Detection of DEX AGN Group (DEX AGN ) and autologous T cells for 12 hours, the expression of intracellular cytokine IFN-γ in T cells;

[0072] D Control group (NC), DEX group (DEX), DEX AGN Group (DEX AGN ) and autologous T cells for 12 hours, the expression of intracellular cytokine IFN-γ in T cells (statistical graph), (n=3, *P<0.05, **P<0.01, ***P<0.001).

[0073] Figure 7 The control group (NC), DEX group (DEX), and DEX AGN Group (DEX AGN ) and autologous T cells for 12 hours, and T cells and human liver cancer cell line Hep G2 were co-cultured for 48 hours. The killing of cancer cells by T cells is as follows:

[0074] A The control group (NC), DEX group (DEX), and DEX group observed under a fluorescence microscope AGN Group (DEX AGN ) The state of activated T cells after co-culture with human liver cancer cell line Hep G2 for 48 hours, multiple: 20 times B DEX, DEX AGN Stimulated T cells were co-cultured with liver cancer cells HepG2 to test the killing percentage of HepG2 cells (n=3, *P<0.05, **P<0.01, ***P<0.001) DETAILED DESCRIPTION

[0075] In order to make those skilled in the art have a clear understanding of the present invention, the following is a detailed description using specific examples. The methods used in each embodiment are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0076] Example 1: Liver cancer exosome vaccine

[0077] The materials and sources used in the following examples are as follows: RPMI 1640 medium (Gibco);

[0078] Serum-free X-VIVO medium (Lonza); H-MHC II (BD); H-MHC I (BD); CD63 (BD); CD81 (BD); H-CD54 (BD); H-CD80 (BD); H-CD86 (BD); granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech, USA); tumor necrosis factor-α (TNF-α) (PeproTech, USA); interleukin-4 (IL-4) (PeproTech, USA).

[0079] The preparation method of the present invention comprises the following steps:

[0080] 1) Dendritic cell culture

[0081] 1. Isolate mononuclear cells from peripheral blood, 3×10 6 cells / mL cell density is moderate.

[0082] 2. Culture the cells in RPMI 1640 medium for 2 hours to allow the cells to adhere.

[0083] After 3.2 hours, gently aspirate the medium and wash once with PBS to remove suspended cells.

[0084] 4. Collect the non-adherent lymphocytes in the supernatant, centrifuge at 500×g for 5 minutes, and freeze. The collected lymphocytes can be used for subsequent mixed leukocyte reaction experiments.

[0085] 5. Culture the cells with X-VIVO complete medium (containing GM-CSF and IL-4), change half the medium on the second day, and change the full medium on the fifth day and add TNF-α to the X-VIVO medium to stimulate the maturation of dendritic cells.

[0086] 6. On the 8th day, the supernatant secreted by mature dendritic cells can be harvested. The medium is replaced by half every two days and the supernatant is collected. Characterization of the obtained mature dendritic cells shows that the cells after 8 days have obvious dendritic morphology and grow in clusters. The results are as follows Figure 1 As shown. Under transmission electron microscopy, DEX presents a typical round cup-shaped double-layer membrane structure with a diameter of less than 100 nm ( Figure 2 ). Nanoparticle tracking analysis uses a laser light source to illuminate the nanoparticle suspension and detect the scattered light of the nanoparticles. The concentration of nanoparticles is calculated by counting the number of scattered particles. When the extracted DEX is identified under nanoparticle tracking analysis, it can be found that the entire particle is concentrated within 100nm, with a peak of 94.5nm, and the detected particle density is 6.0×10 6 particles / ml( Figure 3 ).

[0087] 2) Extraction of exosomes

[0088] The collected dendritic cell supernatant was centrifuged at room temperature, 500×g, 5min, to remove residual cells and debris; the supernatant was transferred to a new centrifuge tube, taking care not to suck up the bottom precipitate; 4℃, 2000×g, 10min, centrifuged to obtain the supernatant, remove large cell secretions and residual debris; 10000×g centrifuged for 30min to remove larger vesicles, and the supernatant was transferred to an ultracentrifuge tube with a pipette, and an ultracentrifuge rotor was selected, and the supernatant was removed and resuspended with 10mL pre-cooled PBS, and an ultracentrifuge rotor was selected, and then centrifuged again at 110000×g for 70min, and the supernatant was removed and resuspended with 100μL pre-cooled PBS to obtain exosomes. Exosomes can be stored for a long time in a -80℃ refrigerator, or in a 4℃ refrigerator for up to one week. The obtained exosomes were characterized, and the results are as follows. Figure 2As shown. By imaging flow cytometry, DC cell surface molecules such as histocompatibility complex (MHC-I and MHC-II) related to immune activation and co-stimulatory factors CD80, CD86, and CD54 were enriched on DEX. The results are shown in Figure 4 shown.

[0089] 3) Peptide-modified dendritic cell exosomes

[0090] The exosomes obtained after separation and purification were surface modified with liver cancer specific peptides and immune adjuvants. 0.05-0.1 mg / mL of liver cancer specific antigen peptide hAFP 158-166 、hGPC3 144-152 , and the HMGN1 functional domain N1ND that enhances the immunogenicity of dendritic cells were co-incubated with dendritic cell exosomes at 0-4°C for 12-24 hours, and then filtered and sterilized to obtain a peptide-modified liver cancer exosome vaccine. The exosomes after co-incubation were detected by imaging flow cytometry for hAFP 158-166 、hGPC3 144-152 The binding efficiency of three short peptides N1ND is shown in the following figure. Figure 5 shown.

[0091] Embodiment 2:

[0092] DEX AGN Evaluation of immune activation ability

[0093] 1. Obtaining human lymphocytes: After the cells extracted from human PBMC adhere to the wall for two hours, the suspended cells that are not attached to the wall are lymphocytes. The separated peptide-modified liver cancer exosomes are co-cultured with the corresponding lymphocytes.

[0094] 2. In vitro T cell activation ability: Figure 6 The intracellular factor IFNγ was detected by flow cytometry to analyze the in vitro T cell activation ability. AGN After 12 hours of co-incubation, the cells were resuspended by centrifugation and labeled with fluorescent antibodies for CD3+T cells, CD4+T cells, and CD8+T cells, respectively. After staining for 30 minutes, the cells were washed, 200ul of permeabilization solution was added, the membrane was permeated and fixed at 4°C for 30 minutes, washed twice with washing solution, 500×g, resuspended by centrifugation, and IFN-γ was labeled with fluorescent antibodies. After staining for 30 minutes, the cells were washed and resuspended, and detected by flow cytometry. The results showed that DEX and DEX AGN Compared with the control group, significantly more IFN-γ was secreted, which has the ability to activate the immune system. DEX and DEX AGN Can effectively enhance the immune function of T cells, such as Figure 6 shown.

[0095] Embodiment 3:

[0096] DEX AGN Antigen-specific killing response

[0097] Detecting the tumor-killing ability of human lymphocytes: hAFP 158-166 The positive hepatocellular carcinoma cell line HepG2 cells were used as target cells (T) and compared with autologous DEX and DEX AGN The co-cultured lymphocytes are effector cells (E), and the co-culture is carried out at a ratio of T:E = 1:20 for 48 hours. The lactate dehydrogenase content in the culture supernatant is detected, which is the extent of tumor cell killing by effector cells, DEX AGN Immunized autologous T cells can significantly kill HepG2 cells. Figure 7 shown.

[0098]

Claims

1. A method for preparing a dendritic cell exosome vaccine, comprising the following steps: (1) Cultivation of primary dendritic cells: Mononuclear cells were isolated from peripheral blood, 3×10 6 cells / mL The cell density is moderate; culture with RPMI 1640 medium for 2 hours to allow cells to adhere; after 2 hours, gently aspirate the medium and gently wash once with PBS to remove suspended cells; Collect the non-adherent lymphocytes in the supernatant, centrifuge at 500×g for 5 min, and freeze. The collected mixed lymphocytes can be used for subsequent experiments; culture the cells with X-VIVO complete medium, add 1000 U / mL rmIL-4 and 1000 U / mL rmGM-CSF induction factors, and change half of the medium on the second day; (2) Stimulate dendritic cell maturation: On the 5th day, the medium was completely replaced and TNF-α, which stimulates the maturation of dendritic cells, was added to the X-VIVO medium. On the 8th day, the supernatant secreted by mature dendritic cells could be harvested, and the medium was replaced by half every two days, and the supernatant was collected. (3) Extraction of exosomes: The collected dendritic cell supernatant was centrifuged at room temperature, 500×g for 5 minutes to remove residual cells and debris; the supernatant was transferred to a new centrifuge tube; the supernatant was centrifuged at 4°C, 2000×g for 10 minutes, and the large cell secretions and residual debris were removed; the supernatant was transferred to an ultracentrifuge tube with a pipette, and a 40Ti ultracentrifuge was selected. The supernatant was removed and resuspended with 10 mL of pre-cooled PBS, and a 40Ti ultracentrifuge was selected. The supernatant was removed and resuspended with 10 mL of pre-cooled PBS, and then the supernatant was removed and resuspended with 100 μL of pre-cooled PBS to obtain exosomes. (4) Peptide-modified dendritic cell exosomes: The exosomes obtained after separation and purification were surface modified with liver cancer specific peptides and immune adjuvants, and 0.05-0.1 mg / mL of liver cancer specific antigen short peptide hAFP was added. 158-166 、hGPC3 144-152 , and the HMGN1 functional domain N1ND that enhances the immunogenicity of dendritic cells are co-incubated with dendritic cell exosomes at 0-4°C for 12-24 hours, filtered and sterilized to obtain a peptide-modified liver cancer exosome vaccine; Among them, hAFP 158-166 A short peptide, whose amino acid sequence is as shown in SEQ ID NO.1; Among them, hGPC3 144-152 A short peptide, whose amino acid sequence is as shown in SEQ ID NO.2; The immunoadjuvant N1ND has an amino acid sequence as shown in SEQ ID NO.3; Among them, the connection method of each part of the dendritic cell exosome vaccine is as follows: DEX carries DC major histocompatibility complex MHC I / II, MHC I / II and hAFP 158-166 、hGPC3 144-152 Antigen peptide fragments combine to form MHC I / II-antigen peptide complexes, which are recognized by T cell receptors and presented on the cell surface for T cells to recognize and trigger immune responses. Among them, the amino acid sequence of N1ND has a FAM fluorescent group connected to the left end and a cholesterol polyethylene glycol active ester that can be inserted into the plasma membrane connected to the right end.

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