CN107496912B - Antigen presenting signal group combining hepatitis B virus antigen peptide with liver cancer cell antigen information and application thereof - Google Patents
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Abstract
The invention discloses an antigen presenting signal group of Hepatitis B Virus (HBV) antigen peptide combined with liver cancer cell antigen information and application thereof, wherein the signal group consists of an HBV core antigen 18-27 peptide segment, an HBV pre-S2 antigen 44-53 peptide segment and a human liver cancer cell strain HepG2 lysate, and the mass ratio of the HBV core antigen 18-27 peptide segment, the HBV pre-S2 antigen 44-53 peptide segment and the human liver cancer cell strain HepG2 lysate is 0.5-1:0.5-1:1 to 2. The DC cell induced by the signal group becomes a DC vaccine carrying multiple specific hepatitis B virus related liver cancer antigen signals, and can activate the proliferation and the function of tumor specific T cells, thereby strengthening the effect of the tumor specific T cells on resisting hepatitis B liver cancer.
Description
Technical Field
The invention relates to the technical field of specific cellular immunotherapy of hepatitis B virus-related liver cancer, in particular to an antigen presenting signal group combining hepatitis B virus antigen peptide and liver cancer cell antigen information and application thereof, in particular to application of activating dendritic cells to induce T cells.
Background
Compared with the continuous decline of the overall mortality of the cancer in the world, the incidence and mortality of the liver cancer rise year by year, and the latest epidemiological data show that the incidence of the liver cancer is 78.2 million people, the death cases are 74.5 ten thousand, the average 5-year survival rate is lower than 5 percent, and the overall survival time is only 10 months; china accounts for 55 percent of liver cancer worldwide and becomes the country with the most number of liver cancer diseases and deaths, more than 85 percent of patients have HBV infection history, and almost all cancer cells contain HBV genes. However, the progress of treatment means is not great in 20 years, common methods include operations (tumor resection and transplantation) and local treatment (radio frequency, ablation, microwave, freezing, absolute alcohol injection, hepatic artery chemoembolization and TACE), 50-60% of patients have already reached the middle and late stages in initial diagnosis, no operation chance exists, the survival rate of the local treatment curative effect is increased by 14% at most in 2 years compared with that of the patients without treatment, and the liver failure is caused by serious patients with certain toxic and side effects, so that the liver cancer becomes a heavy economic burden and a health killer of people in China.
Traditional anticancer drugs are basically ineffective when tumor resistance recurs and progresses, and have high cytotoxicity, and the problem can be avoided to a certain extent by antitumor immunotherapy, wherein tumor-specific T cells have the most potential, T cells can find tumors for attack through 'wandering inspection' in blood, and recent research proves that the cancer T cell immunotherapy can continuously relieve some hematological tumors and improve the overall survival rate; however, the low content of T cells in tumors limits the successful expansion of immunotherapy. The core theory of tumor immunology indicates that Dendritic Cells (DCs) which are responsible for the maximum antigen presentation function of human bodies are key promoters of T cell activation and proliferation, play an important role in the stages of T cell maturation, differentiation and proliferation, and the activated DCs can effectively activate the anti-tumor immune effect of the T cells, so that the function of the T cells is enhanced by restoring the DCs. In recent years, some clinical studies utilize vaccines to treat middle and late stage tumors, including in vitro DC loaded with tumor antigens for reinfusion or inducing in-tumor DC to present endogenous antigens, suggesting that improving antigen presentation and promoting functional T cells have certain potential and prospect.
According to the core theory of chronic HBV infection, the body is immune tolerant to virus and the hepatitis B liver cancer has a large amount of lymphocyte infiltration, which suggests that the control of tumor progression and the attack/elimination of cancer cells depend on a sufficient number of powerful specific T cells, and the treatment of hepatitis B virus-associated liver cancer (HBV-HCC) by generating or enhancing immune response has huge potential. At present, the curative effect of clinical research on immunotherapy around some cancers is initially shown, but because the interaction between tumor cells and immune cells and the mutual balance are very obvious, the number and the function of induced specific T cells are not complete, and the specific T cells cannot well play an inhibiting role in solid tumors. The main technical difficulties are as follows: (1) HBV-HCC has highly heterogeneous tumor antigens, has diversity, complexity and variability, and can help cancer cells to easily escape from the monitoring of the immune system; (2) the immunosuppressive microenvironment formed inside HBV-HCC makes it difficult for immune cells to break their protective net.
Disclosure of Invention
The invention aims to solve the main technical difficulties of poor curative effect of the existing immunotherapy of solid tumors represented by liver cancer, including the antigen characteristics of high heterogeneity, diversity, complexity and variability of the solid tumors, and provides a group of antigen presenting signal groups (COST) of hepatitis B virus antigen peptides combined with liver cancer cell antigen information. The DC cell induced by the signal group becomes a DC vaccine (COST-DC) carrying multiple specific hepatitis B virus related liver cancer antigen signals, and can activate the proliferation and the function of tumor specific T cells, thereby strengthening the effect of the tumor specific T cells on resisting hepatitis B liver cancer.
Another objective of the invention is to provide a DC vaccine carrying multiple antigen signals of hepatitis B virus-associated liver cancer.
It is still another object of the present invention to provide a killer T cell for hepatitis B and liver cancer.
It is still another object of the present invention to provide a COST-DC-CTL cell preparation for treating hepatitis B and liver cancer.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a group of antigen presenting signal groups of hepatitis B virus antigen peptide combined with liver cancer cell antigen information comprises HBV core antigen 18-27 peptide segments, HBV pre-S2 antigen 44-53 peptide segments and human liver cancer cell strain HepG2 lysate, wherein the mass ratio of the HBV core antigen 18-27 peptide segments, the HBV pre-S2 antigen 44-53 peptide segments and the human liver cancer cell strain HepG2 lysate is 0.5-1:0.5-1:1 to 2.
The HBV core antigen 18-27 peptide segment is abbreviated as HBcAg, the amino acid sequence thereof is FLPSDFFPSV, HBV pre-S2 antigen 44-53 peptide segment is abbreviated as HBPreS2, the amino acid sequence thereof is SILSKTGDPV, and the lysate of the human liver cancer cell strain HepG2 is abbreviated as HepG 2-Lys. Hepatitis B virus related liver cancer may also be referred to as hepatitis B liver cancer.
The DC vaccine carrying multiple specific hepatitis B virus related liver cancer antigen signal is prepared through adding the hepatitis B virus antigen peptide and the antigen presenting signal group of liver cancer cell antigen information into mature mocC cell separated from peripheral blood and cultured to obtain the vaccine.
A DC vaccine carrying multiple specificity hepatitis B virus related liver cancer antigen signals is prepared by the following steps: adding the antigen presenting signal group (COST) of the hepatitis B virus antigen peptide combined with the antigen information of the liver cancer cell into the mature modC cell cultured to the 6 th day by using AIM-V culture solution, wherein the final concentration of the COST is 24ug/ml, supplementing the AIM-V culture solution the next day, and recovering and obtaining the vaccine after culturing for 2 days.
A killer T cell for hepatitis B and liver cancer comprises the following specific steps: the DC vaccine carrying multiple specific hepatitis B virus related liver cancer antigen signals prepared in the above way is subjected to adoptive culture with T cells separated from peripheral blood, and then hepatitis B liver cancer killer T cells (COST-CTL) can be obtained.
A killer T cell for hepatitis B and liver cancer comprises the following specific steps: the number of the cells is 2-5 x106The DC vaccine carrying the antigen signals of the multiple specificity hepatitis B virus related liver cancer is subjected to adoptive culture with T cells at 8 days after separation and amplification from peripheral blood, wherein the number of the T cells is 2-5 x107At 37 ℃ and 5% CO2Co-culturing in an incubator for 2 days, namely 10 days; supplementing 5-10ml of AIM-V culture solution, wherein the culture solution contains IL-2 with the concentration of 300 IU/ml and 1.5ml of albumin with the mass volume concentration of 20%; day 12: supplementing 5-10ml of AIM-V culture solution, wherein the culture solution contains IL-2 with the concentration of 300 IU/ml and albumin with the mass volume concentration of 20% by 1.5 ml; day 14: obtaining viral tumorsAntigen co-activated DC induces specific T cell to obtain hepatitis B liver cancer killing T cell (COST-CTL).
A COST-DC-CTL cell preparation for treating hepatitis B liver cancer is prepared by the following steps: mixing the obtained killer T cells of hepatitis B liver cancer with the DC vaccine carrying the antigen signals of the multiple specificity hepatitis B virus related liver cancer, and transferring the mixture into physiological saline containing 1% human serum albumin to obtain the COST-DC-CTL cell preparation for treating the hepatitis B liver cancer.
A COST-DC-CTL cell preparation for treating hepatitis B liver cancer is prepared by mixing the obtained hepatitis B liver cancer killer T cells with a DC vaccine carrying antigen signals of multiple specific hepatitis B virus related liver cancer in a cell number ratio of 8-18: 1 (namely 8-18 COST-CTL cells are matched with 1 COST-DC cell) to obtain 'multi-weapon combined antigen presentation signal group DC vaccine induced specific tumor killer T cells (COST-DC-CTL)', and transferring 100ml of physiological saline containing 1% of human serum albumin to prepare the cell preparation for treatment.
More preferably, the ratio of the number of mixed cells of the hepatitis b liver cancer killer T cells and the DC vaccine carrying the antigen signals of the multiple specific hepatitis b virus-associated liver cancer is 10: 1.
when the specific COST-DC-CTL cell preparation prepared by the invention is used for treating liver cancer patients, the specific application method is that the specific COST-DC-CTL cell preparation is directly infused into tumors through a hepatic artery route or is back infused through a peripheral vein route.
In order to maximize the therapeutic effect of COST-DC-CTL cell preparation, the tumor microenvironment of the patient can be improved 2 days before the return transfusion by using 250mg/m Cyclophosphamide (CY)21 time of intravenous drip; cell return can be administered systemically by intravenous infusion or locally by hepatic artery infusion into the patient.
The invention provides a scheme for treating hepatitis B virus-related liver cancer, which particularly comprises the following steps of strengthening HBV-HCC specific immunity: in vitro multiple antigen-loaded DCs: after inducing peripheral blood mononuclear cell derived dendritic cells (modC), co-stimulating the modC by using two groups of HBV antigen peptides (HBcAg and PreS 2) and human liver cancer cell strain (HepG 2) lysate (containing a plurality of liver cancer antigens: AFP, GPC3 and PCA) to obtain activated DC cells which are directionally induced to mature. In vivo self tumor antigen stimulation of DC: surgery or local treatment can promote the release of the tumor antigen of the patient and provide more specific immunity source for DC; treatment produces DNA damage and inflammation that amplifies antigen presentation. ③ treatment with Cyclophosphamide (CY) can improve the immunosuppressive internal environment of tumor.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a group of antigen presenting signal groups (COST) combining hepatitis B virus antigen peptides with hepatoma cell antigen information for the first time. The DC cell induced by the signal group becomes a DC vaccine (COST-DC) carrying multiple specific hepatitis B virus related liver cancer antigen signals, and can activate the proliferation and the function of tumor specific T cells, thereby strengthening the effect of the tumor specific T cells on resisting hepatitis B liver cancer.
Drawings
The upper panel in FIG. 1 is a mature DC cell (flow cytometry results); the lower panel shows the expression rate of the DC maturation marker.
FIG. 2 shows that mature DCs can express multiple hepatoma protein antigens and hepatitis B virus genes simultaneously.
FIG. 3 shows ELISPOT detection of high cytokine secretion and expansion of T cells cultured in vitro.
FIG. 4 shows the inhibition of Trges by cyclic phosphoramides (CY).
FIG. 5 shows that mature DCs from normal liver and peripheral blood have higher T cell activating effect than DCs from cancer tissues.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Preparation of AIM-V Medium: the medium contained IL-4 (interleukin 4) at a concentration of 1000IU/ml and GM-CSF (colony stimulating factor) at a concentration of 1000 IU/ml.
Example 1
1. And (3) separating and performing amplification culture on the peripheral blood DC and the T cells.
(1) Obtaining peripheral blood mononuclear cells: the method comprises the steps of operating according to a program set by a specification by using an mcs + portable blood cell collector, circulating peripheral blood by 2000-2500 ml, collecting peripheral blood mononuclear cells by 50ml, centrifuging by a 5810R centrifuge for 20 minutes at 500G, collecting upper plasma, subpackaging into 1.5ml centrifuge tubes, and freezing and storing as conventional biochemical detection. Physiological saline 1 for lower layer cells: 1 diluting, transferring into a 50ml centrifuge tube containing 25ml lymphocyte separation liquid, centrifuging for 30 minutes by using a 5810R centrifuge of 450G, carefully absorbing a middle white cell layer by using a disposable pipette, transferring into a 50ml centrifuge tube containing 40ml physiological saline, gently and uniformly blowing, centrifuging for 15 minutes by 250G, discarding supernatant, adding 45ml physiological saline, gently and uniformly blowing, centrifuging for 15 minutes by 250G, and discarding supernatant; after repeating the washing 3 times, AIM-V medium was added and the cells were counted.
(2) DC and T cell isolation: adding AIM-V culture medium 4ml into T25 culture flask, and packaging separated mononuclear cells into culture flask with cell concentration controlled at 2x106Per ml, 37 ℃ and 5% CO2And (4) incubating for 120min in a saturated humidity incubator, wherein the suspended cells are T cells, and the rest adherent cells are mononuclear cells (immature DC).
(3) Monocyte induction dendritic cell (moDC) maturation culture: adding 4ml of AIM-V culture medium into adherent cells, wherein the culture medium contains IL-4 with the concentration of 1000IU/ml and GM-CSF with the concentration of 1000 IU/ml; after 3 days, 2ml of AIM-V culture medium was added, and the mixture was incubated at 37 ℃ with 50% CO2The cells were cultured in an incubator to day 6 to mature into monocyte-induced dendritic cells (modcs). See figure 1, upper panel: flow cytometry techniques confirmed the DC maturation marker; the following figures: DC were confirmed to successfully induce and express multiple epitopes.
(4) T cell expansion culture: transferring the suspension cells, i.e., T cells, to a T75 flask containing 4ml of AIM-V medium, and washing with AIM-V medium 3 times in an amount of 2 ml/time(ii) a Then adding OKT3 to a final concentration of 1. mu.g/ml and anti-CD 28 to a final concentration of 1. mu.g/ml, and placing at 37 ℃ with 50% CO2Culturing in an incubator; day 2: the T cells are supplemented with 5-10ml of AIM-V culture solution, wherein the culture solution contains IL-2 with the concentration of 300 IU/ml and 1.5ml of albumin with the mass volume concentration of 20%; day 4: the T cells were supplemented with 5-10ml of AIM-V medium containing 300 IU/ml of IL-2 and 1.5ml of albumin at a concentration of 20% by volume, and continued at 37 deg.C with 50% CO2Culturing in an incubator until day 8 to obtain proliferated T cells.
2. The preparation of the dual hepatitis B virus antigen peptide co-stimulation stock solution is carried out in a biological safety cabinet.
(1) Taking 1mg of hepatitis B core antigen (HBcAg) 18-27 peptide fragment (amino acid sequence: FLPSDFFPSV) and 1mg of hepatitis B surface pre-S2 antigen (HBPreS 2) 44-53 (amino acid sequence: SILSKTGDPV Australia, Mimotopes Pty Ltd) powder from a low-temperature refrigerator at-80 ℃, throwing the powder to the bottom of a tube by high-speed centrifugation, adding 25ul of DMSO into each tube, diluting to 40mg/ml of preservation solution, and preserving in a refrigerator at-80 ℃ to obtain HBcAg stimulation stock solution and HBPreS2 stimulation stock solution.
(2) Taking out 1.5ul HBcAg stimulating stock solution and 1.5ul HBPreS2 stimulating stock solution, and mixing to obtain HBV co-stimulating stock solution; supplementing AIM-V to 5ml, working concentration of peptide fragment is 24ug/ml, packaging, and storing at 4 deg.C.
3. The preparation of multiple liver cancer antigen peptide stimulating stock solution, all operations are carried out in a biological safety cabinet.
(1) HepG2 cell line culture: hepatoma cell line HepG2 was purchased from Shanghai cell bank of Chinese academy of sciences, DMEM with 10% FBS at 37 ℃ and 5% CO2The cells are cultured in the cell culture box, and the cells are observed to grow to about 90 percent of the area of the culture bottle, so that the next experiment can be carried out.
(2) Preparing a protein lysate: working on ice, in a bottle of 25cm2The flask of (3) was then filled with 250ul of RIPA in a 1.5ML centrifuge tube, and the protease inhibitor cocktail was added and mixed until the final concentration of cocktail was 0.4 mg/ML.
(3) Cell lysis, cell protein lysate extraction: the cell supernatant in the flask was decanted, washed twice with pre-cooled HBSS, drained and then 250ul of protein lysate was added and left on ice for 15 minutes. The cells were scraped off with a pipette tip and the cell protein lysate was collected in a sterile 1.5ML centrifuge tube. Placing the centrifugal tube in a precooled refrigerated centrifuge, balancing, centrifuging at 4 ℃ and 14000 RPM for 15 minutes, and taking out to see that white protein residues are precipitated at the bottom of the tube. The supernatant was taken to another sterile 1.5ML centrifuge tube and placed on ice to stand.
(4) BCA method for measuring protein concentration of cell protein lysate: the protein quantitative standard solution (2 mg/ml) is diluted by PBS step by step to have the concentration of 0, 125, 250, 500, 1000 and 2000 ng/ul. Calculating the total amount of the detection solution according to the number of samples to be detected, arranging a plurality of holes for the samples to be detected, and preparing the detection solution according to an accurate proportion: solution A1X, and solution B50X. The assay solution was added to a 96-well plate at 200ul per well. Then, 10ul of protein quantitative standard solution and protein sample solution were added to each well. The added 96-well plate was placed in an incubator at 37 ℃ for 30 minutes and then detected in a microplate reader.
(5) Dilution and filtration of protein samples: the stock solution was diluted with the prepared protein lysate, the protein lysate was aspirated out using a 5ML syringe and filtered through a 0.22um filter to a final concentration of 24ug/ML (HepG 2-Lys), aliquoted and stored at 4 ℃.
4. The configuration of the multi-weapon combination antigen presenting signal group (COST) and the preparation of COST-DC vaccine, all operations were performed in a biosafety cabinet.
(1) Multiple arms combination antigen presentation signal group (COST) configuration: taking HBcAg antigen peptide stock solution and HBPreS2 antigen peptide stock solution, and diluting the stock solution from the stock solution concentration of 40mg/ml to the working concentration of 24 ug/ml; diluting HepG2-Lys stock solution obtained in step 4 from a stock solution concentration of 100mg/ml to 24 ug/ml; the COST formula is as follows: HBcAg + HBPreS2 + HepG 2-Lys; the compatibility is as follows: HBcAg PreS2 HepG2-Lys =0.5-1:0.5-1: 1-2; the working concentration was 24 ug/ml.
(2) Preparation of a DC vaccine carrying a multi-weapon combination antigen presenting semaphore (COST-DC): adding COST into MODC cell matured at 6 days, culturing to obtain final concentration of 24ug/ml, supplementing AIM-V culture solution the next day, culturing for 2 days, recovering at 8 days to obtain COST-DC vaccine, detecting CD83, CD86 and HLA-DR expression with flow cytometry, partially preparing specific killer T Cell (CTL) for hepatitis B liver cancer, and partially freezing with liquid nitrogen. FIG. 2 shows that mature DCs induced by COST can successfully carry hepatitis B virus antigen and liver cancer antigen simultaneously.
5. Preparation of killer T cells for hepatitis b liver cancer, all operations were performed in a biosafety cabinet: taking a COST-DC vaccine, wherein the number of cells is 2-5 x106The number of T cells is 2-5 x10 with the T cells on the 8 th day after the expansion7Adoptive culture at 37 deg.C with 5% CO2Co-culturing in an incubator for 2 days, namely 10 days; supplementing 5-10ml of AIM-V culture solution, wherein the culture solution contains IL-2 with the concentration of 300 IU/ml and 1.5ml of albumin with the mass volume concentration of 20%; day 12: supplementing 5-10ml of AIM-V culture solution, wherein the culture solution contains IL-2 with the concentration of 300 IU/ml and albumin with the mass volume concentration of 20% by 1.5 ml; day 14: the obtained virus tumor antigen co-activated DC induces specific T cells, namely polyclonal T cells (COST-CTL) capable of coping with liver cancer cells are obtained. FIG. 3: the polyclonal T cell of the prepared specific liver cancer cell can induce anti-tumor immune factor IFN-r and CD8+ T cell aiming at the cancer cell.
6. Preparation of COST-DC-CTL mixed cell preparation: on day 13, COST-DC vaccine 0.5-1 x10 was removed from liquid nitrogen7Recovering, washing to remove cell debris, and culturing for 1 day while controlling cell number at 2 × 106(ii) a The quantity obtained in the step 5 is 0.5-1 x108 COST-CTL mixing, the total amount of cells is controlled to be 1-2 x108(ii) a Adding into a 50ml centrifuge tube, washing with normal saline for 3-6 times, suspending in 10ml normal saline, adding 20% albumin 1.5ml to prevent ion adhesion, adding into 100ml normal saline with a syringe to obtain COST-DC-CTL cell preparation for treatment, and storing at 4 deg.C. FIG. 5 functional validation of T cells: DC cells in peripheral blood and normal liver tissue sources of a liver cancer patient are activated by COST to induce T cell functions which are obviously higher than those induced by DC cells from cancer tissue sources.
Therapeutic indications and contraindications for COST-DC-CTL cell preparations are as follows:
the method is suitable for people:
(1) the method can be used by liver cancer patients in any state, and the treatment course of the scheme is individualized and comprises the following steps:
(2) the initial diagnosis is a patient with early-stage small liver cancer and a patient who can be subjected to tumor resection, and a personalized specific immune cell treatment course is formulated after the operation, so that the tumor recurrence and metastasis can be prevented.
(3) The initial diagnosis is already the middle and late stage liver cancer patient, and surgical resection cannot be carried out.
(4) Patients who have had relapses or/and metastases.
(5) A patient after partial ablation.
(6) Chemoembolization (TACE) patients.
The forbidden diseases: patients with autoimmune diseases
In order to maximize the therapeutic effect of COST-DC-CTL cell preparation, a single dose of cyclophosphamide for intravenous drip may be administered 2 days before the immunocyte therapy in an amount of 250mg/m2. The effect is to improve the internal environment of the tumor, especially the level of Tregs, and is beneficial to the COST-DC-CTL to exert the optimal antiviral efficacy. FIG. 4 optimization of optimal doses of Cyclophosphamide (CY) effective in reducing the inhibitory factor of T cell activation.
If the patient cannot be surgically resected but can be treated with hepatic artery chemoembolization, COST-DC-CTL cell preparation may be administered locally in the hepatic artery; otherwise, intravenous return is given. Routine examination before and after immune cell therapy: blood routine, biochemical, liver and kidney functions; and observation and recording of vital signs.
Claims (1)
1. A cell preparation, which is prepared by the following method: mixing the hepatitis B liver cancer killer T cells with a DC vaccine carrying antigen signals of multiple specific hepatitis B virus related liver cancer, and transferring the mixture into physiological saline containing 1% of human serum albumin to prepare a cell preparation;
the number ratio of the cells of the hepatitis B liver cancer killer T cells to the cells of the DC vaccine carrying the antigen signals of the multiple specificity hepatitis B virus related liver cancer is 8-18: 1;
the hepatitis B liver cancer killer T cell is prepared by the following method: the DC vaccine carrying the antigen signals of the multiple specificity hepatitis B virus related liver cancer is subjected to adoptive culture with T cells separated from peripheral blood to obtain hepatitis B liver cancer killer T cells;
the DC vaccine carrying the antigen signals of the multiple specificity hepatitis B virus related liver cancer is prepared by the following method: adding an antigen presenting signal group of hepatitis B virus antigen peptide combined with liver cancer cell antigen information into an MODC cell cultured to be mature on the 6 th day by using an AIM-V culture solution, wherein the final concentration of protein in the cracking of an HBV core antigen 18-27 peptide segment, an HBV pre-S2 antigen 44-53 peptide segment and a human liver cancer cell strain HepG2 is 24ug/ml, supplementing the AIM-V culture solution the next day, and recovering and obtaining the vaccine after culturing for 2 days;
the antigen presenting signal group of the combination of the hepatitis B virus antigen peptide and the liver cancer cell antigen information consists of an HBV core antigen 18-27 peptide segment, an HBV pre-S2 antigen 44-53 peptide segment and a human liver cancer cell strain HepG2 lysate, wherein the mass ratio of proteins in the HBV core antigen 18-27 peptide segment, the HBV pre-S2 antigen 44-53 peptide segment and the human liver cancer cell strain HepG2 lysate is 0.5-1:0.5-1: 1-2; the human liver cancer cell strain HepG2 lysate contains a plurality of liver cancer antigens: AFP, GPC3, PCA;
the amino acid sequence of the HBV core antigen 18-27 peptide fragment is FLPSDFFPSV, HBV, and the amino acid sequence of the pre-S2 antigen 44-53 peptide fragment is SILSKTGDPV.
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| Comparative Analysis of Cytotoxic T Lymphocyte Response Induced by Dendritic Cells Loaded with Hepatocellular Carcinoma-Derived RNA or Cell Lysate;Ke Pan等;《Int. J. Biol. Sci.》;20101231;第6卷;639-648 * |
| Cytotoxic T Lymphocyte Response to a Wild Type Hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope;bertoletti a等;《j exp med》;19940930;第180卷;993-943 * |
| Dendritic cells from chronic hepatitis B patients can induce HBV antigen-specific T cell responses;Ruo-Bing Li等;《World J Gastroenterol》;20041231;第10卷(第11期);1578-1582 * |
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