CN102153658A - Tumor antigen, DC (dendritic cell) tumor vaccine and preparation method thereof - Google Patents
Tumor antigen, DC (dendritic cell) tumor vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tumor antigen. The tumor antigen mainly consists of heat shock protein 70 and polymerization peptide, wherein the polymerization peptide is TERT572Y-MAGE-A248V9-HER-2/neu402Y or TERT988Y-MAGE-A248V9-HER-2/neu402Y polymerization peptide sequentially consisting of implicit site abnormal peptide TERT572Y or TERT988Y, MAGE-A248V9 and HER-2/neu402Y. The invention also discloses a DC (dendritic cell) tumor vaccine prepared by using sensitive dendritic cells of the tumor antigen and a preparation method thereof. The DC tumor vaccine can be used for efficiently, safely and specifically treating multiple tumors, and has significance for clinical treatment of tumors.
Description
Technical field
The present invention relates to the tumor biotherapy field, relate in particular to a kind of tumour antigen, the DC tumor vaccine that obtains with this antigen sensibilization dendritic cell and the preparation method of this vaccine.
Background technology
Tumour remains the main killer of serious harm human health, is the second largest common dead factor of industrialized country, receives the concern of government, nonprofit organization and drugmaker.The number that influenced by cancer every year in the global range surpasses 1,000 ten thousand, and expecting the year two thousand twenty according to " report of world's cancer " of the World Health Organization will constantly increase, and annual the morbidity reaches 1,500 ten thousand people, dead 1,000 ten thousand people.The mortality ratio of domestic tumour has risen to first in the city, be second in the rural area.This means that the situation that oncotherapy faces is very severe.Be still based on operation for treatment for cancer clinically at present, chemotherapy and radiation is auxilliary, but the specificity deficiency of treatment usually causes damage to healthy tissues and organ, and operative treatment still exists recurrence of postoperative height and the easy problem that shifts.The immunotherapy of tumor vaccine can produce the specific reaction at tumour, even can remove remaining tumor focus, and the generation immunological memory, become the 4th kind of oncotherapy pattern after operation, chemotherapy and radiation, and tangible complementarity is arranged with three big routine treatments.
Tumor vaccine mainly is a therapeutic vaccine, is a kind of mode of immunotherapy.Wherein tumour-cell vaccine is to utilize patient's self tumour cell, tumour specific antigen and other immunity regulatory cell is treated and prophylaxis of tumours, along with the prevention effect of tumour-cell vaccine to oncotherapy manifests gradually, the continuous maturation of method and means, it has opened up the modern approach of a safe and effective treatment tumour.Anti tumor immune response is to absorb tumour antigen by antigen presenting cell (APC) earlier, by APC tumour antigen is handled and is offered to excite to the T cell.(Dendritic Cell DC) as the most powerful full-time APC of function in the human body, can absorb, handles and offer antigen to dendritic cell efficiently, starts the cell-mediated immune response of T, thereby becomes the key link of anti tumor immune response.The DC of application load tumour antigen, can excitating organism in tumour-specific T cell, thereby effective killing tumor cell and can be set up persistent antineoplastic specificity immunne response.Utilize DC vaccine therapy tumour to become a kind of new tumor biotherapy mode in recent years, be one of knubble biological therapy of at present domestic and international curative effect the best, thereby receive much concern.
In recent years, Chinese scholars is impacted with external tumour antigen and is made its sensitization prepare the DC vaccine from body DC, can effectively resist tumor growth of the same race,, and in clinical treatment, obtain certain effect as bone-marrow-derived lymphocyte knurl, multiple myeloma, prostate tumor, melanoma etc.Although early stage, the exploitation of DC tumor vaccine obtained significant progress in clinical treatment research, and effectively prolonged patient's life, but still exist certain limitation, so far do not have to obtain very special tumor associated antigen epi-position, immunogenicity is not high, immunotolerance etc. and cause the tumour patient postoperative to exist highly problem such as recurrence and easy transfer all needs to be resolved hurrily.
Summary of the invention
The present invention will solve owing to lacking the specific tumour related antigen and make technical problems such as DC tumor vaccine immunogenicity is not high, immunotolerance, and a kind of tumour antigen is provided, and its high specificity can be used for sensitization DC.
In addition, also provide a kind of DC tumor vaccine and preparation method thereof, this DC tumor vaccine can safety, specific treatment kinds of tumors efficiently.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of tumour antigen, described tumour antigen mainly is made up of heat shock protein 70 (Hsp70) and pdef polypeptide, and described pdef polypeptide is by recessive epi-position abnormity peptide TERT
572YOr TERT
988Y, MAGE-A
248V9, HER-2/neu
402YThe TERT of Zu Chenging successively
572Y-MAGE-A
248V9-HER-2/neu
402YOr TERT
988Y-MAGE-A
248V9-HER-2/neu
402YPdef polypeptide, the aminoacid sequence of this pdef polypeptide are YLFFYRKSV-YLEYRQVPV-YLEEITGYL or YLQVNSLQTV-YLEYRQVPV-YLEEITGYL.
In another aspect of this invention, also provide a kind of DC tumor vaccine, this vaccine is to make with above-mentioned tumour antigen sensitization DC.
Preferably, described DC derives from tumour patient self.
Described DC tumor vaccine is a therapeutic vaccine, is used for the treatment of kinds of tumors, comprises lung cancer, prostate cancer, liver cancer, kidney, cancer of the stomach, carcinoma of the pancreas, mammary cancer, ovarian cancer, uterus carcinoma, esophagus cancer etc.
In another aspect of this invention, also provide a kind of preparation method of DC tumor vaccine, may further comprise the steps:
(1) external synthetic tumour antigen, this tumour antigen mainly is made up of Hsp70 and pdef polypeptide, and described pdef polypeptide is by recessive epi-position abnormity peptide TERT
572YOr TERT
988Y, MAGE-A
248V9, HER-2/neu
402YThe TERT of Zu Chenging successively
572Y-MAGE-A
248V9-HER-2/neu
402YOr TERT
988Y-MAGE-A
248V9-HER-2/neu
402YPdef polypeptide, the aminoacid sequence of this pdef polypeptide are YLFFYRKSV-YLEYRQVPV-YLEEITGYL or YLQVNSLQTV-YLEYRQVPV-YLEEITGYL;
(2) extract patient's peripheral blood, and adopt lymphocyte separation medium to separate the acquisition peripheral blood mononuclear cell;
(3) resuspended with serum-free cell culture medium, adjusting cell concn is 2～4 10
6/ mL is inoculated in the orifice plate, and places incubator to cultivate;
(4) cultivate after 2～3 hours, wash plate 1～2 time, add with containing 500～1000U/mL GM-CSF, 500～1000U/mL IL-4,10% autoserum or people AB serum, 1% antibiotic cell culture fluid continuation cultivation with serum-free cell culture medium; Added the DC nutrient solution on the 3rd day;
Add tumour antigen in (5) the 5th days, and be equipped with adjuvant, continue to cultivate, impact the immature DC of sensitization jointly;
Add 500U/mL TNF-α after (6) 12～16 hours and induce the DC maturation;
Collected the DC tumor vaccine in (7) the 7th～8 days.
The final concentration of the tumour antigen that adds in the described step (5) is 5～15mM.
Described tumour antigen is by Hsp70 and the external be combined into of pdef polypeptide, or makes by gene engineering method is recombinant expressed.
Tumour antigen of the present invention, high specificity by the DC tumor vaccine of its sensitization from body DC preparation, is compared with existing tumor vaccine, has the following advantages:
(a) effectively reduce tolerance, improved immunogenicity, all can produce specific immune response at kinds of tumors;
(b) composition is clear and definite, safe;
(c) be equipped with suitable adjuvant, effectively enhancing immunity is replied.
Therefore, DC tumor vaccine of the present invention has great importance for the clinical treatment kinds of tumors.
Embodiment
The present invention is further detailed explanation below in conjunction with embodiment.
In order further to improve tumor vaccine immunogenicity, security and effectively to reduce tolerance, seek specific tumour related antigen and significant for the clinical treatment of tumour with its preparation DC tumor vaccine.
The tumour antigen of the restricted special CTL identification of MHC-I quasi-molecule mainly contains MAGE-3, HER-2/neu etc.; The tumour antigen of the restricted special CTL identification of MHC-II quasi-molecule mainly contains TERT etc.; Antigen and tumor type, transfer and the prognosis of these MHC molecule Restricted CTL identifications have certain relation, should activate CD4+, CD8+ T cell and B cellullar immunologic response simultaneously and treat the most effective vaccine of tumour clinically.Tumour antigen of the present invention mainly is made up of Hsp70 and pdef polypeptide, and described pdef polypeptide is by recessive epi-position abnormity peptide TERT
572YOr TERT
988Y, MAGE-A
248V9, HER-2/neu
402YThe TERT of Zu Chenging successively
572Y-MAGE-A
248V9-HER-2/neu
402YOr TERT
988Y-MAGE-A
248V9-HER-2/neu
402YPdef polypeptide, the aminoacid sequence of this pdef polypeptide are YLFFYRKSV-YLEYRQVPV-YLEEITGYL or YLQVNSLQTV-YLEYRQVPV-YLEEITGYL.Tumour antigen of the present invention is united use with MHC-I class and the restricted tumour antigen of MHC-II quasi-molecule, can more effectively activate CD4+, CD8+ T cell and B cellullar immunologic response, and Hsp70 wherein makes immune effect better.
Utilize tumour antigen of the present invention to be equipped with adjuvant and impact sensitization autologous dendritic cell (DC) jointly, the DC tumor vaccine of preparation gained, the efficient activating cytotoxic T-lymphocyte (CTL) of energy, killing tumor cell specifically, clinical treatment tool to kinds of tumors improves significantly, and effectively suppresses the development and the transfer of tumour.
The preparation method of DC tumor vaccine of the present invention may further comprise the steps:
(1) external synthetic tumour antigen, this tumour antigen mainly is made up of Hsp70 and pdef polypeptide, and described pdef polypeptide is by recessive epi-position abnormity peptide TERT
572YOr TERT
988Y, MAGE-A
248V9, HER-2/neu
402YThe TERT of Zu Chenging successively
572Y-MAGE-A
248V9-HER-2/neu
402YOr TERT
988Y-MAGE-A
248V9-HER-2/neu
402YPdef polypeptide, the aminoacid sequence of this pdef polypeptide are YLFFYRKSV-YLEYRQVPV-YLEEITGYL or YLQVNSLQTV-YLEYRQVPV-YLEEITGYL;
(2) extract patient's peripheral blood, and adopt lymphocyte separation medium to separate the acquisition peripheral blood mononuclear cell;
(3) resuspended with serum-free cell culture medium, adjusting cell concn is 2～4 10
6/ mL is inoculated in the orifice plate, and places incubator to cultivate;
(4) cultivate after 2～3 hours, wash plate 1～2 time, add with containing 500～1000U/mL GM-CSF, 500～1000U/mL IL-4,10% autoserum or people AB serum, 1% antibiotic cell culture fluid continuation cultivation with serum-free cell culture medium; Added the DC nutrient solution on the 3rd day;
Add tumour antigen in (5) the 5th days, and be equipped with adjuvant, continue to cultivate, impact the immature DC of sensitization jointly;
Add 500U/mL TNF-α after (6) 12～16 hours and induce the DC maturation;
Collected the DC tumor vaccine in (7) the 7th～8 days.
The final concentration of the tumour antigen that adds in the described step (5) is 5～15mM.
Described tumor antigen peptide is by Hsp70 and the external be combined into of pdef polypeptide, or makes by gene engineering method is recombinant expressed.
Embodiment 1 preparation DC tumor vaccine
(1) external synthetic tumour antigen, this tumour antigen mainly is made up of Hsp70 and pdef polypeptide, and described pdef polypeptide is by recessive epi-position abnormity peptide TERT
572YOr TERT
988Y, MAGE-A
248V9, HER-2/neu
402YThe TERT of Zu Chenging successively
572Y-MAGE-A
248V9-HER-2/neu
402YOr TERT
988Y-MAGE-A
248V9-HER-2/neu
402YPdef polypeptide, the aminoacid sequence of this pdef polypeptide are YLFFYRKSV-YLEYRQVPV-YLEEITGYL or YLQVNSLQTV-YLEYRQVPV-YLEEITGYL;
(2) extract patient's peripheral blood 200 mL, adopt lymphocyte separation medium Ficoll-Hypaque(density 1.077) separation acquisition peripheral blood mononuclear cell;
(3) resuspended with serum-free RPMI1640 nutrient solution, adjusting cell concn is 2～4 10
6/ mL is inoculated in 6 orifice plates, and every hole 2mL places 37 ℃, 5%CO
2Cultivate in the incubator;
(4) behind cultivation 2～3h, light rolling 6 orifice plates, non-adherent cell is removed in suction, wash plate 1～2 time with serum-free RPMI1640 nutrient solution again, add with containing 500～1000U/mL GM-CSF, 500～1000U/mL IL-4,10% autoserum or people AB serum, 1% antibiotic RPMI1640 nutrient solution continuation cultivation; 3d adds DC nutrient solution 2～3mL;
(5) 5d adding tumour antigen (5～15mM), be equipped with adjuvant, continue to cultivate, impact the immature DC of sensitization jointly;
Add 500U/mL TNF-α behind (6) 12～16h and induce the DC maturation;
(7) the 7th～8d collect the DC tumor vaccine.
Embodiment 2
Clinical transplantation DC tumor vaccine
The strict aseptic technique of migration process.
(1) gets greater than 1 10
7The DC tumor vaccine, resuspended with the physiological saline that contains 5% autologous plasma, mixing, subcutaneous injection or venoclysis feed back in the tumour patient body.
(2) residue DC tumor vaccine is with 1.2～1.5 * 10
7Cell/, strict frozen by stem cell cryopreservation step, totally 2～3, be used for the back cell recovery after, feed back the patient.
(3) injection is a course of treatment 3～4 times, one the course of treatment 20d～1 month.
PRELIMINARY RESULTS shows that this DC vaccine all has therapeutic action to most of tumour patients.
The extracorporeal anti-tumor function of embodiment 3 sensitization DC tumor vaccines
Separate the peripheral blood from patients with lung cancer mononuclearcell, put 37 ℃, 5%CO
2After cultivating 2h in the incubator, attached cell is used to cultivate DC, non-adherent cell suspends with the RPMI1640 nutrient solution that contains 5%FBS, adopt nylon hair post partition method to separate and obtain the T lymphocyte, the DC vaccine of tumour antigen sensitization and the T lymphocyte of acquisition are pressed 1:20, and adding IL-2(600U/mL) co-cultivation 3d obtains CTL, the action effect cell.With human lung adenocarcinoma cell line A549, human small cell lung carcinoma cell strain NCI-H446, the sclc cell line NCI-H460 of the National People's Congress or people's lung squamous cell cancer cell strain NCI-H520 as target cell.Than 10:1,20:1 and 50:1 mix the effector cell with target cell, put 37 ℃, 5%CO by the different targets of imitating
2Co-cultivation 24h in the incubator is with the killing activity of LDH kit measurement CTL to various lung carcinoma cells.
The result shows that tumour antigen sensitization DC vaccine can be induced the peripheral blood from patients with lung cancer cytotoxic T cell specifically external, effectively kills and wounds lung carcinoma cell.
The extracorporeal anti-tumor function of embodiment 4 sensitization DC tumor vaccines
Separate the patients with prostate cancer peripheral blood mononuclear cell, put 37 ℃, 5%CO
2After cultivating 2h in the incubator, attached cell is used to cultivate DC, non-adherent cell suspends with the RPMI1640 nutrient solution that contains 5%FBS, adopt nylon hair post partition method to separate and obtain the T lymphocyte, the DC vaccine of tumour antigen sensitization and the T lymphocyte of acquisition are pressed 1:20, and adding IL-2(600U/mL) co-cultivation 3d obtains CTL, the action effect cell.With Human Prostate Cancer Cells strain PC-3 or DU-145 as target cell.Than 10:1,20:1 and 50:1 mix the effector cell with target cell, put 37 ℃, 5%CO by the different targets of imitating
2Co-cultivation 24h in the incubator is with the killing activity of LDH kit measurement CTL to prostate cancer cell.
The result shows that tumour antigen sensitization DC vaccine can be induced patients with prostate cancer peripheral blood cells poison T lymphocyte, effectively killing prostate cancer cells specifically external.
The model mouse anti-tumor in vivo effect of embodiment 5 sensitization DC tumor vaccines
Bring out BALB/c mouse lung cancer according to bibliographical information by diethylnitrosamine (DEN), set up the lung cancer model mouse, with tumour antigen sensitization DC vaccine (1x10
6) go into model mouse through tail vein injection, inject continuously 3～4 times, at interval 4d～1w.
The result shows that this DC vaccine brings out model mouse CTL, effectively kills and wounds lung carcinoma cell, obviously suppresses the lung cancer of mouse.
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. a tumour antigen is characterized in that, described tumour antigen mainly is made up of heat shock protein 70 and pdef polypeptide, and described pdef polypeptide is by recessive epi-position abnormity peptide TERT
572YOr TERT
988Y, MAGE-A
248V9, HER-2/neu
402YThe TERT of Zu Chenging successively
572Y-MAGE-A
248V9-HER-2/neu
402YOr TERT
988Y-MAGE-A
248V9-HER-2/neu
402YPdef polypeptide, the aminoacid sequence of this pdef polypeptide are YLFFYRKSV-YLEYRQVPV-YLEEITGYL or YLQVNSLQTV-YLEYRQVPV-YLEEITGYL.
2. a DC tumor vaccine is characterized in that, this vaccine is to make with claim 1 described tumour antigen sensitization dendritic cell.
3. DC tumor vaccine according to claim 2 is characterized in that described dendritic cell derives from tumour patient self.
4. DC tumor vaccine according to claim 2 is characterized in that, described vaccine is a tumor therapeutic vaccine.
5. DC tumor vaccine according to claim 2 is characterized in that, described tumour comprises: lung cancer, prostate cancer, liver cancer, kidney, cancer of the stomach, carcinoma of the pancreas, mammary cancer, ovarian cancer, uterus carcinoma or esophagus cancer.
6. the preparation method of a DC tumor vaccine is characterized in that, may further comprise the steps:
(1) external synthetic tumour antigen, this tumour antigen mainly is made up of heat shock protein 70 and pdef polypeptide, and described pdef polypeptide is by recessive epi-position abnormity peptide TERT
572YOr TERT
988Y, MAGE-A
248V9, HER-2/neu
402YThe TERT of Zu Chenging successively
572Y-MAGE-A
248V9-HER-2/neu
402YOr TERT
988Y-MAGE-A
248V9-HER-2/neu
402YPdef polypeptide, the aminoacid sequence of this pdef polypeptide are YLFFYRKSV-YLEYRQVPV-YLEEITGYL or YLQVNSLQTV-YLEYRQVPV-YLEEITGYL;
(2) extract patient's peripheral blood, and adopt lymphocyte separation medium to separate the acquisition peripheral blood mononuclear cell;
(3) resuspended with serum-free cell culture medium, adjusting cell concn is 2～4 ' 10
6/ mL is inoculated in the orifice plate, and places incubator to cultivate;
(4) cultivate after 2～3 hours, wash plate 1～2 time, add and contain 500～1000U/mL GM-CSF, 500～1000U/mL IL-4,10% autoserum or people AB serum, 1% antibiotic cell culture fluid continuation cultivation with serum-free cell culture medium; Added the DC nutrient solution on the 3rd day;
Add tumour antigen in (5) the 5th days, and be equipped with adjuvant, continue to cultivate, impact the immature DC of sensitization jointly;
Add 500U/mL TNF-α after (6) 12～16 hours and induce the DC maturation;
Collected the DC tumor vaccine in (7) the 7th～8 days.
7. preparation method according to claim 6 is characterized in that, the final concentration of the tumour antigen that adds in the described step (5) is 5～15mM.
8. according to claim 6 or 7 described preparation methods, it is characterized in that described tumour antigen is by heat shock protein 70 and the external be combined into of pdef polypeptide, or make by gene engineering method is recombinant expressed.
9. preparation method according to claim 6 is characterized in that described DC derives from tumour patient self.
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| CN103386123A (en) * | 2012-05-08 | 2013-11-13 | 刘江秋 | Method and application of MEK used as costimulatory factor for preparing novel tumor vaccine |
| CN103599528A (en) * | 2013-12-04 | 2014-02-26 | 深圳市合一康生物科技有限公司 | Method for preparing human dendritic cell vaccine |
| CN114051414A (en) * | 2019-06-11 | 2022-02-15 | 瓦克松生物技术公司 | Combination of markers for predicting response to Vx-001 |
| CN117402218A (en) * | 2023-12-15 | 2024-01-16 | 上海惠盾因泰生物科技有限公司 | Individualized dendritic cell vaccine for Survivin positive tumor and preparation method thereof |
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| US20080254051A1 (en) * | 2005-05-09 | 2008-10-16 | Kosmatopoulos Kostantinos Kostas | Use of Native Peptides and Their Optimized Derivatives For Vaccination |
| WO2007073768A1 (en) * | 2005-12-23 | 2007-07-05 | Vaxon Biotech | Immunogenic polypeptide composed of tumor antigen-derived optimized cryptic peptides, and uses thereof |
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| CN103386123A (en) * | 2012-05-08 | 2013-11-13 | 刘江秋 | Method and application of MEK used as costimulatory factor for preparing novel tumor vaccine |
| CN103599528A (en) * | 2013-12-04 | 2014-02-26 | 深圳市合一康生物科技有限公司 | Method for preparing human dendritic cell vaccine |
| CN103599528B (en) * | 2013-12-04 | 2015-08-05 | 深圳市合一康生物科技股份有限公司 | The preparation method of human dendritic cell vaccine |
| CN114051414A (en) * | 2019-06-11 | 2022-02-15 | 瓦克松生物技术公司 | Combination of markers for predicting response to Vx-001 |
| CN117402218A (en) * | 2023-12-15 | 2024-01-16 | 上海惠盾因泰生物科技有限公司 | Individualized dendritic cell vaccine for Survivin positive tumor and preparation method thereof |
| CN117402218B (en) * | 2023-12-15 | 2024-02-20 | 上海惠盾因泰生物科技有限公司 | Individualized dendritic cell vaccine for Survivin positive tumor and preparation method thereof |
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Application publication date: 20110817 |