CN103357001A - Preparation method of tumour antigen - Google Patents
Preparation method of tumour antigen Download PDFInfo
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- CN103357001A CN103357001A CN2013103126581A CN201310312658A CN103357001A CN 103357001 A CN103357001 A CN 103357001A CN 2013103126581 A CN2013103126581 A CN 2013103126581A CN 201310312658 A CN201310312658 A CN 201310312658A CN 103357001 A CN103357001 A CN 103357001A
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Abstract
The invention provides a preparation method of a tumor antigen. The preparation method includes: inoculating tumor cells and carrying out culture; inoculating virus with cell lysis property and carrying out culture, and taking a culture supernatant and separating matters with the molecular weight of no less than 20 kDa as the tumor antigen. The invention also provides the tumor antigen prepared by the preparation method, the application of the tumor antigen and the like.
Description
Technical field
The invention belongs to immune medical domain, particularly, the present invention relates to tumor antigen, its preparation and application etc.
Background technology
The cell biological treatment is the novel cancer method of using the autoimmune system, has the treatment pattern of significant curative effect.It uses Protocols in Molecular Biology and cell engineering to learn a skill, with biological preparation the cell that gathers in the patient body is cultivated and processed external, feed back to again in the patient body, thereby excite, the immunologic function of enhancing body self, reach the purpose that monitors and kill sick cell or repair damaged cell.
There has been at present relative literature to report that the employing cell biological treats to process patient tumors.For example, Chinese patent 01139230.4 has been reported a kind of autoimmune cell for the treatment of tumor and preparation method thereof, comprises preparation autologous tumor tissue mRNA; By from the body blood system from the preparation mononuclear cell; Add cytokine and induce the expansion of antigen presenting cell; Namely get again the antigen presenting cell of load tumor antigen with tumor mRNA transfection antigen presenting cell, that is the autoimmune cell for the treatment of tumor.
And for example, Chinese patent 200710139687.7 has been reported a kind of preparation method of self-body large intestine cancer vaccine, may further comprise the steps, and the external evoked cultivation of DC obtains ripe DC; Take from the body large intestine cancer cell, it is for subsequent use to obtain tumor antigen; With the tumor antigen for preparing and the DC Mixed culture 24h for preparing, to obtain the DC of self-body large intestine cancer Modified antigen, again with DC and lymphocyte according to 1: 10～1000 quantitative proportion Mixed culture.
Yet, the tumor antigen of prior art preparation, service efficiency is low, can't directly use, and through a large amount of post-processed, so that antigen itself is degraded or inactivation, specific antigen is lost easily easily.For this reason, the inventor gropes through great efforts, the fragment of finding unexpectedly the virolysis tumor cell such as adenovirus and producing evenly and have a strong immunogenicity, be suitable as very much tumor antigen, therefore worked out the preparation method of new tumor antigen, the tumor antigen that obtains directly use in the body, stable curative effect and active high also can externally be finished the isocellular sensitization of DC.
Summary of the invention
The technical problem that will solve of the present invention is the tumor antigen, its preparation and the application that provide new, so that the curative effect for the treatment of (especially directly treatment) tumor is improved.
Particularly, in a first aspect of the present invention, the invention provides a kind of preparation method of tumor antigen, it comprises:
(1) to the inoculation of medium tumor cell, and cultivates;
(2) inoculating cell cracking performance virus in the cultured products that obtains to step (1), and cultivate; With,
(3) get the culture supernatant that step (2) obtains, and isolate molecular weight and be not less than the material of 20kDa (preferably being not less than 25kDa, such as 30kDa) as tumor antigen.
The method of a first aspect of the present invention is in vitro method, does not relate to the living animal treatment step of (comprising the people).The method of therefore preferred a first aspect of the present invention does not comprise the step of obtaining tumor cell.Tumor cell can be from the tumor cell of cultivating, the tumor cell of purchase, the tumor cell that perhaps provides based on Principles in Informed Consent and by doctor or patient.
Preferably in the method for a first aspect of the present invention, tumor is solid tumor, is more preferably liver tumor, lung tumor, breast tumor, cutaneous tumor, tumor of cervix, the cerebral tumor, rhinopharyngeal neoplasm and/or gastric tumor, such as liver tumor and/or breast tumor.
Preferably in the method for a first aspect of the present invention, the condition of cultivation is 35～38 ℃ and 4.5～6%CO
2, preferably 37 ℃ and 5%CO
2The condition of this cultivation is applicable to respectively the cultivation of step (1) and step (2).The condition of the cultivation of these two steps can be different, and is preferably identical.
Preferably in the method for a first aspect of the present invention, the time of the cultivation of step (1) is to be cultured to attached cell to produce, and usually, this time was 4～7 days, such as 5 days.
Can invade (tumor) cell and so that the virus of cell death cracking, can be as lysis sexually transmitted disease (STD) poison of the present invention, such as adenovirus.Have the live virus that copies generation in the supernatant of collecting, produce safety issue, preferably in the method for a first aspect of the present invention, lysis sexually transmitted disease (STD) poison is replication-defective adenoviral.Current, many safe replication-defective adenoviral commercializations have been arranged, can buy from the market easily acquisition.
Preferably in the method for a first aspect of the present invention, the inoculum concentration of lysis sexually transmitted disease (STD) poison is 0.1～10x10
11/ ml is preferably 0.8～2x10
11/ ml is such as 1.0x10
11/ ml.
The time of the cultivation of step (2) wants so that lysis sexually transmitted disease (STD) poison is all invaded (tumor) cell and caused lysis, usually needs more than 20 hours.Preferably in the method for a first aspect of the present invention, the time of the cultivation of step (2) was 24～72 hours, is preferably 45～55 hours, such as 48 hours.
In second aspect, the invention provides the tumor antigen of the method preparation of a first aspect of the present invention.This tumor antigen can be directly as tumor vaccine and clinically prevention or treatment patient's tumor, also can be as prior art, in external so that the sensitization of DC cell or other immunocytes or activation, and then be used for prevention or treatment patient's tumor.The patient that this tumor antigen can be applied to originate (that is, personalized treatment) also can be applied to suffer from other patients of tumor of the same type.
In the third aspect, the invention provides the application of tumor antigen in the preparation anti-tumor drug of a second aspect of the present invention.Wherein, tumor is solid tumor preferably, is more preferably liver tumor, lung tumor, breast tumor, cutaneous tumor, tumor of cervix, the cerebral tumor, rhinopharyngeal neoplasm and/or gastric tumor, such as liver tumor and/or breast tumor.
Beneficial effect of the present invention is, tumor antigen easy to prepare, quick, and production cost is low, and immunogenicity is strong, can directly apply to the patient who comprises personalized treatment and tumor of the same type, and therapeutic effect is good, and is safe and reliable.
For the ease of understanding, below will describe in detail the present invention by specific embodiment.It needs to be noted that instantiation only is in order to illustrate, not consist of limitation of the scope of the invention.Obviously those of ordinary skill in the art can illustrate according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.In addition, the present invention has quoted open source literature, and these documents also are in order more clearly to describe the present invention, and their full text content is all included the present invention in and carried out reference, just look like they full text in description of the present invention repeated description excessively the same.
The specific embodiment
Embodiment 1, the present invention are used for the preparation of the liver tumor vaccine of laboratory animal
According to classical documents (Xu Yuanding, tumor; 2:289,1982) method adopts diethylnitrosamine (DENA) method, and rat is carried out the liver tumor modeling.In brief, with 1% DENA solution gavage, dosage is 70mg/Kg, and weekly, the solution of all the other times 0.25% is in the rat drinking water.Rat produces the hepatocarcinoma lump after 16 weeks.Get liver cancer tissue, then be seeded in the abdominal cavity (method is seen strong mould etc., Journal of Experimental Biology 11:1,1973) of rat.Totally 65 of rats.The identical hepatocarcinoma lump (0.5cm size box) of size is in the parallel abdominal cavity that is inoculated in rat.When the about 2x2x2cm of tumor of rat abdominal cavity, the rat that produces liver tumor is got one at random, in order to prepare tumor antigen of the present invention and contrast antigen.
To downcut for the preparation of the liver tumor tissue of the rat of antigen, after cleaning with physiological saline solution, be cut into the square fritter in the 0.8mm left and right sides with scalpel.Get a part of fritter under aseptic condition, after the normal saline suspension and placing homogenizer homogenate, place 72 ℃ of water-baths 30 minutes, then dissolve in 37 ℃ after placing-80 ℃ of refrigerators to freeze, so freeze to dissolve 3 times, then with 4000rpm centrifugal 30 minutes, get supernatant, measure protein concentration and be 0.5mg/ml with normal saline dilution to protein concentration, prepare thus contrast antigen.
All the other fritters are inoculated in DMEM (liquid) culture medium that contains 15% hyclone (FBS) (can available from PromoCell company) with 37 ℃, 5%CO
2The condition adhere-wall culture, after observing culture dish bottom and formed fine and close attached cell (about 5 days) to microscopy, reach 1.0x10 to wherein inoculating adenovirus Gendicine strain (can be available from the Shenzhen City SaiBaiNuo Gene Technology Co., Ltd, it is replication defect type) to virus quantity
11Then/ml culture medium continues to cultivate 48 hours.Get culture supernatant and be difficult 4000rpm centrifugal 30 minutes, get supernatant, membrane filtration take molecular cut off as 30kDa, collect filtrate and place bag filter to dialyse take normal saline as dialysis solution, measure protein concentration and be 0.5mg/ml with normal saline dilution to protein concentration, prepare thus tumor antigen of the present invention.Get this tumor antigen and carry out the Virus culture test, do not detect the adenovirus that existence is lived.
The rat of modeling success is divided into 3 groups (being the blank group, contrast antigen group, antigen group of the present invention) at random, and antigen treatment group rat of the present invention tumor antigen 1ml of the present invention more than injection in 3 days injects 3 times altogether in the abdominal cavity; Contrast antigen treatment group rat contrast antigen 1 ml more than injection in 3 days injects 3 times altogether in the abdominal cavity; Blank group rat is with the equivalent physiologic saline for substitute.Inject first 30 days afterwards dissection respectively organize rat and measure the volume of the liver tumor that forms.The result is as shown in table 1, although contrast antigen treatment group mean tumour volume reduces to some extent than the blank group, does not possess significance statistically; Antigen treatment group of the present invention has then significantly reduced gross tumor volume with respect to the blank group, and mean tumour volume also contrast antigen treatment group is much smaller.
The therapeutic effect of table 1 liver tumor vaccine relatively
| Group | Gross tumor volume (cm 3) |
| The blank group | 8.5±1.6 |
| Contrast antigen treatment group | 8.1±1.8 |
| Antigen treatment group of the present invention | 2.5±1.2* |
* represent with respect to blank p＜0.05
Embodiment 2 the present invention are used for the preparation of the breast carcinoma vaccine of laboratory animal
According to the method (Akla et al Anticancer research, 23:3761-3766,2003) of document, get MDB-MB-231 breast cancer cell (1x10
6) suspension 1ml, direct inoculation is in the rat breast fat pad.After 16 weeks, rat can form the breast cancer bump of 1.5x1.5x1cm size.Get at random a rat mammary gland tumor, in order to prepare tumor antigen of the present invention and contrast antigen (concrete grammar is with reference to embodiment 1).Rat is divided into 3 groups (blank group, antigen treatment group of the present invention and contrast antigen treatment groups), 25 every group at random.Antigen treatment group rat of the present invention tumor antigen 1ml of the present invention more than injection in 3 days injects 3 times in lump altogether; Contrast antigen treatment group rat contrast antigen 1 ml more than injection in 3 days injects 3 times in lump altogether; Blank group rat is with the equivalent physiologic saline for substitute.Inject first 30 days afterwards measurement respectively organize the melanomatous volume that rat forms.The result is as shown in table 2, and the mean tumour volume of contrast antigen treatment group has significant minimizing than the blank group; Antigen treatment group of the present invention has then not only significantly reduced gross tumor volume with respect to the blank group, and with respect to contrast antigen treatment group significant minimizing is arranged also.
The therapeutic effect of table 2 breast carcinoma vaccine relatively
| Group | Gross tumor volume (cm 3) |
| The blank group | 2.3±0.4 |
| Contrast antigen treatment group | 1.5±0.3* |
| Antigen treatment group of the present invention | 0.7±0.3*# |
* represent with respect to blank p＜0.05; # represents with respect to contrast antigen, p＜0.05.
Claims (10)
1. the preparation method of a tumor antigen, it comprises:
(1) to the inoculation of medium tumor cell, and cultivates;
(2) inoculating cell cracking performance virus in the cultured products that obtains to step (1), and cultivate; With,
(3) get the culture supernatant that step (2) obtains, and isolate molecular weight and be not less than the material of 20kDa (preferably being not less than 25kDa, such as 30kDa) as tumor antigen.
2. preparation method according to claim 1, it does not comprise the step of obtaining tumor cell.
3. preparation method according to claim 1, the condition of wherein cultivating is 35～38 ℃ and 4.5～6%CO2, preferably 37 ℃ and 5%CO2.
4. preparation method according to claim 1, wherein the time of the cultivation of step (1) is to be cultured to attached cell to produce.
5. preparation method according to claim 1, wherein lysis sexually transmitted disease (STD) poison is adenovirus, is preferably replication-defective adenoviral.
6. preparation method according to claim 1, the inoculum concentration of wherein lysis sexually transmitted disease (STD) poison is 0.1～10x1011/ml, is preferably 0.8～2x1011/ml, such as 1.0x1011/ml.
7. preparation method according to claim 1, wherein the time of the cultivation of step (2) was 24～72 hours, is preferably 45～55 hours, such as 48 hours.
8. preparation method according to claim 1, wherein said tumor is solid tumor, is preferably liver tumor, lung tumor, breast tumor, cutaneous tumor, tumor of cervix, the cerebral tumor, rhinopharyngeal neoplasm and/or gastric tumor, for example liver tumor and/or breast tumor.
9. the tumor antigen of arbitrary described preparation method preparation according to claim 1～8.
10. the application of tumor antigen according to claim 9 in the preparation anti-tumor drug.
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|---|---|---|---|
| CN2013103126581A CN103357001A (en) | 2013-07-24 | 2013-07-24 | Preparation method of tumour antigen |
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| CN2013103126581A CN103357001A (en) | 2013-07-24 | 2013-07-24 | Preparation method of tumour antigen |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001070175A2 (en) * | 2000-03-21 | 2001-09-27 | The University Of Virginia Patent Foundation | Osteocalcin promoter directed adenovirus replicaton for therapy |
| CN101168053A (en) * | 2007-11-01 | 2008-04-30 | 史增祥 | Method for preparing self-body large intestine cancer vaccine |
| CN103110939A (en) * | 2012-10-23 | 2013-05-22 | 郑州大学 | Vaccine for inducing specific immunity of tumor and application thereof |
-
2013
- 2013-07-24 CN CN2013103126581A patent/CN103357001A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001070175A2 (en) * | 2000-03-21 | 2001-09-27 | The University Of Virginia Patent Foundation | Osteocalcin promoter directed adenovirus replicaton for therapy |
| CN101168053A (en) * | 2007-11-01 | 2008-04-30 | 史增祥 | Method for preparing self-body large intestine cancer vaccine |
| CN103110939A (en) * | 2012-10-23 | 2013-05-22 | 郑州大学 | Vaccine for inducing specific immunity of tumor and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 崔莹莹等: "冻融肿瘤细胞和肿瘤细胞培养上清对淋巴细胞活化能力的影响", 《肿瘤研究与临床》, vol. 24, no. 5, 30 May 2012 (2012-05-30) * |
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Application publication date: 20131023 |