CN105462926A - Preparation method and application of immunocytoma vaccine - Google Patents
Preparation method and application of immunocytoma vaccine Download PDFInfo
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- CN105462926A CN105462926A CN201511015628.XA CN201511015628A CN105462926A CN 105462926 A CN105462926 A CN 105462926A CN 201511015628 A CN201511015628 A CN 201511015628A CN 105462926 A CN105462926 A CN 105462926A
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Abstract
The invention discloses a preparation method of an immunocytoma vaccine and application of the immunocytoma vaccine in cancer therapy. The preparation method comprises the following steps: in a PBMC (peripheral blood mononuclear cell) culture medium for initial culture, adding a DC cell activation factor, and adding one or a plurality of tumor antigen proteins or long-chain polypeptides, wherein in such process, the tumor antigen is ingested by monocytes, decomposed and presented to the monocyte surface; on the basis of continuing culturing PBMC, adding a cell factor capable of promoting the monocyte to become the DC cell to be activated and mature, one or a plurality of toll-like receptor activating factors and one or a plurality of polypeptides capable of being combined with MHCI on the DC surface; and meanwhile, adding an anti-human CTLA-4 antibody, and coculturing to intercept the inhibiting action of the CTLA-4. The cells cultured by the method can increase tumor-antigen-specific T cells and obviously enhance and promote the capacity of the immunological cells for killing tumor cells, thereby enhancing the curative effect in clinical cancer therapy.
Description
Technical field
The present invention relates to biomedicine technical field, particularly relate to a kind of preparation method and application thereof of IC seedling.
Background technology
Malignant tumour is the disease of class serious harm human health the most, and about three million peoples in the annual whole nation are diagnosed as a new tumour patient, and more than 200 ten thousand people die from cancer.Although can be treated by operation, radiotherapy, chemotherapy, most patient still dies from recurrence and the transfer of tumour.So, how to remove residual minimal neoplastic tissue or tumour cell is then that modern tumor immunology has one of problem to be solved.Basic reason due to cancer generation is the monitoring of tumour cell escape immunocyte and carries out immoderate growth, so raising immune system recognition and killing tumor cell will from fundamentally removing tumour.Along with new technology application clinically, immunotherapy becomes one of most effective means of Therapeutic cancer gradually.
Dendritic cell or antigen presenting cell (DC) known todayly uniquely can activate primality T cell and the strongest professional antigen presenting cells (AntigenPresentingCells, APC) of function.DC not only can activate natural killer sexual cell (NK) and killer T cell (CTL), meanwhile, can also activate B cell, play an important role in inducing cellular immune and humoral immunization.
DC knurl seedling is exactly by the environment in in-vitro simulated body, induced monocyte is divided into ripe DC cell, simultaneously, make the corresponding antigenic information of DC cell loading, comprise the tumor immune response that tumour-specific related antigen, inhibitive ability of immunity antigen etc. carry out excitating organism body internal specific, enter to remove tumour cell.This functional DC carrying tumor associated antigen information is called as DC knurl seedling.
In April, 2010, U.S. FDA approval autologous dendritic cell vaccine (sipuleucel-T, Provenge) treats the asymptomatic metastatic prostate cancer of endocrine therapy failure.The while that its principle being Activation In Vitro DC cell, carrying single tumour antigen PAP in DC surface, then feed back in patient body, cause antineoplastic immune response further.Research shows, compared with placebo, this vaccine therapy group median survival interval extends 4.1 months, and the mortality risk of a year reduces by 22%.
But exist on T cell surface immunosuppressive agent-immunologic test point (immunecheckpoint)-existence, the activation of these immunologic test point energy suppressor T cell, propagation and effector function.And these immunologic test point control stimulate altogether and suppress the balance of signal, and stimulate altogether and suppress signal at maintenance self tolerance, amplitude and the time length aspect of the self-protection of histoorgan and regulatory T cells response have vital role.Two main, and immunologic test point molecule--the albumen 1 (PD1) of cytotoxic T lymphocyte albumen 4 (CTLA4) and apoptosis is the negative regulatory factor of expressing on cytotoxic T cell.The B7 quasi-molecule of the immunologic test point CTLA-4 on T cell surface and DC cell surface combines, the activating molecules CD28 on T cell surface can be stoped to be combined with the B7 quasi-molecule of DC cell surface, and suppression DC cell antigen pass the ability of function and the T cell activation activation of holding further.So, in the process of immune cell activation, utilize CTLA-4 antibody to hinder the combination of CTLA-4 and B7 quasi-molecule, the Antigen-presenting role of DC can be strengthened, improve the T cell ratio of antigen-specific.
Owing to lacking the control to immunosuppression target spot, it is low that the DC knurl seedling Therapeutic cancer ubiquity on Present clinical specificity, feeds back the little weakness of aftereffect.For overcoming the above-mentioned shortcoming of DC vaccine, the innovative point of this patent is: in DC preparation process, add CTLA-4 antibody to increase the activity of DC, and then increases the T cell ratio of antigenic specificity, increases the function of immunity system killing tumor cell after feeding back.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and application thereof of IC seedling, to solve the problems of the technologies described above.
The present invention is by the following technical solutions for achieving the above object:
A preparation method for IC seedling, comprises the steps:
1) in the substratum of initial incubation PBMC, add DC cell activation factor, and add one or more tumor antigen proteins or long-chain polypeptide, be placed in 37 DEG C, 5%CO
2cultivate 12-16 hour in incubator, in the process, tumour antigen is by monocytic cell ingests, and decomposition and submission are to onthe surface of monocytes;
2) on the basis of continuing above-mentioned cultivation PBMC, add and promote that monocyte becomes cytokine and one or more class tongued bell receptor activator of DC cell activation maturation, with can with DC one or more polypeptide of being combined of MHCI on the surface, meanwhile add anti-human CTLA-4 antibody co-cultivation, block the restraining effect of CTLA-4, be placed in 37 DEG C, 5%CO
2cultivate 12 days in incubator.
Described DC cell activation factor is GM-CSF, IL-4.
Described class tongued bell receptor activator is CpG, poly(I:C), IFN-γ, R848.
A kind of IC seedling is in cancer therapy and other clinical application.This IC seedling adds T cells with antigenic specificity ratio, has higher specificity to tumor-cell antigen.
The invention has the beneficial effects as follows: the present invention is by the change to cultivation activation and immunocyte (as DC) technique, both by adding immunologic test point (CTLA-4) antibody in cell cultures or after cell cultures, thus produce the immune mature cell with high antigenic specificity and strong killing tumor cell ability, the function that it strengthens inhibition tumor cell can be predicted, experiment in vitro proves, cultured cells of the present invention can improve the narrow spectrum T cell of tumour antigen, significantly improve and Promote immunity cell killing tumour cell ability, thus improve its curative effect at clinical anticancer.
Accompanying drawing explanation
Fig. 1 is that the cell of cultivation after 14 days detects the comparison diagram of the CD8 positive and the antigen-specific positive by stream type cell analyzer.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is further elaborated.
Embodiment 1
In the substratum of initial incubation PBMC, add DC cell activation factor GM-CSF and IL-4, and many tumor antigen proteins or long-chain polypeptide (0.1ug/ml/each), be placed in 37 DEG C, 5%CO
212-16 hour is cultivated in incubator; Add DC cell activation factor IFN-γ, CpG, Poly(I:C), R848, melanoma tumors antigen small peptide (0.1ug/ml) and anti-human CTLA-4 antibody (KS-0003, Kang Zhi pharmacy) (10ug/ml), be placed in 37 DEG C, 5%CO
2cultivate in incubator, cell continues cultivation and to gather after 12 days analysis.
The test of cell cultivation process cellular constituent change: the composition of the product before cultivating and after cultivating carries out the specific T cell of test antigen by stream type cell analyzer.The T cell pentamer of melanoma tumors antigen-specific detects, and proves to add the ratio that CTLA-4 obviously increases T cells with antigenic specificity.
As shown in Figure 1, CTLA-4 antibody can increase the expression of antigen-specific cellular: cultivate the cell after 12 days detects the CD8 positive and antigen-specific positive expression by stream type cell analyzer, as can be seen from the figure, add CTLA-4 antibody (ks-0003), make the positive rate of antigenic specificity cell (Pentamer+) in CD8 be increased to 6.39% from 5.19%.
As known by the technical knowledge, the present invention can be realized by other the embodiment not departing from its spirit or essential feature.Therefore, above-mentioned disclosed embodiment, with regard to each side, all just illustrates, is not only.Within the scope of the present invention all or be all included in the invention being equal to the change in scope of the present invention.
Claims (6)
1. a preparation method for IC seedling, is characterized in that, comprises the steps.
2.1) in the substratum of initial incubation PBMC, add DC cell activation factor, and add one or more tumor antigen proteins or long-chain polypeptide, be placed in 37 DEG C, 5%CO
2cultivate 12-16 hour in incubator, in the process, tumour antigen is by monocytic cell ingests, and decomposition and submission are to onthe surface of monocytes.
3.2) on the basis of continuing above-mentioned cultivation PBMC, add and promote that monocyte becomes cytokine and one or more class tongued bell receptor activator of DC cell activation maturation, with can with DC one or more polypeptide of being combined of MHCI on the surface, meanwhile add anti-human CTLA-4 antibody co-cultivation, block the restraining effect of CTLA-4, be placed in 37 DEG C, 5%CO
2cultivate 12 days in incubator.
4. the preparation method of a kind of IC seedling as claimed in claim 1, is characterized in that, described DC cell activation factor is GM-CSF, IL-4.
5. the preparation method of a kind of IC seedling as claimed in claim 1, is characterized in that, class tongued bell receptor activator is CpG, poly(I:C), IFN-γ, R848.
6. the IC seedling prepared of the preparation method of IC seedling as claimed in claim 1, is characterized in that, this IC seedling application in cancer therapy.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511015628.XA CN105462926A (en) | 2015-12-31 | 2015-12-31 | Preparation method and application of immunocytoma vaccine |
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| CN201511015628.XA CN105462926A (en) | 2015-12-31 | 2015-12-31 | Preparation method and application of immunocytoma vaccine |
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| CN105462926A true CN105462926A (en) | 2016-04-06 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574149A (en) * | 2016-07-05 | 2018-01-12 | 上海细胞治疗研究院 | A kind of efficient rush maturation method of BMDC and application thereof |
| WO2020038299A1 (en) * | 2018-08-20 | 2020-02-27 | 中国科学院过程工程研究所 | Exosome-based antitumor vaccine |
| CN114099662A (en) * | 2021-10-27 | 2022-03-01 | 山西协策企业管理咨询有限公司 | Monocyte-loaded E6E7 fusion protein vaccine composition and preparation method and application thereof |
| WO2023078305A1 (en) * | 2021-11-02 | 2023-05-11 | 上海细胞治疗集团有限公司 | Super dc expressing immune checkpoint inhibitor and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102782123A (en) * | 2010-02-10 | 2012-11-14 | 伊穆尼肯公司 | Improved composition for inhibiting tumor cell proliferation |
| CN104258415A (en) * | 2014-09-16 | 2015-01-07 | 上海交通大学 | Cell fusion tumor vaccine as well as preparation method and application of tumor vaccine in gastric cancer prevention and treatment |
-
2015
- 2015-12-31 CN CN201511015628.XA patent/CN105462926A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102782123A (en) * | 2010-02-10 | 2012-11-14 | 伊穆尼肯公司 | Improved composition for inhibiting tumor cell proliferation |
| CN104258415A (en) * | 2014-09-16 | 2015-01-07 | 上海交通大学 | Cell fusion tumor vaccine as well as preparation method and application of tumor vaccine in gastric cancer prevention and treatment |
Non-Patent Citations (1)
| Title |
|---|
| 连增志: "细胞毒性T细胞联合PD-L1单抗对肺癌免疫治疗的实验研究", 《中国优秀硕士论文全文数据库 医药卫生科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574149A (en) * | 2016-07-05 | 2018-01-12 | 上海细胞治疗研究院 | A kind of efficient rush maturation method of BMDC and application thereof |
| CN107574149B (en) * | 2016-07-05 | 2022-02-18 | 上海细胞治疗研究院 | Maturation promoting method of dendritic cells and application thereof |
| WO2020038299A1 (en) * | 2018-08-20 | 2020-02-27 | 中国科学院过程工程研究所 | Exosome-based antitumor vaccine |
| CN114099662A (en) * | 2021-10-27 | 2022-03-01 | 山西协策企业管理咨询有限公司 | Monocyte-loaded E6E7 fusion protein vaccine composition and preparation method and application thereof |
| WO2023078305A1 (en) * | 2021-11-02 | 2023-05-11 | 上海细胞治疗集团有限公司 | Super dc expressing immune checkpoint inhibitor and use thereof |
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Application publication date: 20160406 |