CN105695403A - Killer immune cell culturing method and application thereof - Google Patents

Killer immune cell culturing method and application thereof Download PDF

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CN105695403A
CN105695403A CN201511018960.1A CN201511018960A CN105695403A CN 105695403 A CN105695403 A CN 105695403A CN 201511018960 A CN201511018960 A CN 201511018960A CN 105695403 A CN105695403 A CN 105695403A
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cell
cells
tumor
antibody
immunocyte
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岳喜连
刘根桃
韩姝
殷雪
程钊
李晓祥
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Zmks International Cancer Therapy Biotechnologies Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]

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Abstract

The invention discloses a killer immune cell culturing method and application thereof. The method is a novel technology for culturing, activating and amplifying peripheral blood mononuclear immune cells into tumor killer cells, and the tumor killing capacity of T cells is enhanced by adding a PD-1 antibody to inhibit combination of PD-1 on the T cells and tumor cells in the late period of cell culturing or before transfusion through the characteristic that the inhibition effect of an immunosuppression target point on the immune cell functions is inhibited by adding an immune check point antibody; the cells cultured through the method have CTL cells which are targeted on the tumor antigen specificity in high proportion and also have the high capacity on killing the tumor cells. In this way, not only is the defect of an existing technology on the CTL cell culturing effect overcome, but also limitation of an immune checking point on the T cell functions is fully utilized and relieved, therefore, the tumor cell killing capacity of the prepared cells is improved, and the efficacy of the prepared cells on clinical cancer treatment is improved.

Description

The cultural method of a kind of lethal immunocyte and application thereof
Technical field
The present invention relates to biomedicine technical field, particularly relate to cultural method and the application thereof of a kind of lethal immunocyte。
Background technology
Malignant tumor is the disease of a class serious harm human health the most, and the about three million people's diagnosis in the annual whole nation send out tumor patient for new, and more than 200 ten thousand people die from cancer。Although can be treated by operation, radiotherapy, chemotherapy, but most patient still dies from recurrence and the transfer of tumor。So, minimal neoplastic tissue or tumor cell how to remove residual are then that modern tumor immunology has one of problem to be solved。The basic reason occurred due to cancer is the monitoring of tumor cell escape immunocyte and carries out immoderate growth, so improving immune system identification and killing tumor cell will from can fundamentally remove tumor。Along with new technique application clinically, immunotherapy is increasingly becoming one for the treatment of cancer most efficient method。The cardinal principle of immune cell therapy treatment of cancer is dependent on the immunocyte specific killing to tumor, and the specificity and the function that how to improve immunocyte are one of urgent need to solve the problems。
As a class to antigen specific CD8 (ClusterofDifferentiation8) T cell, through dendritic cell (DendriticCell, DC) effective tumor antigen activates, and give antineoplastic specificity, cytotoxic T lymphocyte (CytotoxicTLymphocytes, CTL) is the topmost responsiveness lymphocyte of body antitumor。The functional advantage of CTL is at identical major histocompatibility complex (MajorHistocompatibilityComplex, when MHC) limiting, identify the target cell with direct killing antigen positive specifically, the cancerous cell with specific for tumour antigen can be eliminated pointedly。This antitumaous effect is to being distributed in tissue of patient intraorganic small size tumor tissues and individual tumors cell is particularly important and effective, and last one step that Ye Shi tumor tissue cell is eliminated, its effect in clinical practice is fully confirmed。
But exist on T cell surface immunosuppressive agent-immunologic test point (immunecheckpoint)-existence, these immunologic test points can suppress the activation of T cell, propagation and effector function。And these immunologic test point control stimulate the balance with coinhibitory signals altogether, and stimulate altogether and in the amplitude maintaining self tolerance and regulatory T-cell response and persistent period, there is important function with coinhibitory signals。Two main immunologic test point molecules---the albumen 1 (PD1) of cytotoxic T lymphocyte albumen 4 (CTLA4) and programmed cell death, is the negative growth factor expressed on cytotoxic T cell。Immunologic test point CTLA-4 and the B7 quasi-molecule of DC cell surface on T cell surface combine, and the B7 quasi-molecule of activating molecules CD28 and the DC cell surface on T cell surface can be stoped to be combined, and suppress DC cellular antigens to pass further and hold the ability activated with T cell activation。Immunologic test point PD-1 molecule on T cell surface is combined with PD-L1 or the PD-L2 molecule of tumor cell surface, will stop T cell release cells killer factor (such as IFN), and weaken the function of T cell killing tumor cell, helps tumor cell to escape。So, before narrow spectrum T cell (CTL) feeds back, the PD-L1/L2 on tumor cell of the PD-1 molecule in T cell can be hindered to be combined with PD-1 antibody treated cells, weaken restraining factors, strengthen the ability of T cell killing tumor cell。
It is low that CTL treatment cancer ubiquity on Present clinical specificity, feeds back the little weakness of aftereffect。For overcoming disadvantages mentioned above, the innovative point of this patent is in that: carried out blocking immunity by PD-1 antibody treatment before CTL feeds back and suppresses the function of target spot, and then strengthens the function of T cell killing tumor cell。
Summary of the invention
It is an object of the invention to provide the cultural method of a kind of lethal immunocyte and application thereof, to solve above-mentioned technical problem。
The present invention is by the following technical solutions for achieving the above object:
The cultural method of a kind of lethal immunocyte, comprises the steps:
1) first stage: add the cytokine and one or more tumor antigen proteins or long-chain polypeptide that promote the picked-up of DC cellular antigens in PBMC culture fluid, be placed in 37 DEG C, 5%CO2Cultivating 12-16 hour in incubator, in the process, tumor antigen, by monocytic cell ingests, decomposes and submission to onthe surface of monocytes；
2) second stage: continuing on the basis of first stage cultivation PBMC, promote that mononuclear cell becomes the ripe cytokine of DC cell activation and one or more classes tongued bell receptor activator (TLRLigands), and add and in conjunction with the polypeptide continuation of one or more, 37 DEG C, 5%CO can be placed in by MHCI on DC surface2Incubator is cultivated；
3) phase III: continue to cultivate 12 days and collect after reaching certain quantity, and with anti-human PD-1 antibody pretreatment, be used as the experiment of Cytotoxicity in vitro tumor cell after washing away free antibody or feed back patient。
The described cytokine promoting the picked-up of DC cellular antigens is GM-CSF, IL-4。
Described class tongued bell receptor activator is CpG, poly(I:C), IFN-γ, R848。
By the method for anti-human PD-1 antibody pretreatment in described step 3), adding normal saline to cell concentration is 1x108/ ml, is subsequently adding PD-1 antibody (KS-0002, Kang Zhi pharmacy) to 10ug/ml, and ambient temperatare is put 15 minutes, adds brine once, and adding culture medium to cell concentration is 2x107/ ml, the secretion as CTL fragmentation test or as antigen-specific Interferon is tested。
The method of described CTL fragmentation test, adds immunocyte in melanoma cell strain (M14) culture plate, being placed in 37 DEG C, 5%CO by effect target than 5:12Incubator is cultivated and tests by CCK8 method after co-culturing 5 hours。
The method of the secretion test of described antigen-specific Interferon, adds melanoma specific antigen peptide (1ug/ml) at immunocyte, is placed in 37 DEG C, 5%CO2Incubator is cultivated and after co-culturing 2 hours, carries out test interferon secretion by streaming method。
Utilize the lethal immunocyte of one that said method is cultivated in treatment of cancer and other clinical practices。
The invention has the beneficial effects as follows: the present invention is by cultivating activation and immunocyte (such as CTL, NK) change of technique, both by adding immunologic test point (PD-1) antibody in cultivating at cell or after cell cultivation, due to process through PD-1 antibody in the process that new cell is cultivated, so avoiding the restraining factors of immunologic test point PD-1 in effect process, thus producing that there is high antigenic specificity and the immune mature cell of strong killing tumor cell ability, experiment in vitro proves, the existing secretion for tumor antigen height specificity interferon of new method cultured cells, there is again the killing enhancing the tumor cell positive for tumor antigen, significantly improve and promote immunocyte killing tumor cell ability, it is contemplated that it strengthens the function suppressing tumor cell, thus improving its curative effect at clinical anticancer。
Accompanying drawing explanation
Fig. 1 is immunocyte killing tumor cells target cell activity comparison diagram；
Fig. 2 is the secretion ratio comparison diagram of immunocyte interferon；
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is further elaborated。
Embodiment 1:
In the culture medium 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%CO2Incubator is cultivated 12-16 hour, adds DC cell activation factor IFN-γ, CpG, Poly(I:C), R848, melanoma tumors antigen small peptide (0.1ug/ml), it is placed in 37 DEG C, 5%CO2Cultivating in incubator, cell is gathered after continuing cultivation 12 days。
The test of cell cultivation process cell component change: the composition of the product before cultivating and after cultivating carries out the T cell of test antigenic specificity by stream type cell analyzer。The T cell pentamer of melanoma tumors antigenic specificity detects, it was demonstrated that the ratio of T cells with antigenic specificity。
The cell collected, adding normal saline to cell concentration is 1x108/ ml, is subsequently adding PD-1 antibody (KS-0002, Kang Zhi pharmacy) to 10ug/ml。Ambient temperatare is put 15 minutes, adds brine once, and adding culture medium to cell concentration is 2x107/ ml, the secretion as CTL fragmentation test or as antigen-specific Interferon is tested。
The test of cells against tumor fragmentation test: add immunocyte in melanoma cell strain (M14) culture plate, being placed in 37 DEG C, 5%CO than 5:1 by effect target2Incubator is cultivated and tests by CCK8 method after co-culturing 5 hours。Result shows that adding PD-1 antibody can substantially increase the ability of immunocyte killing tumor cell M14。
The secretion making antigen-specific Interferon is tested by cell: adds melanoma specific antigen peptide (1ug/ml) at immunocyte, is placed in 37 DEG C, 5%CO2Incubator is cultivated and after co-culturing 2 hours, carries out test interferon secretion by streaming method。Result shows that adding PD-1 antibody can substantially increase the ability of immunocyte secretion interferon。
PD-1 antibody can increase the immunocyte killing to target cell as shown in Figure 1: add PD-1 antibody (ks-0002) and the immunocyte killing tumor cells target cell activity of preparation can be made to increase to 35.60% from 20.67%。
PD-1 antibody can increase the secretion of immunocyte interferon as shown in Figure 2: when stimulating with tumor specific antigen, and addition PD-1 antibody (ks-0002) can make the secretion ratio of immunocyte interferon kill increases to 4.77% from 3.02%。
The present invention is by cultivating activation and immunocyte (such as CTL, NK) change of technique, both by adding immunologic test point (PD-1) antibody in cultivating at cell or after cell cultivation, due to process through PD-1 antibody in the process that new cell is cultivated, so avoiding the restraining factors of immunologic test point PD-1 in effect process, thus producing that there is high antigenic specificity and the immune mature cell of strong killing tumor cell ability, experiment in vitro proves, the existing secretion for tumor antigen height specificity interferon of new method cultured cells, there is again the killing enhancing the tumor cell positive for tumor antigen, significantly improve and promote immunocyte killing tumor cell ability, it is contemplated that it strengthens the function suppressing tumor cell, thus improving its curative effect at clinical anticancer。
As known by the technical knowledge, the present invention can be realized by the embodiment of other essence without departing from its spirit or essential feature。Therefore, embodiment disclosed above, for each side, all it is merely illustrative, is not only。All within the scope of the present invention or be all included in the invention in the change being equal in the scope of the present invention。

Claims (4)

1. the cultural method of a lethal immunocyte, it is characterised in that comprise the steps:
1) first stage: add the cytokine and one or more tumor antigen proteins or long-chain polypeptide that promote the picked-up of DC cellular antigens in PBMC culture fluid, it is placed in 37 DEG C, 5%CO2 incubator is cultivated 12-16 hour, in the process, tumor antigen, by monocytic cell ingests, decomposes and submission to onthe surface of monocytes；
2) second stage: continuing on the basis of first stage cultivation PBMC, promote that mononuclear cell becomes the ripe cytokine of DC cell activation and one or more class tongued bell receptor activator, and add and can continue in conjunction with one or more polypeptide by MHCI on DC surface, it is placed in 37 DEG C, 5%CO2 incubator is cultivated；
3) phase III: continue to cultivate 12 days and collect after reaching certain quantity, and with anti-human PD-1 antibody pretreatment, be used as the experiment of Cytotoxicity in vitro tumor cell after washing away free antibody or feed back patient。
2. the cultural method of a kind of lethal immunocyte as claimed in claim 1, it is characterised in that the described cytokine promoting the picked-up of DC cellular antigens is GM-CSF, IL-4。
3. the cultural method of a kind of lethal immunocyte as claimed in claim 1, it is characterised in that class tongued bell receptor activator is CpG, poly(I:C), IFN-γ, R848。
4. the cultural method of a kind of lethal immunocyte as claimed in claim 1, it is characterized in that, by the method for anti-human PD-1 antibody pretreatment in described step 3), adding normal saline is 1x108/ml to cell concentration, is subsequently adding PD-1 antibody (KS-0002, Kang Zhi pharmacy) to 10ug/ml, ambient temperatare puts 15 minutes, adding brine once, adding culture medium to cell concentration is 2x107/ml, and the secretion as CTL fragmentation test or as antigen-specific Interferon is tested。
CN201511018960.1A 2015-12-31 2015-12-31 Killer immune cell culturing method and application thereof Pending CN105695403A (en)

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CN106177931A (en) * 2016-08-25 2016-12-07 河北利同康生物科技有限公司 The double CTL high efficiency that blocks of immune detection point kills the preparation method of cell preparation
CN108048397A (en) * 2017-12-15 2018-05-18 海南博鳌银丰康养国际医院有限公司 It is a kind of to utilize the method for targeting PD-1 antibody silence immunocyte negative regulation receptors
CN108379569A (en) * 2018-05-11 2018-08-10 北京百益宁医学科技有限责任公司 The DC vaccines of efficient load tumour antigen and its method of induced amplification specific for tumour antigen CTL
CN112867922A (en) * 2018-10-03 2021-05-28 国立大学法人长崎大学 Method for predicting effect of immune checkpoint inhibitor
CN113046313A (en) * 2021-03-18 2021-06-29 重庆福美干细胞生物科技发展有限公司 Composition and kit for efficiently inducing and amplifying human peripheral blood killer immune cells and culture method of immune cells
CN114292813A (en) * 2022-03-02 2022-04-08 北京市希波生物医学技术有限责任公司 Culture medium formulations for activation of the global anti-tumor immune system and methods for preparing agonist-activated global immune effector cells

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106177931A (en) * 2016-08-25 2016-12-07 河北利同康生物科技有限公司 The double CTL high efficiency that blocks of immune detection point kills the preparation method of cell preparation
CN106177931B (en) * 2016-08-25 2018-02-02 河北浓孚雨生物科技有限公司 The double preparation methods for blocking CTL high efficiency killing cell preparation of immune detection point
CN108048397A (en) * 2017-12-15 2018-05-18 海南博鳌银丰康养国际医院有限公司 It is a kind of to utilize the method for targeting PD-1 antibody silence immunocyte negative regulation receptors
CN108379569A (en) * 2018-05-11 2018-08-10 北京百益宁医学科技有限责任公司 The DC vaccines of efficient load tumour antigen and its method of induced amplification specific for tumour antigen CTL
CN112867922A (en) * 2018-10-03 2021-05-28 国立大学法人长崎大学 Method for predicting effect of immune checkpoint inhibitor
CN113046313A (en) * 2021-03-18 2021-06-29 重庆福美干细胞生物科技发展有限公司 Composition and kit for efficiently inducing and amplifying human peripheral blood killer immune cells and culture method of immune cells
CN114292813A (en) * 2022-03-02 2022-04-08 北京市希波生物医学技术有限责任公司 Culture medium formulations for activation of the global anti-tumor immune system and methods for preparing agonist-activated global immune effector cells
CN114292813B (en) * 2022-03-02 2022-11-08 北京市希波生物医学技术有限责任公司 Culture medium formulations for activating the global anti-tumor immune system and methods for preparing agonist-activated global immune effector cells

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