CN117003831A - NK cells amplified by polypeptide induction and application of NK cells in cell therapy - Google Patents
NK cells amplified by polypeptide induction and application of NK cells in cell therapy Download PDFInfo
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- CN117003831A CN117003831A CN202311009965.2A CN202311009965A CN117003831A CN 117003831 A CN117003831 A CN 117003831A CN 202311009965 A CN202311009965 A CN 202311009965A CN 117003831 A CN117003831 A CN 117003831A
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- 238000002659 cell therapy Methods 0.000 title abstract description 6
- 230000006698 induction Effects 0.000 title abstract description 4
- 238000000338 in vitro Methods 0.000 claims abstract description 18
- 230000004663 cell proliferation Effects 0.000 claims abstract description 15
- 230000001737 promoting effect Effects 0.000 claims abstract description 10
- 230000002147 killing effect Effects 0.000 claims abstract description 7
- 230000002708 enhancing effect Effects 0.000 claims abstract description 6
- 230000022534 cell killing Effects 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
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- 238000002360 preparation method Methods 0.000 claims description 5
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- 108010056995 Perforin Proteins 0.000 description 4
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 4
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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- 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
- C12N5/0646—Natural killers cells [NK], NKT cells
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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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
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Abstract
The invention provides NK cells amplified by polypeptide induction and application of NK cells in cell therapy. The polypeptide provided by the invention can promote NK cell proliferation in vitro and can also improve the killing power of NK cells, so that the polypeptide can be used for promoting NK cell proliferation in vitro and enhancing the tumor killing activity of NK cells, and can be used for preparing a cell culture medium capable of promoting NK cell proliferation in vitro and enhancing the tumor killing activity of NK cells.
Description
Technical Field
The invention relates to the technical field of NK cells, in particular to an NK cell with polypeptide induced amplification and application thereof in cell therapy.
Background
Natural killer cells (natural killer cell, NK cells) are derived from bone marrow lymphoid stem cells, whose differentiation and development depend on the bone marrow and thymus microenvironment, and are predominantly distributed in bone marrow, peripheral blood, liver, spleen, lung and lymph nodes. NK cells, unlike T, B cells, are a class of lymphocytes that can nonspecifically kill tumor cells and virus-infected cells without prior sensitization.
Cell immunotherapy is also called adoptive cell immunotherapy, which refers to the in vitro use of IL-2 to culture lymph node cells with anti-tumor cell property in the body of a tumor patient, when the lymph node cells are amplified to a certain quantity, the lymph node cells with anti-tumor cell property are returned to the body of the patient, and the lymphocytes with anti-tumor cell property can be utilized to kill or kill tumor cells, thereby achieving the anti-tumor purpose. The cell immunotherapy is mainly applied to natural killer cells, cytokine-induced killer cells, tumor-infiltrating lymphocytes, cytotoxic T lymphocytes and the like.
Among them, NK cells are important immune cells of the body, and are involved not only in anti-tumor, anti-viral infection and immunomodulation, but also in the development of hypersensitivity and autoimmune diseases in some cases, capable of recognizing target cells, killing mediators. Cellular immunotherapy has high requirements on the input amount of immune cells such as NK, and therefore, a sufficient amount of NK cells needs to be obtained in vitro before the input. Therefore, how to promote NK cell proliferation and increase the killing power of tumor cells in vitro has become an important research direction in the field of cellular immunotherapy.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides NK cells amplified by polypeptide induction and application of NK cells in cell therapy. The polypeptide provided by the invention can promote NK cell proliferation in vitro and improve the killing power of NK cells, so that the polypeptide can be used in the field of cell therapy.
The object of the present invention is first to provide a polypeptide, which is characterized in that it comprises the following sequence QNEMWI NDTEDENMLHPHGHGPMRKMPMKE.
Preferably, the polypeptide comprises a polypeptide variant which has more than 90% similarity to QNEMWI NDTEDENMLHPHGHGPMRKMPMKE and which has the same function.
Preferably, the N-and C-termini of the polypeptides are not chemically modified polypeptides.
Further preferred are polypeptides whose N-and C-termini are chemically modified.
Further preferably, the N-terminus is modified by acetylation and the C-terminus is modified by amidation.
It is another object of the present invention to provide the use of the above polypeptide for the preparation of a product for promoting proliferation of NK cells in vitro.
It is another object of the present invention to provide the use of a polypeptide for the preparation of a product for enhancing NK cell killing.
It is another object of the present invention to provide the use of a polypeptide for preparing an NK cell in vitro medium which can promote proliferation and enhance killing of NK cells in vitro.
It is a further object of the present invention to provide a product or medium for promoting proliferation of NK cells in vitro, characterized in that said product or medium contains the polypeptide according to any one of claims 1-8.
Preferably, the concentration of the polypeptide is 10-20. Mu.M.
The invention has the following advantages: the polypeptide provided by the invention can promote NK cell proliferation in vitro and can also improve the killing power of NK cells, so that the polypeptide can be used for promoting NK cell proliferation in vitro and enhancing the tumor killing activity of NK cells, and can be used for preparing a cell culture medium capable of promoting NK cell proliferation in vitro and enhancing the tumor killing activity of NK cells.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
A polypeptide that promotes NK cell proliferation, said polypeptide having the amino acid sequence QNEMWINDTEDENMLHPHGHGPMRKMPMKE. The polypeptide is synthesized by a solid phase synthesis technology, each amino acid is sequentially connected by adopting an Fmoc-N end protection strategy according to a resin solid phase synthesis method, fmoc-protecting groups are sequentially removed during the process, then peptide is cut, a crude product is obtained, and the crude product is separated and purified by a C18 reverse phase silica gel column, so that the purity of the crude product is not lower than 98%.
Example 2
2.1NK cell isolation and culture
10mL of fresh healthy adult peripheral blood was withdrawn, heparin anticoagulated, peripheral blood mononuclear cells were isolated by a conventional method using lymphocyte separation solution (purchased from Solarbio), and cells were resuspended using MACS buffer (purchased from Miltenyi, germany). NKCell Biotin-Antibody Cocktail and NK cell MicroBead Cocktail (available from Miltenyi Corp., germany) were added in sequence for cell positive sorting, NK cell-enriched cell suspension was collected and examined for CD3 by flow cytometry - CD56 + CD16 + NK cell purity. CD3 was cultured using NK cell medium (available from Shanghai Gei Biotech Co., ltd.) - CD56 + CD16 + NK cells were resuspended to a cell density of 1X 10 5 Cell suspension/mL, inoculated in culture flask at 37℃in 5% CO 2 Culturing under the condition, changing liquid half amount every 2 days, continuously culturing for 1 week, and collecting NK cells.
2.2NK cell proliferation Activity assay
Collecting NK cells after 1 week of culture, inoculating into 96-well plate with 100 μl of culture medium per well, and adjusting cell density to 1×10 5 And/or holes. After 12h, 100. Mu.L of NK cell culture medium containing 10. Mu.M and 20. Mu.M of polypeptide was added to each of treatment groups 1-2, the control group was added with an equal amount of NK cell culture medium containing no polypeptide, and the blank group was a single NK cell culture medium with 8 wells each. 37 ℃ and 5% CO 2 After 48h of incubation, 50. Mu.L of CCK-8 solution was added to each well and incubated at 37℃in 5% CO 2 After incubation for 4 hours under the condition, the absorbance (OD) value of each well at 490nm wavelength was measured, and the cell proliferation rate was calculated according to the formula.
Proliferation rate = (treatment OD value-blank OD value)/(control OD value-blank OD value) ×100%.
2.3NK cell killing Activity assay
Collecting NK cells continuously cultured for 1 week, washing with PBS, and re-suspending with NK cell culture medium to obtain cell density of 5×10 6 Cell suspension/mL, inoculated in 24-well plate, cultured per wellBased on 2mL. After 24h, the medium was changed, wherein NK cell medium containing 10. Mu.M and 20. Mu.M polypeptides was sequentially added to treatment groups 1-2, and an equal amount of NK cell medium containing no polypeptide was added to control group, each group was repeated 6 times. 37 ℃ and 5% CO 2 After 48h incubation, cells were collected, washed with PBS, resuspended, and grown at 2X 10 6 The individual/tube was added to a flow tube, and fluorescein isothiocyanate-labeled perforin mab, granzyme B mab, CD107a mab, incubated in the dark for 30min, washed with pbs, and expression levels of granzyme B, perforin, CD107a in NK cells were detected with a flow cytometer.
The test results are as follows:
the NK cell proliferation activity analysis results showed that the NK cell proliferation activity of treatment groups 1-2 was significantly higher than that of the control group, and that the high concentration of polypeptide treatment group 2 had a stronger proliferation activity for promoting NK cells than that of the low concentration of polypeptide treatment group 1, as shown in table 1. It is illustrated that the polypeptides of the present invention are effective in promoting NK cell proliferation.
TABLE 1NK cell proliferative Activity
| Value-added rate (%) | |
| Treatment group 1 | 165±4.56 |
| Treatment group 2 | 278±3.97 |
| Control group | 100±2.45 |
The NK cell killing activity analysis results show that, as shown in Table 2, granzyme B, perforin and CD107a are all marker molecules showing the NK cell killing activity, and the NK cell killing activity can be intuitively reflected by analyzing the marker molecules. Under the treatment of the polypeptide, the expression level of the NK cell killing activity related marker molecules is obviously increased and is obviously higher than that of a control group. Further proved that the polypeptide plays a role in improving NK cell killing activity.
TABLE 2 granzyme B, perforin, CD107a expression levels
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A polypeptide comprising the sequence QNEMWINDTEDENMLHPHGHGPMRKMPMKE.
2. The polypeptide of claim 1, wherein the polypeptide comprises a polypeptide variant having greater than 90% similarity to QNEMWINDTEDENMLHPHGHGPMRKMPMKE and having the same function.
3. The polypeptide according to claim 1 or 2, characterized in that: a polypeptide whose N-and C-termini are not chemically modified.
4. The polypeptide according to claim 1 or 2, characterized in that: a polypeptide whose N and C termini are chemically modified.
5. The polypeptide of claim 4, wherein the N-terminus is modified by acetylation and the C-terminus is modified by amidation.
6. Use of a polypeptide according to any one of claims 1 to 5 for the preparation of a product for promoting proliferation of NK cells in vitro.
7. Use of a polypeptide according to any one of claims 1 to 5 for the preparation of a product for enhancing NK cell killing.
8. Use of the polypeptide according to any one of claims 1 to 5 for the preparation of an NK cell in vitro culture medium which promotes NK cell proliferation and enhances killing thereof in vitro.
9. A product or medium for promoting proliferation of NK cells in vitro, comprising the polypeptide of any one of claims 1-8.
10. The product or culture medium of claim 9, wherein the polypeptide is present at a concentration of 10 to 20 μm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118147066A (en) * | 2024-05-09 | 2024-06-07 | 山东省泉溪生物技术有限公司 | Efficient NK cell culture method |
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| CN114369140A (en) * | 2021-12-27 | 2022-04-19 | 周桂英 | Polypeptide and application thereof in NK cell culture and tumor cell immunotherapy |
| KR20220063446A (en) * | 2020-11-10 | 2022-05-17 | 주식회사 티제이씨라이프 | Method of promoting differentiation or proliferation during the differentiation process of peripheral blood mononuclear cells into natural killer cells |
| CN114835779A (en) * | 2022-06-13 | 2022-08-02 | 陈飞 | Polypeptide and application thereof in promoting proliferation of human natural killer cells and preparing human natural killer cell culture medium |
| CN115261319A (en) * | 2022-08-30 | 2022-11-01 | 溯玄(上海)生物技术有限公司 | Polypeptide-induced amplified NK (Natural killer) cell and application thereof in cell therapy |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220063446A (en) * | 2020-11-10 | 2022-05-17 | 주식회사 티제이씨라이프 | Method of promoting differentiation or proliferation during the differentiation process of peripheral blood mononuclear cells into natural killer cells |
| CN114369140A (en) * | 2021-12-27 | 2022-04-19 | 周桂英 | Polypeptide and application thereof in NK cell culture and tumor cell immunotherapy |
| CN114835779A (en) * | 2022-06-13 | 2022-08-02 | 陈飞 | Polypeptide and application thereof in promoting proliferation of human natural killer cells and preparing human natural killer cell culture medium |
| CN115261319A (en) * | 2022-08-30 | 2022-11-01 | 溯玄(上海)生物技术有限公司 | Polypeptide-induced amplified NK (Natural killer) cell and application thereof in cell therapy |
Non-Patent Citations (1)
| Title |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118147066A (en) * | 2024-05-09 | 2024-06-07 | 山东省泉溪生物技术有限公司 | Efficient NK cell culture method |
| CN118147066B (en) * | 2024-05-09 | 2024-07-30 | 山东省泉溪生物技术有限公司 | NK cell culture method |
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