CN102559600A - Artificial antigen presenting cell and application thereof in NK (natural killer) cell amplification - Google Patents
Artificial antigen presenting cell and application thereof in NK (natural killer) cell amplification Download PDFInfo
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Abstract
The invention provides an in vitro amplification method for efficient and highly cytotoxic natural killer (NK) cells. Novel artificial antigen presenting cells 4-1BBL-mIL-21-aAPC, such as 4-1BBL-mIL-21-K562 cells and the like, are constructed through stably expressing 4-1BB ligands (4-1BBL) and membrane immobilized interleukin 21 (mIL-21) on the surfaces of cell membranes, and by using the novel artificial antigen presenting cells as feeder cells for amplification, the NK cells are directly amplified from peripheral blood lymphocytes. Flow cytometry, cytotoxicity test and the like suggest that the amplified cells NK have high purity and strong cytotoxicity and have obvious killing effect on tumor cells.
Description
Technical field
The present invention relates to the immunotherapy of disease field, relate in particular to the method for NK cell expansion ex vivo in the treatment of NK cellular immunization.
Background technology
NK cell (natural killer cell; Nk cell) is one type of large granular lymphocyte; Be the important bridge of body initial (innate) immunity and follow-up (adaptive) immunity, in body anti-infectious disease and malignant tumour immunosurveillance process, play an important role.Secondary cases NK Transplanted cells can produce good anti-tumour effect; And himself is seldom repelled by acceptor; And can not cause any GVHD (Graft Versus Host Disease); Therefore, Secondary cases NK cell therapy (Adoptive NK cell therapy) has become one of the most popular at present and the most promising treating malignant tumor method.
Although the treatment of Secondary cases NK cellular immunization has good anti-tumour effect and great application prospect; But the treatment of NK cellular immunization still is difficult to carry out routine clinical uses; Wherein main obstacle is that in peripheral blood lymphocyte (PBMC), the NK cell is a very little colony; Only account for 10～15% of human peripheral lymphocyte, be difficult to satisfy huge clinical needs.For solving this difficult problem, at first to quantitatively guarantee the effective supply of NK cell, therefore, explore the important development trend that various effective NK cell expansion ex vivo (Ex vivo expansion) method has become the treatment of NK cellular immunization.
(artificial antigen presenting cell aAPC) carries out a kind of important method that the NK cell expansion ex vivo has become the NK cell amplification as feeder cell (feeder cell) to the using artificial antigen presenting cell.K562 cell that the investigator uses genetic modification is arranged as artificial antigen submit cell in research in the past; As use fixedly IL15 (membrane-bound IL-15 of 4-1BBL (CD137L) and film; MIL-15) the K562 cell that transforms carries out the NK cell amplification; This method can obtain higher Cytotoxic NK cell, but NK cell proliferation is restricted, 5-6 after week cell no longer increase.
Summary of the invention
The objective of the invention is, a kind of artificial antigen submit cell is provided.
Second purpose of the present invention is that a kind of preparation method of artificial antigen submit cell is provided.
The 3rd purpose of the present invention is that the application of a kind of artificial antigen submit cell in the NK cell amplification is provided.
The 4th purpose of the present invention is that a kind of NK methods for cell expansion is provided.
In order to solve first purpose of the present invention, the invention provides a kind of artificial antigen submit cell, said artificial antigen submit cell is 4-1BBL-mIL-21-aAPC, its surface of cell membrane stably express 4-1BB part and film be interleukin-22 1 (mIL-21) fixedly.
As one preferred, said artificial antigen submit cell is the 4-1BBL-mIL-21-K562 cell.
In order to solve second purpose of the present invention, the invention provides the preparation method of artificial antigen submit cell, may further comprise the steps:
(1) makes up the transposon that 4-1BBL expresses;
(2) 4-1BBL transposon and the corresponding cell of transposase SB11 cotransfection, the fluidic cell sorting obtains 4-1BBL stably express cell 4-1BBL-aAPC;
(3) import on the 4-1BBL-aAPC basis that step (2) obtains that (membrane-bound IL-21 mIL-21), sets up 4-1BBL-mIL-21-aAPC at the IL-21 of surface of cell membrane stably express.
As one preferred, said artificial antigen submit cell is the 4-1BBL-mIL-21-K562 cell.
In order to solve the 3rd purpose of the present invention, the invention provides the application of artificial antigen submit cell in the NK cell amplification.
In order to solve the 4th purpose of the present invention, the invention provides a kind of NK methods for cell expansion, utilize above-mentioned artificial antigen submit cell to increase, may further comprise the steps:
(1) makes up the transposon that 4-1BBL expresses;
(2) 4-1BBL transposon and the corresponding cell of transposase SB11 cotransfection, the fluidic cell sorting obtains 4-1BBL stably express cell 4-1BBL-aAPC;
(3) import on the 4-1BBL-aAPC basis that step (2) obtains that (membrane-bound IL-21 mIL-21), sets up 4-1BBL-mIL-21-aAPC at the IL-21 of surface of cell membrane stably express;
(4) artificial antigen submit cell of lethal exposure or MTC treatment step (3) acquisition is pressed 2:1 and is mixed with peripheral blood lymphocyte, in NK cell amplification nutrient solution, cultivates altogether;
After (5) 1 weeks, repetitive stimulation 1 time, continuous several weeks are to obtain the NK cell of q.s.
As one preferred, said artificial antigen submit cell is the 4-1BBL-mIL-21-K562 cell.
The invention has the advantages that; For setting up a kind of NK cell expansion ex vivo method of efficient and high cell toxicity; We are through at the fixing interleukin-22 1 (mIL-21) of surface of cell membrane stably express 4-1BB part (4-1BBL) and film, and structure novel artificial antigen presenting cell 4-1BBL-mIL-21-aAPC is like the 4-1BBL-mIL-21-K562 cell; With these feeder cell as amplification, the NK cell directly increases from peripheral blood lymphocyte.Flow cytometry, cell toxicity test etc. show that the NK cell purity height and the cytotoxicity that are increased are strong, have tangible tumor cytotoxicity effect.NK methods for cell expansion provided by the invention is simple, easy handling; Amplification efficiency is good, purity is high; Amplification longer duration, tumor cytotoxicity effect is obvious.
Description of drawings
Fig. 1 is 4-1BBL and the expression of mIL-21 in 4-1BBL-mIL-21-K562 artificial antigen submit cell.
Fig. 2 by the 4-1BBL-mIL-21-K562 artificial antigen submit cell the purity (W representative amplification all numbers) of amplification NK cell.
Fig. 3 by 4-1BBL-mIL-21-K562 artificial antigen submit cell and 4-1BBL-mIL-15-K562 artificial antigen submit cell amplification NK cell breeding ratio.
Fig. 4 is the expression of receptor (all numbers of W representative amplification, cell before the 0 W representative amplification) before and after the NK cell amplification.
Fig. 5 by 4-1BBL-mIL-21-K562 artificial antigen submit cell and 4-1BBL-mIL-15-K562 artificial antigen submit cell the tumor cytotoxicity efficient (increasing different donors the 21st day) of amplification NK cell.
Embodiment
Below in conjunction with accompanying drawing the preparation method of 4-1BBL-mIL-21-K562 provided by the invention and the embodiment of NK methods for cell expansion are elaborated.
The preparation method of embodiment 1, artificial antigen submit cell 4-1BBL-mIL-21-K562
1. from Open Biosysems (Thermo Fisher Scientific; CO USA) buys 4-1BBL/PCR4 TOPO plasmid, pcr amplification; Insert the Nhe I-XhoI site of GlySer-EGFP (CoOp)-pSBSO sleeping beauty expression vector then, make up the 4-1BBL/pSBSO transposon.
2. with 4-1BBL/pSBSO transposon and transposase SB11 cotransfection K562 cell, the fluidic cell sorting obtains the K562 cell (4-1BBL-K562) of 4-1BBL stably express.
3. with IL-21-Fc (CoOp)-pSBSO and transposase SB11 cotransfection 4-1BBL-K562 cell, the 4-1BBL-mIL-21-K562 artificial antigen submit cell is set up in the fluidic cell sorting.
1. from Open Biosysems (Thermo Fisher Scientific; CO USA) buys 4-1BBL/PCR4 TOPO plasmid, pcr amplification; Insert the Nhe I-XhoI site of GlySer-EGFP (CoOp)-pSBSO sleeping beauty expression vector then, make up the 4-1BBL/pSBSO transposon.
2. with 4-1BBL/pSBSO transposon and transposase SB11 cotransfection K562 cell, the fluidic cell sorting obtains the K562 cell (4-1BBL-K562) of 4-1BBL stably express.
3. with IL-21-Fc (CoOp)-pSBSO and transposase SB11 cotransfection 4-1BBL-K562 cell, 4-1BBL-mIL-21-K562 artificial antigen submit cell is set up in the fluidic cell sorting.
4. with IL-15-Fc (CoOp)-pSBSO and transposase SB11 cotransfection 4-1BBL-K562 cell, 4-1BBL-mIL-15-K562 artificial antigen submit cell is set up in the fluidic cell sorting.
5. with lymphocyte separation medium separation of human peripheral blood lymphocyte.
6.100Gy the artificial antigen submit cell that radiation exposure makes up.
7. the artificial antigen submit cell that shone mixes by 2:1 with lymphocyte, in the RPMI1640 nutrient solution that contains 10%FBS, 1% P/S, 2 mM L-glutaminate, 50U/ml IL-2 in 5% CO
2Cultivate altogether in the incubator, changed fresh culture once in per 2 days.
8. cell was 1 time after 6 and 7 program repetitive stimulations increased set by step weekly.
9. in different time points, detect NK cell purity (see figure 2), cell number (see figure 3), surface receptor and express (see figure 4) and tumour cell killed and wounded the effectiveness (see figure 5).
Through making up the novel artificial antigen presenting cell, with these feeder cell as amplification, the NK cell directly increases from peripheral blood lymphocyte.Flow cytometry, cell toxicity test etc. show that the expanded cells NK of institute purity height and cytotoxicity are strong, have tangible tumor cytotoxicity effect.
The above only is a preferred implementation of the present invention, should be pointed out that for those skilled in the art; Under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching,, otherwise realize 4-1BBL and mIL-21 expression at surface of cell membrane like alternative K562 cells such as cell 721.221 cells with other type; Amplification condition and program are improved or the like, and these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (7)
1. an artificial antigen submit cell is characterized in that, said artificial antigen submit cell is 4-1BBL-mIL-21-aAPC, its surface of cell membrane stably express 4-1BB part and mIL-21.
2. a kind of artificial antigen submit cell according to claim 1 is characterized in that, said artificial antigen submit cell is the 4-1BBL-mIL-21-K562 cell.
3. the preparation method of the described artificial antigen submit cell of claim 1 is characterized in that, may further comprise the steps:
(1) makes up the transposon that 4-1BBL expresses;
(2) 4-1BBL transposon and the corresponding cell of transposase SB11 cotransfection, the fluidic cell sorting obtains 4-1BBL stably express cell 4-1BBL-aAPC;
(3) IL-21 that on the 4-1BBL-aAPC basis that step (2) obtains, imports at the surface of cell membrane stably express sets up 4-1BBL-mIL-21-aAPC.
4. the preparation method of artificial antigen submit cell according to claim 3 is characterized in that, said artificial antigen submit cell is the 4-1BBL-mIL-21-K562 cell.
5. claim 1 or 2 application of described artificial antigen submit cell in the NK cell amplification.
6. a NK methods for cell expansion is characterized in that, utilizes the said artificial antigen submit cell of claim 1 to increase, and may further comprise the steps:
(1) makes up the transposon that 4-1BBL expresses;
(2) 4-1BBL transposon and the corresponding cell of transposase SB11 cotransfection, fluidic cell sorting, the cell 4-1BBL-aAPC of acquisition 4-1BBL stably express;
(3) IL-21 that on the 4-1BBL-aAPC basis that step (2) obtains, imports at the surface of cell membrane stably express sets up 4-1BBL-mIL-21-aAPC;
(4) artificial antigen submit cell of lethal exposure or MTC treatment step (3) acquisition is pressed 2:1 and is mixed with peripheral blood lymphocyte, in NK cell amplification nutrient solution, cultivates altogether;
After (5) 1 weeks, repetitive stimulation 1 time, continuous several weeks are to obtain the NK cell of q.s.
7. a kind of NK methods for cell expansion according to claim 6 is characterized in that said artificial antigen submit cell is the 4-1BBL-mIL-21-K562 cell.
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| CN 201110449527 CN102559600A (en) | 2011-12-29 | 2011-12-29 | Artificial antigen presenting cell and application thereof in NK (natural killer) cell amplification |
| CN201210237023.5A CN102911918B (en) | 2011-12-29 | 2012-07-10 | Gene engineering cell and application thereof in NK (Nature Killer) cell proliferation |
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| CN102911918B (en) | 2014-12-10 |
| CN102911918A (en) | 2013-02-06 |
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