CN101386840A - Construction method of CD3<->CD56<+>NK cell high-efficient multiplication culture system - Google Patents
Construction method of CD3<->CD56<+>NK cell high-efficient multiplication culture system Download PDFInfo
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- CN101386840A CN101386840A CNA2008101550732A CN200810155073A CN101386840A CN 101386840 A CN101386840 A CN 101386840A CN A2008101550732 A CNA2008101550732 A CN A2008101550732A CN 200810155073 A CN200810155073 A CN 200810155073A CN 101386840 A CN101386840 A CN 101386840A
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Abstract
The invention discloses a method for constructing CD3<->CD56<+>NK cell efficient culture system, and mainly provides a culture system for adhering invitro mass amplified CD3<->CD56<+>NK cells which consist of anti-CD137mAb and IL-15 by using Poly-D-Lysine or Poly-L-Lysine as adhesion agent. Compared with the prior other methods, the method obviously has the advantages of simple and reasonable method, easy promotion, less required raw materials, low cost, shorter preparation cycle, high amplification efficiency, high killing efficiency to tumor cells and so on. Experiments prove that the capability of killing A549 of the CD3<->CD56<+>NK cells which are amplified by the method is increased by one time compared with CD3<->CD56<+>NK cells which are amplified by other known cytokines or cytokine combination in the same cell concentration.
Description
Technical field
The present invention relates to the CD3 that can be used for the treatment of tumor disease that a kind of Poly-D-Lysine sticks mouse anti human CD137 monoclonal antibody anti-CD137 mAb and recombinant human cytokine IL-15 composition
-CD56
+The construction process of the amplification cultivation system of NK cell belongs to a kind of amplification method of biological products.
Background technology
The NK cell was found in 1975, belonged to large granular lymphocyte on form, derived from marrow, accounted for 5%-10% of total number of peripheral blood.It has the broad-spectrum anti-tumor cytosis, and is particularly more obvious to lymphoma and leukemia cell's effect, is the first line of defence of antineoplastic immune, at CD3
-CD56
+The lethal effect of the responsive target cell of NK (as the K562 cell), this killing ability is not subjected to target cell whether to express the influence of MHC; CD3
-CD56
+The NK cell still connects the important step of the natural immunity and acquired immunity.Excite in the process of ctl response CD3 at DC
-CD56
+The NK cell can bring into play similar TH cell subsidiary function (Mailliard RB, et al.J.Immunol, 2003,171:2366).Recent studies show that CD3
-CD56
+NK cell and dendritic cell (DC) interact, can improve the ability that DC excites CD4+TH1 reaction and ctl response (Adam C, et al.Blood, 2005,106:338).Even CD3
-CD56
+The NK cell also has the ability of direct activated T cell.
Because CD3
-CD56
+The unique effect of NK cell in immunne response, it is used in the tumour cell treatment and more and more comes into one's own.Yet, CD3
-CD56
+How NK cell comparatively small amt in vivo obtains the CD3 of sufficient amount
-CD56
+The NK cell becomes the bottleneck of its clinical application of restriction, and the CD3 of present method amplification
-CD56
+NK is still very limited to the lethal effect of solid tumor.In addition, discover CD3 in the tumour patient body
-CD56
+The NK cell quantity increases its function and descends on the contrary, CD3
-CD56
+The often cancer-prone just people of the people that the NK cell is maximum, the intravital CD3 of this explanation tumour patient
-CD56
+May there be defective in the NK cell.Therefore, how to improve CD3
-CD56
+The quantity of NK cell and its function that strengthens become the difficult problem of puzzlement medical profession.
Nearest studies show that, costimulatory molecules CD137 has brought into play important role in NK cell proliferation and function adjusting.People CD137 is TNF acceptor (TNFR) superfamily member, and molecular weight is the I type transmembrane glycoprotein of 30kD.Under the condition of depositing the TCR signal, the CD137 signal can provide the costimulatory signal of collaborative CD28, keeps the active state of cell, suppresses the generation of AICD.Studies show that in recent years, CD137 also is expressed in the surface of NK cell and has participated in the function adjusting of NK cell.Discoveries such as Wilcox excite CD3
-CD56
+CD137 molecule on the NK cell can promote the secretion of NK cell proliferation and IFN-γ, and this activated CD3
-CD56
+The NK cell by and the killing activity of the interaction enhanced CT L of CTL (Wilcox RA, et al.J Immunol, 2002,169:4230).Other has the research report, the application of CD137 activated NK cell in leukemia treating, they with transfection people 4-1BB Ligand (4-1BBL) and IL-15 gene and express membranous type 4-1BBL and the K562 human leukemia cell of IL-15 molecule and human peripheral are cultivated, obtained CD3
-CD56
+The enrichment of NK cell, this CD3
-CD56
+The NK cell to all kinds of blood system cell strains have very strong lethal effect (Shi J, et al.Blood, 2005,106:3392).The The above results prompting, the collaborative IL-15 of CD137 agonist activates CD3
-CD56
+The approach of NK cell (CD137-IL-15-NK) has and helps solve CD3
-CD56
+The difficult problem that the NK cell is used in the tumour cell treatment.Also need to solve series of theories and technical problem but how the CD137-IL-15-NK cell moves towards clinical application from the laboratory.At first the mode of action of CD137 and IL-15 needs to improve, adopt retrovirus 4-1BBL and IL-15 transfection simultaneously to be arrived the method for tumor cell line at present, though but it is comparatively loaded down with trivial details to improve the amplification efficiency method, be unfavorable for promoting, adopt retrovirus that certain risk is arranged and the transgenic cell introduced in culture system has also restricted CD3
-CD56
+The separation and purification of NK cell.
Summary of the invention
The objective of the invention is weak point, utilize the collaborative IL-15 of anti-CD137 mAb to activate CD3 for the transgenic cell that overcomes the retroviral method cultivation of above-mentioned use
-CD56
+The approach of NK cell provides a kind of simple structure, easy handling, and the amplification efficiency height obviously improves the kill rate of lung adenocarcinoma cell line A549, to CD3
-CD56
+The NK cell is applied to the significant CD3 of clinical immunotherapy
-CD56
+The construction process of NK cell high-efficient amplification cultivation system.
CD3 of the present invention
-CD56
+The construction process of NK cell high-efficient amplification cultivation system, mainly provide a kind of by Poly-D-Lysine as binder, stick external a large amount of amplification CD3 that anti-CD137 mAb and IL-15 constitute
-CD56
+The culture systems of NK cell, its key step comprises:
(1) gets an amount of binder Poly-D-Lysine or Poly-L-Lysine with distilled water diluting to 10%, add plastic culture plate, culture dish or culturing bottle, sop up the binder diluent after hatching 5-10min, binder is attached on plastic culture plate, culture dish or the culturing bottle;
(2) getting an amount of anti-CD137 mAb or CD137L recombinant protein and IL-15 adds in the damping fluid simultaneously, be diluted to concentration and be respectively 1-10 μ g/ml and 10-100ng/ml, to have in the plastic culture of Poly-D-Lysine or Poly-L-Lysine plate, culture dish or the culturing bottle sticking of preparation in anti-CD137 mAb and the IL-15 mixed diluting liquid adding above-mentioned steps (1), 4 ℃ are spent the night, wrap by anti-CD137mAb and IL-15 on plastic culture plate, culture dish or the culturing bottle that binder was handled, the diluent of plastic plate is removed in suction, and is stand-by;
(3) peripheral blood that picks up from the healthy volunteer separates acquisition mononuclearcell (PBMC) through Ficoll, adjusts concentration to 3 * 10 of PBMC with serum free medium
6/ ml adds 6 hole plastic culture plates and cultivates 2-3h, draws suspension cell, above-mentioned suspension cell or adjust concentration to 1 * 10 with serum free medium from the high purity N K cell that sorting wherein obtains
6/ ml adds Poly-D-Lysine or Poly-L-Lysine and handled and wrapped by on the culture plate of anti-CD137 mAb and IL-15, culture dish or the culturing bottle, and adds IL-2 simultaneously, cultivates after 3-6 days the CD3 after collecting cell must increase
-CD56
+The NK cell.
CD3 with method amplification cultivation of the present invention
-CD56
+The CD3 of NK cell and other method amplification cultivation of existing usefulness
-CD56
+The NK cell is compared, and obviously has the following advantages and effect:
(1) simple in structure, be easy to promote.Adopt retrovirus that 4-1BBL and IL-15 transfection are simultaneously arrived tumor cell line, stimulate the NK cell with this cell strain then, though can improve amplification efficiency, but this method is comparatively loaded down with trivial details, and the transgenosis cell strain cycle that obtains stably express 4-1BBL and IL-15 is longer, and starting material required for the present invention are few, and cost is low, the preparation method is simple, and the cycle is also shorter.
(2) retrovirus has the potential pathogenic risk, and the CD3 of amplification
-CD56
+Sneaked into the CD3 that transgenic cell has also caused this method to increase in the NK cell
-CD56
+The NK cell can't directly drop into clinical use.And the present invention has overcome above-mentioned defective.
(3) amplification efficiency height.Compare with other known cytokines or combination of cytokines expanding effect, the present invention has higher amplification efficiency.
(4) killing-efficiency height.CD3 with other known cytokines or combination of cytokines amplification
-CD56
+The NK cell is compared, under identical cell concn, and the CD3 of the present invention's amplification
-CD56
+The NK cell is enhanced about more than once to the killing-efficiency of lung adenocarcinoma cell line A549.
Description of drawings
Fig. 1 is the human peripheral PBMC proliferating cells analysis of accounts figure that adopts 8 kinds of methods to cultivate;
It is 2 * 10 that PBMC adopts serum free medium to be adjusted to concentration
5/ ml, and interpolation 500IU/ml IL-2, add then to wrap after Poly-D-Lysine handles and wrap again by (Poly-D-Lysine+immobilized anti-CD137 mAb+immobilized IL-15+IL-2) in the culture plate of anti-CD137 mAb and IL-15, cultivated the propagation of cell counting analysis of cells 3 days.Poly-D-Lysine handles the back bag by homotype control mice IgG1 (Poly-D-Lysine+IgG1), IL-2, anti-CD137 mAb, IL-15, anti-CD137 mAb and IL-15 (anti-CD137 mAb+IL-15), direct coated anti-CD137 mAb and IL-15 (immobilized anti-CD137 mAb+immobilized IL-15) and direct coated anti-CD137 mAb and IL-15 add IL-2 (immobilized anti-CD137 mAb+immobilizedIL-15+IL-2) again and organize in contrast.
Fig. 2 adopts 8 kinds of method cultivator peripheral bloods CD3 after PBMC3 days
-CD56
+The flow cytometry figure of NK cell content;
Collect the cell that different methods was cultivated 3 days, mark anti-CD3-ECD and anti-CD56-PC5 fluorescence antibody.Adopt flow cytometry CD3 then
-CD56
+The percentage composition of NK cell.Different treatment group is respectively: Poly-D-Lysine+IgG1 (1), anti-CD137 mAb (2), IL-2 (3), IL-15 (4), anti-CD137mAb+IL-15 (5), immobilized anti-CD137 mAb+immobilized IL-15 (6), immobilizedanti-CD137 mAb+immobilized IL-15+IL-2 (7), Poly-D-Lysine+immobilized anti-CD137mAb+immobilized IL-15+IL-2 (8).
Fig. 3 is the CD3 that adopts 8 kinds of method culture purified
-CD56
+The cell counting analysis chart of NK cell proliferation;
Adopt negative selection of magnetic bead or selected by flow cytometry apoptosis to obtain CD3
-CD56
+The NK cell.Being adjusted to concentration with serum free medium is 2 * 10
5/ ml, and add 500IU/ml IL-2, add then in the culture plate of preparation among the embodiment 2, cultivated the propagation of cell counting analysis of cells 3 days.Poly-D-Lysine+IgG1, anti-CD137 mAb, IL-2, IL-15, anti-CD137 mAb+IL-15, immobilized anti-CD137 mAb+immobilized IL-15, immobilizedanti-CD137 mAb+immobilized IL-15+IL-2 organizes in contrast.
Fig. 4 adopts serum lactic dehydrogenase (LDH) method for releasing to measure the killing activity analysis chart of CIK cell to target cell.
According to CytoTox
Non-Radioactive Cytotoxicity test kit specification sheets, get the NK cell of cultivating 3 days, imitate target than all being made as 10:1 for every group, to be in the A549 cell tryptase enzymic digestion of logarithmic phase, tongue expects that indigo plant refuses to dye method counting (viable count〉95%), adjusts cell concn, add 96 orifice plates, every hole 50 μ l are imitated target than adding effector cell, every hole 50 μ l according to difference, while laying effect cell and target cell nature release aperture, substratum nature release aperture, the maximum release aperture of target cell, volume correction control wells, every pore volume 100 μ l, all establishing 3 multiple holes, is centrifugal 4min under the condition of 250g at acceleration, places 37 ℃ again, concentration is 5% CO
2In hatch 4h, before reaction finishes during 45min, adding 10 μ l lysates in every hole of the maximum release aperture of target cell.After reaction finished, 50 μ l supernatants were drawn in every hole and 50 μ lLDH enzyme reaction solutions place another 96 new orifice plates, and room temperature lucifuge reaction 30min adds reaction terminating liquid 50 μ l, and microplate reader detects its OD value.Killing activity %=(measuring pipe OD value-target cell nature releasing tube OD value-effector cell's nature releasing tube OD value)/(the maximum releasing tube OD of target cell value-target cell nature releasing tube OD value) * 100% (detail is with reference to the cytotox 96 non-radioa ctive cytotoxicity assay kit specification sheetss of U.S. Promega company).
Embodiment
The present invention relates to tumor necrosis factor superfamily member's conjugated protein or polypeptide and cytokine, more particularly, the present invention relates to the construction process of the NK cell expansion system of anti-CD137 mAb and IL-15 composition.
Said in this specification sheets " CD137 acceptor " is meant expressed protein on the antigen activated T cell surface; The CD137 part be express on some mammalian cell can with CD137 receptor-specific bonded protein.
Term " anti-CD137 antibody " can be CD137 specific monoclonal or polyclonal antibody or their immunologic competence part.Term " CD137L " comprises complete CD137 part, solubility CD137 part and comprises the fusion rotein of the functionally active part of CD137 part.Among the present invention, preferably monoclonal antibody, particularly anti-CD137mAb.
According to the preferred embodiments of the invention, adopt commercialization anti-CD137 mAb, IL-15, IL-2,24 hole plastic culture plate and tamanori Poly-D-Lysine.
The water-reducible Poly-D-Lysine solution-treated of aseptic distillation plastic culture plate will wrap then by anti-CD137 mAb and IL-15.Cultivate PBMC with this culture plate of handling then, amplification and enrichment CD3
-CD56
+The NK cell.Experiment showed, PBMC that the present invention cultivates not only cell quantity be significantly improved and CD3 wherein
-CD56
+The percentage composition of NK cell also obviously rises.Obtain highly purified CD3 if desired
-CD56
+The CD3 that the then direct amplification purification of NK cell is crossed
-CD56
+The NK cell.
In order to identify the CD3 of amplification system amplification of the present invention
-CD56
+The biological function of NK cell, the present invention has carried out the CD3 of amplification
-CD56
+The NK cell is to human lung adenocarcinoma cell line A549 killing experiments in vitro.Experiment showed, the CD3 that adopts method amplification of the present invention
-CD56
+NK cell and the CD3 that increases with other known cytokines or combination of cytokines
-CD56
+The NK cell is compared, and under identical cell concn, the kill capability of A549 is enhanced about more than once.
Below describe the present invention for example in detail by non-limiting example.Those skilled in the art can not deviate under essence spirit of the present invention and the principle prerequisite, change or change some technology contents or the sport technique segment of describing in this specification sheets.But one will understand that change that these are parallel or change all will be included within the claim scope that awaits the reply of the present invention.
Embodiment 1: the preparation of the plastic culture plate of high adhesion
1.Poly-D-Lysine or Poly-L-Lysine solution adopts sterile purified water ddH
2O 1:10 dilutes this Poly-L-Lysine Solution.
2.Poly-D-Lysine or Poly-L-Lysine diluent (300 μ l) adds in plastic culture plate or culture dish or the culturing bottle room temperature placement 5-10 minute.
3. inhale Poly-D-Lysine or the Poly-L-Lysine diluent that goes in plastic plate, culture dish or the culturing bottle, ambient temperature overnight dried for standby.
The bag quilt of embodiment 2:anti-CD137 mAb and IL-15
1.Anti-CD137 mAb and IL-15 add phosphoric acid buffer (PBS) simultaneously, are diluted to concentration and are respectively 1-10 μ g/ml and 10-100ng/ml.
2. above-mentioned anti-CD137 mAb and IL-15 mixed diluting liquid (300 μ l) are added in the plastic culture plate or culture dish or culturing bottle of preparation among the embodiment 1,4 ℃ are spent the night.
3. inhale the diluent that removes plastic plate or culture dish or culturing bottle, stand-by.
The cultivation of embodiment 3:NK cell
1. peripheral blood mononuclear cell (PBMC), CD3
-CD56
+The preparation of NK cell: get normal volunteer's peripheral blood 100ml (deriving from Red Cross blood station, Nanjing), separate (Ficoll) and obtain PBMC.After washing cell, be diluted to 3 * 10 with the RPMI1640 substratum that contains 10% calf serum
6/ ml.Place 6 well culture plates (2ml/ hole) to cultivate 2 hours (5%CO
2, 37 ℃) and sucking-off suspension cell gently, promptly obtain PBMC.CD3
-CD56
+The NK cell adopts that magnetic bead is negative to be selected or selected by flow cytometry apoptosis obtains (purity〉95%) from PBMC.
2.NK the amplification of cell and enrichment
It is 1 * 10 that PBMC adopts the RPMI1640 that contains 10% calf serum to be adjusted to concentration
5/ ml, and add 500IU/mlIL-2, add then in the culture plate of preparation among the embodiment 2, to cultivate 3-6 days, collecting cell is the CD3 after the amplification
-CD56
+The NK cell.The phenotype of collecting cell counting and employing flow cytometry identification of cell, experimental result as shown in Figure 1, ordinate zou among the figure is a cell quantity, X-coordinate is the differential responses group, its preceding 7 column figure represent the control group result, article 8, column figure is result of the present invention, and visible amplification system of the present invention can promote the cell total amount obviously to increase, and CD3 wherein
-CD56
+The percentage composition of NK cell significantly increases, and control group all can not reach above-mentioned expanding effect (as shown in Figure 2).The different treatment group of among Fig. 2 1～8 expression is respectively: Poly-D-Lysine+IgG1 (2-1), anti-CD137 mAb (2-2), IL-2 (2-3), IL-15 (2-4), anti-CD137 mAb+IL-15 (2-5), immobilizedanti-CD137 mAb+immobilized IL-15 (2-6), immobilized anti-CD137 mAb+immobilizedIL-15+IL-2 (2-7), Poly-D-Lysine+immobilized anti-CD137 mAb+immobilized IL-15+IL-2 (2-8 is a design sketch of the present invention).This shows that amplification system of the present invention can not only promote the CD3 among the PBMC
-CD56
+NK cell proliferation can also improve CD3
-CD56
+The content of NK cell reaches enrichment CD3
-CD56
+The effect of NK cell.
3. highly purified CD3 increases
-CD56
+The NK cell
Adopt negative selection of magnetic bead or selected by flow cytometry apoptosis to obtain highly purified CD3
-CD56
+The NK cell adopts the culture plate amplification CD3 for preparing among the embodiment 2 then
-CD56
+The NK cell adds 500IU/ml IL-2 in the substratum, supported collecting cell, the phenotype of counting and employing flow cytometry identification of cell 3-6 days.Experimental result as shown in Figure 3, the ordinate zou among the figure is a cell quantity, X-coordinate is the differential responses group, its preceding 7 column figure represent the control group result, the 8th column figure is result of the present invention, visible amplification system of the present invention can promote CD3
-CD56
+NK cell total amount obviously increases.
Embodiment 4:CD3
-CD56
+The NK cell is to the killing experiments of tumour cell
According to CytoTox
Non-Radioactive Cytotoxicity test kit specification sheets, get the NK cell of cultivating 6 days, imitate target than all being made as 10:1 for every group, to be in the A549 cell tryptase enzymic digestion of logarithmic phase, tongue expects that indigo plant refuses to dye method counting (viable count〉95%), adjusts cell concn, adds 96 orifice plates, every hole 50 μ l, every hole 50 μ l, while laying effect cell and target cell nature release aperture, substratum nature release aperture, the maximum release aperture of target cell, the volume correction control wells, every pore volume 100 μ l all establish 3 multiple holes, the centrifugal 4min of 250g, placing 37 ℃, concentration is 5% CO
2In hatch 4h, the every hole of 45min target cell maximum release aperture adds 10 μ l lysates before reaction finishes.After reaction finished, 50 μ l supernatants were drawn in every hole and 50 μ l LDH enzyme reaction solutions place another 96 new orifice plates, and room temperature lucifuge reaction 30min adds reaction terminating liquid 50 μ l,, microplate reader detects its OD value.Killing activity %=(measuring pipe OD value-target cell nature releasing tube OD value-effector cell's nature releasing tube OD value)/(the maximum releasing tube OD of target cell value-target cell nature releasing tube OD value) * 100% (detail is with reference to the cytotox 96 non-radioactive cytotoxicityassay kit specification sheetss of U.S. Promega company).The result shows that as shown in Figure 4 the ordinate zou among the figure is a kill rate, and X-coordinate is the differential responses group, and its preceding 7 column figure represent the control group result, and the 8th column figure is result of the present invention, as seen uses the CD3 of method amplification of the present invention
-CD56
+The NK cell to the kill rate of A549 apparently higher than other experimental group.
Claims (1)
1. CD3
-CD56
+The establishment method of NK cell high-efficient amplification cultivation system is characterized in that comprising the steps:
(1) gets an amount of binder Poly-D-Lysine or Poly-L-Lysine with distilled water diluting to 10%, add plastic culture plate, culture dish or culturing bottle, sop up the binder diluent after hatching 5-10min, binder is attached on plastic culture plate, culture dish or the culturing bottle;
(2) getting an amount of anti-CD137mAb or CD137L recombinant protein and IL-15 adds in the damping fluid simultaneously, be diluted to concentration and be respectively 1-10 μ g/ml and 10-100ng/ml, to have in the plastic culture of Poly-D-Lysine or Poly-L-Lysine plate, culture dish or the culturing bottle sticking of preparation in anti-CD137mAb and the IL-15 mixed diluting liquid adding above-mentioned steps (1), 4 ℃ are spent the night, wrap by anti-CD137mAb and IL-15 on plastic culture plate, culture dish or the culturing bottle that binder was handled, the diluent of plastic plate is removed in suction, and is stand-by;
(3) peripheral blood that picks up from the healthy volunteer separates acquisition mononuclearcell (PBMC) through Ficoll, adjusts concentration to 3 * 10 of PBMC with serum free medium
6/ ml adds 6 hole plastic culture plates and cultivates 2-3h, draws suspension cell, above-mentioned suspension cell or adjust concentration to 1 * 10 with serum free medium from the high purity N K cell that sorting wherein obtains
6/ ml adds Poly-D-Lysine or Poly-L-Lysine and handled and wrapped by on the culture plate of anti-CD137mAb and IL-15, culture dish or the culturing bottle, and adds IL-2 simultaneously, cultivates after 3-6 days the CD3 after collecting cell must increase
-CD56
+The NK cell.
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| CN102356154A (en) * | 2008-10-24 | 2012-02-15 | 株式会社美迪赛 | Method for efficiently proliferating and differentiating natural killer cells from umbilical cord blood |
| CN102600454A (en) * | 2011-11-19 | 2012-07-25 | 江苏省人民医院 | Application of CD137L in preparation of medicine for treating non-small cell lung cancer |
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| CN102352342A (en) * | 2011-09-30 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | Method for amplifying cytokine induced kill cells (CIK) and CIK cell preparation |
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| CN102600454A (en) * | 2011-11-19 | 2012-07-25 | 江苏省人民医院 | Application of CD137L in preparation of medicine for treating non-small cell lung cancer |
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| CN105101978A (en) * | 2013-03-27 | 2015-11-25 | 株式会社日本生物治疗研究所 | Method for producing NK cell-enhancing blood product |
| WO2015014291A1 (en) * | 2013-08-02 | 2015-02-05 | 北京赛诺泰生物科技有限公司 | Lymph cell amplification and activation method via serum-free cultivation |
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| CN107083362A (en) * | 2017-06-19 | 2017-08-22 | 金浩范 | A kind of autologous NK cells preparation method and the kit for implementing this method |
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