CN101735982A - Method for amplifying lymphocyte by interleukin 15 receptor and interleukin 2 complex - Google Patents
Method for amplifying lymphocyte by interleukin 15 receptor and interleukin 2 complex Download PDFInfo
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Abstract
The invention discloses a technology in the aspect of immunology, in particular to a method for amplifying natural killer (NK) cells and CD8 lymphocyte by interleukin 2 and interleukin 15 receptor (IgG1 Fc) and interleukin 2 complex. A polypeptide complex is cultured by an interleukin 2 and the interleukin 15 receptor which are protein having the expression function, and the aim of changing the immune state is achieved by the lymphocyte or a lymphocyte precursor cell activated and amplified by the polypeptide complex. The patient is helped to resist a tumour, viruses and bacteria by improving the immunity through the interleukin 15 receptor (IgG1 Fc) and interleukin 2 complex, and the realizing effect is good. The invention has wide prospect in the aspect of immunology.
Description
Technical field
The invention belongs to field of immunology, specifically be meant the use interleukin-22, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture amplifying natural killer cell (NK) and CD8 lymphocyte.
Technical background
In the past studies show that interleukin-22 and interleukin 15 have participated in the NK cell, obtain the congenital immunity power of dendritic cell and cd8 cell and the day after tomorrow activation process of answering property immunizing power.Yet, very limited with increase the in vitro and in vivo effect of these cells of interleukin 15 or interleukin-22 in the past.
Interleukin 15 and interleukin-22 combine by the special receptor with CD8 and NK lymphocytic cell surface, activate CD8 and NK cell.Interleukin-22, interleukin 15 and interleukin 15 acceptor have very strong expression in activated dendritic cell and monocyte.Interleukin-22, interleukin 15 can form the heterodimer mixture with the interleukin 15 receptors bind respectively and activate CD8 and NK cell.
The biological action of interleukin-22 and interleukin 15 with and interleukin 15 acceptor (IgG1 Fc) form heterodimer direct relation arranged.Interleukin-22, the biological activity of the heterodimer mixture that interleukin 15 and interleukin 15 acceptor (IgG1 Fc) form is much higher than interleukin-22 and interleukin 15.Because the interleukin 15 biological effect need realize with receptor complex by forming part, therefore still can't make full use of in external or body, increase NK and cd8 cell of interleukin 15 at present and strengthen congenital immunity power and answering property of acquisition day after tomorrow immunizing power.In fact, directly handle NK and cd8 cell with the interleukin 15 acceptor, though can form interleukin 15 and interleukin 15 acceptor heterodimer, this heterodimer has antagonistic action to interleukin 15.The invention solves this problem, the invention provides a kind of can be at amplification in vitro NK and cd8 cell, the method for strengthening immunity.NK and cd8 cell with this method amplification can be resisted tumour with helping patient, virus and bacterium.
Summary of the invention
The present invention proposes a kind of method of effective amplifying lymphocyte.
The present invention is achieved by following technical proposals:
Interleukin-22 and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture has important effect to lymphocyte survival and amplification.The lymphocyte of amplification comprises the NK cell, cd8 cell and NKT cell.These cells have important medical functions, because they can discern the cell with destroyed tumor cell and pathogenic infection, and comprising virus infection, as hepatitis B virus, hepatitis A virus (HAV) and virus of AIDS.
The present invention utilizes interleukin-22, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture, and amplifying lymphocyte and/or lymphocyte precursor cell comprise the NK cell, cd8 cell and NKT cell.The method that the present invention utilizes is amplifying lymphocyte and/or lymphocyte precursor cell effectively, is that ideal strengthens the immunoreactive method of patient.
Interleukin-22 among the present invention and interleukin 15 acceptor are these proteinic whole polypeptide, wherein also comprise the polypeptide composition that possesses function in these protein.Interleukin 15 acceptor among the present invention (IgG1 Fc) is the associating polypeptide of interleukin 15 acceptor and immunoglobulin fc region territory (IgG1 Fc), and this zone of IgG1 Fc comprises but is not limited only to CD80, CD86, B7-H1, B7-H2, B7-H3 and B7-H4 have active zone.Thereby the active region is the All Ranges that can activate the IgG1 of interleukin 15 acceptor activated lymphocyte amplification behind the formation associating albumen.
Lymphocyte among the present invention comes from peripheral blood and marrow.Lymphocyte among the present invention can be the NK cell, cd8 cell and NKT cell; Also can be the NK cell, the various combination of cd8 cell and NKT cell; Also can be the NK cell, cd8 cell, NKT cell and other lymphocytic various combination; Also can be that interleukin-22 and interleukin 15 acceptor (IgG1Fc)-interleukin-22 mixture are injected into all leukocytic summations in the blood that is acted on behind the human vas.Interleukin-22 among the present invention and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture derives from the people, but be not limited only to the people, as injection or handle to as if other species, as mouse, rat, monkey or the like also can be the interleukin-22 of other species, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture.
The invention provides a scheme that can be used for making medicine, the medicine of making according to this scheme can have following described multiple use.This scheme comprises interleukin-22, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture.Utilize the lymphocyte (comprising the NK cell, cd8 cell and NKT cell) of this programme amplification to be suitable for and the various sights that need to strengthen patient's immunizing power.For example, amplification NK cell infects effective especially to the treatment bacterium in fungi or the parasite.For example, amplification NK cell is effective especially to the treatment tumour, as acute myelocytic leukemia, and chronic lymphatic knurl leukemia, tumor of prostate, malignant melanoma and nephrocyte malignant tumour, but be not limited only to above-mentioned tumour.The NK of amplification and NKT cell are effective especially to the treatment virus infection, as hepatitis B virus, and hepatitis A virus (HAV) and virus of AIDS but be not limited only to above-mentioned virus infection.The NK and the NKT cell of amplification can significantly improve antigenic action effect.
Except as otherwise noted, the implication of all technology and science vocabulary is the implication of the personage's common sense with common skill in the industry among the present invention.Being defined in all molecular biology books of Essential Terms can be found, for example, and Benjamin Lewin, Genes VIII, Oxford University Press, 2004 (ISBN 0-13-145140-5);
Interleukin 15 acceptor among the present invention (IgG1 Fc) is the protein of a biologically active, the polypeptide of being made up of interleukin 15 acceptor and IgG1 Fc.The IgG1 Fc that the present invention uses picks out polypeptide from gamma immune sphaeroprotein (Ig1) commonly used; yet; the gamma immune sphaeroprotein that the IgG1 Fc that the present invention uses is not limited to use always, the gamma-globulin Fc zone of other classifications form the polypeptide zone that similar functions is arranged of associating polypeptide also within the scope of protection of the invention with the interleukin 15 acceptor.
In order to ensure for a long time, the production recombinant interleukin of high yield (interleukin-22, interleukin 15 acceptor) and other polypeptide, the present invention has adopted protein stabilized expression system.For example, clone has been transcribed stably express interleukin-22 polypeptide expression carrier, contains viral promotors and selectable marker gene in this carrier, as antibiotics resistance gene.After the heterogenous expression carrier enters host cell, activate promotor with appropriate methods, culturing cell promptly can obtain the polypeptide of high level expression after for some time.
The polypeptide products that is secreted in substratum or the supernatant liquor can come purifying and separate by the technology that the personage was familiar with common skill in the industry.These technology include but not limited to following listed method: ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography method, chromatography and lectin.If necessary, the scheme of protein renaturation can be used to help purifying expressed proteins polypeptide.If necessary, high performance liquid chromatography (HPLC) can be made last purification step and be further purified protein.
Interleukin-(interleukin-22, interleukin 15 acceptor) and other polypeptide also can be by standard the method for manipulation nucleotide coding sequence produce.For example, with specific, unspecific rite-directed mutagenesis or other Protocols in Molecular Biologies are produced functional equivalent, but primary structure polypeptide inequality.Do not influence proteinic biochemical characteristic if simple one or more amino acid is revised, these guard the protein of replacement generation also within the scope of protection of the invention by amino acid.
Interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture can be by obtaining interleukin-22 and interleukin 15 acceptor (IgG1Fc) co-cultivation.Polypeptide is dissolved in the aqueous solution, as buffer salt solution or cell culture fluid.According to mole number 1.5: 1 or 2: 1 or 3: 1, or bigger ratio (for example, 5: 1 even 10: 1) added interleukin and interleukin-2-receptor (IgG1 Fc).Selecting to guarantee that forming maximum mixtures is target of ratio, the personage with common skill in the industry can rule of thumb judge.
Understanding some at interleukin 15 acceptor (IgG1 Fc) polypeptide of host cell inner expression among the present invention is secreted in the cell culture supernatant with dimeric form.Therefore, can be by in supernatant liquor, adding the spontaneous formation receptor-ligand of the mode mixture of interleukin-22.
Interleukin-22 among the present invention and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture can be used to the population of amplifying lymphocyte.The cell portable patient of amplification in vitro.For example, interleukin-22 and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture activates specific cells amplification and survival in order successively, comprises the NK cell, CD8 and NKT cell.This method is approximately than interleukin II, or interleukin 15 acceptor (not containing IgG1 Fc)-interleukin 15 mixture is more effective more than 10 times.
Interleukin-22 and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture activates and expanded cells in order, comprises the NK cell, and CD8 and NKT cell can be used for carrying out autotransplantation, also can carry out heteroplastic transplantation.Under the histocompatibility antigen that is subjected to contributor (grownup or minor) and blood antigen and contributor's (grownup or minor) histocompatibility antigen and prerequisite that blood antigen is complementary, the contributor is activated and expanded cells can be injected into by the mode of intravenous drip by in contributor's the blood vessel.In some applications, clone (for example, NK clone, cd8 cell system, NKT clone) also can be passed through interleukin-22, and interleukin 15 and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture activates successively in order and increases.It also is protection scope of the present invention that expanded cells system is used for the clinical treatment purpose.
Lymphocyte or lymphocyte precursor cell can be from deriving from any tissue that includes big amount lymphocyte and precursor cell.For example, peripheral blood (comprising Cord blood), marrow, spleen and lymphoglandula.Lymphocyte or lymphocyte precursor cell are taken from peripheral blood or marrow usually.In other are used (for example, experimental study), spleen and/or lymphoglandula also are suitable sources.
For example, lymphocyte and precursor cell thereof can be by taking out peripheral blood and/or marrow obtains.Lymphocyte/precursor cell can further obtain the NK cell, cd8 cell, NKT cell through purifying.Purifying NK cell, cd8 cell, the step of NKT cell and technology are that the personage with common skill in the industry is familiar with.Lymphocyte can come erythrocyte in separating blood or the marrow by the mode of density gradient centrifugation, lymphocyte and other cells.NK cell, cd8 cell, NKT cell can utilize antibody-mediated affine preparation method to be further purified, for example the antibodies paramagnetic particle method.The purifying lymphocyte does not also require that the lymphocyte of purifying is definitely pure, and is meant that the cell of purifying is higher than its ratio shared in coming source tissue.Generally, the lymphocyte of purifying at least total number of representatives 50%, perhaps 60%, perhaps higher.The NK cell of purifying, cd8 cell, the NKT cell suspension is in a suitable physiological buffer solution, then suspension growth in but be not limited in the following nutrient solution, Eagle ' s nutrient solution adds 10% human serum, RPMI 1640 adds 10% human serum, and (Ham ' s) Nutrientmixtures adds 10% human serum to F-10.Human serum also can replace with 10% calf serum, but human serum is to the NK cell, cd8 cell, and the expanding effect of NKT cell is better.
The lymphocyte of purifying and/or lymphocyte precursor cell suspension grow in the cell culture fluid, add interleukin-22 and cultivate 1-2 after week, and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture is to activate and amplification NK cell cd8 cell, NKT cell.Generally in order to reduce cost, the present invention adopts minimum doses to come amplifying lymphocyte.The dosage of interleukin-22 and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture 50 skins rub/be raised to 1 receive rub/liter between.In some cases, the dosage of interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture rub at 100 skins at least/liter more than.In some cases, be necessary to carry out the processing of high dosage, activate concentration and be about 0.2,0.3,0.5,0.75 or 1 receive rub/liter.In addition, interleukin-22 and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture is injected directly into human body, is used for the NK cell that increases in vivo, cd8 cell, during the NKT cell, used dosage 0.5 skin rub/be raised to 0.5 receive rub/liter between.Can transplant to lymphocytic contributor itself or be subjected to the contributor at the lymphocyte of amplification in vitro, to strengthen born or the antigen specific immune reaction.Generally, the lymphocyte of amplification is by being dissolved in the method intravenous drip of normal saline solution.
Beneficial effect: the present invention helps patient and resists tumour, opposing virus and bacterium by interleukin 15 acceptor (IgG1 Fc)-immunizing power of interleukin-22 mixture raising patient.
Figure of description
Fig. 1 interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture synoptic diagram.
Fig. 2 NK cells of human beings amplification in vitro linear graph shows the NK cell at interleukin-22, interleukin 15, and interleukin 15 acceptor (IgG1Fc)-interleukin-22 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture effect lower linear increases.
Fig. 3 people's cd8 cell amplification in vitro linear graph shows cd8 cell at interleukin-22, interleukin 15, and interleukin 15 acceptor (IgG1Fc)-interleukin-22 mixture, interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture effect lower linear increases.
Fig. 4 people NK, cd8 cell amplification in vitro linear graph shows NK, cd8 cell increases in interleukin 15 acceptor (IgG1 the Fc)-interleukin-22 mixture effect lower linear of different concns.
Fig. 5 human peripheral CD3-lymphocyte amplification in vitro stream shows the instrument data plot, human peripheral CD3-lymphocyte is at interleukin-22, and the ratio phenomenal growth of NK cell (CD56+ and CD56+/CD16+) after 7 days and 14 days is cultivated in interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture effect down.
Fig. 6 human peripheral lymphocyte and myelolymphocyte amplification in vitro stream of cells are shown the instrument data plot, human peripheral lymphocyte and myelolymphocyte are at interleukin-22, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture effect was cultivated 14 days down, the lymphocytic ratio phenomenal growth of NK and CD8.
The NK cell of Fig. 7 amplification in vitro shows the instrument data plot to SK-N-AS solid tumor clone lethal effect stream, NK cells of human beings is at interleukin-22, or interleukin-22 and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture effect cultivate down after 14 days, with tumour cell according to 1: 4 ratio co-cultivation 6 hours.
The NK cell of Fig. 8 amplification in vitro shows the instrument data plot to solid tumor clone SK-N-AS cell killing effect stream, NK cells of human beings is at interleukin-22, or interleukin-22 and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture effect cultivate down after 14 days, with tumour cell according to 1: 4 ratio co-cultivation 6 hours.
The NK of Fig. 9 amplification in vitro and cd8 cell are to K562 blood tumor cell lethal effect figure.People NK and cd8 cell be at interleukin-22, and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture effect was cultivated down after 14 days, with the K562 blood tumor cell according to different ratio co-cultivation 6 hours.
The NK of Figure 10 amplification in vitro and cd8 cell are to chronic lymphocytic leukemia tumour cell (B cell) lethal effect figure.People NK and cd8 cell be at interleukin-22, and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture effect was cultivated down after 14 days, with chronic lymphocytic leukemia B tumour cell according to different ratio co-cultivation 6 hours.
Embodiment
Below enforcement of the present invention is specified:
Interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture amplification in vitro lymphocyte.
The healthy people's of donation NK and cd8 cell are cultivated in RPMI1640, are aided with 10% human serum.In nutrient solution, add interleukin-22 after three days, cultivated behind adding interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture (Fig. 1) or interleukin 15 acceptor (IgG1 the Fc)-interleukin-22 mixture some days.Interleukin-22 and interleukin-15 are organized in contrast, as shown in Figures 2 and 3.NK and cd8 cell are at interleukin-22, and under the effect of interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture, quantity significantly increases.And cellular control unit is at interleukin-22, under the independent effect of interleukin 15, increase to be far from experimental group obvious.The increase of cell quantity can be measured by the method for inserting thymidine.Cell continued to cultivate 12 hours after thymidine inserted, and then the variation that begins to measure cell quantity.Receive 1 and to rub/to rise that NK and cd8 cell quantity entered logarithmic phase in the time of 7 days under interleukin 15 acceptor (IgG1 the Fc)-interleukin-22 mixture of concentration and interleukin 15 acceptor (IgG1 the Fc)-interleukin 15 mixture effect, arrive plateau in the time of 14 days, increased by 10 times approximately.NK and cd8 cell be minimum receive 0.01 rub/liter interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture effect under can begin to increase.The concentration of the growth of NK and cd8 cell and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture is proportional, and the concentration of mixture is high more, and it is high more that NK and cd8 cell increase, as shown in Figure 4.
Interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture not only can amplification purification NK and cd8 cell, NK and cd8 cell in can also increase not purified peripheral blood lymphocyte and the marrow.Receive 1 and to rub/to rise the interleukin-22 of concentration, under interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture effect, peripheral blood lymphocyte surpasses 70% CD3-lymphocyte and becomes NK cell (CD56+) in the time of the 7th day, surpass 90% CD3-lymphocyte in the time of the 14th day and become NK cell (CD56+) (Fig. 5).Receive 1 and to rub/to rise the interleukin-22 of concentration, under interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture effect, the NK cell in peripheral blood lymphocyte and the marrow surpassed 60% in the time of the 14th day, and cd8 cell surpasses 45% and 50% respectively) (Fig. 6).
Interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture activated NK cell is to the splitting action of tumour cell
Studies show that to the injected in mice IL-15 that suffers from tumour in the past can strengthen the anti-tumor capacity of mouse, delays the life-span of mouse.The present invention adopts interleukin-22, the method of interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture and interleukin 15 acceptor (IgG1Fc)-interleukin 15 mixture activated NK cell and tumour cell co-cultivation is studied with interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture and is carried out antineoplastic feasibility.
After healthy donor's NK cell separated from lymphocyte, add interleukin-22 successively, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture or interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture activates 14 days.SK-N-AS, K562 and CD19+ chronic lymphocytic leukemia B cell are used as the cracked target cell.SK-N-AS is the clone of neuroblastoma, and neuroblastoma is a solid tumor.K562 is a B cell tumour clone, and this clone is blood tumor cell system.The SK-N-AS cell surface has CD56 and GD2 surface antigen to express.Activated NK cell and SK-N-AS cell are according to 1: 4 ratio co-cultivation after 6 hours, and be fixing, adds antibody staining.Interleukin-22 activated NK cell in contrast.Interleukin-22, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture activated NK cell is significant stronger to the splitting action of SK-N-AS than only using interleukin-22 activated NK cell.After the scission reaction, the ratio of SK-N-AS from control group surpass 80% reduce to below 31% of experimental group (Fig. 7, Fig. 8).
Interleukin-22, interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture activated NK cell to the cracking ability of K562 than activated cd8 cell height (Fig. 9) under the same terms.As the effector cell: when the ratio of target cell was 4: 1, the cracking ability of NK cell was 4 times of cd8 cell.This shows that in vivo activated NK cell may play a part bigger than cd8 cell to the killing and wounding in the process of tumour cell.Interleukin-22, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture activated NK cell has significant lethal effect (Figure 10) to the B cell that extracts from chronic lymphocytic leukemia patient peripheral blood.Mixture activated cd8 cell also also has certain lethal effect to patient's B cell.Identical with the lethal effect trend to K562, the cracking ability of activated NK cell is 2 times of cd8 cell.
Above-mentioned experiment shows interleukin-22, interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture and interleukin 15 acceptor (IgG1 Fc)-interleukin 15 mixture activated NK cell and cd8 cell be to the solid tumor cell, blood tumor cell and all have tangible lethal effect from the patient's tumor cell.This prompting interleukin-22 and interleukin 15 acceptor (IgG1 Fc)-interleukin-22 mixture can be used for clinical oncotherapy.
Claims (9)
1. the method for interleukin 15 acceptor-interleukin-22 mixture amplifying lymphocyte is characterized in that:
(1) interleukin-22 and interleukin 15 acceptor are these proteinic whole polypeptide or comprise the polypeptide composition that possesses function in these protein;
Interleukin-22 and interleukin 15 acceptor adopt protein stabilized expression system, and wherein clone has been transcribed stably express interleukin-22 polypeptide expression carrier, contains viral promotors and selectable marker gene in the carrier;
After the heterogenous expression carrier enters host cell, activate promotor, promptly can obtain polypeptide behind the culturing cell;
Again polypeptide is used successively: ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, the hydroxylapatite chromatography method, chromatography and lectin carry out purifying, get final product mixture;
Perhaps, polypeptide is dissolved in the aqueous solution, wherein the aqueous solution is buffer salt solution or cell culture fluid, and the mole number of water and solute is 1.5～10: 1 in the aqueous solution; And then add interleukin and interleukin-2-receptor, form mixture;
Perhaps, the preparation cell culture fluid is again by adding the spontaneous formation receptor-ligand of the mode mixture of interleukin-22 in supernatant liquor;
(2) interleukin 15 acceptor-interleukin-22 mixture is activated and the lymphocyte or the lymphocyte precursor cell of amplification, wherein the dosage of interleukin 15 acceptor-interleukin-22 mixture rub at 100 skins/liter～1 receive rub/liter.
2. method according to claim 1 is characterized in that described interleukin 15 acceptor is the associating polypeptide in interleukin 15 acceptor and immunoglobulin fc region territory.
3. method according to claim 2, the zone that it is characterized in that described interleukin 15 acceptor is CD80, CD86, B7-H1, B7-H2, B7-H3 and B7-H4 have active zone; Thereby or form the active region that to activate the IgG1 that interleukin 15 acceptor activated lymphocyte increases behind the associating albumen.
4. method according to claim 3, the zone that it is characterized in that described interleukin 15 acceptor is CD80, CD86, B7-H1, B7-H2, B7-H3 and B7-H4 have active zone.
5. method according to claim 1 is characterized in that described interleukin 15 acceptor is to pick out polypeptide from the gamma immune sphaeroprotein.
6. method according to claim 1 is characterized in that described polypeptide being dissolved in the aqueous solution, and wherein the aqueous solution is buffer salt solution or cell culture fluid, and the mole number of water and solute is 1.5～3: 1 in the aqueous solution.
7. method according to claim 1 is characterized in that described lymphocyte or lymphocyte precursor cell are the NK cell of purifying, and one or more in cd8 cell or the NKT cell mix, and wherein purifying is meant more than 50% of lymphocyte total number of representatives;
The NK cell of purifying, cd8 cell, the NKT cell places physiological buffer solution;
Nutrient solution wherein is: Eagle ' s nutrient solution adds 10% human serum or 10% calf serum, or RPMI 1640 adds 10% human serum or 10% calf serums, or F-10 (Ham ' s) Nutrient mixtures adds 10% human serum or 10% calf serum.
8. method according to claim 7, it is characterized in that described nutrient solution wherein is: Eagle ' s nutrient solution adds 10% human serum, or RPMI 1640 adds 10% human serum, or F-10 (Ham ' s) Nutrient mixtures adds 10% human serum.
9. method according to claim 1, it is characterized in that the dosage of described interleukin 15 acceptor-interleukin-22 mixture rubs at 50 skins/liter～1 receive rub/liter.
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| CN104694472A (en) * | 2015-02-13 | 2015-06-10 | 杭州易文赛生物技术有限公司 | Method for amplifying and cryopreserving natural killer cells |
| CN104673750B (en) * | 2015-02-13 | 2018-04-27 | 杭州易文赛生物技术有限公司 | A kind of method of natural killer cells amplification and a kind of culture media composition |
| CN105647863A (en) * | 2016-03-15 | 2016-06-08 | 浙江大学 | Method for amplifying and activating LAK cells by non-fetus bovine serum |
| CN105647863B (en) * | 2016-03-15 | 2020-08-25 | 浙江大学 | Method for amplifying and activating LAK cells without fetal bovine serum |
| WO2019019998A1 (en) | 2017-07-25 | 2019-01-31 | 江苏恒瑞医药股份有限公司 | Il-15 protein complex pharmaceutical composition and uses thereof |
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