CN105647863A - Method for amplifying and activating LAK cells by non-fetus bovine serum - Google Patents
Method for amplifying and activating LAK cells by non-fetus bovine serum Download PDFInfo
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Abstract
The invention discloses a method for amplifying and activating LAK cells by non-fetus bovine serum. The method has the advantage that compared with the existing fetal bovine medium, the risk brought by adopting fetus bovine serum can be avoided; compared with the existing serum-free medium, the purity of amplified and activated immune cells is higher, the amplification efficiency is higher, and the ability to kill tumor is stronger. The purity of amplified and activated immune cells, the amplification efficiency, and the ability to kill tumor by the method are equivalent to those of the existing fetal calf serum medium. The immune cells amplified by the method on the aspects of controlling and relieving progresses of a part of diseases, such as cancer, communicable disease, diabetes, or organ transplantation and hepatitis B have a wide prospect.
Description
Technical field
The invention belongs to field of immunology, specifically refer to use the host cell having transfected multiple proteins, the method that associating multiple protein combined effect directed expansion activates LAK cell.
Background technology
LAK cell is the class cell mass that Lymphokine produces, the cell such as including CIK, NK, NKT, T. LAK cell therapy is to kinds of tumors, for instance: the oncotherapys such as pulmonary carcinoma, head and neck squamous cell cancer, sarcoma cancer and acute leukemia have obvious curative effects. Current LAK cell therapy is mainly used to and performs the operation, chemotherapy and radiation is used in combination the Problems Concerning Their Recurrence solving tumor. LAK cell quantity and killing ability are directly related with curative effect, have problems in that acquisition high-purity and sufficient amount of CIK cell and NK cell are extremely difficult in treatment. The LAK cell purity low (20%-30%) of the serum-free medium amplification that tradition uses; CIK cell killing ability after amplification is low, it is impossible to meet clinical demand. Although adopting hyclone cultured cells purity and quantitatively disclosure satisfy that the demand of clinic, but it is because hyclone and is likely to containing bovine viral diarrhea virus (BovineViralDiarrheaVirus, BVDV), the reason such as bovine spongiform encephalopathy Protein virus (BSE), foot and mouth disease (FMD) and antibiotic pollution, adopt the technique of serum-free medium progressively in the industry cycle to reach common understanding. Before the present inventor invention adopt transfection CD8 ��-interleukin-22 1, CD14, CD19, CD86, CD163 and CD137L protein K562 cell (hereinafter referred to as host cell) be used for expanding NK cell. Host cell is combined the serum-free technique directed expansion that tradition uses and activates CIK cell and NK cell by the present invention, solves serum-free number of processes and the low problem of purity, avoids the problem adopting hyclone to bring potential threat to patient simultaneously.
Summary of the invention
The present invention is directed to the deficiency in existing serum-free culture immunocyte technology, it is proposed to a kind of significantly more efficient method, it is achieved the amplification of LAK cell.
The present invention is achieved by following technical proposals: a kind of without the hyclone amplification activation lymphocytic method of LAK, use the host cell having transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F, combine one or more protein and jointly lymphocyte is carried out amplification activation, obtain LAK lymphocyte;Described a-protein comprises the function fragment of CD8 �� and the function fragment of interleukin-22 1; PROTEIN B comprises the function fragment of CD14; Protein C comprises the function fragment of CD19; 3-protein d comprises the function fragment of CD86; Protein E comprises the function fragment of CD163; Protein F comprises the function fragment of CD137L.
Further, described LAK lymphocyte is CIK lymphocyte, the protein of associating includes a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1, the function fragment that described a-protein 1 comprises the function fragment of interleukin-22, PROTEIN B 1 comprises the function fragment of IL-1 ��, protein C 1 comprises IFN-�� function fragment, 3-protein d 1 comprise CD3mAb; Described lymphocyte is selected from one or more in peripheral blood lymphocyte, cord blood lymphocytes cell, CIK cell, or be one or more and other the lymphocytic combination in peripheral blood lymphocyte, cord blood lymphocytes cell, CIK cell, or for one or more in interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, interleukin-22, IL-1 ��, IFN-��, CD3mAb according to arbitrarily acting on CIK lymphocyte produced by the blood in human vas than the mixture formed.
Further, said method comprising the steps of:
(1) to transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F host cell carry out lethal process; Described lethal process is selected from strong acid, highly basic, irradiation (dosage is 100Gy-1000Gy).
(2) containing in lymphocytic culture fluid, the host cell after step 1 processes and a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1 co-cultivation 7 days are added; Make described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F amount be 50pM-1000pM. A-protein 1, PROTEIN B 1, protein C 1 amount be 10-1000IU/ milliliter, the amount of 3-protein d 1 is in 10-1000 nanograms/milliliter; Described host cell and lymphocytic usage ratio (quantity ratio) are host cell: lymphocyte=1-10:1, it is more preferred to, for host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1.
(3) by the medium centrifugal after cultivation, LAK lymphocyte precipitate is obtained, and resuspended with culture fluid;
(4) host cell after radiation treatment is added, making host cell and lymphocytic usage ratio (quantity ratio) is host cell: lymphocyte=1-10:1, it is highly preferred that be host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1 cultivation 7 days; By the medium centrifugal after cultivation, obtain the lymphocyte precipitate of survival, namely obtain LAK lymphocyte;
Wherein, described culture fluid is formed by by the human serum of GT-T551 and 10 volume of 100 volumes.
Further, described LAK lymphocyte is NK cell; The protein of associating is a-protein 1, and a-protein 1 comprises the function fragment of interleukin-22. Described lymphocyte is selected from one or more in peripheral blood lymphocyte, cord blood lymphocytes cell, NK cell, or be one or more and other the lymphocytic combination in peripheral blood lymphocyte, cord blood lymphocytes cell, NK cell, or for one or more in interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, interleukin-22 according to arbitrarily acting on NK lymphocyte produced by the blood in human vas than the mixture formed.
Further, said method comprising the steps of:
(1) to transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F host cell carry out lethal process; Described lethal process is selected from strong acid, highly basic, irradiation (dosage is 100Gy-1000Gy).
(2) containing in lymphocytic culture fluid, the host cell after step 1 processes and a-protein 1 co-cultivation 7 days are added; Make described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F amount be 50pM-1000pM. The amount of a-protein 1 is 10-1000IU/ milliliter, described host cell and lymphocytic usage ratio (quantity ratio) are host cell: lymphocyte=1-10:1, it is highly preferred that be host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1.
(3) by the medium centrifugal after cultivation, the lymphocyte precipitate of survival is obtained, and resuspended with culture fluid;
(4) host cell after radiation treatment is added, making host cell and lymphocytic usage ratio (quantity ratio) is host cell: lymphocyte=1-10:1, it is highly preferred that be host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1 cultivation 7 days; Medium centrifugal after cultivation, obtains the lymphocyte precipitate of survival, namely obtains NK lymphocyte;
Wherein, described culture fluid is made up of the human serum of GT-T551 and 1 volume of 100 volumes.
Further, described host cell is K562 cell.
Further, described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F are the protein of purification or are immediately expressed at surface of cell membrane by host cell, and a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1 are the protein of purification; Described purification refers to that mass fraction is more than 90%.
Further, described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F respectively CD8 ��-interleukin-22 1 complex, CD14, CD19, CD86, CD163 and CD137L, wherein, CD8 �� is the memebrane protein expressed on host cell membrane, CD8 �� makes interleukin-22 1 express on cell membrane after connecting interleukin-22 1, becomes transmembrane protein; CD14, CD19, CD86 and CD137 are memebrane protein.
Further, transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F host cell obtained by protein stabilized expression system.
A kind of lymphocytic application of LAK, described application is, LAK lymphocyte is used for preparing the medicine for the treatment of cancer, infectious disease, diabetes or organ transplantation.
Beneficial effects of the present invention:
(1) present invention is on K562 cell, has transfected CD8 ��-interleukin-22 1, CD137L, CD14, CD19, CD86, CD163. On K562 cell, CD14, CD86, CD163 of transfection contribute to activating LAK cell recognition and killing the tumor-associated macrophages of M2 type in microenvironment; Transfection CD19 contributes to activating LAK cell recognition and killing B cell.
(2) present invention provides amplification in vitro and activates the method that the method ratio of CIK cell does not add host cell, and the quantity of amplification improves 2.1 times; After amplification, the purity of the CIK cell in PBMC brings up to 75.6% from 3.5%. Results of animal shows: with do not add host cell amplification CIK cell compared with, adopt the present invention amplification CIK cell significantly improve the killing ability to mouse interior tumor.
(3) present invention provides amplification in vitro and activates the method that the method ratio of NK cell does not add host cell, and the quantity of amplification improves 2.2 times;After amplification, the purity of the NK cell in PBMC brings up to 90.5% from 5.5%. Results of animal shows: compared with the NK cell with traditional scheme amplification, adopts the NK cell of present invention amplification to significantly improve the killing ability to mouse interior tumor.
(4) immunity that the present invention cultivates, by host cell, interleukin-22, IL-1 ��, IFN-��, CD3mAb etc., CIK and the NK cell raising patient that amplification activates helps patient to resist tumor, opposing virus and antibacterial.
Accompanying drawing explanation
CIK and NK cell expansion ex vivo linear graph in Fig. 1 .PBMC. A.CIK cell increases (culture fluid is 1% human serum, is labeled as GT551H3+ host cell) in host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect lower linear. CIK cell in IL-1 ��, IFN-��, CD3mAb and interleukin-22 amplification PBMC is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification CIK cell is as comparison (being labeled as 1640+ hyclone). B.NK lymphocyte increases at host cell and interleukin-22 combined effect lower linear. Interleukin-22 amplification NK cell is as comparison; Hyclone amplification NK cell is as comparison.
CIK cell in vitro amplification Flow cytometry data figure in Fig. 2 .PBMC. After PBMC cell is cultivated 14 days under host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect, the ratio of CIK cell (CD3+/CD56+) significantly increases (culture fluid is 1% human serum, is labeled as GT551H3+ host cell). IL-1 ��, IFN-��, CD3mAb and interleukin-22 amplification CIK cell are as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification CIK cell is as comparison (being labeled as 1640+ hyclone).
NK cell expansion ex vivo Flow cytometry data figure in Fig. 3 .PBMC. After PBMC cell is cultivated 14 days under host cell and interleukin-22 combined effect, the ratio of NK cell (CD3-/CD56+) significantly increases (culture fluid is 1% human serum, is labeled as GT551H3+ host cell). Interleukin-22 amplification NK cell is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification NK cell is as comparison (being labeled as 1640+ hyclone).
Fig. 4 .CIK cell killing tumor cell line figure. PBMC cell is after host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect are cultivated 14 days, the CIK cell the amplified lethal effect (culture fluid is 1% human serum, is labeled as GT551H3+ host cell) to K562, PC3, A549, HepG2 tumor cell. In IL-1 ��, IFN-��, CD3mAb and interleukin-22 amplification PBMC, CIK cell is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification CIK cell is as comparison (being labeled as 1640+ hyclone).
Fig. 5 .CIK cell killing TAMs cell. PBMC cultivates after 14 days in host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect, the lethal effect (culture fluid is 1% human serum, is labeled as GT551H3+ host cell) to TAMs cell of the CIK cell after amplification. In IL-1 ��, IFN-��, CD3mAb and interleukin-22 amplification PBMC, CIK cell is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification CIK cell is as comparison (being labeled as 1640+ hyclone).
Fig. 6 .PBMC cell after host cell and interleukin-22 combined effect are cultivated 14 days, the NK cell the amplified lethal effect (culture fluid is 1% human serum, is labeled as GT551H3+ host cell) to K562, PC3, A549, HepG2 tumor cell.In interleukin-22 amplification PBMC, NK cell is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification NK cell is as comparison (being labeled as 1640+ hyclone).
Fig. 7 .NK cell killing TAMs cell. PBMC after host cell and interleukin-22 combined effect are cultivated 14 days, the lethal effect (culture fluid is 1% human serum, is labeled as GT551H3+ host cell) to TAMs cell of the NK cell after amplification. In interleukin-22 amplification PBMC, NK cell is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification NK cell is as comparison (being labeled as 1640+ hyclone).
Fig. 8 .CIK cell killing mouse interior tumor cytological map. PBMC cell is after host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect are cultivated 14 days, and CIK cell kills mouse interior tumor cell (culture fluid is 1% human serum, is labeled as GT551H3+ host cell); In IL-1 ��, IFN-��, CD3mAb and interleukin-22 amplification PBMC, CIK cell is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification CIK cell is as comparison (being labeled as 1640+ hyclone).
Fig. 9 .CIK cell therapy mice with tumor survival curve. The CIK cell treatment mice with tumor of host cell, IL-1 ��, IFN-��, CD3mAb amplification, mice with tumor survival curve (culture fluid is 1% human serum, is labeled as GT551H3+ host cell). In IL-1 ��, IFN-��, CD3mAb and interleukin-22 amplification PBMC, CIK cell is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification CIK cell is as comparison.
Figure 10 .NK cell killing mouse interior tumor cytological map. After PBMC host cell and interleukin-22 combined effect are cultivated 14 days, NK cell killing mouse interior tumor cell (culture fluid is 1% human serum, is labeled as GT551H3+ host cell) after amplification. In interleukin-22 amplification PBMC, NK cell is as comparison; Hyclone amplification NK cell is as comparison (being labeled as 1640+ hyclone).
Figure 11 .NK cell therapy mice with tumor survival curve. The NK cell therapy mice with tumor of host cell and interleukin-22 amplification, mice with tumor survival curve (culture fluid is 1% human serum, is labeled as GT551H3+ host cell). In interleukin-22 amplification PBMC, NK cell is as comparison (culture fluid is 1% human serum, is labeled as GT551H3); Hyclone amplification NK cell is as comparison (being labeled as 1640+ hyclone).
Detailed description of the invention
The invention discloses a kind of method expanding activated lymphocyte without hyclone, use the host cell having transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F, associating multiple protein confrontation lymphocyte carries out amplification and activates, and obtains LAK lymphocyte; Wherein lymphocytic amplification and activation are had important effect by the host cell after transfection, the LAK cell of available sufficient amount and function, tumor cell in recognizable and destroyed tumor microenvironment, the mononuclear cell of M2 type and CD19+B cell, strengthen patient's immunoreation. Described a-protein comprises the function fragment of CD8 �� and the function fragment of interleukin-22 1; PROTEIN B comprises the function fragment of CD14; Protein C comprises the function fragment of CD19; 3-protein d comprises the function fragment of CD86; Protein E comprises the function fragment of CD163; Protein F comprises the function fragment of CD137L;Wherein, CD8 �� function fragment is the memebrane protein expressed on host cell membrane, and CD8 �� function fragment makes interleukin-22 1 function fragment express on cell membrane after connecting interleukin-22 1 function fragment, become transmembrane protein; CD14 function fragment, CD19 function fragment, CD86 function fragment and CD137 function fragment are memebrane protein.
Described LAK lymphocyte includes CIK lymphocyte and NK lymphocyte etc., when expanding CIK lymphocyte, the albumen of associating includes a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1, the function fragment that wherein a-protein 1 comprises the function fragment of interleukin-22, PROTEIN B 1 comprises the function fragment of IL-1 ��, protein C 1 comprises IFN-�� function fragment, 3-protein d 1 comprise CD3mAb. When expanding NK lymphocyte, the protein of associating includes a-protein 1.
A-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F can respectively CD8 ��-interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, it is also possible to be the derived protein of the function fragment being respectively provided with CD8 ��-interleukin-22 1, CD14, CD19, CD86, CD163, CD137L; In like manner, a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1 can respectively interleukin-22, IL-1 ��, IFN-��, CD3mAb, it is also possible to for being respectively provided with the derived protein of interleukin-22, IL-1 ��, IFN-��, CD3mAb function fragment; CD8 ��-interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, interleukin-22, IL-1 ��, IFN-��, CD3mAb function fragment be the known general knowledge of this area.
CD8 ��-interleukin-22 1, CD14, CD19, CD86, CD163 and CD137L can also be produced by the method handling nucleotide coding sequence of standard. Such as, with specific, unspecific rite-directed mutagenesis or other Protocols in Molecular Biologies produce functional equivalent, but the polypeptide that primary structure differs. If the amendment of simple one or more aminoacid does not affect the biochemical characteristic of protein, these replace the protein produced also within the scope of protection of the invention by conservative.
Above-mentioned host cell can be K562 cell.
Described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F dosage be 50pM-1000pM. A-protein 1, PROTEIN B 1, protein C 1 dosage be 10-1000IU/ milliliter, the dosage of 3-protein d 1 is in 10-1000 nanograms/milliliter, described host cell and lymphocytic usage ratio (quantity ratio) are host cell: lymphocyte=1-10:1, more preferably, for host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1.
Described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F are the protein of purification or are immediately expressed at surface of cell membrane by host cell, and a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1 are the protein of purification; Described purification refers to that mass fraction is more than 90%. On cell membrane, the CD8 ��-interleukin-22 1 of high level expression, CD14, CD19, CD86 and CD137L can come purification and separation by the technology that one of ordinary skill scholar is familiar with that has in the industry. These technology include but not limited to method set forth below: ammonium sulfate or ethanol precipitation, acid extraction, anion or cation-exchange chromatography, hydrophobic interaction chromatography, affinity chromatograph, hydroxyapatite chromatography, chromatography and agglutinin.If it is necessary, the scheme of protein renaturation can be used to the protein polypeptide helping purification to express. If it is necessary, high performance liquid chromatography (HPLC) can make last purification step is further purified protein. The present invention understands some be secreted in cell culture supernatant at the CD8 ��-interleukin-22 1 of host cell inner expression, CD14, CD19, CD86, CD163 and CD137L. The one of ordinary skill scholar that has in the industry can verify according to common Measurement for Biochemistry. Be secreted into the cross-film interleukin-22 1 in cell culture supernatant, CD14, CD19, CD86, CD163 and CD137L can also pass through said method purification and separation.
Cell membrane is that CD8 ��-interleukin-22 1, CD14, CD19, CD86, CD163 and CD137L provide solid phase support, and described solid phase support includes but is not limited only to metal, glass, plastics, polymer, granule, molecule, phospholipid, phospholipid bilayer, cell membrane and analog. It is important that the surface of solid phase can adhere to above-mentioned protein.
Described lymphocyte is selected from one or more in peripheral blood lymphocyte, cord blood lymphocytes cell, LAK cell, or be one or more and other the lymphocytic combination in peripheral blood lymphocyte, cord blood lymphocytes cell, LAK cell, or for one or more in interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, interleukin-22, IL-1 ��, IFN-��, CD3mAb according to arbitrarily acting on LAK lymphocyte produced by the blood in human vas than the mixture formed. Proportionate relationship therein can be arbitrarily than. Peripheral blood lymphocytes in the present invention comes from peripheral blood, Cord blood and bone marrow, is an off the tissue of human body. In other application (such as, experimentation), spleen and/or lymph node are also suitable sources.
Such as, lymphocyte can obtain by taking out peripheral blood and/or bone marrow. Lymphocyte can obtain LAK cell further across purification. The one of ordinary skill scholar that has that the step of purification LAK and technique are in the industry is familiar with. Lymphocyte can separate the erythrocyte in blood or bone marrow, lymphocyte and other cells by the mode of density gradient centrifugation. LAK cell can utilize antibody-mediated affine preparation method to be further purified, for instance antibodies paramagnetic particle method. The lymphocyte that purification lymphocyte is not required for purification is definitely pure, and refers to that the cell of the purification ratio more shared in derived tissues than it is higher. Under normal circumstances, the 50% of the lymphocyte of purification at least total number of representatives, or 60%, or higher.
In order to ensure for a long time, the production recombinant interleukin2 1 of high yield, CD14, CD19, CD86, CD163 and CD137L, present invention employs protein stabilized expression system. Building protein stabilized expression system is have, in the industry, the technology that one of ordinary skill scholar generally adopts, and concrete grammar can refer to (Imai etc. Leukemia, 2004,18,676-684). Such as, K562 cell line transcrypted the expression vector of stably express CD8 ��-interleukin-22 1, CD14, CD19, CD86 and CD137L, containing viral promotors and selectable marker gene in this carrier, such as antibiotics resistance gene. CD8 �� is a kind of memebrane protein expressed on cell membrane, and CD8 �� gene makes interleukin-22 1 express on cell membrane after connecting interleukin-22 1 gene, becomes transmembrane protein; CD14, CD19, CD86, CD163 and CD137L are memebrane protein.After heterogenous expression carrier enters host cell, activate promoter by appropriate method, after cultivating cell a period of time, namely can obtain the interleukin-22 1 of high level expression on cell membrane, CD14, CD19, CD86 and CD137L polypeptide. These methods are to have, in the industry, the method that one of ordinary skill scholar is generally understood that.
Interleukin-22 in the present invention, interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, IL-1 ��, IFN-��, CD3mAb derive from people, but it is not limited only to people, as injection or process to as if other species, such as mice, rat, monkey etc., it is also possible to be the interleukin-22 of other species, interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, IL-1 ��, IFN-�� and CD3mAb.
Except as otherwise noted, in the present invention, all of technology and scientific terms are meant that have, in the industry, the implication that one of ordinary skill scholar is generally understood that. The definition of Essential Terms can be found in all molecular biology books, for instance, BenjaminLewin, GenesVIII, OxfordUniversityPress, 2004 (ISBN0-13-145140-5).
Above-mentioned amplification activates the lymphocytic method of LAK, specifically may comprise steps of:
(1) adopt existing technology that host cell is transfected, then pass through the host cell after ultracentrifugal method collects transfection, and to transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F host cell carry out lethal process, described lethal process be selected from strong acid, highly basic, irradiation (dosage is 100Gy-1000Gy); The dosage of described irradiation is 100Gy-1000Gy.
(2) containing in lymphocytic culture fluid, the host cell after step 1 processes and a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1 co-cultivation 7 days are added; Make described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F dosage be 50pM-1000pM. A-protein 1, PROTEIN B 1, protein C 1 dosage be 10-1000IU/ milliliter, the dosage of 3-protein d 1 is in 10-1000 nanograms/milliliter; Described host cell and lymphocytic usage ratio (quantity ratio) are host cell: lymphocyte=1-10:1, it is more preferred to, for host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1.
(3) by the medium centrifugal after cultivation, the lymphocyte precipitate of survival is obtained, and resuspended with culture fluid;
(4) host cell after radiation treatment is added, making host cell and lymphocytic usage ratio (quantity ratio) is host cell: lymphocyte=1-10:1, it is highly preferred that be host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1 cultivation 7 days; After cultivating, the host cell after irradiation breaks, and by the medium centrifugal after cultivation, obtains the lymphocyte precipitate of survival, namely obtains CIK lymphocyte;
Wherein, described culture fluid is made up of the human serum of GT-T551 and 1 volume of 100 volumes;
The available sufficient amount of method that the present invention utilizes and the LAK cell of function, can be applicable to identify and the mononuclear cell of tumor cell in destroyed tumor microenvironment, M2 type and CD19+B cell, strengthen patient's immunoreation. The LAK lymphocyte prepared can be used for the medicine of preparation treatment cancer, infectious disease, diabetes or organ transplantation.
Described cancer is preferably the one in hepatocarcinoma, pulmonary carcinoma, breast carcinoma, gastric cancer, rectal cancer, acute myeloid leukemia, chronic lymphatic tumor leukemia, tumor of prostate, malignant melanoma and renal cell carcinoma or at least two;
Described infectious disease is preferably antibacterial, virus, fungus or parasitic infection, it is more preferred to for hepatitis B virus, hepatitis A virus (HAV) and HIV (human immunodeficiency virus) infection;
Described diabetes are type i diabetes or type ii diabetes, it is more preferred to for type i diabetes;
Described organ transplantation is lung transplantation, renal transplantation, liver transplantation, it is more preferred to for liver transplantation.
The present invention adopts the LAK cell that host cell expands, it is possible to be used for carrying out autotransplantation, it is also possible to carry out heteroplastic transplantation. Under the premise matched by the histocompatibility antigen of contributor (adult or minor) and the histocompatibility antigen of blood antigen and contributor (adult or minor) and blood antigen, contributor is activated and the cell that expands can be injected into by the blood vessel of contributor by the mode of intravenous drip. In some applications, LAK cell line can also be undertaken activating and expanding by host cell. The cell line of amplification is also protection scope of the present invention for preparing the medicine for the treatment of disease.
Technical scheme is further illustrated below in conjunction with accompanying drawing and by detailed description of the invention.
Embodiment 1 expands CIK and the NK cell in not purified peripheral blood lymphocyte
The amplification scheme of the present invention is possible not only to CIK and the NK cell of amplification purification, it is also possible to expand CIK and the NK cell without purification.
The PBMC of the healthy people of donation cultivates in GT-T551, is aided with the human serum of 1%. Co-cultivation 7 days after addition host cell (according to Leukemia such as Imai, method described in 2004,18,676-684 is to, after K562 cell transfecting, carrying out radiation lethal), IL-1 ��, IFN-��, CD3mAb in culture fluid. It is centrifuged after 7 days, resuspended with the culture fluid of equivalent, add the host cell through 100Gy irradiation, be further cultured for 7 days, as shown in Figure 1A. Do not add the amplification scheme of host cell as a control group. CIK cell is under the combined effect of host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22, and quantity dramatically increases, and purity significantly improves. CIK cell quantity entered exponential phase when 7 days, 14 days time about increase 2100 times. The amplification effect that CIK cell grows is significantly stronger than and does not add host cell group by host cell, increases 5.1 times when 14 days. As in figure 2 it is shown, under host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect, when the 14th day, the lymphocyte of 76.3% becomes CIK cell, the matched group peripheral blood lymphocyte not adding host cell has 2.3% cell to become CIK cell.
The PBMC of the healthy people of donation cultivates in GT-T551, is aided with the human serum of 1%. Culture fluid adds co-cultivation 7 days after host cell (according to Leukemia such as Imai, method described in 2004,18,676-684 to after K562 cell transfecting, carries out radiation lethal) and interleukin-22. It is centrifuged after 7 days, resuspended with the culture fluid of equivalent, add the host cell through 100Gy irradiation, be further cultured for 7 days, as shown in Figure 1B. Do not add the amplification scheme of host cell as a control group. NK cell is under the combined effect of host cell and interleukin-22, and quantity dramatically increases, and purity significantly improves. NK cell quantity entered exponential phase when 7 days, 14 days time about increase 2100 times. The amplification effect of NK Growth of Cells is significantly stronger than and does not add host cell group by host cell, increases 5.1 times when 14 days. As it is shown on figure 3, under host cell and interleukin-22 combined effect, when the 14th day, the lymphocyte of 76.3% becomes NK cell, the matched group peripheral blood lymphocyte not adding host cell has 2.3% cell to become NK cell.
The splitting action of CIK and the NK cells against tumor cells system after embodiment 2 amplification and to mouse interior tumor.
Research in the past shows, can strengthen the anti-tumor capacity of mice to tumor model injected in mice CIK cell, delay the life-span of mice. The present embodiment adopts the CIK cell adding host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect amplification in GT-T551, by comparing CIK cell antineoplastic effect, studies the CIK cell antineoplastic feasibility after amplification. After the lymphocyte of healthy donor is separated, add host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect amplification CIK cell, when the 7th day and the 14th day, be separately added into host cell, dosage reference example one used. Do not add the CIK cell of host cell amplification as a control group. The lethal effect of K562, PC3, A549, HepG2 cell is obviously enhanced by the CIK cell that the CIK cell of host cell amplification expands than unused host cell, the lethal effect of tumor-associated macrophages is also then obviously enhanced, p < 0.05, as shown in Figure 4 and Figure 5.
The anti-tumor capacity of mice can be strengthened to tumor model injected in mice NK cell, delay the life-span of mice. The present embodiment adopts the NK cell adding host cell and interleukin-22 combined effect amplification in GT-T551, by comparing the effect of NK cell anti-tumor, studies the CIK cell antineoplastic feasibility after amplification. After the lymphocyte of healthy donor is separated, add host cell and interleukin-22 combined effect amplification NK cell, when the 7th day and the 14th day, be separately added into host cell, dosage reference example one used. Do not add the NK cell of host cell amplification as a control group. The lethal effect of K562, PC3, A549, HepG2 cell is obviously enhanced by the CIK cell that the NK cell of host cell amplification expands than unused host cell, the lethal effect of tumor-associated macrophages is also then obviously enhanced, p < 0.05, as shown in Figures 6 and 7.
The PC3 cell line of external use Lentivirus system constructing Fluc reporter gene stably express. FlucLentivirus system is purchased from SystemBiosciences (U.S., California), and concrete construction method refers to product introduction. PC3-Fluc cells i is injected into severe immune deficiency mice (NOD/SCID), the growing state of fluorescence detector detection tumor, random packet after 21 days. CIK cell after amplification is according to 2x106Cell/every mice is from tail vein injection, biweekly. Stop treatment after treatment surrounding continuously, observe therapeutic effect. Fig. 8 and Figure 10 shows the fluorescence signal of tumor reporter gene after treatment 14 days. The CIK cell of host cell amplification and the growth of NK cells against tumor cells have certain inhibitory action, the CIK cell expanded than unused host cell and NK cell that the lethal effect of PC3 tumor cell is strong. Fig. 9 and 11 show, the survival rate extending mice is had certain effect by CIK and the NK cell of unused host cell amplification. And compared with matched group, significantly improve the cure rate to tumor by the CIK cell of host cell amplification and NK cell, significantly extend the survival rate (p < 0.01) of mice. The CIK cell and the NK cell that add host cell amplification in this prompting GT-T551 may be used for clinical oncotherapy.
Providing a scheme that can be used to make medicine in an embodiment of the present invention, the medicine made according to this scheme can have multiple use disclosed below.This scheme includes: add the CIK cell of host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 combined effect amplification in GT-T551; CIK cell and NK cell that the NK cell adding host cell and interleukin-22 combined effect amplification in GT-T551 utilizes this programme to expand are applicable to strengthen the various sights of patient's immunity. Such as, the CIK cell of amplification and NK cells for therapeutic administration tumor are effective especially, such as acute myeloid leukemia, chronic lymphatic tumor leukemia, tumor of prostate, malignant melanoma and renal cell carcinoma, but are not limited only to above-mentioned tumor. Such as, infect in the CIK cell of amplification and NK cells for therapeutic administration antibacterial, fungus or parasite effective especially. The CIK cell of amplification and NK cells for therapeutic administration viral infection are effective especially, such as hepatitis B virus, hepatitis A virus (HAV) and HIV (human immunodeficiency virus) but be not limited only to above-mentioned viral infection. The CIK cell of amplification and NK cell can significantly improve the action effect of antigen.
The CIK cell adding host cell, IFN-��, CD3mAb and interleukin-22 amplification in the present invention in GT-T551 can transplant patient; The NK cell adding host cell and interleukin-22 amplification in GT-T551 can also transplant patient. Such as, the method that host cell, IL-1 ��, IFN-��, CD3mAb and interleukin-22 amplification activate CIK cell is better than the method without host cell amplification CIK cell, and the efficiency of amplification is high 2.1 times, and the quantity of cell can reach 1.0x1010Above, the demand of clinical treatment is met. The method that host cell and interleukin-22 amplification activate NK cell is better than the method without host cell amplification NK cell, and the efficiency of amplification is high 2.2 times, and the quantity of cell can reach 1.0x1010Above,
Applicant states, the present invention illustrates detailed features and the method for the present invention by above-described embodiment, but the invention is not limited in above-mentioned detailed features and method, does not namely mean that the present invention has to rely on above-mentioned detailed features and method could be implemented. Person of ordinary skill in the field it should be understood that; any improvement in the present invention; the equivalence of material selected by the present invention and step is replaced and auxiliary material and the increase of step, concrete way choice etc., all fall within protection scope of the present invention and open scope.
Claims (10)
1. one kind is activated the lymphocytic method of LAK without hyclone amplification, it is characterized in that, use the host cell having transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F, combine one or more protein and jointly lymphocyte is carried out amplification activation, obtain LAK lymphocyte; Described a-protein comprises the function fragment of CD8 �� and the function fragment of interleukin-22 1; PROTEIN B comprises the function fragment of CD14; Protein C comprises the function fragment of CD19; 3-protein d comprises the function fragment of CD86; Protein E comprises the function fragment of CD163; Protein F comprises the function fragment of CD137L.
2. method according to claim 1, it is characterized in that, described LAK lymphocyte is CIK lymphocyte, the protein of associating includes a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1, the function fragment that described a-protein 1 comprises the function fragment of interleukin-22, PROTEIN B 1 comprises the function fragment of IL-1 ��, protein C 1 comprises IFN-�� function fragment, 3-protein d 1 comprise CD3mAb; Described lymphocyte is selected from one or more in peripheral blood lymphocyte, cord blood lymphocytes cell, CIK cell, or be one or more and other the lymphocytic combination in peripheral blood lymphocyte, cord blood lymphocytes cell, CIK cell, or for one or more in interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, interleukin-22, IL-1 ��, IFN-��, CD3mAb according to arbitrarily acting on CIK lymphocyte produced by the blood in human vas than the mixture formed.
3. method according to claim 2, it is characterised in that said method comprising the steps of:
(1) to transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F host cell carry out lethal process; Described lethal process is selected from strong acid, highly basic, irradiation (dosage is 100Gy-1000Gy).
(2) containing in lymphocytic culture fluid, the host cell after step 1 processes and a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1 co-cultivation 7 days are added; Make described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F amount be 50pM-1000pM. A-protein 1, PROTEIN B 1, protein C 1 amount be 10-1000IU/ milliliter, the amount of 3-protein d 1 is in 10-1000 nanograms/milliliter; Described host cell and lymphocytic usage ratio (quantity ratio) are host cell: lymphocyte=1-10:1, it is more preferred to, for host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1.
(3) by the medium centrifugal after cultivation, LAK lymphocyte precipitate is obtained, and resuspended with culture fluid;
(4) host cell after radiation treatment is added, making host cell and lymphocytic usage ratio (quantity ratio) is host cell: lymphocyte=1-10:1, it is highly preferred that be host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1 cultivation 7 days; By the medium centrifugal after cultivation, obtain the lymphocyte precipitate of survival, namely obtain LAK lymphocyte;
Wherein, described culture fluid is formed by by the human serum of GT-T551 and 1 volume of 100 volumes.
4. method according to claim 1, it is characterised in that described LAK lymphocyte is NK cell; The protein of associating is a-protein 1, and a-protein 1 comprises the function fragment of interleukin-22. Described lymphocyte is selected from one or more in peripheral blood lymphocyte, cord blood lymphocytes cell, NK cell, or be one or more and other the lymphocytic combination in peripheral blood lymphocyte, cord blood lymphocytes cell, NK cell, or for one or more in interleukin-22 1, CD14, CD19, CD86, CD163, CD137L, interleukin-22 according to arbitrarily acting on NK lymphocyte produced by the blood in human vas than the mixture formed.
5. method according to claim 4, it is characterised in that said method comprising the steps of:
(1) to transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F host cell carry out lethal process; Described lethal process is selected from strong acid, highly basic, irradiation (dosage is 100Gy-1000Gy).
(2) containing in lymphocytic culture fluid, the host cell after step 1 processes and a-protein 1 co-cultivation 7 days are added; Make described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F amount be 50pM-1000pM. The amount of a-protein 1 is 10-1000IU/ milliliter, described host cell and lymphocytic usage ratio (quantity ratio) are host cell: lymphocyte=1-10:1, it is highly preferred that be host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1.
(3) by the medium centrifugal after cultivation, the lymphocyte precipitate of survival is obtained, and resuspended with culture fluid;
(4) host cell after radiation treatment is added, making host cell and lymphocytic usage ratio (quantity ratio) is host cell: lymphocyte=1-10:1, it is highly preferred that be host cell: lymphocyte=1-4:1, it is most preferred that be 1: 1 cultivation 7 days;Medium centrifugal after cultivation, obtains the lymphocyte precipitate of survival, namely obtains NK lymphocyte;
Wherein, described culture fluid is made up of the human serum of GT-T551 and 1 volume of 100 volumes.
6. method according to claims 1 to 5, it is characterised in that described host cell is K562 cell.
7. method according to claims 1 to 5, it is characterized in that, described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F are the protein of purification or are immediately expressed at surface of cell membrane by host cell, and a-protein 1, PROTEIN B 1, protein C 1,3-protein d 1 are the protein of purification; Described purification refers to that mass fraction is more than 90%.
8. method according to claims 1 to 5, it is characterized in that, described a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F respectively CD8 ��-interleukin-22 1 complex, CD14, CD19, CD86, CD163 and CD137L, wherein, CD8 �� is the memebrane protein expressed on host cell membrane, CD8 �� makes interleukin-22 1 express on cell membrane after connecting interleukin-22 1, becomes transmembrane protein; CD14, CD19, CD86 and CD137 are memebrane protein.
9. method according to claims 1 to 3, it is characterised in that transfected a-protein, PROTEIN B, protein C, 3-protein d, protein E, protein F host cell obtained by protein stabilized expression system.
10. the lymphocytic application of LAK that method according to any one of a claim 1-8 prepares, it is characterised in that described application is, is used for preparing the medicine for the treatment of cancer, infectious disease, diabetes or organ transplantation by LAK lymphocyte.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4438032A (en) * | 1981-01-30 | 1984-03-20 | The Regents Of The University Of California | Unique T-lymphocyte line and products derived therefrom |
| CN1323072A (en) * | 2001-06-11 | 2001-11-21 | 吴桐 | Waste battery treating method |
| CN101735982A (en) * | 2009-12-31 | 2010-06-16 | 浙江中赢控股集团有限公司 | Method for amplifying lymphocyte by interleukin 15 receptor and interleukin 2 complex |
| CN102154207A (en) * | 2011-03-28 | 2011-08-17 | 浙江中赢干细胞生物工程股份有限公司 | Method for amplifying and activating lymphocyte by using CD8 alpha-interleukin 21-CD137 compound |
| CN103232973A (en) * | 2012-03-12 | 2013-08-07 | 浙江中赢方舟生物工程股份有限公司 | Method for amplification and activation of NK cells by K562 cells |
-
2016
- 2016-03-15 CN CN201610145951.7A patent/CN105647863B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4438032A (en) * | 1981-01-30 | 1984-03-20 | The Regents Of The University Of California | Unique T-lymphocyte line and products derived therefrom |
| CN1323072A (en) * | 2001-06-11 | 2001-11-21 | 吴桐 | Waste battery treating method |
| CN101735982A (en) * | 2009-12-31 | 2010-06-16 | 浙江中赢控股集团有限公司 | Method for amplifying lymphocyte by interleukin 15 receptor and interleukin 2 complex |
| CN102154207A (en) * | 2011-03-28 | 2011-08-17 | 浙江中赢干细胞生物工程股份有限公司 | Method for amplifying and activating lymphocyte by using CD8 alpha-interleukin 21-CD137 compound |
| CN103232973A (en) * | 2012-03-12 | 2013-08-07 | 浙江中赢方舟生物工程股份有限公司 | Method for amplification and activation of NK cells by K562 cells |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695014A (en) * | 2020-12-23 | 2021-04-23 | 杭州中赢生物医疗科技有限公司 | Culture system and culture method for amplifying NK cells |
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