WO2017147894A1 - 增强对异常细胞杀伤力的组合物及其应用 - Google Patents

增强对异常细胞杀伤力的组合物及其应用 Download PDF

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WO2017147894A1
WO2017147894A1 PCT/CN2016/075595 CN2016075595W WO2017147894A1 WO 2017147894 A1 WO2017147894 A1 WO 2017147894A1 CN 2016075595 W CN2016075595 W CN 2016075595W WO 2017147894 A1 WO2017147894 A1 WO 2017147894A1
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cells
cell
natural killer
composition
killer cells
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PCT/CN2016/075595
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French (fr)
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张明杰
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张明杰
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Priority to CN201680000083.1A priority Critical patent/CN109153974A/zh
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  • the present invention relates to the field of biomedical, in particular, the present invention relates to compositions and their use, and more particularly to a composition, use of the composition, a method of treating a patient, a system for treating a patient, A method and apparatus for enhancing the lethality of abnormal cells.
  • Natural killer (NK) cells are important immune cells of the body, not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in hypersensitivity and autoimmune diseases in some cases.
  • NK cells The role of NK cells is broad-spectrum and non-specific; for specific tumor cells, the targeting is not strong; like rockets, some shells may not hit the target and are wasted.
  • T cells are able to find tumors and settle there by "walking and patrolling" in the blood, and in theory, T cells can be expanded in vitro to achieve clinically demanding numbers while providing a more durable anti-tumor effect.
  • T cell reinfusion has significant benefits because antibodies can neither pass through the blood-brain barrier (BBB) nor achieve sustained and effective anti-tumor concentrations within solid tumors.
  • BBB blood-brain barrier
  • TIL Tumor-infiltrating Lymphocytes
  • ACT adoptive cell therapy
  • CTLs cytotoxic T cells
  • CTL can be isolated from peripheral blood, expanded in vitro/specifically conferred to the patient, and returned to the patient with a tumor-derived polypeptide on the MHC-I of the CTL, so that it can “lock” and activate it.
  • TCR T cell receptor
  • TCR allows T cells to proliferate and produce anti-tumor properties.
  • CMV cytomegalovirus
  • TIL and CTL are antigen-specific T lymphocytes with specific effects; they are effective in anticancer antiviral, especially antigen-specific cancers.
  • TAA Tumor associated antigens
  • TAA Tumor associated antigens
  • tumor cells usually mutate in order to escape the pressure of the immune system. It is not uncommon for CTL to be off target.
  • the present invention is directed to solving one of the above technical problems at least to some extent or to provide a commercial choice.
  • MHC class I molecules are expressed on the surface of most normal cells in the body, which forms the polymorphism of tissues and organs between different individuals in the human population.
  • Inhibitory receptors on NK cells distinguish between self or allogeneic by identifying MHC class I molecules on the cell surface.
  • Cellular cancer or infection of the virus often causes MHC class I molecules to down-regulate, and the killing mechanism of activated NK cells is eliminated; while some cells are cancerous or virus-infected, MHC class I molecules are not down-regulated but are up-regulated, which is cancer.
  • NK cells can't do anything about it.
  • TIL and CTL requires information transmitted by antigen presenting cells (APCs), which is the antigen-MHC complex formed by antigenic determinants and MHC molecules.
  • APCs antigen presenting cells
  • MHC molecule When the MHC molecule is down-regulated, it will affect the formation of the antigen-MHC complex, thereby impairing the function of TIL and CTL. That is to say, from the point of view of immune recognition, if NK cells or TIL/CTL are used alone, immune escape may occur.
  • a method of treating a patient comprising the step of administering to the patient natural killer cells (NK) and at least one of the following T lymphocytes: tumor infiltrating lymphocytes (TIL) and cytotoxic T cells (CTL), Natural killer cells comprise highly active natural killer cells (HANK) obtained by activating natural killer cells in vivo by empty cells of the cell with cytokines.
  • NK natural killer cells
  • TIL tumor infiltrating lymphocytes
  • CTL cytotoxic T cells
  • Natural killer cells comprise highly active natural killer cells (HANK) obtained by activating natural killer cells in vivo by empty cells of the cell with cytokines.
  • HANK cells were obtained by the inventors by activating in vitro culture to activate NK cells derived from the body. It should be noted that the so-called HANK cells are not ordinary in vivo NK cells. Ordinary NK cells are not only small in number, but are usually in a state of inhibition, and the activity of killing abnormal cells is low, for example, the activity of killing cancer cells or virus-infected cells is low. In addition, the present invention does not limit the source or preparation manner of the combined administration of TIL and/or CTL, for example, it can be isolated from solid tumor, pleural effusion or ascites, and then expanded and activated in vitro.
  • the cytokine carried by the cell vacant shell may be a cytokine which is naturally expressed on the surface of the cell itself, or may be expressed by a genetic engineering method, for example, by transient transfection or stable expression. On the cell surface, it can also be a cytokine that adsorbs or crosslinks on the cell surface.
  • the cells for preparing the cell vesicle may be primary cells such as peripheral blood mononuclear cells (PBMC), or may be passage cells such as K562 cells.
  • PBMC peripheral blood mononuclear cells
  • NK cells comprising HANK cells and TIL and/or CTL
  • administration of NK cells can enhance the efficacy of NK cells and TIL and/or CTL, not only Mutual regulation of secreted cytokines enhances their lethality, and on the one hand, TIL/CTL can effectively eliminate tumors and infections in which MHC class I molecules are not down-regulated and escape NK cell killing, and on the other hand, NK cells are effectively cleared.
  • NK cells and T cells and DC cells can regulate each other and enhance immune response through multiple pathways; 2) When combined, the two divisions work together and synergistically, NK cells focus on clearing MHC class I molecules Down-regulated cancer cells and virus-infected cells, TIL/CTL can focus on clearing cancer cells and virus-infected cells that are not down-regulated by MHC molecules; 3) NK cells exert anti-cancer anti-viral infection through innate, broad-spectrum non-specific response Its role is the body's first line of defense against cancer and anti-virus. TIL/CTL specifically targets cancerous and virally infected cells through acquired secondary immune responses.
  • the method of this aspect of the invention may also have at least one of the following additional technical features:
  • the so-called patient has at least one of cancer and microbial infections.
  • the so-called patient is a solid tumor patient with pleural effusion and/or ascites, and the patient is said to have NK cells and TIL comprising HANK cells.
  • the so-called patient is a solid tumor patient without pleural and ascites, and the patient is said to have NK cells and CTLs comprising HANK cells.
  • the present invention does not limit the manner in which TIL and CTL are prepared.
  • TIL can be isolated from solid tumors of surgical resection or tissue biopsy using conventional TIL preparation methods, and then expanded in vitro to obtain TIL.
  • a DC-CTL method, a ⁇ T-CTL method, a PBMC mixed culture method, or the like can be used.
  • CTLs are antigen-specific, it is generally necessary to separately prepare CTLs specific for different antigens.
  • the present invention does not limit the types of antigens and antigens that can be used in the preparation of CTLs, and the antigens that can be used include, but are not limited to, polypeptides.
  • Fragments and proteins which may be natural or genetically recombinant polypeptides or proteins, may be soluble or particulate; particulates include, but are not limited to, nanoparticles (including adsorbed, encapsulated, and cross-linked) Associated with a virus (such as an adenovirus, an adeno-associated virus, etc.) or a cell surface (such as natural and recombinant trophoblasts, freshly isolated or passaged tumor cells, etc.), tumor cell membrane extracts, etc. .
  • a virus such as an adenovirus, an adeno-associated virus, etc.
  • a cell surface such as natural and recombinant trophoblasts, freshly isolated or passaged tumor cells, etc.
  • the inventors advocate that it is preferable to apply TIL to patients with pleural effusion or ascites, and to patients with solid tumors without pleural and ascites, especially those with viral infections.
  • Patients with cancer associated with viral infection are preferentially administered with CTL. For example, it is more convenient to separate TIL from pleural fluid and ascites than to separate and culture TIL from solid tumors.
  • the chest and ascites usually contain A large number of T cells and tumor cells not only obtain a large amount of TIL, but also the chest or abdomen is like a natural culture bag, which provides an excellent reaction condition for tumor cells with T cells. Good specificity for a particular individual's tumor cells is only relatively small in number and relatively low in activity. After the collection, it is the most suitable immune cell for treating the individual by a large amount of amplification and activation treatment under in vitro culture conditions.
  • the ratio of the number of highly active natural killer cells and the number of TIL and/or CTL in the natural killer cells administered to the patient in combination is 0.5 to 2.
  • the doctor or the drug instruction will give the amount according to the patient's condition.
  • In vitro tests and animal tests show that the combined administration of the ratio of effector cells can significantly enhance the effect of treating cancer.
  • the total number of cells (effector cells) administered to the patient in combination is at least 1 fold the number of diseased cells (target cells).
  • the HANK cell is obtained by in vitro activation of at least one of NK cells derived from the body: the patient's own NK cells, the patient's semi-matched NK cells, and an allogeneic NK cell.
  • the so-called patient's haplotype NK cells refer to NK cells from the relatives of the patient. For example, collecting the patient's own peripheral blood NK cells, preferably before routine treatment; collecting peripheral blood cells of the patient's relatives, ie, semi-matched peripheral blood NK cells or unrelated individuals, NK cells or cord blood NK cells are not treated. Restriction, as long as the blood transfusion infectious blood test is qualified.
  • the so-called cell vesicles can be derived from natural cells or from engineered cells.
  • Engineering cells refer to the modification or recombination of the genetic material of the host cell by genetic engineering technology or cell fusion technology to obtain cells with stable inheritance and unique traits.
  • the cytokine carried by the cell vacant shell comprises at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL.
  • the cell vacant shell carries IL-15, 4-1BBL and IL-21 cytokines.
  • it can be used to efficiently activate NK cells derived from the body in vitro to obtain HANK cells.
  • preparing the cell empty shell comprises: washing the cells to obtain washed cells; passing the washed cells through a hypotonic treatment or other methods commonly used in the industry to obtain the Cell Empty shell.
  • the inventors have found that the cell empty shell can be obtained quickly and efficiently by using the method, and the method is simple in operation, easy to control, and easy to realize mass production.
  • the cytokine carried by the cell vacant shell may be a cytokine expressed on the surface of the cell itself, or may be expressed in a cell by genetic engineering methods, for example, by transient transfection or stable expression.
  • the surface may also be a cytokine that adsorbs or crosslinks on the cell surface.
  • the cell for preparing the cell vesicle may be a primary cell such as PBMC or a passage cell such as K562 cell or the like.
  • the washing treatment comprises: suspending the cells in an isotonic solution to obtain a cell suspension; and centrifuging the cell suspension to obtain the washed cells.
  • the isotonic solution is pre-cooled to 4 degrees Celsius before suspending the cells in an isotonic solution.
  • the isotonic solution is an isotonic phosphate buffer (PBS) having a pH of 7.4.
  • PBS isotonic phosphate buffer
  • the hypotonic treatment comprises: suspending the washed cells in a hypotonic solution according to a predetermined volume ratio, and allowing the obtained cell suspension to stand for 2 hours to obtain a cell hypotonic treatment.
  • the cell hypotonic treatment is subjected to centrifugation to obtain the cell empty shell.
  • the predetermined volume ratio is 1:40
  • the hypotonic solution is a hypotonic Tris hydrochloric acid buffer.
  • the hypotonic solution is pre-cooled to 4 degrees Celsius before suspending the washed cells in a hypotonic solution.
  • obtaining the HANK cell comprises: isolating a monocyte from a peripheral blood, the PBMC comprising the in vivo NK cell; culturing the PBMC with a medium to which the cell vacant is added
  • the NK cells in the PBMC are activated by amplification to obtain the HANK cells, and the cell empty shell is prepared by the method for preparing a cell empty shell in any of the above embodiments.
  • a large amount of expansion of NK cells derived from the body by the technique in vitro can efficiently obtain a large amount of HANK, and then return the HANK to the patient for treatment of HANK cells, and the test proves that multiple cancers are compared. Good curative effect.
  • the NK cells comprise at least 50% of said HANK cells.
  • the NK cells comprise at least 90% of the HANK cells.
  • the PBMC is cultured in a medium supplemented with a cell empty shell to amplify the in vivo NK cells in the activated PBMC to obtain HANK cells, including:
  • the PBMCs were cultured in an X-Vivo15 serum-free medium containing 200 IU/ml IL-2, the cell empty shell and 5% autologous plasma for 12-20 days, the number of empty cells added to the monocytes The ratio of the number is 1:1.
  • HANK cells Preferably, in order to efficiently obtain high content of HANK cells, at least once on days 4-8 of the culture.
  • the co-administration is: sequentially administering or simultaneously administering the natural killer cells and the T lymphocytes to the patient in an order.
  • the combination of NK cells containing HANK and TIL/CTL can be used in the order of TIL/CTL after NK cells, NK cells after TIL/CTL, or both NK cells and TIL/CTL cells.
  • TIL/CTL can be used in the course of 1-2 weeks after the use of NK cells, following the process of in vivo immune response production, and preferably within 1-2 weeks after the use of NK cells.
  • the present invention provides a composition comprising natural killer cells and at least one of the following T lymphocytes: tumor infiltrating lymphocytes and cytotoxic T cells, said natural killer cells A highly active natural killer cell is obtained which is obtained by activating a natural killer cell in vivo by utilizing a cell empty envelope with a cytokine.
  • the HANK cells and the so-called T lymphocytes in this combination are mutually synergistic, and in vitro cell assays and in vivo mouse experiments indicate that significant effects can be obtained by using the composition against abnormal cells or treating diseases.
  • the so-called abnormal cells include tumor cells, virus-infected cells, and the like, and diseases include cancer, viral infection, and immune diseases.
  • the composition is suitable for use in treating a patient having at least one of a cancer and a microbial infection, the patient being a mammal.
  • the composition administered to the patient is NK cells and TIL comprising HANK cells.
  • the composition administered to the patient is the NK cells and CTLs comprising HANK cells.
  • NK cells and TIL are preferable to apply to patients with pleural effusion or ascites, and to solid tumor patients without pleural and ascites, especially viruses.
  • sexually infected persons and cancer patients with viral infections are preferentially administered with NK cells and CTL.
  • the chest and ascites usually contain A large number of T cells and tumor cells not only obtain a large amount of TIL, but also the chest or abdomen is like a natural culture bag, which provides an excellent reaction condition for tumor cells with T cells. Good specificity for a particular individual's tumor cells is only relatively small in number and relatively low in activity. After the collection, it is the most suitable immune cell for treating the individual by a large amount of amplification and activation treatment under in vitro culture conditions.
  • the ratio of the number of HANK cells in the NK cells to the number of TILs and/or CTLs is 0.5 to 2.
  • the doctor's or composition instructions will give a suggested amount of use based on the patient's condition. In vitro and animal studies have shown that combined administration of this ratio of effector cells can significantly enhance killer cells or treat cancer.
  • the present invention is not limited to the individual source used to amplify activated NK cells.
  • the HANK cells are obtained by in vitro activation of at least one of the following NK cells derived from the body: the patient's own NK cells, the patient's semi-matched NK cells, and unrelated allogeneic NK cells.
  • the so-called patient's haplotype NK cells refer to NK cells from the relatives of the patient. For example, collecting the patient's own peripheral blood NK cells, preferably before routine treatment; collecting peripheral blood cells of the patient's relatives, ie, semi-matched peripheral blood NK cells or unrelated individuals, NK cells or cord blood NK cells are not treated. Restriction, as long as the blood transfusion infectious blood test is qualified.
  • the so-called cell vesicles can be derived from natural cells or from engineered cells.
  • Engineering cells refer to the modification or recombination of the genetic material of the host cell by genetic engineering technology or cell fusion technology to obtain cells with stable inheritance and unique traits.
  • the cytokine carried by the cell vacant shell comprises at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL.
  • the cell vacant shell carries IL-15, 4-1BBL and IL-21 cytokines.
  • it can be used to efficiently activate NK cells derived from the body in vitro to obtain HANK cells.
  • the NK cells comprise at least 50% of HANK cells.
  • the NK cells comprise at least 90% of the HANK cells.
  • the composition has a significantly enhanced killing power against abnormal cells or a therapeutic effect on diseases compared to a single cell.
  • preparing the cell empty shell comprises: washing the cells to obtain washed cells; passing the washed cells through a hypotonic treatment or other methods commonly used in the industry to obtain the The cell is empty.
  • the inventors have found that the cell empty shell can be obtained quickly and efficiently by using the method, and the method is simple in operation, easy to control, and easy to realize mass production.
  • the cytokine carried by the cell vacant shell may be a cytokine expressed on the surface of the cell itself, or may be expressed in a cell by genetic engineering methods, for example, by transient transfection or stable expression.
  • the surface may also be a cytokine that adsorbs or crosslinks on the cell surface.
  • the cell for preparing the cell vesicle may be a primary cell such as PBMC or a passage cell such as K562 cell or the like.
  • the washing treatment comprises: suspending the cells in an isotonic solution to obtain a cell suspension; and centrifuging the cell suspension to obtain the washed cells.
  • the isotonic solution is pre-cooled to 4 degrees Celsius before suspending the cells in an isotonic solution.
  • the isotonic solution is an isotonic phosphate buffer (PBS) having a pH of 7.4.
  • PBS isotonic phosphate buffer
  • the hypotonic treatment comprises: suspending the washed cells in a hypotonic solution according to a predetermined volume ratio, and allowing the obtained cell suspension to stand for 2 hours to obtain a cell hypotonic treatment.
  • the cell hypotonic treatment is subjected to centrifugation to obtain the cell empty shell.
  • the predetermined volume ratio is 1:40
  • the hypotonic solution is a hypotonic Tris salt. Acid buffer.
  • the hypotonic solution is pre-cooled to 4 degrees Celsius before suspending the washed cells in a hypotonic solution.
  • obtaining the HANK cell comprises: isolating a monocyte from a peripheral blood, the PBMC comprising the in vivo NK cell; culturing the PBMC with a medium to which the cell vacant is added
  • the NK cells in the PBMC are activated by amplification to obtain the HANK cells, and the cell empty shell is prepared by the method for preparing a cell empty shell in any of the above embodiments.
  • a large amount of expansion of NK cells derived from the body by the technique in vitro can efficiently obtain a large amount of HANK, and then return the HANK to the patient for treatment of HANK cells, and the test proves that multiple cancers are compared. Good curative effect.
  • the PBMC is cultured in a medium supplemented with a cell vacant to amplify the in vivo NK cells in the activated PBMC to obtain HANK cells, including:
  • the PBMCs were cultured in an X-Vivo15 serum-free medium supplemented with 200 IU/ml IL-2, the cell empty shell and 5% autologous plasma for 12-20 days, the number of empty shells added to the single core The ratio of the number of cells is 1:1.
  • the X-Vivo15 serum-free medium and the cell vesicles are added at least once on days 4-8 of the culture.
  • the present invention provides the use of the composition of the above aspect or any of the embodiments for treating a patient, enhancing the killing power against abnormal cells, and/or preparing a medicament for treating a patient suffering from A disease of the tumor and/or microbial infection, the abnormal cell being a tumor cell and/or a microbial infected cell.
  • the present invention provides a method for enhancing the lethality of an abnormal cell, which comprises combining the natural killer cells and T lymphocytes in the composition of any of the above embodiments to kill the abnormality.
  • the steps of the cell are the technical features and advantages of the composition of one or both of the embodiments of the invention.
  • the total number of cells (effector cells) in the combined use composition is at least 1 fold the number of abnormal cells (target cells).
  • target cells abnormal cells
  • the combined use is to contact the natural killer cells and the T lymphocytes in contact with the abnormal cells, either sequentially or simultaneously.
  • the combination of NK cells containing HANK and TIL/CTL can be used in the order of TIL/CTL after NK cells, NK cells after TIL/CTL, or both NK cells and TIL/CTL cells.
  • the immune response can be generated in vivo The procedure is to first NK post TIL/CTL, and it is best to use TIL/CTL within 1-2 weeks after using NK cells.
  • a device for enhancing the lethality of an abnormal cell for carrying out the method for enhancing the killing power of abnormal cells in one or both of the above-described embodiments of the present invention.
  • the device comprises an administration unit comprising the composition of any of the above embodiments for use in combination with natural killer cells and T lymphocytes in the composition.
  • the total number of cells (effector cells) in the combined use composition is at least 1 fold the number of abnormal cells (target cells).
  • effector cells abnormal cells
  • the combined use is to contact the natural killer cells and the T lymphocytes in contact with the abnormal cells, either sequentially or simultaneously.
  • the combination of NK cells containing HANK and TIL/CTL can be used in the order of TIL/CTL after NK cells, NK cells after TIL/CTL, or both NK cells and TIL/CTL cells.
  • TIL/CTL can be used in the course of 1-2 weeks after the use of NK cells, following the process of in vivo immune response production, and preferably within 1-2 weeks after the use of NK cells.
  • a system for treating a patient having a tumor and/or a microbial infection comprising: a drug delivery module comprising the above aspect or any of the embodiments a composition for the combined administration of natural killer cells and T lymphocytes in said composition to said patient.
  • the total number of cells (effector cells) of the composition administered to the patient is at least 1 time the number of abnormal cells (target cells).
  • the co-administration is: sequentially administering or simultaneously administering the natural killer cells and the T lymphocytes to the patient in an order.
  • a combination of NK cells containing HANK and TIL/CTL can be administered to patients with TIL/CTL after NK cells, NK cells after TIL/CTL, and NK cells and TIL/CTL cells. Apply to the patient at the same time.
  • TIL/CTL can be used in the course of 1-2 weeks after the use of NK cells, following the process of in vivo immune response production, and preferably within 1-2 weeks after the use of NK cells.
  • NK cells In the various aspects of the invention described above, based on the inventors' consideration of the mechanism of action of NK cells and TIL/CTL, the two are combined and the compositions are used in combination to combat tumor cells or virus-infected cells.
  • the composition that is, the combination of NK cells containing HANK cells and TIL and/or CTL, for example, injection into a patient, can enhance the efficacy of NK cells and TIL and/or CTL, not only through secreted cytokines. Regulation enhances their own lethality, and on the one hand, TIL/CTL can effectively eliminate tumors in which MHC class I molecules are not down-regulated and escape NK cell killing. Infection, on the other hand, NK cells effectively remove tumors and infections in which MHC-I molecules are down-regulated and escape TIL/CTL; thus, tumor- and micro-organized cells have nowhere to escape and can be completely eliminated.
  • Fig. 1 is a graph showing the results of killing effect of HANK cells in combination with CTL cells on SUDHL-4 lymphoma cells in one embodiment of the present invention.
  • Figure 2 is a graphical representation of the results of treatment of mouse lymphoma with HANK cells in combination with CTL cells in one embodiment of the invention.
  • Figure 3 is a graph showing the results of killing effect of HANK cells in combination with CTL cells on MDA-MB-435 breast cancer cells in one embodiment of the present invention.
  • Figure 4 is a graph showing the effect of HANK cells in combination with CTL cells on mouse breast cancer in one embodiment of the present invention.
  • Fig. 5 is a graph showing the results of killing effect of HANK cells in combination with CTL cells on HepG2 hepatoma cells in one embodiment of the present invention.
  • Figure 6 is a graphical representation of the efficacy of HANK cells in combination with CTL cells on liver cancer in mice in one embodiment of the invention.
  • Figure 7 is a graphical representation of the ADCC effect of HANK cells in combination with CTL cells on JEV-infected BHK cells in one embodiment of the invention.
  • Figure 8 is a graphical representation of the efficacy of HANK cells in combination with CTL cells in JEV-infected mice in one embodiment of the invention.
  • RPMI 8866 which is a human B lymphoblastoid cell line according to the following procedure. details as follows:
  • RPMI8866 cells were suspended in 3-fold amount of isotonic PBS pre-cooled to 4 ° C and pH 7.4, centrifuged at 1500 rpm for 10 minutes at 4 ° C, the supernatant was removed, and washing was repeated 1-3 times to obtain washed RPMI 8866.
  • RPMI 8866 cells were added to a hypotonic Tris-HCl buffer pre-cooled to 4 ° C and a concentration of 10 mmol/L at a ratio of 1:40, while slowly stirring, then, The mixture was allowed to stand in a refrigerator at 4 ° C for 2 hours to completely lyse the cells; then, the cells were centrifuged at 9000 rpm for 10 minutes at 4 ° C to precipitate the cells, and the washing was further repeated and centrifuged 3-5 times.
  • RPMI8866 cell empty shell namely RPMI 8866-empty shell. Then, it is divided into 2 ⁇ 10 7 cell empty shells/ml, and is frozen in a refrigerator at minus 80 degrees Celsius; or after being freeze-dried, it is stored frozen in a refrigerator at 4 degrees Celsius.
  • the general preparation method includes the following steps:
  • PBMC can be cultured in X-Vivo15 serum-free medium supplemented with IL-2 and RPMI 8866-shell and 1%-5% autologous plasma, which can activate and activate NK cells in large quantities;
  • the number of NK cells can be expanded by hundreds to thousands of times, and the purity of NK cells can be increased from 10% in PBMC to more than 90%;
  • the activated NK cells expanded in vitro are HANK cells, which can be used freshly or frozen in a -80 degree refrigerator or liquid nitrogen for use as effector cells in subsequent in vitro killing assays, in animal experiments alone or Combined with monoclonal antibody, it plays a role in anti-cancer and anti-virus.
  • lymphocyte complete medium containing about 200 IU/ml IL-2 and autologous plasma 1-10% was added to the isolated PBMC to prepare a cell suspension (about 5 ⁇ 10 6 cells). Lymphocytes), added to the T75 flask; simultaneously added 2 ⁇ 10 7 RPMI 8866 - empty shell, placed in a saturated humidity, 37 ° C, 5.0% CO 2 incubator;
  • each culture bag contains There are about 640 ml of the complete lymphocyte culture medium
  • NK cells in PBMC can be cultured at different times depending on the number of cells required, for example, continuous culture for 18 days or 20 days.
  • mice were inoculated with S180 sarcoma cells: ICR females, 4-6 weeks old, 18-22 g mice, 10 inoculated 2X10 ⁇ 5 S180 ascites cancer cells in the left side, normal drinking water, diet, and 10 days of feeding;
  • TIL TIL in S180 ascites cancer mice: The mice were sacrificed, 3 ml of ascites was aseptically taken, 3 ml of red blood cell lysate was added, and the cells were centrifuged at 1000 rpm for 5 minutes at room temperature for 2 minutes. Lymphocytes were separated by lymphocyte separation, and cultured in X-Vivo15 medium containing 1000 U/ml IL-2 for 15 days of activation and activation, which was S180 sarcoma cell-specific TIL, and collected for use.
  • the tumor antigens (leukocyte cell line SUDHL-4, breast cancer MDA-MB-435 cell line, liver cancer HepG2 cells) in the following examples are corresponding tumor cell freeze-thaw lysates; JEV antigen is a recombinant E protein.
  • CTLs are antigen specific, that is, for different antigens, it is necessary to separately prepare CTLs specific for them. Although there are many methods for preparing CTL, such as DC-CTL method, ⁇ T-CTL method, PBMC mixed culture method, etc., each method has its own advantages and disadvantages, but the preparation of CTL by any one method is inseparable from the specificity. Sex antigen.
  • the PBMC mixed culture method is taken as an example to illustrate the preparation process of antigen-specific CTL.
  • the main difference is that the loaded antigens are different, and the other steps are basically the same.
  • CTL medium-1 serum-free medium, 2-5% autologous plasma and IL2 (100 U/ml);
  • CTL medium-2 CTL medium-1 and the following cytokines: IL7 (10 ng/ml), IL4 (1,666 U/ml).
  • PHA-PBMC PHA-activated PBMC
  • PBMC is divided into two, such as 1/7 and 6/7;
  • the antigen-stimulated PHA-PBMC were suspended in CTL medium-3 (IL-4/7) at 2X 10 ⁇ 6 cells per ml.
  • the fluid should not be more than 3X 10 ⁇ 6;
  • a cell line corresponding to TIL or CTL specificity was used as a target cell for each measurement, and K562 cells were used as a positive control cell. 6X10 ⁇ 5 effector cells and 3X10 ⁇ 5 target cells are required;
  • test set the maximum release hole, negative control hole, HANK hole, TIL/CTL hole and HANK+TIL/CTL hole;
  • the cells were gently pipetted with a 100 ⁇ L sampler to suspend the released calcein calcein; centrifuged at 100 g for 5 min to pellet the cells. Gently pipet 100 ⁇ L of the supernatant and transfer to a new plate to prevent foaming. If there is foam formation, use a needle to pierce;
  • a) Establishment of a tumor model of human diffuse large B-cell lymphoma subcutaneous inoculation of 10 ⁇ 7 cells in SCID mice can successfully establish a diffuse large B-cell lymphoma (DLBCL) xenograft model, and the tumor formation rate 70%, the histological performance of the tumor is similar to human DLBCL.
  • DLBCL diffuse large B-cell lymphoma
  • SUDHL-4 is a human GCB-like DLBCL cell line.
  • the cells with an initial density of 2.5 ⁇ 10 ⁇ 5/ml were placed in a T25 cell culture flask containing RPMI1640 medium containing 10% FBS, 100 U/ml penicillin, 100 ⁇ g/ml glutamine, and 30 ⁇ g/ml glutamine.
  • mice Female, 5 weeks old, weighing 16-20g, were randomly divided into groups. The feeding and experiment were carried out in a SPF class rat room with constant temperature (20-26 °C) and constant humidity (50%-56%). . The mice were placed in a laminar flow box with a cover mouse box, the air was filtered by medium efficiency, and the standard pellet feed was fed, and all the items in contact with the rats were previously sterilized;
  • mice (10 per group) cell suspension containing 107 cells were inoculated subcutaneously on one side of the rib 0.1ml; normal control group (10 per group) were injected subcutaneously in the right side of the rib 0.1ml PBS.
  • mice During the experiment, the general condition, tumor formation and tumor growth of the mice were observed daily. Body weight and tumor length and diameter were measured daily, and tumor volume was calculated (calculation method: ⁇ /6 ⁇ length ⁇ width ⁇ height); when the tumor reached 1200 mm 3 , it was regarded as the human end point. After the mice were anesthetized and the neck was sacrificed, the tumors were observed at various parts of the body surface; then the animals were dissected and the internal organs and lymph node metastasis were observed.
  • Human breast cancer MDA-MB-435 cell line nude mouse xenograft model fully humidified with 37 U/ml gentamicin, 10% inactivated calf serum MEM medium at 37 ° C and 5% CO 2
  • the human breast cancer cell line MDA-MB-435 was used for further use.
  • MDA-MB-435 human breast cancer cells were inoculated into the fat pad of the second nipple on the left side of nude mice, and the inoculum amount was 0.1 ml/cell (the number of cells was 1 ⁇ 10 6 /piece). After 2 weeks, the subcutaneous lesions were clearly randomized when they reached the growth of the mass. The administration time was 10 weeks, and the animals were sacrificed by cervical dislocation 3 days after the withdrawal.
  • JEV SA14 is administered through the brain of the suckling rat for 3 consecutive generations of poisoning
  • mice 8 g of Balb/c mice of 3 weeks old were used, and the brain suspension of JEV-infected rats was inoculated intraperitoneally, and the measurement was 10 ⁇ 5 LD50;
  • S180 ascites cancer mouse model 2X 10 5/0.2 ml of S180 sarcoma cells in logarithmic growth phase were inoculated subcutaneously in mice.
  • In vitro killing test According to the second example, HANK cells and SUDHL-4 cell-specific CTLs were used as effector cells, K562 cells and SUDHL-4 cells were used as target cells, and HANK and SUDHL-4 cell-specific CTL pairs were determined. The killing effect of a target cell.
  • the results are shown in Figure 1.
  • the killing rate of HANK cells to K562 cells was about 88%, and the killing rate of SUDHL-4 cells was about 47%.
  • SUDHL-4 cell-specific CTL did not affect the killing effect of HANK cells on K562 cells.
  • the killing effect on SUDHL-4 cells was greatly increased, from 47% to 77%.
  • the tumor volume of the tumor-bearing mice was about 538 mm ⁇ 3.
  • the tumor-bearing mice were divided into 4 groups of 10 animals each. One group was treated with HANK cells alone, and each mouse was intravenously infused with 1X 10 ⁇ 7HANK cells; the other group was treated with SUDHL-4 specific CTL cells alone, and each mouse was intravenously infused with 1X 10 ⁇ 7 SUDHL-4 specificity.
  • group 3 is treated with HANK cells combined with SUDHL-4 specific CTL cells, intravenously infused with HANK cells and SUDHL-4 specific CTL cells 1X10 ⁇ 7, and group 4 is saline saline; each group is once a week.
  • the results are shown in Figure 2: 14 days after treatment, the saline control group had a tumor volume of 1232 mm ⁇ 3, and the HANK cell treatment was performed.
  • the tumor volume of the treatment group was 255 mm ⁇ 3, the SUDHL-4 specific CTL cell treatment group was 422 mm ⁇ 3, and the HANK cell+SUDHL-4 specific CTL cell combination treatment group was 88 mm ⁇ 3.
  • In vitro killing test HANK cells and MDA-MB-435 specific CTL cells were used as effector cells, K562 cells and MDA-MB-435 cells were used as target cells, and HANK cells and MDA-MB-435 specific CTL cells were determined. The killing effect of two target cells.
  • the results are shown in Figure 3.
  • the killing rate of HANK cells on K562 cells was about 87%, and the killing rate on MDA-MB-435 cells was about 63%.
  • the MDA-MB-435-specific CTL cells did not affect HANK cells to K562.
  • the killing effect of the cells but greatly enhanced the killing effect on MDA-MB-435 cells, from 63% to about 91%.
  • the tumor volume of the tumor-bearing mice was about 311 mm ⁇ 3.
  • the tumor-bearing mice were divided into 4 groups of 10 animals each.
  • One group was treated with HANK cells alone, and each mouse was intravenously infused with 1 ⁇ 10 ⁇ 7HANK cells; the other group was treated with MD-MB-435-specific CTL cells alone, and each mouse was intravenously infused with 1 ⁇ 10 ⁇ 7 CTL cells;
  • Group 3 was treated with HANK cells combined with MD-MB-435-specific CTL cells, intravenously infused with HANK cells plus MD-MB-435-specific CTL cells 1X10 ⁇ 7;
  • Group 4 was saline control groups, each group It is once a week.
  • Fig. 4 14 days after treatment, the tumor volume of the saline control group was 724 mm ⁇ 3, the tumor volume of the HANK cell treatment group was 121 mm ⁇ 3, and the MD-MB-435 specific CTL cell treatment group was 312 mm ⁇ 3, HANK cells+ The MD-MB-435 specific CTL cell combination treatment group was 51 mm ⁇ 3. The smaller the tumor volume after treatment, the better the efficacy.
  • HANK cells and HepG2 cell-specific CTL cells were used as effector cells, K562 cells and HepG2 cells were used as target cells, and the killing effects of HANK cells and HepG2-specific CTL cells on two target cells were determined.
  • the results are shown in Figure 5.
  • the killing rate of HANK cells to K562 cells is about 90%, and the killing rate of HepG2 cells is about 50%.
  • HepG2 cell-specific CTL cells do not affect the killing effect of HANK cells on K562 cells, but The killing effect on HepG2 cells was increased from 50% to 87%.
  • the tumor volume of the tumor-bearing mice was about 435 mm ⁇ 3.
  • the tumor-bearing mice were divided into 4 groups of 10 animals each.
  • One group was treated with HANK cells alone, and each mouse was intravenously infused with 1X10 ⁇ 7HANK cells; the other group was treated with HepG2 cell-specific CTL cells alone, and each mouse was intravenously infused with 1X10 ⁇ 7 HepG2 cell-specific CTL cells;
  • Group 3 is treated with HANK cells in combination with HepG2 cell-specific CTL cells, veins Infusion of HANK and CTL each 1X10 ⁇ 7;
  • Group 4 is saline control group; each group is once a week;
  • the tumor volume of the saline control group was 944 mm ⁇ 3
  • the tumor volume of the HANK cell treatment group was 188 mm ⁇ 3
  • the HepG2 cell-specific CTL cell treatment group was 433 mm ⁇ 3
  • the HANK cell + HepG2 cell was specific.
  • the CTL cell combination treatment group was 82 mm ⁇ 3. The smaller the tumor volume after treatment, the better the efficacy.
  • HANK cells combined with CTL cells for treatment of Japanese encephalitis
  • HANK cells and JEV-specific CTL cells were used as effector cells, K562 cells and JEV-infected BHK cells were used as target cells, and the killing effect of HANK cells and JEV-specific CTL cells on the two target cells was determined.
  • the results are shown in Figure 7.
  • the killing rate of HANK cells to K562 cells is about 90%, and the killing rate of JEV-BHK cells is about 60%.
  • JEV-specific CTL does not affect the killing effect of HANK cells on K562 cells, but The killing effect on JEV-BHK cells was increased from 60% to 89%.
  • mice were close to death.
  • the infected mice were divided into 4 groups of 10 animals each.
  • One group was treated with HANK cells alone, and each mouse was intravenously infused with 1X10 ⁇ 7HANK cells; the other group was treated with JEV-specific CTL alone, and each mouse was intravenously infused with JEV-specific CTL cells 1X10 ⁇ 7;
  • Group 3 It is a combination of HANK cells and JEV-specific CTL cells, intravenously infused with HANK cells plus JIV-specific CTL cells 1X10 ⁇ 7;
  • Group 4 is a saline control group; each group is once a week.
  • Fig. 8 14 days after the treatment, all the mice in the saline control group died, the survival rate of the mice in the HANK cell treatment group was 70%, and the survival rate in the JEV-specific CTL cell treatment group was 68%. HANK cells + JEV The survival rate of the mice in the specific CTL cell combination treatment group was 95%. The higher the protection rate, the better the efficacy.
  • HANK group On the next day after subcutaneous inoculation of S180 sarcoma cells, HANK group was injected with 1 ⁇ 10 ⁇ 7/0.2 ml of HANK cells at the site of inoculated tumor cells for 6 consecutive days; TIL group was injected with TIL cells at the site of inoculated tumor cells. 1X10 ⁇ 7/0.2ml for 6 consecutive days; HANK+TIL group injected HANX cells 1X10 ⁇ 7/0.2ml at the site of inoculated tumor cells for 2 consecutive days, and injected TIL cells 1X10 ⁇ 7/0.2ml on the third day.
  • each injection of HANK cells was 1X10 ⁇ 7/0.2ml for 2 consecutive days, and on the sixth day, TX cells were injected with 1X 10 ⁇ 7/0.2ml.
  • the control group was injected with the same volume of physiological saline, and the rats were killed after 6 days of continuous injection. Remove the tumor mass and perform histopathological sectioning.
  • necrosis 1/3 or less of the tumor is (+), 1/3-1/2 of the tumor is (++), and 2/3 or more of the tumor is (+++); Lymphocyte (LC) infiltration: 1-2 LC per field of view (+), 3-5 LC per field of view (++), 5 or more LC per field (+++); 3) Neutral Granulocytes (WBC): 1-2 WBCs per field of view (+), each 3-5 WBCs are (++), and more than 5 WBCs per field of view (+++).
  • LC Lymphocyte
  • WBC Neutral Granulocytes
  • the tumor block (3.10 ⁇ 0.48) g in the control group was determined to have a tumor inhibition rate of 0; the tumor block (1.58 ⁇ 0.3) g in the HANK group and the tumor inhibition rate was 65.16%; the tumor block in the HANK group (0.52 ⁇ 0.2) g, The tumor inhibition rate was 83.23%, and there was a significant difference between the three groups (P ⁇ 0.01).
  • lymphocytes, WBC, and tumor cell necrosis in the treatment group were significantly higher than those in the control group, as shown in Table 1.

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Abstract

一种增强对异常细胞杀伤力的组合物和制药用途，所述组合物由自然杀伤细胞和以下一种T淋巴细胞组成：肿瘤浸润性淋巴细胞和细胞毒性T细胞，自然杀伤细胞包含高活性自然杀伤细胞，高活性自然杀伤细胞通过利用带有细胞因子的细胞空壳体外活化体内自然杀伤细胞而获得。将包含HANK细胞的NK细胞与TIL和/或CTL联合给予患者，HANK细胞和TIL或CTL之间能够互相增强对异常细胞的杀伤力。

Description

组合物及其应用 技术领域
本发明涉及生物医疗领域，具体的，本发明涉及组合物及其应用，更具体的，本发明涉及一种组合物、组合物的用途、一种治疗患者的方法、一种治疗患者的系统、一种增强对异常细胞杀伤力的方法和装置。
背景技术
自然杀伤(natural killer，NK)细胞是机体重要的免疫细胞，不仅与抗肿瘤、抗病毒感染和免疫调节有关，而且在某些情况下参与超敏反应和自身免疫性疾病的发生。
NK细胞的作用是广谱的、非特异性的；对具体肿瘤细胞来说，靶向性不强；就像火箭炮一样，有些炮弹可能打不到靶子上，被浪费了。
T细胞能够通过在血液中“游走巡查”找到肿瘤并定居在那里，而理论上来说，T细胞既能够在体外扩增以达到临床要求的数目，又能够提供较持久的抗瘤效果。和抗体治疗相比，T细胞回输有明显的的好处，因为抗体既不能透过血脑屏障(BBB)又不能在实体瘤内部达到持续有效的抗肿瘤浓度。
最早研究并见成效的T细胞治疗是肿瘤浸润性淋巴细胞(Tumor-infiltrating Lymphocytes，TIL)。TIL也是最早的过继细胞治疗(Adoptive Cell Therapy，ACT)回输细胞。一开始的做法就是将TIL从肿瘤细胞中分离出来，在体外用IL-2扩增，再系统回输给淋巴细胞清除(lymphodepleted)的进展期黑色素瘤患者。结果让人惊讶：TIL能够通过MHC-I和T细胞受体(TCR)的相互作用识别细胞内的肿瘤抗原，患者临床反应率50-70％，甚至有22％见到了肿瘤完全退缩。近期，在TIL上加上了指引T细胞找瘤的“归巢导航”，让更多的T细胞汇聚。TIL在恶性黑色素瘤中给人带来了惊喜。
而另外一种T细胞疗法，细胞毒性T细胞(cytotoxic T cell，CTL)则在病毒相关的肿瘤中取得进展。同样的，CTL可以从外周血中被分离出来，体外扩增/赋予肿瘤特异性后回输给患者，CTL的MHC-I上有肿瘤来源的多肽，这样就能够“锁定”并激活和它结合的T细胞受体(TCR)，让T细胞增殖并产生抗肿瘤的特性。比如，利用针对巨细胞病毒(CMV)特异性的CTL治疗胶质母细胞瘤已经进入临床实验的探索阶段。
TIL和CTL是抗原特异性的T淋巴细胞，作用特异；在抗癌抗病毒，特别是抗原明确的癌症上，疗效显著。但是，由于很多肿瘤细胞的特异性抗原仍不清楚，体外制备CTL时 常常采用肿瘤相关抗原(Tumor associated antigens,TAA)，因此，特异性往往大打折扣。再加上肿瘤细胞为了逃避免疫系统的压力，通常会发生变异，CTL脱靶的现象并不少见。
如何有效或者更有效地利用NK细胞和T淋巴细胞治疗疾病，具有重要的意义。
发明内容
本发明旨在至少在一定程度上解决上述技术问题之一或者提供一种商业选择。
以下认识和猜想是发明人作出本发明的基础：体内既有先天的广谱的非特异性免疫系统，也有获得的特异性免疫系统，二者各有其特殊用处，在体内一起发挥作用。体内的绝大部分正常细胞表面都表达MHC-I类分子，从而形成了人群中不同个体之间组织器官的多型性。NK细胞上的抑制性受体通过识别细胞表面的MHC-I类分子来区分是自我还是异体。细胞癌变或感染病毒后往往会引起MHC-I类分子下调，激活NK细胞的杀伤机制被清除；而有些细胞癌变或病毒感染后，MHC-I类分子不但不下调反而会上调，此即为癌症和病毒逃逸免疫系统的机制。在这种情况下，NK细胞就无能为力了。而和NK细胞的作用机制相反，TIL和CTL的形成需要接受抗原呈递细胞(APC)传递的信息，这种信息就是抗原决定簇和MHC分子形成的抗原-MHC复合物。MHC分子下调的时候，会影响抗原-MHC复合物的形成，进而削弱TIL及CTL的功能。也就是说，从免疫识别的角度出发，假如单独使用NK细胞或TIL/CTL，都有可能会发生免疫逃逸。因而可以说，在用体外制备的免疫细胞进行过继免疫治疗时，单独强调先天性非特异免疫而忽视特异性免疫很可能是片面的、不可靠的或者说效果有待提高的；反之也一样。发明人猜想联合先天的广谱的非特异性免疫细胞以及获得的特异性免疫细胞，对两种类型的细胞经过体外处理，很可能能够全面地、最大限度地发挥免疫系统的抗癌抗病毒等保护作用。
依据本发明的一方面，提供一种治疗患者的方法。所述患者患有疾病，所述方法包括联合给予患者自然杀伤细胞(NK)和以下至少一种T淋巴细胞的步骤：肿瘤浸润性淋巴细胞(TIL)和细胞毒性T细胞(CTL)，所述自然杀伤细胞包含高活性自然杀伤细胞(HANK)，所述高活性自然杀伤细胞是通过带有细胞因子的细胞空壳体外活化体内自然杀伤细胞而获得的。
所称的HANK细胞是发明人通过体外培养扩增活化来自体内的NK细胞获得的。需要说明的是，所称的HANK细胞，非普通的体内NK细胞。普通的NK细胞不但数量少，而且通常处于抑制状态，杀伤异常细胞的活性低，例如杀伤癌细胞或病毒感染细胞的活性低。另外，本发明对联合给予的TIL和/或CTL的来源或制备方式不作限制，例如可以分离自实体瘤、胸水或腹水，进而体外扩增活化。
需要说明的是，所述细胞空壳带有的细胞因子，可以是细胞本身在其表面天然表达的细胞因子，也可以是通过基因工程方法，例如利用瞬时转染或稳定表达将特定细胞因子表达在细胞表面，还可以是在细胞表面吸附或交联的细胞因子。另外，用于制备细胞空壳的细胞可以为原代细胞，例如外周血单核细胞(PBMC)，也可以为传代细胞，例如K562细胞等。
利用本发明的这一方面的方法，将包含HANK细胞的NK细胞和TIL和/或CTL联合施与患者，例如注射到患者体内，能够使NK细胞和TIL和/或CTL的疗效互相增强，不但通过分泌的细胞因子互相调节提高了各自的杀伤力，而且一方面可以通过TIL/CTL有效清除那些MHC-I类分子没有下调而逃逸NK细胞杀伤的肿瘤及感染，另一方面通过NK细胞有效清除那些MHC-I分子下调而逃逸TIL/CTL的肿瘤及感染；从而使肿瘤和微生物感染不管怎么变化都无处可逃，达到彻底清除的效果。具体表现包括：(1)NK细胞和T细胞及DC细胞可以通过多种通路互相调节、增强免疫应答；2)联合使用时，二者分工以及相互增效，NK细胞重点清除MHC-I类分子下调的癌细胞及病毒感染的细胞，TIL/CTL能够重点清除MHC分子没有下调的癌细胞及病毒感染的细胞；3)NK细胞通过先天的、广谱的非特异性没有应答发挥抗癌抗病毒感染的作用，是机体抗癌抗病毒的第一道防线，TIL/CTL则通过获得性二次免疫应答特异地定点清除癌变及病毒感染的细胞。
根据本发明的实施例，本发明的这一方面的方法还可以至少具有以下附加技术特征之一：
所称的患者患有的疾病至少为癌症和微生物感染中的一种。根据本发明的一个实施例，所称的患者为具有胸水和/或腹水的实体瘤患者，联合给予该患者所述包含HANK细胞的NK细胞和TIL。根据本发明的另一个实施例，所称患者为没有胸腹水的实体瘤患者，联合给予该患者所述包含HANK细胞的NK细胞和CTL。
本发明对TIL和CTL的制备方式不作限制。例如，可以利用传统的TIL制备方法，从手术切除或组织活检的实体瘤中分离T细胞，然后在体外培养扩增，获得TIL。制备CTL可以利用DC-CTL法、γδT-CTL法、PBMC混合培养法等。因CTL具有抗原特异性，针对不同的抗原，一般需要分别制备对其具有特异性的CTL，本发明对制备CTL时可以使用的抗原种类及抗原性质不作限制，可以使用的抗原包括但不限于多肽片段和蛋白质，可以是天然的也可以是基因重组的多肽或蛋白，可以是可溶性的也可以是颗粒性的；颗粒性的包括但不限于和纳米颗粒结合的(包括吸附的、包裹的、交联的)，表达在病毒(比如腺病毒，腺病毒相关病毒等)或细胞表面(比如天然的及基因重组的滋养细胞、新鲜分离的或传代的肿瘤细胞等)的，肿瘤细胞膜提取物等等。
根据试验，基于TIL和CTL分离培养的相对难易和效率，发明人主张，对有胸水或腹水的患者优选联合施与TIL，对没有胸腹水的实体瘤患者、特别是病毒性感染者及有病毒感染有关的癌症患者，优先联合施与CTL。比如，与从实体瘤中分离培养TIL相比，从胸水及腹水中分离TIL则更方便，一方面因为临床上往往把胸腹水中的细胞作为废品丢弃了；另一方面，胸腹水中通常含有大量的T细胞及肿瘤细胞，不但比较容易地获得大量的TIL，而且胸腔或腹腔就象一个天然的培养袋，为T细胞提供了一个绝佳的与肿瘤细胞反应的条件；这种TIL具有很好的对特定个体的肿瘤细胞的特异性，只是数量相对较少，活性相对较低。采集后通过在体外培养条件下大量扩增活化等处理措施，将是最适合对该个体进行治疗的免疫细胞。
根据本发明的一个较佳实施例，所述联合给予患者的自然杀伤细胞中的高活性自然杀伤细胞的数目和TIL和/或CTL的数目的比例为0.5至2。治疗患者时，医生或者药物说明书会根据患者情况给出使用量，体外试验以及动物试验表明，联合给予该比例的效应细胞，能够显著地增强治疗癌症的效果。
根据本发明的一个较佳实施例，联合给予所述患者的细胞(效应细胞)总数为疾病细胞(靶细胞)数目的至少1倍。体外试验以及动物试验表明，基于患者的疾病发展情况，基于患者病灶异常细胞的数量，对患者输入该比例的效应细胞，能够显著地增强治疗的效果。
本发明对于用来扩增活化的NK细胞的个体来源不作限制。根据本发明的一个实施例，所述HANK细胞是通过体外活化至少以下一种来自体内的NK细胞获得的：所述患者自身的NK细胞、所述患者的半相合的NK细胞和无关异体的NK细胞。所称的患者的半相合NK细胞指来自患者亲属的NK细胞。例如，采集患者自身的外周血NK细胞，最好是在常规治疗前；采集患者亲属的，即半相合的外周血NK细胞或者无关个体的外周血NK细胞或脐带血NK细胞则不受治疗此限制，只要输血传染病血筛检测合格就可以。
所称细胞空壳可以来自天然细胞，也可以来自工程细胞。工程细胞是指采用基因工程技术或细胞融合技术对宿主细胞的遗传物质进行修饰改造或重组，获得具有稳定遗传的独特性状的细胞。根据本发明的实施例，所述细胞空壳带有的细胞因子至少包括IL-4、IL-7、IL-15、IL-21、CD19、CD64、CD86和4-1BBL中的一种。较佳的，所述细胞空壳带有IL-15、4-1BBL和IL-21细胞因子。如此，能够用于高效地在体外扩增活化来自体内的NK细胞，获得HANK细胞。
根据本发明的实施例，制备所述细胞空壳包括：将细胞进行洗涤处理，获得经过洗涤的细胞；将所述经过洗涤的细胞通过低渗处理或行业内常用的其它方法，以获得所述细胞 空壳。发明人发现，利用该方法能够快速有效地制备获得所述细胞空壳，且该方法操作简单、容易控制，易于实现大规模生产。
需要说明的是，所述细胞空壳带有的细胞因子，可以是在细胞本身表面表达的细胞因子，也可以是通过基因工程方法，例如利用瞬时转染或稳定表达将特定细胞因子表达在细胞表面，还可以是在细胞表面吸附或交联的细胞因子。另外，用于制备细胞空壳的细胞可以为原代细胞，例如PBMC，也可以为传代细胞，例如K562细胞等。
根据本发明的实施例，所述洗涤处理包括：将所述细胞悬浮于等渗溶液中，获得细胞悬液；将所述细胞悬液进行离心处理，以获得所述经过洗涤的细胞。
根据本发明的一个较佳实施例，在将所述细胞悬浮于等渗溶液中之前，预先将所述等渗溶液预冷至4摄氏度。
根据本发明的一个实施例，所述等渗溶液为pH为7.4的等渗磷酸盐缓冲液(PBS)。
根据本发明的一个实施例，所述低渗处理包括：按照预定体积比例将所述经过洗涤的细胞悬浮于低渗溶液中，将所得到的细胞悬液静置2小时，获得细胞低渗处理物；将所述细胞低渗处理物进行离心处理，获得所述细胞空壳。
根据本发明的一个实施例，所述预定体积比例为1：40，所述低渗溶液为低渗Tris盐酸缓冲液。由此，有利于提高细胞低渗裂解的效率。
根据本发明的一个实施例，在将所述经过洗涤的细胞悬浮于低渗溶液中之前，预先将所述低渗溶液预冷至4摄氏度。
根据本发明的实施例，获得所述HANK细胞，包括：从外周血中分离出单核细胞，所述PBMC包含所述体内NK细胞；利用添加了所述细胞空壳的培养基培养所述PBMC，以扩增活化所述PBMC中的NK细胞，获得所述HANK细胞，利用上述任一实施例中的制备细胞空壳的方法制备获得所述细胞空壳。根据本发明的实施例，在体外通过该技术大量扩增活化来自体内的NK细胞，能够高效的获得大量HANK，然后将HANK回输给患者进行HANK细胞治疗，试验证明对多种癌症都有比较好的疗效。
根据本发明的实施例，所述NK细胞至少包含50％的所述HANK细胞。较佳的，所述NK细胞至少包含90％的所述HANK细胞。
为了达到上述HANK的含量，根据本发明的一个较佳实施例，所述利用添加了细胞空壳的培养基培养PBMC，以扩增活化PBMC中的体内NK细胞，获得HANK细胞，包括：在添加了200IU/ml IL-2、所述细胞空壳和5％自身血浆的X-Vivo15无血清培养液中培养所述PBMC 12-20天，所述细胞空壳的添加数目与所述单核细胞的数目的比例为1:1。
较佳的，为了高效获得高含量的HANK细胞，在所述培养的第4-8天，至少添加一次 所述X-Vivo15无血清培养液和所述细胞空壳。
根据本发明的实施例，所述联合给予为：不分顺序先后给予或者同时给予所述患者所述自然杀伤细胞和所述T淋巴细胞。例如，包含HANK的NK细胞和TIL/CTL的联合使用，使用的顺序可以先NK细胞后TIL/CTL，也可以先TIL/CTL后NK细胞，还可以NK细胞和TIL/CTL细胞同时使用。根据本发明的一个较佳实施例，可按照体内免疫应答产生的过程，先NK后TIL/CTL，并且最好是在使用NK细胞后的1-2周内再使用TIL/CTL。
依据本发明的另一方面，本发明提供一种组合物，所述组合物由自然杀伤细胞和以下至少一种T淋巴细胞组成：肿瘤浸润性淋巴细胞和细胞毒性T细胞，所述自然杀伤细胞包含高活性自然杀伤细胞，所述高活性自然杀伤细胞通过利用带有细胞因子的细胞空壳体外活化体内自然杀伤细胞而获得。
该组合中的HANK细胞和所称的T淋巴细胞能够互相增效，体外细胞试验和小鼠体内试验表明，用该组合物对抗异常细胞或者治疗疾病，能够获得显著的效果。所称的异常细胞包括肿瘤细胞、病毒感染的细胞等，所称的疾病包括癌症、病毒感染和免疫疾病。
该组合物适于用于治疗患有癌症和微生物感染中的至少一种疾病的患者，所称患者为哺乳动物。根据本发明的一个实施例，对于具有胸水和/或腹水的实体瘤患者，联合给予该患者的组合物为包含HANK细胞的NK细胞和TIL。根据本发明的另一个实施例，对于没有胸腹水的实体瘤患者，联合给予该患者的组合物为所述包含HANK细胞的NK细胞和CTL。基于TIL和CTL分离培养的相对难易和制备效率，较佳的，发明人主张，对有胸水或腹水的患者优选联合施与NK细胞和TIL，对没有胸腹水的实体瘤患者、特别是病毒性感染者及有病毒感染有关的癌症患者，优先联合施与NK细胞和CTL。比如，与从实体瘤中分离培养TIL相比，从胸水及腹水中分离TIL则更方便，一方面因为临床上往往把胸腹水中的细胞作为废品丢弃了；另一方面，胸腹水中通常含有大量的T细胞及肿瘤细胞，不但比较容易地获得大量的TIL，而且胸腔或腹腔就像一个天然的培养袋，为T细胞提供了一个绝佳的与肿瘤细胞反应的条件；这种TIL具有很好的对特定个体的肿瘤细胞的特异性，只是数量相对较少，活性相对较低。采集后通过在体外培养条件下大量扩增活化等处理措施，将是最适合对该个体进行治疗的免疫细胞。
根据本发明的一个较佳实施例，该组合物中，NK细胞中的HANK细胞的数目和TIL和/或CTL的数目的比例为0.5至2。治疗患者时，医生或者组合物说明书会根据患者情况给出建议的使用量，体外试验以及动物试验表明，联合给予该比例的效应细胞，能够显著地增强杀伤细胞或者治疗癌症的效果。
本发明对于用来扩增活化的NK细胞的个体来源不作限制。根据本发明的一个实施例， 所述HANK细胞是通过体外活化至少以下一种来自体内的NK细胞获得的：所述患者自身的NK细胞、所述患者的半相合的NK细胞和无关异体的NK细胞。所称的患者的半相合NK细胞指来自患者亲属的NK细胞。例如，采集患者自身的外周血NK细胞，最好是在常规治疗前；采集患者亲属的，即半相合的外周血NK细胞或者无关个体的外周血NK细胞或脐带血NK细胞则不受治疗此限制，只要输血传染病血筛检测合格就可以。
所称细胞空壳可以来自天然细胞，也可以来自工程细胞。工程细胞是指采用基因工程技术或细胞融合技术对宿主细胞的遗传物质进行修饰改造或重组，获得具有稳定遗传的独特性状的细胞。根据本发明的实施例，所述细胞空壳带有的细胞因子至少包括IL-4、IL-7、IL-15、IL-21、CD19、CD64、CD86和4-1BBL中的一种。较佳的，所述细胞空壳带有IL-15、4-1BBL和IL-21细胞因子。如此，能够用于高效地在体外扩增活化来自体内的NK细胞，获得HANK细胞。
根据本发明的实施例，该组合物中，所述NK细胞至少包含50％的HANK细胞。较佳的，所述NK细胞至少包含90％的所述HANK细胞。如此，相较于单种细胞，组合物对异常细胞的杀伤力或者对疾病的治疗效果显著增强。
根据本发明的实施例，制备所述细胞空壳包括：将细胞进行洗涤处理，获得经过洗涤的细胞；将所述经过洗涤的细胞通过低渗处理或行业内常用的其它方法，以获得所述细胞空壳。发明人发现，利用该方法能够快速有效地制备获得所述细胞空壳，且该方法操作简单、容易控制，易于实现大规模生产。
需要说明的是，所述细胞空壳带有的细胞因子，可以是在细胞本身表面表达的细胞因子，也可以是通过基因工程方法，例如利用瞬时转染或稳定表达将特定细胞因子表达在细胞表面，还可以是在细胞表面吸附或交联的细胞因子。另外，用于制备细胞空壳的细胞可以为原代细胞，例如PBMC，也可以为传代细胞，例如K562细胞等。
根据本发明的实施例，所述洗涤处理包括：将所述细胞悬浮于等渗溶液中，获得细胞悬液；将所述细胞悬液进行离心处理，以获得所述经过洗涤的细胞。
根据本发明的一个较佳实施例，在将所述细胞悬浮于等渗溶液中之前，预先将所述等渗溶液预冷至4摄氏度。
根据本发明的一个实施例，所述等渗溶液为pH为7.4的等渗磷酸盐缓冲液(PBS)。
根据本发明的一个实施例，所述低渗处理包括：按照预定体积比例将所述经过洗涤的细胞悬浮于低渗溶液中，将所得到的细胞悬液静置2小时，获得细胞低渗处理物；将所述细胞低渗处理物进行离心处理，获得所述细胞空壳。
根据本发明的一个实施例，所述预定体积比例为1：40，所述低渗溶液为低渗Tris盐 酸缓冲液。由此，有利于提高细胞低渗裂解的效率。
根据本发明的一个实施例，在将所述经过洗涤的细胞悬浮于低渗溶液中之前，预先将所述低渗溶液预冷至4摄氏度。
根据本发明的实施例，获得所述HANK细胞，包括：从外周血中分离出单核细胞，所述PBMC包含所述体内NK细胞；利用添加了所述细胞空壳的培养基培养所述PBMC，以扩增活化所述PBMC中的NK细胞，获得所述HANK细胞，利用上述任一实施例中的制备细胞空壳的方法制备获得所述细胞空壳。根据本发明的实施例，在体外通过该技术大量扩增活化来自体内的NK细胞，能够高效的获得大量HANK，然后将HANK回输给患者进行HANK细胞治疗，试验证明对多种癌症都有比较好的疗效。
为了达到预定的HANK的含量，根据本发明的一个较佳实施例，所述利用添加了细胞空壳的培养基培养PBMC，以扩增活化PBMC中的体内NK细胞，获得HANK细胞，包括：在添加了200IU/ml IL-2、所述细胞空壳和5％自身血浆的X-Vivo15无血清培养液中培养所述PBMC 12-20天，所述细胞空壳的添加数目与所述单核细胞的数目的比例为1:1。
较佳的，为了高效获得高含量的HANK细胞，在所述培养的第4-8天，至少添加一次所述X-Vivo15无血清培养液和所述细胞空壳。
依据本发明的再一方面，本发明提供上述一方面或者任一实施例中的组合物在治疗患者、增强对异常细胞杀伤力和/或制备治疗患者的药物中的用途，所述患者患有肿瘤和/或微生物感染类的疾病，所述异常细胞为肿瘤细胞和/或微生物感染的细胞。上述对本发明一方面或者任一实施例中的组合物的技术特征和优点的描述，同样适用于该用途，在此不再赘述。
依据本发明的又一方面，本发明提供一种增强对异常细胞杀伤力的方法，该方法包括联合使用上述任一实施例的组合物中的自然杀伤细胞和T淋巴细胞，以杀伤所述异常细胞的步骤。上述对本发明一方面或者任一实施例中的组合物的技术特征和优点的描述，同样适用于该方法，在此不再赘述。
根据本发明的一个实施例，联合使用的组合物中的细胞(效应细胞)总数为异常细胞(靶细胞)数目的至少1倍。体外试验以及动物试验表明，基于病灶异常细胞的数量，输入该比例的效应细胞，对异常细胞杀伤力的效果显著。
根据本发明的实施例，所述联合使用为：不分顺序先后或者同时使所述自然杀伤细胞和所述T淋巴细胞接触所述异常细胞。例如，包含HANK的NK细胞和TIL/CTL的联合使用，使用的顺序可以先NK细胞后TIL/CTL，也可以先TIL/CTL后NK细胞，还可以NK细胞和TIL/CTL细胞同时使用。根据本发明的一个较佳实施例，可按照体内免疫应答产生 的过程，先NK后TIL/CTL，并且最好是在使用NK细胞后的1-2周内再使用TIL/CTL。
依据本发明的一方面，提供一种增强对异常细胞杀伤力的装置，该装置用以实施上述本发明一方面或者任一实施例中的增强对异常细胞杀伤力的方法。该装置包括施与单元，所述施与单元包含上述任一实施例中的组合物，用以联合使用所述组合物中的自然杀伤细胞和T淋巴细胞。上述对本发明一方面或者任一实施例中的增强对异常细胞杀伤力的方法的技术特征和优点的描述，同样适用于该装置，在此不再赘述。
根据本发明的实施例，联合使用的组合物中的细胞(效应细胞)总数为异常细胞(靶细胞)数目的至少1倍。体外试验以及动物试验表明，基于病灶异常细胞的数量，输入该比例的效应细胞，对异常细胞杀伤力的效果显著。
根据本发明的实施例，所述联合使用为：不分顺序先后或者同时使所述自然杀伤细胞和所述T淋巴细胞接触所述异常细胞。例如，包含HANK的NK细胞和TIL/CTL的联合使用，使用的顺序可以先NK细胞后TIL/CTL，也可以先TIL/CTL后NK细胞，还可以NK细胞和TIL/CTL细胞同时使用。根据本发明的一个较佳实施例，可按照体内免疫应答产生的过程，先NK后TIL/CTL，并且最好是在使用NK细胞后的1-2周内再使用TIL/CTL。
依据本发明的一方面，还提供一种治疗患者的系统，所述患者患有肿瘤和/或微生物感染，该系统包括：给药模块，所述给药模块含有上述一方面或者任一实施例中的组合物，用以联合给予所述患者所述组合物中的自然杀伤细胞和T淋巴细胞。
根据本发明的实施例，联合给予患者的组合物的细胞(效应细胞)总数为异常细胞(靶细胞)数目的至少1倍。体外试验以及动物试验表明，基于患者的疾病发展情况，基于患者病灶异常细胞的数量，对患者输入该比例的效应细胞，能够显著地增强治疗的效果。
根据本发明的实施例，所述联合给予为：不分顺序先后给予或者同时给予所述患者所述自然杀伤细胞和所述T淋巴细胞。例如，将包含HANK的NK细胞和TIL/CTL的联合施与患者，施与的顺序可以先NK细胞后TIL/CTL，也可以先TIL/CTL后NK细胞，还可以NK细胞和TIL/CTL细胞同时施与至患者体内。根据本发明的一个较佳实施例，可按照体内免疫应答产生的过程，先NK后TIL/CTL，并且最好是在使用NK细胞后的1-2周内再使用TIL/CTL。
上述本发明的各个方面，基于发明人考虑NK细胞和TIL/CTL的作用机理，组合二者，将组合物联合使用以对抗肿瘤细胞或病毒感染细胞。将组合物，即将包含HANK细胞的NK细胞和TIL和/或CTL联合施与患者，例如注射到患者体内，能够使NK细胞和TIL和/或CTL的疗效互相增强，不但通过分泌的细胞因子互相调节提高了各自的杀伤力，而且一方面可以通过TIL/CTL有效清除那些MHC-I类分子没有下调而逃逸NK细胞杀伤的肿瘤及 感染，另一方面通过NK细胞有效清除那些MHC-I分子下调而逃逸TIL/CTL的肿瘤及感染；从而使肿瘤和微生物感染的细胞不管怎么变化都无处可逃，能够被彻底清除。
附图说明
结合下面附图对实施例的描述将更明显和更容易地理解本发明所述的和/或附加的方面及其优点，其中：
图1是本发明的一个实施例中HANK细胞联合CTL细胞对SUDHL-4淋巴瘤细胞的杀伤效应的结果示意图。
图2是本发明的一个实施例中HANK细胞联合CTL细胞治疗小鼠淋巴瘤的结果示意图。
图3是本发明的一个实施例中的HANK细胞联合CTL细胞对MDA-MB-435乳腺癌细胞的杀伤效应的结果示意图。
图4是本发明的一个实施例中的HANK细胞联合CTL细胞对小鼠乳腺癌的疗效。
图5是本发明的一个实施例中的HANK细胞联合CTL细胞对HepG2肝癌细胞的杀伤效应的结果示意图。
图6是本发明的一个实施例中的HANK细胞联合CTL细胞对小鼠肝癌的疗效的示意图。
图7是本发明的一个实施例中的HANK细胞联合CTL细胞对JEV感染的BHK细胞的ADCC效应的示意图。
图8是本发明的一个实施例中的HANK细胞联合CTL细胞对JEV感染小鼠的疗效的示意图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例一
1、细胞空壳的制备
按照以下步骤，采用传代细胞系RPMI 8866制备细胞空壳，其中，RPMI 8866是人B淋巴母细胞系。具体如下：
将RPMI8866细胞悬浮于3倍量预冷至4摄氏度pH 7.4的等渗PBS中，于4摄氏度1500转/分钟下离心10分钟，除去上清，重复洗涤1-3次，得到经过洗涤的RPMI 8866细胞；然后将经过洗涤的RPMI 8866细胞按体积比为1：40的比例加入预冷至4摄氏度、浓度为10mmol/L的低渗Tris盐酸缓冲液中，边加边缓慢搅拌，接着，将得到的混合液于4摄氏度的冰箱中静置2小时，使细胞完全裂解；随后，于4摄氏度9000转/分钟下离心10分钟，使细胞空壳沉淀，进一步重复洗涤、离心3-5次，即得RPMI8866细胞空壳，即RPMI 8866-空壳。然后，按2×107个细胞空壳/ml的浓度分装，于零下80摄氏度冰箱冷藏；或经冰冻干燥后，于4摄氏度冰箱冷冻保存。
2、HANK细胞制备
2.1一般的制备方法包括如下步骤：
1)采集健康人外周抗凝血，用淋巴细胞分离液分离PBMC；
2)在添加了IL-2及RPMI 8866-空壳和1％-5％自身血浆的X-Vivo15无血清培养液中培养PBMC，可以使其中的NK细胞大量扩增活化；
3)培养14天左右时，NK细胞数目可以扩增上百至上千倍，NK细胞纯度可以从PBMC中的10％作用增加至90％以上；
这种在体外扩增活化的NK细胞就是HANK细胞，可以新鲜使用，也可以在-80度冰箱或液氮中冻存备用，在后续的体外杀伤试验中作为效应细胞，在动物试验中单独或与单抗联合应用，发挥抗癌抗病毒的作用。
2.2利用RPMI 8866-空壳刺激PBMC中NK细胞扩增，共对3个正常人的外周血单核细胞进行了测试，具体如下：
1)自体血浆制备：抽取抗凝外周血50ml，700G，20min室温离心；吸取血浆，置于水浴锅中56℃，30min；然后4℃静置15min；最后4℃，900G，离心30min，取自体血浆4℃保存备用。
2)取血浆后的细胞层加入D-PBS混匀，用淋巴细胞分离液800G，20min分离PBMC；
3)选T75培养瓶1个，向分离出的PBMC加入40ml淋巴细胞完全培养液(含有约200IU/ml IL-2、自体血浆1-10％)制成细胞悬液(约5×106个淋巴细胞)，加入到T75培养瓶；同时加入2×107RPMI 8866-空壳，置于饱和湿度、37℃、5.0％CO2培养箱中培养；
4)在所述培养的大约第四天，补加约40ml淋巴细胞完全培养液；
5)在所述培养的第七天左右，将所述T75培养瓶中的细胞转移到培养袋中，补加所述淋巴细胞完全培养液至约400ml，并添加约8×107个前面所得到的细胞空壳；
6)在所述培养的第十天左右，将培养物传代至两个培养袋中，其中，每个培养袋中含 有约640ml所述淋巴细胞完全培养液；以及
7)在所述培养的第十二天左右，收集培养产物，测试用不同方法对PBMC中NK细胞的扩增活化效果。其中，根据所需细胞的数量，可以对PBMC中NK细胞进行不同时间的培养，例如可连续培养18天或20天等。
3、制备TIL
包括以下步骤：
小鼠接种S180肉瘤细胞：ICR雌性4-6周龄18-22g小鼠10只，左侧腹腔接种2X10^5个S180腹水癌细胞，正常饮水、饮食，饲养10天；
S180腹水癌小鼠TIL制备：处死小鼠，无菌取腹水3ml，加红细胞裂解液3ml，室温作用2min后1000rpm离心5分钟。用淋巴细胞分离液分离淋巴细胞，在在含1000U/ml IL-2的X-Vivo15培养液中培养扩增活化15天，即为S180肉瘤细胞特异性TIL，收集备用。
4、抗原及其制备
下面示例中的肿瘤抗原(白细胞细胞系SUDHL-4、乳腺癌MDA-MB-435细胞株、肝癌HepG2细胞)为相应瘤细胞冻融法裂解液；JEV抗原为基因重组E蛋白。
5、制备CTL
CTL具有抗原特异性，也就是说，针对不同的抗原，需要分别制备对其具有特异性的CTL。尽管制备CTL的方法有很多种，比如DC-CTL法、γδT-CTL法、PBMC混合培养法等，每种方法都有其固有的优缺点，但使用任何一种方法制备CTL都离不开特异性抗原。
下面以PBMC混合培养法为例，说明抗原特异性CTL的制备流程。其中主要的区别是加载的抗原不同，其它步骤则基本相同。
5.1CTL培养液
CTL培养液-1:无血清培养液，2-5％自身血浆和IL2(100U/ml)；
CTL培养液-2:CTL培养液-1和下列细胞因子：IL7(10ng/ml)，IL4(1,666U/ml)。
5.2PHA活化的PBMC(PHA-PBMC)
1)调整PBMC为2X10^6/ml；
2)PBMC一分为二，例如1/7和6/7；
3)取1/7的一份PBMC，向每2X10^6/ml PBMC，加5ug PHA，在CTL培养液-1中培养，每3天补充培养液。
5.3丝裂霉素处理PHA-PBMC
1)制备丝裂霉素C(25ug/ml)溶液；
2)在准备作为刺激细胞当天(第10天)，按每1X10^7PHA-PBMC 500ul丝裂霉素C溶 液的量，培养30分钟以上，彻底洗涤4次，计数，调整为1X10^7/ml；
3)使用当天(第10天)给PHA-PBMC加载抗原：
i.把细胞置于15(50)ml毫升的离心管中，离心去上清，
ii.加抗原，向每15X 10^6PBMC中加每个抗原100ng，
iii.37℃孵育30-60分钟，
iv.把受抗原刺激的PHA-PBMC悬浮在CTL培养液-3(IL-4/7)中，每毫升2X 10^6个细胞。
5.4CTL制备
1)取6/7的一份PBMC，置于15ml毫升的离心管中，离心去上清；
2)加抗原，向每15X 10^6PBMC中加每个抗原100ng；
3)37℃孵育30-60分钟；
4)把受抗原刺激的PBMC悬浮在CTL培养液-2中，每毫升2X 10^6个细胞；
5)第5天补液，维持细胞浓度不要超过3X 10^6；
6)第10天收集细胞，悬浮在CTL培养液-3中，每毫升2X 10^6个细胞。
5.5CTL扩增
1)把CTL与PHA-PBMC按1:1合培养；
2)第3天及5天补加CTL培养液-3；
3)第7天收获。
实施例二
体外杀伤试验，一般方法：
1)测定前一天复苏一管HANK效应细胞，及一管抗原特异性TIL或CTL，在含有IL-2及5％胎牛血清的X-Vivo15中培养；
2)每次测定时使用一种与TIL或CTL特异性相对应的细胞系作为靶细胞，并以K562细胞作为阳性对照细胞。需要6X10^5效应细胞细胞和3X10^5靶细胞；
3)用NKEM培养液稀释Calcein-AM，配制CAM液；
4)把10^6靶细胞悬浮于1mL CAM液。37℃培养1小时，适时晃动。然后用NKEM培养液洗涤2次，每次1200rpm离心5min。计数，调整浓度为1X10^5/mL；
5)把效应细胞(分HANK、TIL/CTL单独及HANK+TIL/CTL三组)稀释为总数为1X10^6/mL，在U形底96孔细胞培养板上设3孔，每孔加靶细胞200uL，对应效靶细胞比例(E:T)10:1；
6)试验设最大释放孔、阴性对照孔、HANK孔、TIL/CTL孔及HANK+TIL/CTL孔；
7)向最大释放孔加100uL 2％Triton X-100，其余孔加100uL完全培养液；
8)把效应细胞做5次倍比稀释，最后一个稀释度的效靶比例(E:T)为0.3125:1；
9)每孔加100μL靶细胞，100g离心1min，引导细胞接触。37℃ 5％CO2浮箱培养4小时；
10)用100μL加样器轻轻吸打细胞，以悬浮释放的钙黄绿素calcein；100g离心5min，以沉淀细胞。轻轻吸取上清液100μL，转移至一个新培养板，防止产生泡沫。如果有泡沫形成，就用针刺破；
11)用荧光读板仪(激发光485nm,发射光530nm)读板；
12)计算特异性细胞毒性百分比。[(试验组-自然释放组)/(最大释放组-自然释放组)]X 100。
实施例三
建立小鼠肿瘤及乙脑模型，一般方法：
a)人弥漫性大B细胞淋巴瘤小鼠肿瘤模型的建立：采用SCID小鼠皮下接种10^7细胞可成功地建立人人弥漫性大B细胞淋巴瘤(DLBCL)移植瘤模型，成瘤率70％，肿瘤的组织学表现类似于人DLBCL。
1)细胞培养：SUDHL-4为人GCB样DLBCL细胞株。将初始密度为2.5×10^5/ml细胞置于含10％FBS、100U/ml青霉素、100μg/ml链霉素、30μg/ml谷氨酰胺的RPMI1640培养液的T25细胞培养瓶中，在37℃、5％CO2、饱和湿度孵箱中培养；待2-3d第1次传代后，转入T75细胞培养瓶，以后根据细胞生长情况适时移入T150细胞培养瓶；
2)SPF级SCID小鼠，雌性，5周龄，体重16-20g随机分组，饲养及实验均在恒温(20-26℃)、恒湿(50％-56％)的SPF级鼠房内进行。小鼠置于层流架带盖鼠盒中，空气经中效过滤，喂食标准颗粒饲料，与鼠接触的一切物品均事先经灭菌处理；
3)细胞接种:取对数生长期的SUDHL-4细胞，用无血清PBS漂洗2遍后，悬浮于无血清PBS。
实验组小鼠(每组10只)在其一侧肋部皮下接种0.1ml含107细胞的细胞悬液；正常对照组(每组10只)在右侧肋部皮下注射0.1ml PBS。
4)荷瘤鼠指标检测:实验期间每日观察小鼠一般情况、成瘤及肿瘤生长情况。每日测量体重和肿瘤长短径和高度，并计算肿瘤体积(计算方法：π/6×长×宽×高)；当瘤体达到1200mm3时视为人道终点。小鼠经麻醉并拉颈处死后，先观察体表各部位成瘤情况；然后解剖动物，观察各内脏器官和淋巴结转移情况。
b)人乳腺癌MDA-MB-435细胞株裸鼠移植瘤模型：用含50U/ml庆大霉素、10％灭活 小牛血清MEM培养液，在37℃和5％C02充分湿化条件下培养人乳腺癌细胞株MDA-MB-435备用。采纳BALB/C雌性裸小鼠，SPF级，体重20±2g；35～40日龄，取对数生长的体外培养的乳腺癌细胞MDA-MB-435，细胞浓度调整为1×107/ml。无菌条件下，将MDA-MB-435人乳腺癌细胞接种于裸小鼠左侧第二乳头的脂肪垫，接种量为0.1ml/只(细胞数为1×106/只)。2周后皮下明显触及肿块生长时随机分组。给药时间为10周，停药后3天将动物脱颈处死。
c)小鼠肝癌模型：给裸鼠皮下注射1000万个HepG2细胞，14天开始治疗。以立方厘米(mm3)为单位的肿瘤大小的计算公式是：(a)X(b)X 0.5；其中a是肿瘤长度，b是肿瘤宽度。
d)小鼠乙脑模型：
1)病毒株：JEV SA14经乳鼠脑内连续传3代增毒后供用；
2)小鼠感染：选用8克左右3周龄的Balb/c小鼠，用JEV感染的鼠脑悬液经腹腔进行接种，计量为10^5LD50；
3)以感染小鼠存活率计算疗效。
e)S180腹水癌小鼠模型：在小鼠皮下接种处于对数生长期的S180肉瘤细胞2X 10个^5/0.2ml。
实施例四
HANK细胞联合CTL细胞治疗白血病
1、体外杀伤试验：参照实施例二进行，以HANK细胞及SUDHL-4细胞特异性CTL为效应细胞，K562细胞及SUDHL-4细胞为靶细胞，测定HANK及SUDHL-4细胞特异性CTL对2种靶细胞的杀伤作用。
结果如图1所示：HANK细胞对K562细胞的杀伤率为88％左右，对SUDHL-4细胞的杀伤率为47％左右；SUDHL-4细胞特异性CTL不影响HANK细胞对K562细胞的杀伤作用，但大大提高了对SUDHL-4细胞的杀伤作用，从47％上升为77％左右。
2、体内保护试验：接种SUDHL-4细胞后第15天，荷瘤小鼠肿瘤体积约538mm^3。把荷瘤小鼠分为:4组，每组10只。一组单用HANK细胞治疗，每只小鼠静脉输注1X 10^7HANK细胞；另一组单用SUDHL-4特异性CTL细胞治疗，每只小鼠静脉输注1X 10^7SUDHL-4特异性CTL细胞；第3组是HANK细胞联合SUDHL-4特异性CTL细胞治疗，静脉输注HANK细胞及SUDHL-4特异性CTL细胞各1X10^7，第4组是生理盐水照组；每组都是每周一次。
结果如图2所示：治疗后14天，生理盐水对照组肿瘤体积1232mm^3，HANK细胞治 疗组肿瘤体积255mm^3，SUDHL-4特异性CTL细胞治疗组422mm^3，HANK细胞+SUDHL-4特异性CTL细胞联合治疗组88mm^3，治疗后荷瘤体积越小表明疗效越好。
实施例五
HANK细胞联合CTL细胞治疗乳腺癌
1、体外杀伤试验：以HANK细胞及MDA-MB-435特异性CTL细胞为效应细胞，K562细胞及MDA-MB-435细胞为靶细胞，测定HANK细胞及MDA-MB-435特异性CTL细胞对2种靶细胞的杀伤作用。
结果如图3所示：HANK细胞对K562细胞的杀伤率为87％左右，对MDA-MB-435细胞的杀伤率为63％左右；MDA-MB-435特异性CTL细胞不影响HANK细胞对K562细胞的杀伤作用，但大大提高了对MDA-MB-435细胞的杀伤作用，从63％上升为91％左右。
2、体内保护试验：接种MD-MB-435细胞后第15天，荷瘤小鼠肿瘤体积约311mm^3。把荷瘤小鼠分为:4组，每组10只。一组单用HANK细胞治疗，每只小鼠静脉输注1X 10^7HANK细胞；另一组单用MD-MB-435特异性CTL细胞治疗，每只小鼠静脉输注1X 10^7CTL细胞；第3组是HANK细胞联合MD-MB-435特异性CTL细胞治疗，静脉输注HANK细胞加MD-MB-435特异性CTL细胞各1X10^7；第4组是生理盐水对照组，每组都是每周一次。
结果如图4所示：治疗后14天，生理盐水对照组肿瘤体积724mm^3，HANK细胞治疗组肿瘤体积121mm^3，MD-MB-435特异性CTL细胞治疗组312mm^3，HANK细胞+MD-MB-435特异性CTL细胞联合治疗组51mm^3。治疗后荷瘤体积越小表明疗效越好。
实施例六
HANK细胞联合CTL细胞治疗肝癌
1、体外杀伤试验：以HANK细胞及HepG2细胞特异性CTL细胞为效应细胞，K562细胞及HepG2细胞为靶细胞，测定HANK细胞及HepG2特异性CTL细胞对2种靶细胞的杀伤作用。
结果如图5所示：HANK细胞对K562细胞的杀伤率为90％左右，对HepG2细胞的杀伤率为50％左右；HepG2细胞特异性CTL细胞不影响HANK细胞对K562细胞的杀伤作用，但大大提高了对HepG2细胞的杀伤作用，从50％上升为87％左右。
2、体内保护试验：接种HepG2细胞后第15天，荷瘤小鼠肿瘤体积约435mm^3。把荷瘤小鼠分为:4组，每组10只。一组单用HANK细胞治疗，每只小鼠静脉输注1X10^7HANK细胞；另一组单用HepG2细胞特异性CTL细胞治疗，每只小鼠静脉输注1X10^7HepG2细胞特异性CTL细胞；第3组是HANK细胞联合HepG2细胞特异性CTL细胞治疗，静脉 输注HANK及CTL各1X10^7；第4组是生理盐水对照组；每组都是每周一次；
结果如图6所示：治疗后14天，生理盐水对照组肿瘤体积944mm^3，HANK细胞治疗组肿瘤体积188mm^3，HepG2细胞特异性CTL细胞治疗组433mm^3，HANK细胞+HepG2细胞特异性CTL细胞联合治疗组82mm^3。治疗后荷瘤体积越小表明疗效越好。
实施例七
HANK细胞联合CTL细胞治疗乙脑
1、体外杀伤试验：以HANK细胞及JEV特异性CTL细胞为效应细胞，K562细胞及JEV感染的BHK细胞为靶细胞，测定HANK细胞及JEV特异性CTL细胞对2种靶细胞的杀伤作用。
结果如图7所示：HANK细胞对K562细胞的杀伤率为90％左右，对JEV-BHK细胞的杀伤率为60％左右；JEV特异性CTL不影响HANK细胞对K562细胞的杀伤作用，但大大提高了对JEV-BHK细胞的杀伤作用，从60％上升为89％左右。
2、体内保护试验:JEV感染后第5天，部分小鼠已经接近死亡。把感染小鼠分为:4组，每组10只。一组单用HANK细胞治疗，每只小鼠静脉输注1X10^7HANK细胞；另一组单用JEV特异性CTL治疗，每只小鼠静脉输注JEV特异性CTL细胞1X10^7；第3组是HANK细胞联合JEV特异性CTL细胞治疗，静脉输注HANK细胞加JIV特异性CTL细胞各1X10^7；第4组是生理盐水对照组；每组都是每周一次。
结果如图8所示：治疗后14天，生理盐水对照组小鼠全部死亡，HANK细胞治疗组小鼠存活率70％，JEV特异性CTL细胞治疗组小鼠存活率68％，HANK细胞+JEV特异性CTL细胞联合治疗组小鼠存活率95％。保护率越高，疗效越好。
实施例八
HANK细胞联合TIL治疗S180肉瘤
1、体内治疗试验：皮下接种S180肉瘤细胞的次日起，HANK组在接种瘤细胞部位处注射HANK细胞1X10^7个/0.2ml，连续6天；TIL组在接种瘤细胞部位处注射TIL细胞1X10^7个/0.2ml，连续6天；HANK+TIL组在接种瘤细胞部位处注射HANK细胞1X10^7个/0.2ml，连续2天，第三天注射TIL细胞1X10^7个/0.2ml，第四条和第五天各注射HANK细胞1X10^7个/0.2ml，连续2天，第六天注射TIL细胞1X 10^7个/0.2ml。对照组注射同样体积的生理盐水，连续注射6天后杀鼠。去肿瘤块秤重，并做组织病理切片。
病理切片观察标准：1)坏死：占肿瘤1/3以下为(+)，占肿瘤1/3-1/2以下为(++)，占肿瘤2/3以上为(+++)；2)淋巴细胞(LC)浸润：每视野1-2个LC为(+)，每视野3-5个LC为(++)，每视野5个以上LC为(+++)；3)中性粒细胞(WBC)：每视野1-2个WBC为(+)，每 视野3-5个WBC为(++)，每视野5个以上WBC为(+++)。
结果：对照组肿瘤块(3.10±0.48)g，将其定为抑瘤率0；HANK组肿瘤块(1.58±0.3)g，抑瘤率65.16％；HANK组肿瘤块(0.52±0.2)g，抑瘤率83.23％，三组比较有显著差异(P≤0.01)。
治疗组淋巴细胞、WBC数、及肿瘤细胞坏死程度均明显高于对照组，如表1所示。
表1 不同组别小鼠的病理形态变化
Figure PCTCN2016075595-appb-000001
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (54)

  1. 一种治疗患者的方法，所述患者患有疾病，其特征在于，所述方法包括联合给予患者自然杀伤细胞和以下至少一种T淋巴细胞的步骤：肿瘤浸润性淋巴细胞和细胞毒性T细胞，
    所述自然杀伤细胞包含高活性自然杀伤细胞，
    所述高活性自然杀伤细胞是通过带有细胞因子的细胞空壳体外活化体内自然杀伤细胞而获得的。
  2. 权利要求1的方法，其特征在于，所述疾病选自癌症和微生物感染中的至少一种。
  3. 权利要求1的方法，其特征在于，所述患者为具有胸水和/或腹水的实体瘤患者，联合给予所述患者所述自然杀伤细胞和所述肿瘤浸润性淋巴细胞。
  4. 权利要求3的方法，其特征在于，所述肿瘤浸润性淋巴细胞来自所述患者的胸水和/或腹水。
  5. 权利要求1的方法，其特征在于，所述自然杀伤细胞至少包含50％的所述高活性自然杀伤细胞。
  6. 权利要求1的方法，其特征在于，所述自然杀伤细胞至少包含90％的所述高活性自然杀伤细胞。
  7. 权利要求1的方法，其特征在于，联合给予所述患者的自然杀伤细胞中的高活性自然杀伤细胞的数目和所述T淋巴细胞的数目的比例为0.5至2。
  8. 权利要求1的方法，其特征在于，联合给予所述患者的细胞总数为疾病细胞数目的至少1倍。
  9. 权利要求1的方法，其特征在于，所述高活性自然杀伤细胞是通过体外活化以下至少一种体内自然杀伤细胞而获得的：所述患者的自然杀伤细胞、所述患者的半相合的自然杀伤细胞和无关异体的自然杀伤细胞。
  10. 权利要求1的方法，其特征在于，所述细胞空壳来自天然细胞和/或工程细胞。
  11. 权利要求10中的方法，其特征在于，所述细胞空壳带有的细胞因子至少包括IL-4、IL-7、IL-15、IL-21、CD19、CD64、CD86和4-1BBL中的一种。
  12. 权利要求10的方法，其特征在于，所述细胞空壳带有IL-15、4-1BBL和IL-21细胞因子。
  13. 权利要求10的方法，其特征在于，通过以下步骤制备所述细胞空壳：
    将细胞进行洗涤处理，获得经过洗涤的细胞；
    将所述经过洗涤的细胞进行低渗处理，以获得所述细胞空壳。
  14. 权利要求13的方法，其特征在于，所述洗涤处理包括：
    将所述细胞悬浮于等渗溶液中，获得细胞悬液；
    将所述细胞悬液进行离心处理，以获得所述经过洗涤的细胞。
  15. 权利要求14的方法，其特征在于，在将所述细胞悬浮于等渗溶液中之前，将所述等渗溶液预冷至4摄氏度。
  16. 权利要求14的方法，其特征在于，所述等渗溶液为pH为7.4的等渗磷酸盐缓冲液。
  17. 权利要求13的方法，其特征在于，所述低渗处理包括：
    按照预定体积比例将所述经过洗涤的细胞悬浮于低渗溶液中，将所得到的细胞悬液静置2小时，获得细胞裂解物；
    将所述细胞裂解物进行离心处理，获得所述细胞空壳。
  18. 权利要求17的方法，其特征在于，所述预定体积比例为1：40。
  19. 权利要求17的方法，其特征在于，在将所述经过洗涤的细胞悬浮于低渗溶液中之前，预先将所述低渗溶液预冷至4摄氏度。
  20. 权利要求17的方法，其特征在于，所述低渗溶液为低渗Tris盐酸缓冲液。
  21. 权利要求1的方法，其特征在于，通过以下步骤获得所述高活性自然杀伤细胞：
    从外周血中分离出单核细胞，所述单核细胞包括所述体内自然杀伤细胞；
    利用添加了通过权利要求13-20任一方法制备的细胞空壳的培养基培养所述单核细胞，以扩增活化所述单核细胞中的体内自然杀伤细胞，获得所述高活性自然杀伤细胞。
  22. 权利要求21的方法，其特征在于，所述利用添加了细胞空壳的培养基培养单核细胞，以扩增活化单核细胞中的体内自然杀伤细胞，获得高活性自然杀伤细胞，包括：
    在添加了200IU/ml IL-2、所述细胞空壳和5％自身血浆的X-Vivo15无血清培养液中培养所述单核细胞12-20天，所述细胞空壳的添加数目与所述单核细胞的比例为1:1。
  23. 权利要求22的方法，其特征在于，在所述培养的第四至第八天，至少添加一次所述X-Vivo15无血清培养液和所述细胞空壳。
  24. 权利要求1-23任一方法，其特征在于，所述联合给予为：
    不分顺序先后给予或者同时给予所述患者所述自然杀伤细胞和所述T淋巴细胞。
  25. 一种组合物，其特征在于，所述组合物由自然杀伤细胞和以下至少一种T淋巴细胞组成：肿瘤浸润性淋巴细胞和细胞毒性T细胞，
    所述自然杀伤细胞包含高活性自然杀伤细胞，
    所述高活性自然杀伤细胞通过利用带有细胞因子的细胞空壳体外活化体内自然杀伤细胞而获得。
  26. 权利要求25的组合物，所述组合物由所述自然杀伤细胞和所述肿瘤浸润性淋巴细胞组成，所述肿瘤浸润性淋巴细胞来自患者的胸水和/或腹水，所述患者患有肿瘤和/或微生物感染。
  27. 权利要求25的组合物，其特征在于，所述自然杀伤细胞至少包含50％的所述高活性自然杀伤细胞。
  28. 权利要求25的组合物，其特征在于，所述自然杀伤细胞至少包含90％的所述高活性自然杀伤细胞。
  29. 权利要求25的组合物，其特征在于，所述自然杀伤细胞中的高活性自然杀伤细胞的数目和所述T淋巴细胞的数目的比例为0.5至2。
  30. 权利要求25的组合物，其特征在于，所述高活性自然杀伤细胞是通过体外活化以下至少一种体内自然杀伤细胞而获得的：自体的自然杀伤细胞、自体的半相合的自然杀伤细胞和无关异体的自然杀伤细胞。
  31. 权利要求25的组合物，其特征在于，所述细胞空壳来自天然细胞和/或工程细胞。
  32. 权利要求31的组合物，其特征在于，所述细胞空壳带有的细胞因子至少包括IL-4、IL-7、IL-15、IL-21、CD19、CD64、CD86和4-1BBL中的一种。
  33. 权利要求31的组合物，其特征在于，所述细胞空壳带有IL-15、4-1BBL和IL-21细胞因子。
  34. 权利要求31的组合物，其特征在于，通过以下步骤制备所述细胞空壳：
    将细胞进行洗涤处理，获得经过洗涤的细胞；
    将所述经过洗涤的细胞进行低渗处理，以获得所述细胞空壳。
  35. 权利要求34的组合物，其特征在于，所述洗涤处理包括：
    将所述细胞悬浮于等渗溶液中，获得细胞悬液；
    将所述细胞悬液进行离心处理，以获得所述经过洗涤的细胞。
  36. 权利要求35的组合物，其特征在于，在将所述细胞悬浮于等渗溶液中之前，将所述等渗溶液预冷至4摄氏度。
  37. 权利要求35的组合物，其特征在于，所述等渗溶液为pH为7.4的等渗磷酸盐缓冲液。
  38. 权利要求34的组合物，其特征在于，所述低渗处理包括：
    按照预定体积比例将所述经过洗涤的细胞悬浮于低渗溶液中，将所得到的细胞悬液静置2小时，获得细胞裂解物；
    将所述细胞裂解物进行离心处理，获得所述细胞空壳。
  39. 权利要求38的组合物，其特征在于，所述预定体积比例为1：40。
  40. 权利要求38的组合物，其特征在于，在将所述经过洗涤的细胞悬浮于低渗溶液中之前，预先将所述低渗溶液预冷至4摄氏度。
  41. 权利要求38的组合物，其特征在于，所述低渗溶液为低渗Tris盐酸缓冲液。
  42. 权利要求25的组合物，其特征在于，通过以下步骤获得所述高活性自然杀伤细胞：
    从外周血中分离出单核细胞，所述单核细胞包括所述体内自然杀伤细胞；
    利用添加了通过权利要求34-41任一组合物中的细胞空壳的培养基培养所述单核细胞，以扩增活化所述单核细胞中的体内自然杀伤细胞，获得所述高活性自然杀伤细胞。
  43. 权利要求42的组合物，其特征在于，所述利用添加了细胞空壳的培养基培养单核细胞，以扩增活化单核细胞中的体内自然杀伤细胞，获得高活性自然杀伤细胞，包括：
    在添加了200IU/ml IL-2、所述细胞空壳和5％自身血浆的X-Vivo15无血清培养液中培养所述单核细胞12-20天，所述细胞空壳的添加数目与所述单核细胞的比例为1:1。
  44. 权利要求43的组合物，其特征在于，在所述培养的第四至第八天，至少添加一次所述X-Vivo15无血清培养液和所述细胞空壳。
  45. 权利要求25-44任一组合物在治疗患者、增强对异常细胞杀伤力和/或制备治疗患者的药物中的用途，所述患者患有肿瘤和/或微生物感染疾病，所述异常细胞为肿瘤细胞和/或微生物感染的细胞。
  46. 一种增强对异常细胞杀伤力的方法，其特征在于，包括联合使用权利要求25-44任一组合物中的自然杀伤细胞和T淋巴细胞以杀伤所述异常细胞的步骤。
  47. 权利要求46的方法，其特征在于，联合使用的组合物中的细胞总数为异常细胞数目的至少1倍。
  48. 权利要求46的方法，其特征在于，所述联合使用为：
    不分顺序先后或者同时使所述自然杀伤细胞和所述T淋巴细胞接触所述异常细胞。
  49. 一种增强对异常细胞杀伤力的装置，其特征在于，
    所述装置包括施与单元，所述施与单元包含权利要求25-44任一组合物，用以联合使用所述组合物中的自然杀伤细胞和T淋巴细胞。
  50. 权利要求49的装置，其特征在于，联合使用的组合物的细胞总数为异常细胞数目的至少1倍。
  51. 权利要求49的装置，其特征在于，所述联合使用为：
    不分顺序先后或者同时使所述自然杀伤细胞和所述T淋巴细胞接触所述异常细胞。
  52. 一种治疗患者的系统，所述患者患有肿瘤和/或微生物感染，其特征在于，所述系统包括：
    给药模块，所述给药模块含有权利要求25-44任一组合物，用以联合给予所述患者所述组合物中的自然杀伤细胞和T淋巴细胞。
  53. 权利要求52的系统，其特征在于，联合给予患者的组合物的细胞总数为异常细胞数目的至少1倍。
  54. 权利要求52的系统，其特征在于，所述联合给予为：
    不分顺序先后给予或者同时给予所述患者所述自然杀伤细胞和所述T淋巴细胞。
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