CN103243072A - Method for amplifying and activating lymphocyte cells by CD 8 alpha-interleukin 21 fragment-CD137 complex - Google Patents
Method for amplifying and activating lymphocyte cells by CD 8 alpha-interleukin 21 fragment-CD137 complex Download PDFInfo
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Abstract
The invention relates to the field of immunology and specifically relates to a method for amplifying and activating natural killer (NK) cells to become lymphokine activated killer (LAK) cells through the action of a CD 8 alpha-interleukin 21 fragment-CD137 complex. The method disclosed by the invention has the advantages that compared with existing similar complexes, the capability of amplifying and activating the lymphocyte cells is stronger and the efficiency is higher through the CD 8 alpha-interleukin 21 fragment-CD137 complex. The method disclosed by the invention has broad prospects in the aspect of immunotherapy.
Description
Technical field
The present invention relates to a kind of field of immunology, specifically refer to use the K562 cell of CD8 α-interleukin-22 1 fragment-CD137 mixture transfection and lower concentration interleukin-22 acting in conjunction amplification to activate killer cell (LAK) cell that natural killer cell (NK) becomes the lymphokine activation.
Background technology
Nineteen eighty-two Grimm etc. reports at first in the peripheral blood lymphocyte and added the interleukin-22 vitro culture 4-6 days that can induce a kind of non-specific killer cell, this class cell can kill and wound multiple to the insensitive tumour cell of NK.This class cell is called as the killer cell (LAK) that lymphokine activates.In November, 1984, Rosenberg study group ratified down tumour patients such as first Application IL-2 and LAK Synergistic treatment 25 routine renal cell carcinomas, melanoma, lung cancer, colorectal carcinoma through U.S. food and drug inspection office (FDA).Wherein 11 routine tumours are dwindled above 50%, 1 routine melanoma and are disappeared fully.IL-2 and LAK cell Synergistic treatment 222 routine tumour patients had been summed up by this study group in 1988, wherein 16 routine patient tumors metastasis disappear fully, 26 routine patient tumors disappear more than 50%, and this therapy is more remarkable to metastatic renal cell cancer, melanoma, colorectal carcinoma and non_hodgkin lymphoma patient's curative effect.Subsequently studies show that the LAK cell to mammary cancer, bladder cancer, liver cancer and acute leukemia are treated also obvious curative effects.The LAK cell therapy is mainly used to unite use with chemotherapy, radiotherapy in the U.S. at present.
The LAK cell quantity is directly related with curative effect, and the problem that exists in the treatment is that a large amount of LAK cells of acquisition are very difficult.The multiple limited (100 times) that the method for the high dosage interleukin-22 activation growth of adopting usually can increase, and need from patient body, gather the dosage that a large amount of peripheral blood (500 milliliter) can tentatively reach treatment, this is a very big physiological load to patient.The interleukin-22 expense costliness of high dosage is again very big economical load to patient.The present LAK cell multiple that studies show that interleukin-22 activation growth is limited relevant with the cell telomerase activation reduction after the interleukin-22 amplification.It also may be the limited major reason of the interior back effect of LAK cell input patient body that the high dosage interleukin-22 activates growth that telomerase activation reduces.The invention solves this problem, the invention provides and a kind ofly can and activate the method for NK at amplification in vitro.The required interleukin-22 of this method has only 1/10th of common employing method, the high 10-100 of the efficient of amplification doubly, the LAK cell telomerase activation after the amplification is to adopt five times of method usually.LAK cell with this method amplification significantly strengthens than method expanded cells purity and activity with common employing, quantitatively can reach the needs of clinical treatment, can resist tumour, virus and bacterium with helping patient.
CN201110075736.1. disclose the method for a kind of CD8 of employing α-interleukin-22 1-CD137 mixture amplification activated lymphocyte, but its amplification and activation effect are still waiting to improve.And the CD8 α that adopts in this invention and interleukin-22 1 fragment are longer, and it protein denaturation inactivation, immunostimulating occur in actual applications easily than problems such as height, and lower in the extensive production efficiency of expressing when producing.
Summary of the invention
The object of the present invention is to provide a kind of method of effective amplifying lymphocyte.
The present invention is achieved by following technical proposals:
The invention provides a kind of method of the activated lymphocyte that increases, described method is used CD8 α-interleukin-22 1 fragment-CD137 mixture and interleukin-22 acting in conjunction amplification activated lymphocyte, preferably, CD8 α, interleukin-22 1 fragment and the CD137 in described CD8 α-interleukin-22 1-CD137 mixture comprises the complete amino acid sequence of SEQ ID NO:1, SEQ ID NO:1 and CD137 respectively.
In the method for amplification activated lymphocyte of the present invention, can add twice or more times described CD8 α-interleukin-22 1 fragment-CD137 mixture, preferably, the dosage of described CD8 α-interleukin-22 1-CD137 mixture is at 50pM～1nM.
In the method for amplification activated lymphocyte of the present invention, described CD8 α-interleukin-22 1 fragment-CD137 mixture can for purifying or expressed immediately by host cell.
In the method for amplification activated lymphocyte of the present invention, described lymphocyte can be for purifying or unpurified, preferably, described lymphocyte is that the NK cell of combination, purifying of the NK cell of NK cell, purifying of peripheral blood lymphocyte, purifying and peripheral blood lymphocyte or peripheral blood lymphocyte and other lymphocytic combination or interleukin-22 and/or interleukin-22 1 and/or CD137 mixture are injected into all white corpuscles in the blood that acts on behind the human vas, and preferably described purifying refers to that the NK cell is more than 50% of total cellular score.
In the method for amplification activated lymphocyte of the present invention, described CD8 α-interleukin-22 1 fragment-CD137 mixture can adopt protein stabilized expression system, preferably, contain viral promotors and selectable marker gene in the expression vector of described CD8 α-interleukin-22 1 fragment-CD137 mixture.
In the method for amplification activated lymphocyte of the present invention, described host cell can be the K562 cell; Preferably, when described CD8 α-interleukin-22 1 fragment-when the CD137 mixture is expressed immediately by the K562 cell, described K562 cell and lymphocytic usage ratio are the K562 cell: lymphocyte=1～10:1, more preferably, described K562 cell and lymphocytic usage ratio are the K562 cell: lymphocyte=1～4:1.
In the method for amplification activated lymphocyte of the present invention, the lowest dose level of described interleukin-22 can be 50 units per ml, preferably, adds twice or more times described interleukin-22.
The method of amplification activated lymphocyte of the present invention can comprise:
(1) in containing described lymphocytic nutrient solution, the K562 cell of the process irradiation of adding expression described CD8 α-interleukin-22 1 fragment-CD137 mixture and interleukin-22 co-cultivation 7 days;
(2) medium centrifugal after will cultivating obtains cell precipitation, and uses with the nutrient solution of step (1) moderate resuspended; With
(3) add described K562 cell, cultivated 7 days;
Wherein, preferably, described nutrient solution is: the Eagle nutrient solution adds 10% human serum or 10% calf serum; Or RPMI1640 adds 10% human serum or 10% calf serum; Or F-10 (Ham ' s) Nutrient mixtures adds 10% human serum or 10% calf serum; Preferably, the dosage of described irradiation is 100Gy-1000Gy.
In the method for amplification activated lymphocyte of the present invention, the lymphocyte that increases by described method can be dissolved in normal saline solution, is preferably used for intravenous drip.
As preferably, the interleukin-22 described in the aforesaid method, interleukin-22 1 fragment and CD137 are from the human body isolated cells.
As preferably, the lymphocyte of the amplification described in the aforesaid method is that the NK cell of combination, purifying of the NK cell of NK cell, purifying of peripheral blood lymphocyte, purifying and peripheral blood lymphocyte or peripheral blood lymphocyte and other lymphocytic combination or interleukin-22 and/or interleukin-22 1 fragment and/or CD137 mixture are injected into all white corpuscles in the blood that acts on behind the human vas;
Wherein purifying refers to more than 50% of NK cell total number of representatives.
As preferably, the nutrient solution described in the aforesaid method is: Eagle ' s nutrient solution adds 10% human serum or 10% calf serum; Or RPMI1640 adds 10% human serum or 10% calf serum; Or F-10 (Ham ' s) Nutrient mixtures adds 10% human serum or 10% calf serum.
As preferably, the dosage of striding film interleukin-22 1 fragment-CD137 mixture described in the aforesaid method is at 50pM～1nM; Wherein K562 cell and lymphocytic usage ratio the following is, the K562 cell: lymphocyte=1～10: 1; As better selection, add in the described K562 cell and stride film interleukin-22 1 fragment-CD137 mixture; K562 cell: lymphocyte=1～4: 1.
As preferably, host K562 cell described in the aforesaid method is striden film interleukin-22 1 fragment-CD137 mixture through what namely obtain amplifying lymphocyte behind strong acid, highly basic or the high dosage irradiation, strong acid described in the present invention, highly basic are that those skilled in the art such as the vitriol oil, hydrochloric acid, nitric acid think that acid enough strong acid gets final product, and high dosage irradiation also is the known irradiation dose of those of ordinary skill in the industry, generally refers to below the above 1000Gy of irradiation 100Gy.
As preferably, the LAK cell of striding film interleukin-22 1 fragment-CD137 mixture amplification described in the aforesaid method is used further to intravenous drip by being dissolved in normal saline solution.
The K562 cell of CD8 α-interleukin-22 1 fragment-CD137 transfection is to lymphocyte and NK increases and activation has important effect.The LAK cell that amplification and activation back produce has important medical functions.The available sufficient amount of method that the present invention utilizes and the LAK cell of quality are desirable identification and the cell of destroyed tumor cell and pathogenic infection, strengthen the immunoreactive method of patient.The LAK cell can be identified the cell with destroyed tumor cell and pathogenic infection, comprising virus infection, as hepatitis B virus, hepatitis A virus (HAV) and virus of AIDS.Tumor type includes but not limited to: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, the cancer that acquired immune deficiency syndrome (AIDS) is relevant, anus cancer, astrocytoma, atypia monster/voluntary muscle sample knurl, middle Chinese catalpa neural system knurl, rodent cancer-skin carcinoma (the plain knurl of non-black), cholangiocarcinoma, bladder cancer, bone tumor, osteosarcoma, malignant fibrous histiocytoma, brain stem glioma, cerebral tumor, mammary cancer, tumor of bronchus, central nerve neuroma, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), chronic myeloproliferative disease, colorectal carcinoma, large bowel cancer, craniopharyngioma, cutaneous T cell lymphoma-cutaneous T cell lymphoma, breast ductal carcinoma in situ, embryo's property tumour, the central nervous system knurl, carcinoma of endometrium, ependymoma, the esophageal carcinoma, the olifactory nerve parent cell, the Ewing sarcoma family tumor, the extracranial germ cell knurl, the outer sexual cell tumour of sexual gland, the extrahepatic bile ducts tumour, cancer eye, the bone fibres histiocytoma, osteosarcoma, carcinoma of gallbladder, cancer of the stomach, gastrointestinal associated cancers, gastrointestinal stromal tumor-adult soft tissue sarcoma, gonioma, gestational trophoblastic tumor, neurospongioma, hairy cell leukemia, the incidence cancer, cardiac tumor, liver cancer, histiocytosis, Langerhans cell, Hodgkin lymphoma, hypopharyngeal cancer, the intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi sarcoma, kidney, langerhans cell histiocytosis, laryngocarcinoma, leukemia, lip and oral carcinoma, liver cancer, lobular carcinoma in situ, cancer, lymphoma, macroglobulinemia, male breast carcinoma, malignant fibrous histiocytoma of bone, osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, malignant mesothe, transitivity squama cancer tumor colli, oral carcinoma, multiple internal secretion knurl syndromes, multiple myeloma/plasma cell tumor, cutaneous T cell lymphoma, myelodysplastic syndrome, myeloproliferative disorder/bone marrow proliferative tumour, chronic myelocytic leukemia, myelocytic leukemia, multiple myeloma, myeloproliferative disease, the sinunasal tumour, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, nonsmall-cell lung cancer, oral carcinoma, lip pharynx cancer, osteosarcoma, the malignant fibrous histiocytoma bone, the ovarian cancer carcinoma of the pancreas, papilloma, chromaffinoma, nasal sinus and CARCINOMA OF THE NASAL CAVITY, parathyroidoma, penile cancer, the pharynx cancer, pheochromocytoma, the pineal gland parenchymal tumor, pituitary tumor, plasma cell tumor/multiple myeloma, the pleura pulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, the rectum cancer, nephrocyte (kidney) cancer, renal plevis and ureter, transitional cell carcinoma, respiratory cancer, retinoblastoma, rhabdosarcoma, salivary tumor transformation, skin carcinoma, small cell lung cancer, intestinal tumor, soft tissue sarcoma, squama cancer tumor colli, original neuroectodermal tumors, t cell lymphoma, carcinoma of testis, laryngocarcinoma, thymoma and thymic carcinoma, thyroid carcinoma, trophoblastic tumor, ureter and transitional cell carcinoma of renal pelvis, urethral carcinoma, uterus carcinoma, sarcoma of uterus, carcinoma of vagina, carcinoma vulvae, Walden this special macroglobulinemia and nephroblastoma.
Interleukin-22 1 fragment, CD137, CD8 α and interleukin-22 among the present invention is whole polypeptide of this protein, wherein also comprises the polypeptide composition that possesses function in these protein.Peripheral blood lymphocyte among the present invention comes from peripheral blood, Cord blood and marrow.Peripheral blood lymphocyte among the present invention can be the NK cell; It also can be the various combination of NK cell and peripheral blood lymphocyte; Also can be NK cell or peripheral blood lymphocyte and other lymphocytic various combination; Also can be that interleukin-22 and/or interleukin-22 1 fragment and/or CD137 mixture are injected into all leukocytic summations in the blood that acts on behind the human vas.
The invention provides a scheme that can be used for making medicine, the medicine of making according to this scheme can have following described multiple use.This scheme comprises interleukin-22, interleukin-22 1 fragment and CD137.Utilize the LAK cell of this programme amplification to be suitable for and the various sights that need to strengthen patient's immunizing power.For example, amplification NK cell is effective especially to the treatment tumour, as acute myelocytic leukemia, chronic lymphatic knurl leukemia, tumor of prostate, malignant melanoma and nephrocyte malignant tumour, but is not limited only to above-mentioned tumour.For example, amplification LAK cell is effective especially to infecting in treatment bacterium, fungi or the parasite.The LAK cell of amplification is effective especially to the treatment virus infection, as hepatitis B virus, hepatitis A virus (HAV) and virus of AIDS but be not limited only to above-mentioned virus infection.The LAK cell of amplification can significantly improve the action effect of antigen.
Except as otherwise noted, the implication of all technology and science vocabulary is the implication of the personage's common sense with common skill in the industry among the present invention.Being defined in all molecular biology books of Essential Terms can be found, for example, and Benjamin Lewin, Genes VIII, Oxford University Press, 2004 (ISBN0-13-145140-5);
In order to ensure for a long time, production recombinant interleukin 21 fragments and the CD137 of high yield, the present invention has adopted protein stabilized expression system.At first make up the expression vector of CD8 α-interleukin-22 1 fragment-CD137, contain viral promotors and selectable marker gene in this carrier simultaneously, as antibiotics resistance gene.CD8 α is a kind of membranin of expressing at cytolemma, CD8 α gene makes interleukin-22 1 fragment and CD137 be expressed in simultaneously on the cytolemma after connecting interleukin-22 1 fragment and CD137 gene, becomes transmembrane protein (below be referred to as stride film interleukin-22 1 fragment-CD137 mixture).Transcribe K562 clone with this expression vector then, activate promotor with appropriate methods, cultivate the K562 cell namely can obtain high level expression on cytolemma after for some time CD8 α-interleukin-22 1 fragment-CD137 mixture.Make up the expression vector of CD8 α-interleukin-22 1 fragment-CD137, the method for transcribing clone is the method for the personage's common sense with common skill in the industry.Wherein make up the following document of method reference of the expression vector of CD8 α-interleukin-22 1 fragment-CD137: Wu.et.al., J Mol Cell Biol (2010) 2 (4): 217-222; Make up protein stabilized expression system and be the institute of the personage with common skill technology of employing usually in the industry.For example, clone has been transcribed the expression vector of stably express CD8 α-interleukin-22 1 fragment-CD137, contains viral promotors and selectable marker gene in this carrier, as antibiotics resistance gene.CD8 α is a kind of membranin of expressing at cytolemma, CD8 α gene makes interleukin-22 1 fragment and CD137 be expressed in simultaneously on the cytolemma after connecting interleukin-22 1 fragment and CD137 gene, becomes transmembrane protein (below be referred to as stride film interleukin-22 1 fragment-CD137 mixture).After the heterogenous expression carrier enters host cell, activate promotor with appropriate methods, culturing cell namely can obtain the polypeptide of high level expression on cytolemma after for some time.These methods are methods of the personage's common sense with common skill in the industry.
The cell that high level expression is striden film interleukin-22 1 fragment-CD137 mixture on cytolemma can be collected by ultracentrifugal method.Cell can include but not limited to following listed method processing by these technology: strong acid, highly basic and irradiation.The film interleukin-22 1 fragment-CD137 mixture of striding on cytolemma can be directly and peripheral blood lymphocyte or NK cell co-cultivation, activates amplification LAK cell, also can purifies and separates after with the peripheral blood lymphocyte co-cultivation, activate amplification LAK cell.
High level expression strides film interleukin-22 1 fragment-CD137 mixture and can come purifying and separate by the technology that the personage was familiar with common skill in the industry on cytolemma.These technology include but not limited to following listed method: ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography method, chromatography and lectin.If necessary, the scheme of protein renaturation can be used to help purifying expressed proteins polypeptide.If necessary, high performance liquid chromatography (HPLC) can be made last purification step and be further purified protein.The film interleukin-22 1 fragment-CD137 mixture of striding of the host cell inner expression among the present invention is understood some and is secreted in the cell culture supernatant.The personage with common skill in the industry can verify according to common Measurement for Biochemistry.The film interleukin-22 1 fragment-CD137 mixture of striding that is secreted in the cell culture supernatant also can be by aforesaid method purifying and separating.
Stride film interleukin-22 1 fragment-CD137 mixture LAK cell that can be used to increase among the present invention.The LAK cell portable patient of amplification in vitro.For example, stride film interleukin-22 1 fragment-CD137 mixture and low dosage interleukin-22 in order successively the amplification method that activates the LAK cell to activate the LAK cell than used usually high dosage interleukin-22 amplification more effective more than 120 times.The LAK cell telomerase activation of striding after film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 are increased is to adopt the amplification of high dosage interleukin-22 to activate more than 5.2 times of LAK cell.Telomerase activation detects and adopts TRAP method (TRAP) (to see Kim NW for details, Piatyszek MA, prowse KR, et al.Science, 1994; 266:2011).It is higher more than 4.7 times than the LAK cytoactive that activates with the amplification of high dosage interleukin-22 to stride the LAK cell that film interleukin-22 1 fragment-amplification of CD137 mixture activates.Stride film interleukin-22 1 fragment-CD137 mixture after three week of amplification, the purity of LAK cell can reach 95%, and the quantity of cell can reach 1.0 * 10
10More than, satisfy the demand of clinical treatment.As seen the every index of amplification method of the present invention all is better than disclosed method among the CN201110075736.1.,
Stride the LAK cell of film interleukin-22 1 fragment-CD137 mixture amplification, can be used for carrying out autotransplantation, also can carry out heteroplastic transplantation.Under the histocompatibility antigen that is subjected to contributor (grownup or minor) and blood antigen and contributor's (grownup or minor) histocompatibility antigen and prerequisite that blood antigen is complementary, the contributor is activated and expanded cells can be injected into by the mode of intravenous drip by in contributor's the blood vessel.In some applications, clone (for example, NK clone, NKT clone) also can be by striding the film interleukin-22 1 fragment-activation of CD137 mixture and amplification.It also is protection scope of the present invention that expanded cells system is used for the clinical treatment purpose.
Lymphocyte or lymphocyte precursor cell can be from deriving from any tissue that includes big amount lymphocyte and precursor cell.For example, peripheral blood (comprising Cord blood), marrow, spleen and lymphoglandula.Lymphocyte or lymphocyte precursor cell are taken from peripheral blood or marrow usually.In other are used (for example, experimental study), spleen and/or lymphoglandula also are suitable sources.
For example, lymphocyte can be by taking out peripheral blood and/or marrow obtains.Lymphocyte can further obtain NK cell and NKT cell through purifying.Purifying NK cell, the step of NKT cell and technology are that the personage with common skill in the industry is familiar with.Lymphocyte can come erythrocyte in separating blood or the marrow by the mode of density gradient centrifugation, lymphocyte and other cells.NK cell and NKT cell can utilize antibody-mediated affine preparation method to be further purified, for example the antibodies paramagnetic particle method.The purifying lymphocyte does not also require that the lymphocyte of purifying is definitely pure, and refers to that the cell of purifying is higher than its ratio shared in coming source tissue.Generally, the lymphocyte of purifying at least total number of representatives 50%, perhaps 60%, perhaps higher.The NK cell of purifying and NKT cell suspension are in a suitable physiological buffer solution, then suspension growth in but be not limited in the following nutrient solution, Eagle ' s nutrient solution adds 10% human serum, RPMI1640 adds 10% human serum, and F-10 (Ham ' s) Nutrient mixtures adds 10% human serum.Human serum also can replace with 10% foetal calf serum, but human serum is better to the expanding effect of LAK cell.
The lymphocyte of lymphocyte and/or purifying and/or lymphocyte precursor cell suspension grow in the cell culture fluid, add and to stride film interleukin-22 1 fragment-CD137 mixture and the low dosage interleukin-22 is cultivated, add weekly and new stride film interleukin-22 1 fragment-CD137 mixture and the low dosage interleukin-22 is cultivated.Generally in order to reduce cost and to avoid infection, the present invention adopts minimum doses to come amplifying lymphocyte.The dosage of interleukin-22 at 50 units/milliliter between 1000 units per ml.In some cases, the dosage of interleukin-22 mixture is at least more than 500 units per ml.In some cases, be necessary to carry out the processing of high dosage, activate concentration and be about 500 units per ml or 1000 units per ml.In addition, stride film interleukin-22 1 fragment-CD137 mixture and be injected directly into human body, when being used for increasing in vivo the LAK cell, used dosage rubs between 0.5 sodium rubs at 0.5 skin.Striding the employing dosage of film interleukin-22 1 fragment-CD137 mixture determines according to striding the expression dosage of film interleukin-22 1 fragment-CD137 mixture in K562.In some cases, according to K562: lymphocyte=1: 1,2: 1,3: 1, film interleukin-22 1 fragment-CD137 mixture was striden in 4: 1 or above adding.In some cases, be necessary to carry out the processing of high dosage, according to K562: film interleukin-22 1 fragment-CD137 mixture is striden in lymphocyte=10: 1 or above adding.Can transplant to lymphocytic contributor itself or be subjected to the contributor at the LAK of amplification in vitro cell, to strengthen born or the antigen specific immune reaction.Generally, the LAK cell of amplification is by being dissolved in the method intravenous drip of normal saline solution.
Beneficial effect: the present invention cultivates the LAK of amplification by striding film interleukin-22 1 fragment-CD137 mixture and low dosage interleukin-22, and its amplification efficiency is higher, and the LAK activity that obtains is higher.
Description of drawings
Fig. 1 strides film interleukin-22 1 fragment-CD137 mixture synoptic diagram, shows the structure of CD8 α-interleukin-22 1 fragment-CD137 mixture and the order that makes up successively;
Fig. 2 A.LAK cell expansion ex vivo linear graph demonstration lymphocyte is being striden film interleukin-22 1 fragment-CD137 mixture, is being striden film interleukin-22 1-CD137 mixture and the growth of low dosage interleukin-22 acting in conjunction lower linear
B. peripheral blood lymphocyte cell expansion ex vivo linear graph demonstration lymphocyte is being striden film interleukin-22 1 fragment-CD137 mixture, is being striden film interleukin-22 1-CD137 mixture and the growth of low dosage interleukin-22 acting in conjunction lower linear.The cell that the high dosage interleukin-22 is handled is as negative control.
Embodiment
Below enforcement of the present invention is specified:
Stride film interleukin-22 1 fragment-CD137 mixture amplification in vitro LAK cell.
Healthy NK cell cultures of contributing the people is aided with 10% human serum in RPMI1640.In nutrient solution, add to express the K562 cell (contrast) of striding film interleukin-22 1-CD137 mixture respectively and express the K562 cell of striding film interleukin-22 1 fragment-CD137 mixture (irradiation, 100Gy) with the low dosage interleukin-22 after cultivation 7 days.Centrifugal after 7 days, resuspended with the nutrient solution of equivalent, add the K562 cell through irradiation, cultivate 7 days (Fig. 1) again.High dosage interleukin-22 (200 units per ml) is also organized in contrast.Shown in Fig. 2 A, the LAK cell is under the acting in conjunction of striding film interleukin-22 1 fragment-CD137 mixture and low dosage interleukin-22, and quantity significantly increases.The increase of cell quantity can be measured by the method for inserting thymidine.Cell continued to cultivate 12 hours after thymidine inserted, and then the variation that begins to measure cell quantity.At K562: lymphocyte=striding of concentration entered logarithmic phase when the LAK cell quantity was at 7 days under film interleukin-22 1 fragment-CD137 mixture effect in 1: 1,14 days the time increased by 1000 times approximately.Stride in adding under the condition of film interleukin-22 1 fragment-CD137 mixture, minimum can the beginning under the interleukin-22 effect of 50 units per ml increases the LAK cell.And the growth of cellular control unit LAK cell under the high dosage interleukin-22 acts on separately significantly is lower than the cell speed of growth of striding under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22.
Stride film interleukin-22 1 fragment-not purified peripheral blood lymphocyte of CD137 mixture amplification
Stride the NK cell that film interleukin-22 1 fragment-CD137 mixture not only can amplification purification, not purified lymphocyte can also increase.The healthy people's of donation PBM cultivates in RPMI1640, is aided with 10% human serum.In nutrient solution, add to express respectively the K562 cell that the K562 cell (contrast) of striding film interleukin-22 1-CD137 mixture and expression stride film interleukin-22 1 fragment-CD137 mixture (irradiation, 100Gy) with the low dosage interleukin-22 after co-cultivation 7 days.Centrifugal after 7 days, resuspended with the nutrient solution of equivalent, add the K562 cell through 100Gy irradiation, cultivate 7 days (Fig. 1) again.High dosage interleukin-22 (200 units per ml) is also organized in contrast.Shown in Fig. 2 B, the LAK cell is under the acting in conjunction of striding film interleukin-22 1 fragment-CD137 mixture and low dosage interleukin-22, and quantity significantly increases.The LAK cell quantity entered logarithmic phase in the time of 7 days, 14 days the time increased by 1200 times approximately.Stride in adding under the condition of film interleukin-22 1 fragment-CD137 mixture, minimum can the beginning under the interleukin-22 effect of 50 units per ml increases the LAK cell.And the growth of cellular control unit LAK cell under the high dosage interleukin-22 acts on separately significantly is lower than the cell speed of growth of striding under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22.
Striding under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22,93% lymphocyte becomes the LAK cell in the time of the 12nd day.Control group under high dosage interleukin-22 (200 units per ml) effect, peripheral blood lymphocyte only have 14% cell become the LAK cell (CD56+, CD16+, CD56+/CD16+).
The activity of LAK cell Telomerase and STAT3 phosphorylation detect
With the cell after the broken amplification of the method for liquid nitrogen multigelation, 10000g is centrifugal, gets supernatant, detects the activity of amplification back LAK cell Telomerase with the TRAP method.Comprising the 20 milli Tris-HCl (pH8.3) that rub/rise in the 50 microlitre TRAP systems, the 1.5 millis MgCl that rubs/rise, 63 millis rub/liter, KCl, 0.005%Tween-20, the 1 milli EDTA that rubs/rise, the 50 millis dNTP that rubs/rise, 0.1 microgram tS, 1 microgram T4,0.1 mg/ml bovine serum albumin, 1～2 microlitre CHAPS, cell extract (containing 6 micrograms) albumen, 0.2～0.4 microlitre [α-
32P] dGTP.Reaction conditions: 23 ℃ 10 minutes, after 94 ℃ of 3 second, add the Taq enzyme, 94 ℃ of 30 second, 50 ℃ of 30 second, 72 ℃ 1.5 minutes, 27 circulations of increasing are got 25 microlitre products and are carried out the 15%PAGE gel electrophoresis.After the PAGE gel electrophoresis radioautograph on the X-mating plate 8 hours to more than two days.With the amplification before compare the activity of the remarkable activated end granzyme of control group high dosage interleukin-22.And the activity of striding the LAK cell Telomerase that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 is significantly higher than the activity (exceeding 5 times) of striding LAK cell Telomerase under the independent effect of film interleukin-22 1-CD137 mixture and low dosage interleukin-22 acting in conjunction (exceeding 8.5%) and control group high dosage interleukin-22.
Embodiment 4
The LAK cell is to the restraining effect of tumour
Stride the LAK cell that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 to the splitting action of tumour cell.
Studies show that to the injected in mice interleukin-22 of suffering from tumour in the past can strengthen the anti-tumor capacity of mouse, delays the life-span of mouse.Present embodiment adopts strides the LAK cell and the method for tumour cell co-cultivation that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22, studies with the LAK cell that increases and carries out antineoplastic feasibility.Present embodiment adopts the method for striding the LAK injection cell mouse interior therapeutic mouse interior tumor that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22, studies the antineoplastic effect of LAK cell of amplification.
After separating in the lymphocyte with healthy donor, adding is striden the LAK cell that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 and was grown 14 days.K562, PC3, A549 and HepG2 are used as the target cell of cracking.K562 is B cell tumour clone, and this clone is blood tumor cell system.PC3 is prostate tumor cells system, and A549 is lung tumor cell system, and HepG2 is tumor cell of liver system.PC3, A549 and HepG2 are solid tumor clone.The LAK cell of amplification and target cell co-cultivation in accordance with the appropriate ratio detect the target cell that survives after 6 hours.The LAK cell of high dosage interleukin-22 amplification is organized in contrast.Striding the LAK cell that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 significantly strengthens than the LAK cell of the control group high dosage interleukin-22 amplification lethal effect to target cell.(table 1)
The PC3 clone of external use Lentivirus system constructing Fluc reporter gene stably express.Fluc Lentivirus system is available from System Biosciences (U.S., California), and concrete construction process sees product introduction for details.Be injected into 32 of serious immunodeficient mouses (NOD/SCID) under the PC3-Fluc cell skin, fluorimetric detector detects growth of tumor situation, random packet after three days.LAK cell after the amplification is according to 1 * 10
7Cell/every mouse is from tail vein injection, biweekly.Stop treatment after continuously around the treatment, observe result for the treatment of.The growth of tumor of LAK of high dosage interleukin-22 amplification has certain restraining effect, and (the average tumor size is 6mm.Stride the LAK cell that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 than the LAK cell of the high dosage interleukin-22 amplification lethal effect strong (the average tumor size is 5.3mm) to the PC3 tumour cell.The LAK cell of interleukin-22 amplification has certain effect to the survival rate that prolongs mouse.And compare with the LAK cell of interleukin-22 amplification, stride the LAK that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 and significantly improved curative ratio to tumour (improving 6%), significant prolongation the survival rate of mouse (improving 12%).The LAK cell that increases under film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 is striden in this prompting can be used for clinical oncotherapy.
Claims (10)
1. the method for the activated lymphocyte that increases, it is characterized in that, use CD8 α-interleukin-22 1 fragment-CD137 mixture and interleukin-22 acting in conjunction amplification activated lymphocyte, preferably, CD8 α, interleukin-22 1 fragment and the CD137 in described CD8 α-interleukin-22 1 fragment-CD137 mixture comprises the complete amino acid sequence of complete fragment, SEQ ID NO:1 and the CD137 of CD8 α respectively.
2. method according to claim 1 is characterized in that, adds twice or more times described CD8 α-interleukin-22 1 fragment-CD137 mixture in the described method, and preferably, the dosage of described CD8 α-interleukin-22 1-CD137 mixture is at 50pM～1nM.
3. method according to claim 1 and 2 is characterized in that, described CD8 α-interleukin-22 1 fragment-CD137 mixture be purifying or expressed immediately by host cell.
4. according to each described method in the claim 1 to 3, it is characterized in that, described lymphocyte is purifying or unpurified, preferably, described lymphocyte is that the NK cell of combination, purifying of the NK cell of NK cell, purifying of peripheral blood lymphocyte, purifying and peripheral blood lymphocyte or peripheral blood lymphocyte and other lymphocytic combination or interleukin-22 and/or interleukin-22 1 and/or CD137 mixture are injected into all white corpuscles in the blood that acts on behind the human vas, and preferably described purifying refers to that the NK cell is more than 50% of total cellular score.
5. according to each described method in the claim 1 to 4, it is characterized in that, described CD8 α-interleukin-22 1 fragment-CD137 mixture adopts protein stabilized expression system, preferably, contain viral promotors and selectable marker gene in the expression vector of described CD8 α-interleukin-22 1 fragment-CD137 mixture.
6. method according to claim 3 is characterized in that, described host cell is the K562 cell; Preferably, when described CD8 α-interleukin-22 1 fragment-when the CD137 mixture is expressed immediately by the K562 cell, described K562 cell and lymphocytic usage ratio are the K562 cell: lymphocyte=1～10:1, more preferably, described K562 cell and lymphocytic usage ratio are the K562 cell: lymphocyte=1～4:1.
7. according to each described method in the claim 1 to 6, it is characterized in that the lowest dose level of wherein said interleukin-22 is 50 units per ml, preferably, add twice or more times described interleukin-22.
8. method according to claim 1 is characterized in that, described method comprises:
(1) in containing described lymphocytic nutrient solution, the K562 cell of the process irradiation of adding expression described CD8 α-interleukin-22 1 fragment-CD137 mixture and interleukin-22 co-cultivation 7 days;
(2) medium centrifugal after will cultivating obtains cell precipitation, and uses with the nutrient solution of step (1) moderate resuspended; With
(3) add described K562 cell, cultivated 7 days;
Wherein, preferably, described nutrient solution is: the Eagle nutrient solution adds 10% human serum or 10% calf serum; Or RPMI1640 adds 10% human serum or 10% calf serum; Or F-10 (Ham ' s) Nutrient mixtures adds 10% human serum or 10% calf serum; Preferably, the dosage of described irradiation is 100Gy-1000Gy.
9. according to each described method among the claim 1-8, it is characterized in that the lymphocyte that increases by described method is dissolved in normal saline solution, is preferably used for intravenous drip.
According to each described method among the claim 1-9 for the preparation of the purposes in the preparation of the medicine for the treatment of cancer or communicable disease, preferably, described cancer is for being selected from by acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, the cancer that acquired immune deficiency syndrome (AIDS) is relevant, anus cancer, astrocytoma, atypia monster/voluntary muscle sample knurl, middle Chinese catalpa neural system knurl, rodent cancer-the skin carcinoma of the plain knurl of non-black, cholangiocarcinoma, bladder cancer, bone tumor, osteosarcoma, malignant fibrous histiocytoma, brain stem glioma, cerebral tumor, mammary cancer, tumor of bronchus, central nerve neuroma, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), chronic myeloproliferative disease, colorectal carcinoma, large bowel cancer, craniopharyngioma, cutaneous T cell lymphoma-cutaneous T cell lymphoma, breast ductal carcinoma in situ, embryo's property tumour, carcinoma of endometrium, ependymoma, the esophageal carcinoma, the olifactory nerve parent cell, the Ewing sarcoma family tumor, sexual cell knurl outside the road, the outer sexual cell tumour of sexual gland, the extrahepatic bile ducts tumour, cancer eye, the bone fibres histiocytoma, carcinoma of gallbladder, cancer of the stomach, gastrointestinal associated cancers, gastrointestinal stromal tumor, adult soft tissue sarcoma, gonioma, gestational trophoblastic tumor, neurospongioma, hairy cell leukemia, the incidence cancer, cardiac tumor, liver cancer, histiocytosis, Langerhans cell, Hodgkin lymphoma, hypopharyngeal cancer, the intraocular melanoma, islet cell tumor, card ripple dilution sarcoma, kidney, langerhans cell histiocytosis, laryngocarcinoma, leukemia, lip and oral carcinoma, liver cancer, lobular carcinoma in situ, cancer, lymphoma, macroglobulinemia, male breast carcinoma, malignant fibrous histiocytoma of bone, medulloblastoma, medulloepithelioma, melanoma, malignant mesothe, transitivity squama cancer tumor colli, oral carcinoma, multiple internal secretion knurl syndromes, myeloproliferative disorder/bone marrow proliferative tumour, chronic myelocytic leukemia, myelocytic leukemia, multiple myeloma, myeloproliferative disease, the sinunasal tumour, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, nonsmall-cell lung cancer, oral carcinoma, lip pharynx cancer, the malignant fibrous histiocytoma bone, ovarian cancer, carcinoma of the pancreas, papilloma, chromaffinoma, nasal sinus and CARCINOMA OF THE NASAL CAVITY, parathyroidoma, penile cancer, the pharynx cancer, pheochromocytoma, the pineal gland parenchymal tumor, pituitary tumor, plasma cell tumor/multiple myeloma, the pleura pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, the rectum cancer, the nephrocyte cancer, renal plevis and carcinoma of ureter, transitional cell carcinoma, respiratory cancer, retinoblastoma, rhabdosarcoma, salivary tumor transformation, skin carcinoma, small cell lung cancer, intestinal tumor, soft tissue sarcoma, squama cancer tumor colli, original neuroectodermal tumors, t cell lymphoma, carcinoma of testis, laryngocarcinoma, thymoma and thymic carcinoma, thyroid carcinoma, trophoblastic tumor, ureter and transitional cell carcinoma of renal pelvis, urethral carcinoma, uterus carcinoma, sarcoma of uterus, carcinoma of vagina, carcinoma vulvae, in the group that this special macroglobulinemia of Walden and the nephroblastoma are formed one or more; And preferably, described communicable disease is selected from one or more in the group of being made up of infectation of bacteria, fungi infestation, parasitic infection, hepatitis B, hepatitis A and acquired immune deficiency syndrome (AIDS).
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