CN103243072B - The method of CD8 α-interleukin-22 1 fragment-CD137 mixture amplification activated lymphocyte - Google Patents
The method of CD8 α-interleukin-22 1 fragment-CD137 mixture amplification activated lymphocyte Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The present invention relates to a kind of field of immunology, specifically refer to use CD8 α-interleukin-22 1 fragment-CD137 mixture to become Tumor-infiltrating lymphocytes (LAK) cell through effect amplification, activation natural killer cell (NK).Advantage of the present invention is: by CD8 α-interleukin-22 1 fragment-CD137 mixture compared with existing similar mixture, and the ability of amplification and activated lymphocyte is stronger, and efficiency is higher.The present invention has broad prospects in immunotherapy.
Description
Technical field
The present invention relates to a kind of field of immunology, specifically refer to that using the K562 cell of CD8 α-interleukin-22 1 fragment-CD137 mixture transfection and lower concentration interleukin-22 acting in conjunction amplification to activate natural killer cell (NK) becomes Tumor-infiltrating lymphocytes (LAK) cell.
Background technology
First nineteen eighty-two Grimm etc. report in peripheral blood lymphocyte and add interleukin-22 vitro culture 4-6 days, and can induce a kind of non-specific killer cell, this kind of cell can kill and wound multiple to the insensitive tumour cell of NK.This kind of cell is called as Tumor-infiltrating lymphocytes (LAK).In November, 1984, Rosenberg study group was under U.S. food and drug inspection office (FDA) approval, the tumour patients such as first Application IL-2 and the routine renal cell carcinoma of LAK Synergistic treatment 25, melanoma, lung cancer, colorectal carcinoma.Wherein 11 routine tumor regression are more than 50%, and 1 routine melanoma disappears completely.Within 1988, this study group summarizes the routine tumour patient of IL-2 and LAK cell Synergistic treatment 222, wherein 16 routine patient tumors metastasis disappear completely, 26 routine patient tumors disappear more than 50%, and the curative effect of this therapy to metastatic renal cell cancer, melanoma, colorectal carcinoma and non_hodgkin lymphoma patient is more remarkable.Research subsequently shows that LAK cell is to mammary cancer, bladder cancer, and liver cancer and acute leukemia carry out treating also have obvious curative effects.Current LAK cell therapy is mainly used to use with chemotherapy, chemotherapy combined radiotherapy in the U.S..
LAK cell quantity is directly related with curative effect, and in treatment, Problems existing is that a large amount of LAK cell of acquisition is very difficult.The multiple limited (100 times) that the method that the high dosage interleukin-22 of usual employing activates growth can increase, and need from patient body, gather the dosage that a large amount of peripheral bloods (500 milliliter) tentatively can reach treatment, this is a very large physiological load to patient.The interleukin-22 somewhat expensive of high dosage is again very large economical load to patient.Current research show interleukin-22 activate growth LAK cell multiple limited increase with interleukin-22 after Cell Telomerase Activity reduce relevant.It also may be the major reason that high dosage interleukin-22 activates that the LAK cell grown inputs limited use after in patient body that telomerase activation reduces.The invention solves this problem, the invention provides a kind of method that can increase and activate NK in vitro.Interleukin-22 needed for the method only has 1/10th of usual employing method, and doubly, the LAK Cell Telomerase Activity after amplification is five times that usually adopt method to the efficiency height 10-100 of amplification.Significantly strengthen than the cell purity increased by the method for usual employing and activity with the LAK cell of the method amplification, quantitatively can reaching the needs of clinical treatment, tumour, virus and bacterium can be resisted with helping patient.
CN201110075736.1. disclose a kind of method adopting CD8 α-interleukin-22 1-CD137 mixture amplification activated lymphocyte, but its amplification and activation effect need to improve.And the CD8 α adopted in this invention and interleukin-22 1 fragment longer, easily there is the problems such as protein denaturation inactivation, immunostimulating are higher in it, and production efficiency is lower in actual applications when extensive Expression product.
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 uses 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 CD137 in described CD8 α-interleukin-22 1-CD137 mixture comprise the complete amino acid sequence of SEQIDNO:1, SEQIDNO:1 and CD137 respectively.
In the method for amplification activated lymphocyte of the present invention, can to add twice or more time 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 immediately expressed by host cell.
In the method for amplification activated lymphocyte of the present invention, described lymphocyte can be purifying or unpurified, preferably, all white corpuscles in the blood that described lymphocyte is peripheral blood lymphocyte, the NK cell of purifying, the NK cell of purifying and the combination of peripheral blood lymphocyte, the NK cell of purifying or peripheral blood lymphocyte and other lymphocytic combination or interleukin-22 and/or interleukin-22 1 and/or CD137 mixture act on after being injected into human vas, preferably described purifying refers to that 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, containing 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 K562 cell; Preferably, when described CD8 α-interleukin-22 1 fragment-CD137 mixture is expressed immediately by K562 cell, described K562 cell and lymphocytic usage ratio are K562 cell: lymphocyte=1 ~ 10:1, more preferably, described K562 cell and lymphocytic usage ratio are 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, to add twice or more time described interleukin-22.
The method of amplification activated lymphocyte of the present invention can comprise:
(1) containing in described lymphocytic nutrient solution, the K562 cell through irradiation of expressing described CD8 α-interleukin-22 1 fragment-CD137 mixture and interleukin-22 co-cultivation 7 days is added;
(2) by the medium centrifugal after cultivation, cell precipitation is obtained, and with resuspended with the nutrient solution of step (1) moderate; With
(3) add described K562 cell, cultivate 7 days;
Wherein, preferably, described nutrient solution is: 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) Nutrientmixtures add 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 increased by described method can be dissolved in normal saline solution, is preferably used for intravenous drip.
As preferably, the cell that the interleukin-22 described in aforesaid method, interleukin-22 1 fragment are separated from human body with CD137.
As preferably, all white corpuscles in the blood that the lymphocyte of the amplification described in aforesaid method is peripheral blood lymphocyte, the NK cell of purifying, the NK cell of purifying and the combination of peripheral blood lymphocyte, the NK cell of purifying or peripheral blood lymphocyte and other lymphocytic combination or interleukin-22 and/or interleukin-22 1 fragment and/or CD137 mixture act on after being injected into human vas;
Wherein purifying refers to more than 50% of NK cell total number of representatives.
As preferably, the nutrient solution described in 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) Nutrientmixtures add 10% human serum or 10% calf serum.
As preferably, the dosage of the cross-film interleukin-22 1 fragment-CD137 mixture described in aforesaid method is at 50pM ~ 1nM; Wherein K562 cell and lymphocytic usage ratio the following is, K562 cell: lymphocyte=1 ~ 10: 1; As better selection, in described K562 cell, add cross-film interleukin-22 1 fragment-CD137 mixture; K562 cell: lymphocyte=1 ~ 4: 1.
As preferably, namely host K562 cell described in aforesaid method obtains the cross-film interleukin-22 1 fragment-CD137 mixture for amplifying lymphocyte after strong acid, highly basic or high dosage irradiation, strong acid described in the present invention, highly basic are that the those skilled in the art such as the vitriol oil, hydrochloric acid, nitric acid think acid enough strong acid, and the irradiation dose of high dosage irradiation also known by the those of ordinary skill in the industry, generally refer to above below the 1000Gy of irradiation 100Gy.
As preferably, the LAK cell of the cross-film interleukin-22 1 fragment-CD137 mixture amplification described in aforesaid method, by being dissolved in normal saline solution, is used further to intravenous drip.
The K562 cell of CD8 α-interleukin-22 1 fragment-CD137 transfection increases to lymphocyte and NK and activates and has important effect.Amplification and the LAK cell produced after activating have important medical functions.The LAK cell of the available sufficient amount of method that the present invention utilizes and quality is the cell of desirable identification and destroyed tumor cell and pathogenic infection, strengthens the immunoreactive method of patient.LAK cell can identify the cell with destroyed tumor cell and pathogenic infection, comprising virus infection, as hepatitis B virus, hepatitis A virus (HAV) and virus of AIDS.The type of tumour 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/rhabdoid tumor, middle Chinese catalpa neural system knurl, rodent cancer-skin carcinoma (non-black melanoma), 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, embryonal tumors, central nervous system knurl, carcinoma of endometrium, ependymoma, the esophageal carcinoma, olfactory neuroblastoma, Ewing sarcoma family tumor, extracranial germ cell knurl, Extragonadal germ cell tumor, Tumors of Extra-hepatic Bile Duct, cancer eye, Bone fibrous 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, incidence cancer, cardiac tumor, liver cancer, histiocytosis, Langerhans cell, Hodgkin lymphoma, hypopharyngeal cancer, 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, metastatic squamous cell carcinoma tumor colli, oral carcinoma, Multiple Endocrine 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, sinunasal tumour, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, nonsmall-cell lung cancer, oral carcinoma, lip pharynx cancer, osteosarcoma, malignant fibrous histiocytoma bone, ovarian cancer carcinoma of the pancreas, papilloma, chromaffinoma, nasal sinus and CARCINOMA OF THE NASAL CAVITY, parathyroidoma, penile cancer, pharynx cancer, pheochromocytoma, pineal gland parenchymal tumor, pituitary tumor, plasma cell tumor/multiple myeloma, pleuropulinonary 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, primitive neuroectodermal tumor, 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 in the present invention are whole polypeptide of this protein, wherein also comprise the polypeptide moiety possessing function in these protein.Peripheral blood lymphocyte in the present invention comes from peripheral blood, Cord blood and marrow.Peripheral blood lymphocyte in the present invention can be NK cell; 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 all leukocytic summations in the blood acted on after interleukin-22 and/or interleukin-22 1 fragment and/or CD137 mixture are injected into human vas.
The invention provides the scheme that can be used for making medicine, multiple use described below the medicine made according to this scheme can have.This scheme comprises interleukin-22, interleukin-22 1 fragment and CD137.The LAK cell utilizing this programme to increase is suitable for and the various sights needing to strengthen patient's immunizing power.Such as, amplification NK cells for therapeutic administration tumour is effective especially, as acute myelocytic leukemia, chronic lymphatic knurl leukemia, tumor of prostate, malignant melanoma and renal cell carcinoma, but is not limited only to above-mentioned tumour.Such as, the interior infection of LAK cells for therapeutic administration bacterium, fungi or parasite of increasing is effective especially.The LAK cells for therapeutic administration virus infection of amplification is effective especially, is not limited only to above-mentioned virus infection as hepatitis B virus, hepatitis A virus (HAV) and virus of AIDS.The LAK cell of amplification can significantly improve the action effect of antigen.
Except as otherwise noted, all in the present invention technology and the implication of scientific terms are the implications that the personage with common skill in the industry understands usually.Being defined in all molecular biology books of Essential Terms can be found, such as, and BenjaminLewin, GenesVIII, OxfordUniversityPress, 2004 (ISBN0-13-145140-5);
In order to ensure for a long time, Restruction interleukin-22 1 fragment of high yield and CD137, present invention employs protein stabilized expression system.First the expression vector of CD8 α-interleukin-22 1 fragment-CD137 is built, simultaneously containing viral promotors and selectable marker gene in this carrier, as antibiotics resistance gene.CD8 α is a kind of membranin of expressing on cytolemma, CD8 α gene makes interleukin-22 1 fragment and CD137 express on cytolemma after connecting interleukin-22 1 fragment and CD137 gene simultaneously, becomes transmembrane protein (being referred to as cross-film interleukin-22 1 fragment-CD137 mixture below).Then transcribe K562 clone with this expression vector, activate promotor by appropriate method, after cultivating K562 cell for some time, namely can obtain CD8 α-interleukin-22 1 fragment-CD137 mixture of high level expression on cytolemma.Build the expression vector of CD8 α-interleukin-22 1 fragment-CD137, the method for transcribing clone is the method that the personage with common skill in the industry understands usually.Wherein build the method for the expression vector of CD8 α-interleukin-22 1 fragment-CD137 with reference to document below: Wu.et.al., JMolCellBiol (2010) 2 (4): 217-222; Building protein stabilized expression system is the technology that the personage with common skill in the industry adopts usually.Such as, clone transcrypted the expression vector of stably express CD8 α-interleukin-22 1 fragment-CD137, containing viral promotors and selectable marker gene in this carrier, as antibiotics resistance gene.CD8 α is a kind of membranin of expressing on cytolemma, CD8 α gene makes interleukin-22 1 fragment and CD137 express on cytolemma after connecting interleukin-22 1 fragment and CD137 gene simultaneously, becomes transmembrane protein (being referred to as cross-film interleukin-22 1 fragment-CD137 mixture below).After heterogenous expression carrier enters host cell, activate promotor by appropriate method, after culturing cell for some time, namely can obtain the polypeptide of high level expression on cytolemma.These methods are methods that the personage with common skill in the industry understands usually.
On cytolemma, the cell of high level expression cross-film interleukin-22 1 fragment-CD137 mixture can be collected by ultracentrifugal method.Method process listed below cell can be included but not limited to by these technology: strong acid, highly basic and irradiation.Cross-film interleukin-22 1 fragment-CD137 mixture on cytolemma directly and peripheral blood lymphocyte or NK cell co-culture, can activate amplification LAK cell, also can after purifies and separates with peripheral blood lymphocyte co-cultivation, activate amplification LAK cell.
On cytolemma high level expression cross-film interleukin-22 1 fragment-CD137 mixture can by the industry have the personage of common skill the technology be familiar with carry out purification and separation.Method listed below these technology include but not limited to: ammonium sulfate or alcohol settling, acid extraction, negatively charged ion or cation-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, chromatography and lectin.If necessary, the scheme of protein renaturation can be used to the protein polypeptide helping purifying expression.If necessary, high performance liquid chromatography (HPLC) can be made last purification step and be further purified protein.The cross-film interleukin-22 1 fragment-CD137 mixture of the host cell inner expression in the present invention is understood some and is secreted in cell culture supernatant.The personage with common skill in the industry can verify according to common Measurement for Biochemistry.The cross-film interleukin-22 1 fragment-CD137 mixture be secreted in cell culture supernatant also can by aforesaid method purification and separation.
Cross-film interleukin-22 1 fragment-CD137 mixture in the present invention can be used to amplification LAK cell.The LAK cell portable patient of amplification in vitro.Such as, the high dosage interleukin-22 amplification that the method ratio of amplification activation LAK cell is usually used successively in order of cross-film interleukin-22 1 fragment-CD137 mixture and low dosage interleukin-22 activates more effective more than 120 times of LAK cell.LAK Cell Telomerase Activity after cross-film interleukin-22 1 fragment-CD137 mixture and low dosage interleukin-22 acting in conjunction amplification adopts the amplification of high dosage interleukin-22 to activate more than 5.2 times of LAK cell.Telomerase activation detects and adopts telomeric repeatamplification protocol (TRAP) (to refer to KimNW, PiatyszekMA, prowseKR, etal.Science, 1994; 266:2011).The LAK cytoactive that the LAK cell ratio high dosage interleukin-22 amplification that the amplification of cross-film interleukin-22 1 fragment-CD137 mixture activates activates is high more than 4.7 times.Cross-film interleukin-22 1 fragment-CD137 mixture increased after three weeks, and the purity of LAK cell can reach 95%, and the quantity of cell can reach 1.0 × 10
10above, the demand of clinical treatment is met.Visible amplification method indices of the present invention is all better than method disclosed in CN201110075736.1.,
The LAK cell of cross-film interleukin-22 1 fragment-CD137 mixture amplification, can be used for carrying out autotransplantation, also can carry out heteroplastic transplantation.Under the prerequisite matched by the histocompatibility antigen of contributor (grownup or minor) and the histocompatibility antigen of blood antigen and contributor (grownup or minor) and blood antigen, the cell that contributor is activated and increases can be injected in the blood vessel by contributor by the mode of intravenous drip.In some applications, clone (such as, NK clone, NKT clone) also can be activated and amplification by cross-film interleukin-22 1 fragment-CD137 mixture.It is also protection scope of the present invention that the clone of amplification is used for clinical treatment object.
Lymphocyte or lymphocyte precursor cell can from deriving from any tissue including large amount lymphocyte and precursor cell.Such as, peripheral blood (comprising Cord blood), marrow, spleen and lymphoglandula.Lymphocyte or lymphocyte precursor cell are taken from peripheral blood or marrow usually.In other application (such as, experimental study), spleen and/or lymphoglandula are also suitable sources.
Such as, lymphocyte can by taking out peripheral blood and/or marrow obtains.Lymphocyte can obtain NK cell and NKT cell further across purifying.Purified NK cells, the step of NKT cell and technique be in the industry have the personage of common skill be familiar with.Lymphocyte can carry out the erythrocyte in separating blood or 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, such as antibodies paramagnetic particle method.Purifying lymphocyte does not require that the lymphocyte of purifying is definitely pure, and refers to that the cell of the purifying ratio more shared in derived tissues than it is higher.Under normal circumstances, the lymphocyte of purifying at least total number of representatives 50%, or 60%, or higher.The NK cell of purifying and NKT cell suspension are in a suitable physiological buffered solution, then suspension growth in but be not limited in following nutrient solution, Eagle ' s nutrient solution adds 10% human serum, RPMI1640 adds 10% human serum, F-10 (Ham ' s) Nutrientmixtures add 10% human serum.Human serum also can replace with 10% foetal calf serum, but the expanding effect of human serum to LAK cell is better.
Lymphocyte and/or the lymphocyte precursor cell suspension of lymphocyte and/or purifying are grown in cell culture fluid, add cross-film interleukin-22 1 fragment-CD137 mixture and the cultivation of low dosage interleukin-22, add weekly new cross-film interleukin-22 1 fragment-CD137 mixture and the cultivation of low dosage interleukin-22.Under normal circumstances in order to reduce costs and avoid infection, the present invention adopts minimum dosage to carry out amplifying lymphocyte.The dosage of interleukin-22 is between 50 units/milliliter to 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 the process carrying out high dosage, activate concentration and be about 500 units per ml or 1000 units per ml.In addition, cross-film interleukin-22 1 fragment-CD137 mixture is injected directly into human body, and when being used for increasing LAK cell in vivo, dosage used rubs between 0.5 sodium rubs at 0.5 skin.The employing dosage of cross-film interleukin-22 1 fragment-CD137 mixture is determined according to the expression dosage of cross-film interleukin-22 1 fragment-CD137 mixture in K562.In some cases, according to K562: lymphocyte=1: 1,2: 1,3: Isosorbide-5-Nitrae: 1 or more adds cross-film interleukin-22 1 fragment-CD137 mixture.In some cases, the process carrying out high dosage is necessary, according to K562: lymphocyte=10: 1 or more adds cross-film interleukin-22 1 fragment-CD137 mixture.The LAK cell increased in vitro can be transplanted to lymphocytic contributor itself or by contributor, to strengthen born or antigen specific immune reaction.Under normal circumstances, 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 cross-film interleukin-22 1 fragment-CD137 mixture and low dosage interleukin-22, and its amplification efficiency is higher, and the LAK activity obtained is higher.
Accompanying drawing explanation
Fig. 1 cross-film interleukin-22 1 fragment-CD137 mixture schematic diagram, the structure showing CD8 α-interleukin-22 1 fragment-CD137 mixture and the order built successively;
Fig. 2 A.LAK cell expansion ex vivo linear graph display lymphocyte increases in cross-film interleukin-22 1 fragment-CD137 mixture, cross-film interleukin-22 1-CD137 mixture and low dosage interleukin-22 acting in conjunction lower linear
B. peripheral blood lymphocyte cell expansion ex vivo linear graph display lymphocyte increases in cross-film interleukin-22 1 fragment-CD137 mixture, cross-film interleukin-22 1-CD137 mixture and low dosage interleukin-22 acting in conjunction lower linear.The cell of high dosage interleukin-22 process is as negative control.
Embodiment
Below enforcement of the present invention is illustrated:
Embodiment 1
Cross-film interleukin-22 1 fragment-CD137 mixture amplification in vitro LAK cell.
The NK cell cultures of healthy donation people, in RPMI1640, is aided with the human serum of 10%.Cultivation is added after the K562 cell (irradiation, 100Gy) of K562 cell (contrast) and the expression cross-film interleukin-22 1 fragment-CD137 mixture of expressing cross-film interleukin-22 1-CD137 mixture and low dosage interleukin-22 respectively 7 days in nutrient solution.Centrifugal after 7 days, resuspended with the nutrient solution of equivalent, add the K562 cell through irradiation, then cultivate 7 days (Fig. 1).High dosage interleukin-22 (200 units per ml) also as a control group.As shown in Figure 2 A, LAK cell is under the acting in conjunction of cross-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 inserting thymidine.After thymidine inserts, cell continues cultivation 12 hours, and then starts the change of measuring cell quantity.At K562: lymphocyte=1: under the cross-film interleukin-22 1 fragment-CD137 mixture effect of 1 concentration, LAK cell quantity entered logarithmic phase 7 days time, 14 days time about increase 1000 times.Under the condition adding cross-film interleukin-22 1 fragment-CD137 mixture, minimum can beginning under the interleukin-22 effect of 50 units per ml increases LAK cell.And the growth of cellular control unit LAK cell under high dosage interleukin-22 independent role is significantly lower than the vitro growth rates under cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22.
Embodiment 2
The peripheral blood lymphocyte that the amplification of cross-film interleukin-22 1 fragment-CD137 mixture is not purified
Cross-film interleukin-22 1 fragment-CD137 mixture not only can the NK cell of amplification purification, can also increase without the lymphocyte of purifying.The PBM of the healthy people of donation cultivates in RPMI1640, is aided with the human serum of 10%.Co-cultivation is added after the K562 cell (irradiation, 100Gy) of K562 cell (contrast) and the expression cross-film interleukin-22 1 fragment-CD137 mixture of expressing cross-film interleukin-22 1-CD137 mixture and low dosage interleukin-22 respectively 7 days in nutrient solution.Centrifugal after 7 days, resuspended with the nutrient solution of equivalent, add the K562 cell through 100Gy irradiation, then cultivate 7 days (Fig. 1).High dosage interleukin-22 (200 units per ml) also as a control group.As shown in Figure 2 B, LAK cell is under the acting in conjunction of cross-film interleukin-22 1 fragment-CD137 mixture and low dosage interleukin-22, and quantity significantly increases.LAK cell quantity entered logarithmic phase 7 days time, 14 days time about increase 1200 times.Under the condition adding cross-film interleukin-22 1 fragment-CD137 mixture, minimum can beginning under the interleukin-22 effect of 50 units per ml increases LAK cell.And the growth of cellular control unit LAK cell under high dosage interleukin-22 independent role is significantly lower than the vitro growth rates under cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22.
Under cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22, when the 12nd day, the lymphocyte of 93% becomes LAK cell.Control group is under high dosage interleukin-22 (200 units per ml) effect, and peripheral blood lymphocyte only has 14% cell to become LAK cell (CD56+, CD16+, CD56+/CD16+).
Embodiment 3
The activity of LAK cellular telomerase and STAT3 phosphorylation detect
With the cell after the method fragmentation amplification of liquid nitrogen multigelation, 10000g is centrifugal, gets supernatant, detects the activity of the rear LAK cellular telomerase of amplification by TRAP method.Comprise 20 millis in 50 microlitre TRAP systems to rub/rise Tris-HCl (pH8.3), 1.5 millis rub/rise MgCl, and 63 millis rub/liter, KCl, 0.005%Tween-20,1 milli rubs/rises EDTA, and 50 millis rub/rise dNTP, 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 DEG C 10 minutes, after 94 DEG C of 3 second, add Taq enzyme, 94 DEG C of 30 second, 50 DEG C of 30 second, 72 DEG C 1.5 minutes, 27 circulations of increasing, get 25 microlitre products and carry out 15%PAGE gel electrophoresis.After PAGE gel electrophoresis, on X-mating plate, radioautograph 8 is little of more than two days.Compared with before amplification, the activity of the remarkable activated end granzyme of control group high dosage interleukin-22.And the activity of the LAK cellular telomerase increased under cross-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 LAK cellular telomerase under cross-film interleukin-22 1-CD137 mixture and low dosage interleukin-22 acting in conjunction (exceeding 8.5%) and control group high dosage interleukin-22 independent role.
Embodiment 4
The restraining effect of LAK cells against tumor
The splitting action of the LAK cells against tumor cells increased under cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22.
Research in the past shows the anti-tumor capacity that can strengthen mouse to the injected in mice interleukin-22 of suffering from tumour, delays the life-span of mouse.The method of the LAK cell that the present embodiment increases under adopting cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 and tumour cell co-cultivation, studies and carries out antineoplastic feasibility with the LAK cell of amplification.The method of the LAK cell infusion Mice Body internal therapy mouse interior tumor that the present embodiment increases under adopting cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22, studies the effect of the LAK cell anti-tumor of amplification.
After separating in the lymphocyte of healthy donor, the LAK Growth of Cells increased under adding cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 14 days.K562, PC3, A549 and HepG2 are used as the target cell of cracking.K562 is B cell tumor cell line, and this clone is blood tumor cell system.PC3 is prostate tumor cells system, and A549 is lung tumor cell system, and HepG2 is hepatoma cell line.PC3, A549 and HepG2 are solid tumor clone.The LAK cell of amplification and target cell in accordance with the appropriate ratio co-cultivation detect the target cell of survival after 6 hours.The LAK cell of high dosage interleukin-22 amplification as a control group.The lethal effect of LAK cell to target cell that the LAK cell increased under cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 increases than control group high dosage interleukin-22 significantly strengthens.(table 1)
The PC3 clone of external use Lentivirus system constructing Fluc reporter gene stably express.FlucLentivirus system is purchased from SystemBiosciences (U.S., California), and concrete construction process refers to product introduction.PC3-Fluc cell subcutaneous injection enters severe immune deficiency mouse (NOD/SCID) 32, and after three days, fluorimetric detector detects the growing state of tumour, random packet.LAK cell after amplification is according to 1 × 10
7cell/every mouse from tail vein injection, biweekly.Stop treatment after continuous treatment surrounding, observe result for the treatment of.The growth of LAK to tumour that high dosage interleukin-22 increases has certain restraining effect, and (average tumor size is 6mm.The LAK cell that the LAK cell increased under cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 increases than high dosage interleukin-22 is to the lethal effect of PC3 tumour cell strong (average tumor size is 5.3mm).The LAK cell of interleukin-22 amplification has certain effect to the survival rate extending mouse.And compared with the LAK cell increased with interleukin-22, the LAK increased under cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 significantly improves the curative ratio (improving 6%) to tumour, the significant prolongation survival rate of mouse (improving 12%).This LAK cell increased under pointing out cross-film interleukin-22 1 fragment-CD137 mixture and the acting in conjunction of low dosage interleukin-22 may be used for clinical oncotherapy.
Claims (14)
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, CD8 α, interleukin-22 1 fragment and CD137 in described CD8 α-interleukin-22 1 fragment-CD137 mixture are respectively the complete fragment of CD8 α, the complete amino acid sequence of SEQIDNO:1 and CD137, described CD8 α-interleukin-22 1 fragment-CD137 mixture is expressed as host cell immediately by K562 cell, and described lymphocyte is NK cell and/or not purified peripheral blood lymphocyte; Described method comprises:
(1) containing in described lymphocytic nutrient solution, the K562 cell through irradiation of expressing described CD8 α-interleukin-22 1 fragment-CD137 mixture and interleukin-22 co-cultivation 7 days is added;
(2) by the medium centrifugal after cultivation, cell precipitation is obtained, and with resuspended with the nutrient solution of step (1) moderate; With
(3) add described K562 cell, cultivate 7 days.
2. method according to claim 1, is characterized in that, to add twice or more time described CD8 α-interleukin-22 1 fragment-CD137 mixture in described method.
3. method according to claim 2, is characterized in that, the dosage of described CD8 α-interleukin-22 1-CD137 mixture is at 50pM ~ 1nM.
4. method according to claim 1, is characterized in that, described purifying refers to that NK cell is more than 50% of total cellular score.
5. method according to claim 1, is characterized in that, described CD8 α-interleukin-22 1 fragment-CD137 mixture adopts protein stabilized expression system.
6. method according to claim 5, is characterized in that, containing viral promotors and selectable marker gene in the expression vector of described CD8 α-interleukin-22 1 fragment-CD137 mixture.
7. method according to claim 1, is characterized in that, described K562 cell and the lymphocytic usage ratio of the described CD8 α-interleukin-22 1 fragment-CD137 mixture of instant expression are K562 cell: lymphocyte=1 ~ 10:1.
8. method according to claim 7, is characterized in that, described K562 cell and the lymphocytic usage ratio of the described CD8 α-interleukin-22 1 fragment-CD137 mixture of instant expression are K562 cell: lymphocyte=1 ~ 4:1.
9. method according to claim 1, is characterized in that, the lowest dose level of wherein said interleukin-22 is 50 units per ml.
10. method according to claim 9, is characterized in that, to add twice or more time described interleukin-22.
11. methods according to claim 1, is characterized in that, wherein, described nutrient solution is: 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) Nutrientmixtures add 10% human serum or 10% calf serum.
12. methods according to claim 1, is characterized in that, wherein, the dosage of described irradiation is 100Gy-1000Gy.
13. methods according to any one of claim 1-12, it is characterized in that, the lymphocyte increased by described method is dissolved in normal saline solution, for intravenous drip.
14. purposes of method in the preparation of medicine being used for the treatment of leukemia, prostate cancer, lung cancer or liver cancer according to any one of claim 1-13.
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