CN105349488A - Combined factor for inducing tumor specific T cells and method for obtaining tumor specific T cells - Google Patents
Combined factor for inducing tumor specific T cells and method for obtaining tumor specific T cells Download PDFInfo
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Abstract
The invention discloses a combined factor for inducing tumor specific T cells and a method for obtaining the tumor specific T cells. The combined factor comprises a tumor specific antigen and cell factors IL-2, IL-7 and IL-15. The method comprises the following steps: suspending peripheral blood PBMC with a fresh serum-free lymphocyte culture solution, wherein the cell density is 2*10<6>-5*10<6>/ ml; adding a stimulus of the combined factor for inducing the tumor specific T cells, conducting culturing in a 5% CO2 culturing box at 37 DEG C for 2-10 days, and separating the IFN gamma positive antigen specific T cells through an IFN gamma magnetic bead enrichment method. The method can be used for obtaining the high-purity tumor antigen specific T cells, and is simple, and low in cost. The prepared tumor antigen specific T cells can be applied to preparation of medicines or vaccines for clinical cell treatment and tumor immunization treatment.
Description
Technical field
The invention belongs to the external evoked separation and Culture field of immunocyte, relate to a kind of connector of inducing tumor-specific T cell and obtain the method for tumor specific T cells.
Background technology
The life of the malignant tumour serious harm mankind is with healthy, and its mortality ratio occupies first of various disease.Estimate according to the World Health Organization (WHO) and American Cancer Society, the whole world increases the cancer new patient of about 1,200 ten thousand every year newly, about has 8,000,000 cancer death (there are 20,000 cancer death every day).China has 1,000 ten thousand cancer patients at present, the common cancers such as liver cancer, lung cancer, mammary cancer and prostate cancer, and sickness rate obviously rises in recent years, and in rejuvenation trend, human health in serious threat.
The large conventional treatments (i.e. operative treatment, radiotherapy and chemotherapy) of tumour existing three in the past few decades in made major contribution for improving the survival rate of tumour patient, but transfer and the Problems Concerning Their Recurrence of tumour cannot be solved all the time, the survival rate of tumour patient also no longer includes in recent years and significantly improves, can say that three large conventional treatmenies have suffered from curative effect bottleneck, in the urgent need to finding the new method that can break through oncotherapy curative effect bottleneck.
Tumor specific cytotoxic T lymphocyte (CTL) i.e. cytotoxic T lymphocyte, receive antigenic information from antigen presenting cell, and through clonal expansion can specific recognition and kill and wound the effector T cell of antigen-specific target cell, CTL is the main mechanism that body removes cancerous tumor cell, in antineoplastic immune process, play Main Function.CTL by the material such as granzyme and pore-forming protein direct killing tumour cell, can not only can also pass through some cytokines of secretion as the killing tumor cell indirectly such as IFN γ and TNF α.Along with biomedicine, the development of genetic engineering technique, tumor specific cytotoxic T lymphocyte treatment comes into one's own in recent years day by day, and demonstrate good application prospect, multiple specific CTL inducing culture scheme is arisen at the historic moment.Obtain good curative effect as far back as Japan in 2000 by adoptive T lymphocyte transplantation treatment liver cancer, recurrence rate reduces, and survival rate improves, and research is published on authorities,medical magazine " lancet "; 2004, the tumor infiltrating lymphocyte (TIL, a class of specific CTL) of professor's Rosenburg research obtained good result on treatment metastasis melanin tumor.Tumor-specific CTL treatment tumor disease has safety, target, efficient feature.This is mainly determined by the φt cell receptor (TCellReceptor, TCR) of T lymphocytic cell surface specific recognition tumour antigen.But the TCR for various antigen in body varies, within 2014, the article of Rosenberg professor delivered by " science " magazine, utilize TCR immune group storehouse degree of depth sequencing technologies from the tumor-infiltrated T lymphocyte populations of transitivity bile duct epithelial cell cancer patient, screen and obtain the TCR sequence of specific for tumour antigen, the DNA of TCR according to sequent synthesis, and by genetic engineering technique transduction on autologous patient T cell surface, obtain good therapeutic action.
Along with progressively carrying out of every clinical application, increasing clinical effectiveness shows that validity that tumor-specific CTL treats is far away higher than other technology.The clinical study that national cancer institute carries out shows, can produce 50% ~ 70% reactivity at metastasis melanin tumor patient conbined usage tumor-specific CTL associating with it IL-2.But the inducing culture of tumor-specific CTL needs to consume a large amount of time, also very high to the requirement of technology, therefore significantly limit this methods for the treatment of in cancer patients's extensive utilization with it.Can increase after having scholar to find to add IL-2 in culture system in early days in animal experiment and there is the cell of cytotoxicity, but research finds that the method inducing cell lethality is strong not, inducing specific CTL inefficiency.Professor Rosenberg etc. develop TIL technical study subsequently, but the problem that til cell is difficult to obtain limits its clinical application.A kind of method effectively can inducing separation tumor-specific CTL how is selected to be current CTL technology problem demanding prompt solution.This research adopts the method for antigen peptide inducing culture associating IFN γ enrichment effectively can obtain tumor-specific CTL, for specific CTL clinical application and subsequently the clone of specific T-cells surface receptor study and establish technical foundation.
Summary of the invention
The object of the present invention is to provide a kind of method of effective inducing tumor-specific CTL.
For achieving the above object, the invention provides a kind of connector for inducing tumor-specific T cell, it is characterized in that, comprise tumour specific antigen, cytokine IL-2, IL-7 and IL-15.
According to embodiments of the invention, described tumour specific antigen is tumor antigen protein, the long peptide of tumour antigen or storehouse, or tumour antigen small peptide or storehouse.
According to embodiments of the invention, in every milliliter of T cell nutrient solution, the consumption of the consumption of tumour specific antigen to be the consumption of 1-50 μ g/ml, IL-2 be 10-6000U/ml, IL-7 is the consumption of 1-50ng/ml, IL-15 is 1-50ng/ml.
According to embodiments of the invention, in every milliliter of T cell nutrient solution, the consumption of the consumption of tumour specific antigen to be the consumption of 2 μ g/ml, IL-2 be 50U/ml, IL-7 is the consumption of 5ng/ml, IL-15 is 5ng/ml.
According to embodiments of the invention, thering is provided a kind of uses the described connector for inducing tumor-specific T cell to carry out inducing culture and the method for IFN γ enrichment acquisition tumor specific T cells, it is characterized in that, step is: be separated patient's peripheral blood PBMC, suspend by fresh serum-free lymphocyte culture fluid, cell density is 2x10
6-5x10
6/ ml; Add the described connector for inducing tumor-specific CTL to stimulate, at 37 DEG C, 5%CO
2cultivate 2-10 days in incubator, the T cells with antigenic specificity being separated the IFN γ positive by IFN γ enrichment with magnetic bead method is tumor specific T cells.
According to embodiments of the invention, the tumor specific T cells that the method that described inducing culture IFN γ enrichment obtain tumor specific T cells prepares is for the preparation of clinical cytology treatment and the medicine of immunotherapy of tumors or the purposes of vaccine.
According to embodiments of the invention, described tumour specific antigen can be synthesis or commercial tumor antigen protein, the long peptide of tumour antigen or peptide storehouse, or tumour antigen small peptide or storehouse.
Described serum-free lymphocyte culture fluid can be AIM-V or other serum free medium of LifeTechnology company.
The T cells with antigenic specificity that the method for the invention prepares is for φt cell receptor clone research and the purposes preparing clinical cytology treatment and immunotherapy of tumors medicine or vaccine.
Adopt the tumor specific T cells prepared by connector of the present invention and method for culturing and separating (tumor-specific CTL cell) to have higher purity, high density inflammatory factor IFN γ can be secreted, there is the TCR spectrum that polymorphism is comparatively single-minded.Applied research fields in clinical tumor immunotherapy plays a significant role by the present invention.
The inducing culture of tumor-specific CTL cell needs a large amount of time to carry out antigen to take turns stimulation more, in the cell mass of induction, CTL content is extremely low, screening and cloning CTL cell is very high to the requirement of technology, and in order to solve the above prior-art problems, technical scheme provided by the invention is:
The invention provides a kind of connector for inducing tumor-specific CTL cell, said composition is adopted to stimulate peripheral blood PBMC can effectively maintain PBMC activity, the propagation of further promotion tumor-specific CTL cell and activity, and then more effectively can secrete antineoplastic immune inflammatory factor IFN γ.
IL-2 of the present invention has the effect promoting its functionally active of multiple T cell propagation maintenance, and IL-7 and IL-15 is the important cytokine maintaining body internal specific CTL cytoactive.IL-7 participates in the lymphocytic maturation of thymus gland T; In human peripheral environment, it reduces the spontaneous apoptosis of CTL by regulation and control anti-apoptotic molecule Bcl-2, promotes the survival of Memory CTL.IL-15 can promote the propagation of Memory CTL cell and strengthen their effector function.The present inventor through a large amount of performing creative labour, surprised discovery, when the consumption of IL-2 is 10-6000U/ml, preferred 50U/ml; The consumption of IL-7 and IL-15 is 1-50ng/ml, and during preferred 5ng/ml, the PBMC of institute's inducing culture contains the specific CTL of higher degree, and energy high-level secretory IFN γ, the degree of non-specific T cell activation is low simultaneously.The application of a large amount of IL-2 will activate non-specific T-cell simultaneously, thus reduces the purity of specific CTL in culture systems.
The consumption of described tumour specific antigen is 1-50 μ g/ml, preferably 2 μ g/ml.When the consumption of tumour specific antigen is 1 μ g/ml, namely research display can successfully induce tumor-specific CTL in the patient bodies such as liver cancer, lung cancer, nasopharyngeal carcinoma, cervical cancer, intestinal cancer.When its consumption is 2 μ g/ml, specific for tumour antigen T cell cultivation effect is better.Unnecessary Financial cost will be greatly improved more than the antigen consumption of 50 μ g/ml.The consumption of IL-2 is 10-6000U/ml, preferred 50U/ml.IL-2 is not obvious to some patients's specific for tumour antigen T cell proliferation function for research display 10U/ml low dosage, and when its consumption is 50U/ml, specific for tumour antigen T cell cultivation effect is better.The propagation of non-T cells with antigenic specificity will be significantly improved more than the IL-2 of 6000U/ml, T cells with antigenic specificity content is significantly reduced, increase the difficulty of specific CTL separation and purification.The consumption of IL-7, IL-15 is 1-50ng/ml, preferred 5ng/ml.Affect the propagation of specific CTL lower than 1ng/ml, increase cost more than 50ng/ml dosage; When its consumption is 5ng/ml, specific for tumour antigen T cell cultivation effect is best.
According to the further feature of composition of the present invention, described lymphocyte culture fluid is serum-free cell culture medium, such as, the serum-free cell culture medium of LifeTechnologies company of the U.S. (
serum-freeMedium).Other is usually used in lymphocytic dulbecco minimum essential medium Dulbecco and also can be used for the present invention.
The nutrient solution of inducing tumor-specific CTL of the present invention, comprises according to the composition for inducing tumor-specific CTL of the present invention and serum-free lymphocyte culture fluid.
Another object of the present invention is to provide a kind of inducing tumor-specific T cell method.
Of the present invention for inducing the method being separated tumor-specific CTL, comprise the following steps: be separated patient's peripheral blood PBMC, suspend by fresh serum-free lymphocyte culture fluid, cell density is 2x10
6-5x10
6/ ml; Add tumour specific antigen (1-50 μ g/ml; Preferably, 2 μ g/ml) and connector (10-6000U/mlIL-2,1-50ng/mlIL-7,1-50ng/mlIL-15; Preferably, 50U/mlIL-2,5ng/mlIL-7,5ng/mlIL-15), at 37 DEG C, 5%CO
2in incubator after inducing culture 2-10 days, be separated the CTL cell of the IFN γ positive by IFN γ enrichment with magnetic bead method.
According to the further feature of method of the present invention, described serum-free lymphocyte culture fluid is the serum-free medium of LifeTechnologies company.
According to the further feature of method of the present invention, the method for described separation tumor-specific CTL is the CTL cell being separated the IFN γ positive by IFN γ enrichment with magnetic bead method.
Another object of the present invention is to provide a kind of high purity tumor-specific CTL.
High purity tumor-specific CTL of the present invention adopts to be separated according to method inducing culture of the present invention the tumor-specific CTL obtained.Experiment of the present invention confirms, in T lymphocyte culture fluid, especially in serum free culture system, by antigen combined IL-2, IL-7 and IL-15 Co stituation, the tumor-specific CTL purity obtained is separated subsequently high with enrichment with magnetic bead, can effectively secrete IFN γ, there is more single-minded TCR polymorphism.This kind of tumor-specific CTL is highly suitable for functional study subsequently and enlarged culturing, and then more effectively induces antitumor reaction and the antineoplastic specificity ctl response of Thl.
Accompanying drawing explanation
Fig. 1 is TCR polymorphism two-dimensional distribution before patient 2 induced concentration;
Fig. 2 is TCR polymorphism two-dimensional distribution after patient 2 induced concentration;
Fig. 3 is TCR polymorphism two-dimensional distribution before patient 2 induced concentration;
Fig. 4 is TCR polymorphism two-dimensional distribution after patient 2 induced concentration;
Fig. 5 is specificity TCR scale map before and after 2 routine patient's induced concentration;
Fig. 6 is CD8 and the two positive cell proportion figure of specificity ten aggressiveness before and after Patients with Cervical Cancer induced concentration.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Be exemplary below by the embodiment be described with reference to the drawings, be intended to for explaining the present invention, and can not limitation of the present invention be interpreted as.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1: tumor specific T cells induced concentration separate instance
1, tumor specific T cells induced concentration is separated
1.1B phase liver cancer patient 2, knows the inside story and informs and sign Informed Consent Form, extract peripheric venous blood 50ml respectively with heparin sodium anticoagulant tube.After lymphocyte separation medium (FreseniusKabiNorgeAS, LymphoprepTM) is separated PBMC, suspend with fresh serum-free medium (AIM-V of LifeTechnology company), cell density is at 2x10
6/ ml.In every milliliter of serum-free medium, add antigen peptide from human hepatocellular carcinoma 2 μ g/ml; IL-250U/ml; IL-7 and IL-15 is respectively 5ng/ml.37 DEG C, 5%CO
2cultivate in incubator.Wherein the cAg epi-position in hepatocellular carcinoma antigen storehouse is FLPSDFFPS (SEQIDNO:1), and Chu Tai bio tech ltd, Shanghai synthesizes.
1.2 cultivated after 72 hours, carried out IFN γ enrichment sorting (MiltenyiBiotec130-054-201).
1.2.1 collect the cell of induction according to the requirement of test kit, 4 DEG C of centrifugal 10min of 300g, abandon supernatant; Break up cell, add the buffer that 10ml is ice-cold, 4 DEG C of centrifugal 10min of 300g, abandon supernatant.
1.2.2 every 1X10
7cell adds the ice-cold AIM-V of 80 μ l and 20 μ lIFN γ capture agents, and mixing, hatches 5 minutes on ice.Add the substratum of 37 DEG C of preheatings, every 1X10
7cell adds 10ml, is placed in 37 DEG C and hatches 45 minutes, within every 5 minutes, turns and shakes once.Cell is placed on ice, and 4 DEG C of centrifugal 10min of 300g, abandon supernatant.Wash once with ice-cold buffer.
1.2.3 every 1X10
7cell adds the IFN γ detection antibody that the ice-cold buffer of 80 μ l and 20 μ lPE marks, and mixing, hatches 10 minutes on ice.The buffer ice-cold with 10ml washes once.
1.2.4 every 1X10
7cell adds the ice-cold buffer of 80 μ l and the anti-PE magnetic bead of 20 μ l, mixing, and refrigeration hatches 15 minutes.The buffer ice-cold with 10ml washes once.Every 1X10
7the buffer that cell 500ul is ice-cold is resuspended.
1.2.5 cross sorting post.The ice-cold buffer rinse of 500 μ l one time of MS type sorting post, adds cell suspension, crosses after post until cell suspension, wash post 3 times with the buffer that 500 μ l are ice-cold.Take off first sorting post to be placed in above second MS sorting post, add the buffer that 1ml is ice-cold, release cell suspension to second sorting post with piston, again wash post 3 times.Take off second sorting post, add 500 μ lAIM-V substratum, release cell suspension with piston.
2, isolated cell TCR polymorphic detection
By the cell harvesting in above-mentioned 1.2.5 step in 1.5mlEpp pipe, 500mlPBS washes one time, centrifugal 5 minutes of 300g, visible cell group, and careful suction abandons supernatant.Break up cell, add 350 μ lRLT reagent (Qiangen company), whirlpool concussion 1-2 minute.Centrifugal 5 minutes of 800g, may be seen indistinctly cell debris, carefully sucts clearly in new Ep pipe, be stored in-80 DEG C.
Sample cold chain transportation carries out TCR polymorphism detection assay to the company of checking order (iRepertoire, the U.S.) and obtains Fig. 1-4.Wherein Fig. 1 is the TCR polymorphism two-dimensional distribution before patient 1 peripheral blood PBMC induced concentration, X-coordinate is wherein hTRBV2 from left to right successively, hTRBV3-1, hTRBV4-1, hTRBV4-2, hTRBV4-3, hTRBV5-1, hTRBV5-4, hTRBV5-5, hTRBV5-6, hTRBV5-8, hTRBV6-1, hTRBV6-2, hTRBV6-3, hTRBV6-4, hTRBV6-5, hTRBV6-6, hTRBV6-8, hTRBV6-9, hTRBV7-2, hTRBV7-3, hTRBV7-4, hTRBV7-6, hTRBV7-7, hTRBV7-8, hTRBV7-9, hTRBV9, hTRBV10-1, hTRBV10-2, hTRBV10-3, hTRBV11-1, hTRBV11-2, hTRBV11-3, hTRBV12-3, hTRBV12-4, hTRBV12-5, hTRBV13, hTRBV14, hTRBV15, hTRBV16, hTRBV18, hTRBV19, hTRBV20-1, hTRBV24-1, hTRBV25-1, hTRBV27, hTRBV28, hTRBV29-1, hTRBV30, ordinate zou is hTRBJ2-7, hTRBJ2-6, hTRBJ2-5, hTRBJ2-4, hTRBJ2-3, hTRBJ2-2, hTRBJ2-1, hTRBJ1-6, hTRBJ1-5, hTRBJ1-4, hTRBJ1-3, hTRBJ1-2, hTRBJ1-1 from below to up successively.Fig. 2 is that patient 1 peripheral blood PBMC is by TCR polymorphism two-dimensional distribution after method enrichment of the present invention; X-coordinate wherein and the same Fig. 1 of ordinate zou.
TCR hypotype polymorphism comparatively horn of plenty before can finding out enrichment from Fig. 1 and Fig. 2, in multiple hypotype, after enrichment, TCR hypotype is mainly hTRBV24-1, hTRBJ1-5, accounts for 31.1% of total TCR.
Fig. 3 is the TCR polymorphism two-dimensional distribution before patient 2 peripheral blood PBMC induced concentration; Fig. 4 is that patient 2 peripheral blood PBMC is by TCR polymorphism two-dimensional distribution after method enrichment of the present invention; Can find out, patient 1 peripheral blood PBMC is more single-minded by TCR polymorphism after method enrichment of the present invention, and after enrichment, narrow spectrum TCR hypotype is hTRBV24-1hTRBJ1-5; Patient 2 peripheral blood PBMC is more single-minded by TCR polymorphism after method enrichment of the present invention, and after enrichment, narrow spectrum TCR hypotype is hTRBV25-1hTRBJ2-5; After 2 routine patient's induced concentration, specificity TCR percentage composition significantly improves that (Fig. 5, wherein lower part shows the segmentation of ordinate zou, is used for amplifying the interval of 0-4.)。The X-coordinate of Fig. 3 is hTRBV2 from left to right successively, hTRBV3-1, hTRBV4-1, hTRBV4-2, hTRBV4-3, hTRBV5-1, hTRBV5-4, hTRBV5-5, hTRBV5-6, hTRBV5-8, hTRBV6-1, hTRBV6-2, hTRBV6-3, hTRBV6-4, hTRBV6-5, hTRBV6-6, hTRBV6-8, hTRBV6-9, hTRBV7-2, hTRBV7-3, hTRBV7-4, hTRBV7-6, hTRBV7-7, hTRBV7-8, hTRBV7-9, hTRBV9, hTRBV10-1, hTRBV10-2, hTRBV10-3, hTRBV11-1, hTRBV11-2, hTRBV11-3, hTRBV12-3, hTRBV12-4, hTRBV12-5, hTRBV13, hTRBV14, hTRBV15, hTRBV16, hTRBV18, hTRBV19, hTRBV20-1, hTRBV24-1, hTRBV25-1, hTRBV27, hTRBV28, hTRBV29-1, hTRBV30, ordinate zou is hTRBJ2-7, hTRBJ2-6, hTRBJ2-5, hTRBJ2-4, hTRBJ2-3, hTRBJ2-2, hTRBJ2-1, hTRBJ1-6, hTRBJ1-5, hTRBJ1-4, hTRBJ1-3, hTRBJ1-2, hTRBJ1-1 from below to up successively.Fig. 4 is that patient 2 peripheral blood PBMC is by TCR polymorphism two-dimensional distribution after method enrichment of the present invention; X-coordinate wherein and the same Fig. 3 of ordinate zou.
Before can finding out enrichment from Fig. 3 and Fig. 4, TCR hypotype is polymorphism comparatively horn of plenty, and in multiple hypotype, after enrichment, TCR hypotype is mainly hTRBV25-1, hTRBJ2-5, accounts for 58.2% of total TCR.
Embodiment 2: cervical cancer specific T-cells induced concentration separation test
1, tumor specific T cells induced concentration is separated
1.1 cervical cancer patients 1, know the inside story and inform and sign Informed Consent Form, extract peripheric venous blood 50ml respectively with heparin sodium anticoagulant tube.After lymphocyte separation medium (FreseniusKabiNorgeAS, LymphoprepTM) is separated PBMC, suspend with fresh serum-free medium (AIM-V of LifeTechnology company), cell density is at 2x10
6/ ml.In every milliliter of serum-free medium, add Cervical Cancer Antigen peptide 2 μ g/ml; IL-250U/ml; IL-7 and IL-15 is respectively 5ng/ml.37 DEG C, cultivate in 5%CO2 incubator.Wherein Cervical Cancer Antigen peptide storehouse, its cAg epi-position is KLPDLCTEL (SEQIDNO:2), and Chu Tai bio tech ltd, Shanghai synthesizes.
1.2 cultivated after 72 hours, carried out IFN γ enrichment sorting (MiltenyiBiotec130-054-201).
1.2.1 collect the cell of induction according to the requirement of test kit, 4 DEG C of centrifugal 10min of 300g, abandon supernatant; Break up cell, add the buffer that 10ml is ice-cold, 4 DEG C of centrifugal 10min of 300g, abandon supernatant.
1.2.2 every 1X10
7cell adds the ice-cold AIM-V of 80 μ l and 20 μ lIFN γ capture agents, and mixing, hatches 5 minutes on ice.Add the substratum of 37 DEG C of preheatings, every 1X10
7cell adds 10ml, is placed in 37 DEG C and hatches 45 minutes, within every 5 minutes, turns and shakes once.Cell is placed on ice, and 4 DEG C of centrifugal 10min of 300g, abandon supernatant.Wash once with ice-cold buffer.
1.2.3 every 1X10
7cell adds the IFN γ detection antibody that the ice-cold buffer of 80 μ l and 20 μ lPE marks, and mixing, hatches 10 minutes on ice.The buffer ice-cold with 10ml washes once.
1.2.4 every 1X10
7cell adds the ice-cold buffer of 80 μ l and the anti-PE magnetic bead of 20 μ l, mixing, and refrigeration hatches 15 minutes.The buffer ice-cold with 10ml washes once.Every 1X10
7the buffer that cell 500ul is ice-cold is resuspended.
1.2.5 cross sorting post.The ice-cold buffer rinse of 500 μ l one time of MS type sorting post, adds cell suspension, crosses after post until cell suspension, wash post 3 times with the buffer that 500 μ l are ice-cold.Take off first sorting post to be placed in above second MS sorting post, add the buffer that 1ml is ice-cold, release cell suspension to second sorting post with piston, again wash post 3 times.Take off second sorting post, add 500 μ lAIM-V substratum, release cell suspension with piston.
2, flow detection and analysis
(wherein ten aggressiveness fluorescence antibodies can the acceptor of conjugated antigen specific T-cells, is the ordinary method of detectable antigens specific T-cells cell before and after enrichment to be contaminated altogether CD8 and ten aggressiveness fluorescence antibodies.The present invention ten aggressiveness fluorescence antibody is purchased from immudex company of the U.S.), flow detection and analysis.After can finding out patient's induced concentration, CD8 and the two positive cell percentage composition of specificity ten aggressiveness significantly improve (Fig. 6).Fig. 6 is CD8 and the two positive cell proportion figure of ten aggressiveness before and after Patients with Cervical Cancer induced concentration.Wherein lower part shows the segmentation of ordinate zou, is used for amplifying the interval of 0-4.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention when not departing from principle of the present invention and aim, revising, replacing and modification.
Claims (6)
1. for a connector for inducing tumor-specific T cell, it is characterized in that, comprise tumour specific antigen, cytokine IL-2, IL-7 and IL-15.
2. according to claim 1 for the connector of inducing tumor-specific T cell, it is characterized in that, described tumour specific antigen is tumor antigen protein, the long peptide of tumour antigen or storehouse, or tumour antigen small peptide or storehouse.
3. according to claim 1 for the connector of inducing tumor-specific T cell, it is characterized in that, in every milliliter of T cell nutrient solution, the consumption of tumour specific antigen is 1-50 μ g/ml, the consumption of IL-2 is 10-6000U/ml, the consumption of IL-7 is the consumption of 1-50ng/ml, IL-15 is 1-50ng/ml.
4. according to claim 1 for the connector of inducing tumor-specific T cell, it is characterized in that, in every milliliter of T cell nutrient solution, the consumption of tumour specific antigen is 2 μ g/ml, the consumption of the consumption of IL-2 to be the consumption of 50U/ml, IL-7 be 5ng/ml, IL-15 is 5ng/ml.
5. one kind is used the method carrying out inducing culture tumor specific T cells described in any one of claim 1-4 for the connector of inducing tumor-specific T cell, it is characterized in that, step is: be separated patient's peripheral blood PBMC, suspend by fresh serum-free lymphocyte culture fluid, cell density is 2x10
6-5x10
6/ ml; The connector for inducing tumor-specific CTL added described in any one of claim 1-4 stimulates, at 37 DEG C, and 5%CO
2cultivate 2-10 days in incubator, the T cells with antigenic specificity being separated the IFN γ positive by IFN γ enrichment with magnetic bead method is tumor specific T cells.
6. according to claim 5 the tumor specific T cells for preparing of method for the preparation of clinical cytology treatment and the medicine of immunotherapy of tumors or the purposes of vaccine.
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| CN113461798A (en) * | 2020-03-18 | 2021-10-01 | 北京鼎成肽源生物技术有限公司 | Fallopian tube cancer target antigen, CTL cell cultured by fallopian tube cancer target antigen in stimulation mode and application of CTL cell |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113461798A (en) * | 2020-03-18 | 2021-10-01 | 北京鼎成肽源生物技术有限公司 | Fallopian tube cancer target antigen, CTL cell cultured by fallopian tube cancer target antigen in stimulation mode and application of CTL cell |
| CN113461799A (en) * | 2020-03-18 | 2021-10-01 | 北京鼎成肽源生物技术有限公司 | Fallopian tube cancer target antigen, CTL cell cultured by fallopian tube cancer target antigen in stimulation mode and application of CTL cell |
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