CN104651311A - Kit for preparing DC-CTL and application of kit - Google Patents
Kit for preparing DC-CTL and application of kit Download PDFInfo
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Abstract
The invention discloses a kit for preparing DC-CTL and an application of the kit. The kit for preparing DC-CTL comprises a reagent for separating single karyocyte, a reagent for inducing DC differentiation and maturation, a tumor antigen activity epitope peptide for sensitizing DC and a reagent for promoting CTL amplification and activation, wherein the reagent for promoting CTL amplification and activation is combination of one or more of an anti-CD3 monoclonal antibody, IFN-gamma, IL-1alpha, PHA, IL-2 and IL-7 and an anti-CD28 monoclonal antibody and anti-ICOS monoclonal antibody. CD8+T cells do not need to be separated from the single karyocyte of peripheral blood by virtue of a flow cytometry or an immunomagnetic bead in preparation of the CTL. The CTL prepared by the method disclosed by the invention is high in purity, high in proliferation capacity, large in number and high in killing activity on tumor cells.
Description
Technical field
The invention belongs to oncotherapy test kit field, especially relate to a kind of test kit and the application thereof of preparing DC-CTL.
Background technology
(1) prior art describes
1. immune cell therapy
In recent decades, oncotherapy achieves larger progress, but still there are some difficult problems: operative treatment can reduce tumor load effectively, but to residual minimum cancer stove and metastatic carcinoma stove less effective; Chemicotherapy can reduce tumor focus and the growth of inhibition tumor cell, but can not effectively distinguish normal cell and tumour cell, and normal tissue toxic side effect is larger.Along with cytobiology and molecular biological fast development, the immune cell therapy that targeting is strong, toxic side effect is little is subject to people and greatly pays close attention to.Immune cell therapy points to input in tumour patient body and has the immunocyte of anti-tumor activity, direct killing tumour cell or excitating organism anti tumor immune response, thus reaches the object for the treatment of tumour.As a kind of novel oncotherapy mode, immune cell therapy has now become the important supplementary means of oncotherapy, which overcome the limitation of routine clinical treatment, effectively can eliminate minimal residual cancer stove, Tumor suppression recurs, improve patient's immunological competence, have broad prospects in clinical therapy of tumor field.
2.DC
Dendritic cell (Dendritic Cell, DC) is found in 1973 by American scholar Steinman, is the immunocyte that the antigen presentation function that finds so far is the strongest, plays a significant role in immunoreactive induction and regulation and control.DC can significant stimulation T cells breed, the initiator of organism adaptation T cell immunne response, play in the induction of adaptability T cell immunne response crucial " Decision-making Function ", therefore it is one of ideal treatment means strengthening tumor-resistant antigen reaction in immunotherapy of tumors, receives much concern at tumor vaccine field.Since Stanford Univ USA medical center Hsu in 1996 etc. report the clinical study of global first term DC tumor vaccine on Nature Medicine, the DC tumor vaccine correlative study of induced tumor antigen-specific effector cell is persistently overheating, and a large amount of clinical studyes is carried out in succession.The DC tumor vaccines such as Sipuleucel-T (Dendreon, the U.S.), CreaVaxRCC (CreaGene, Korea S) and Hybricell (Genoa Biotechnologia, Brazil) obtain listing approval in succession.DC tumor vaccine moves towards clinical by laboratory, its feasibility and security and confirmed the validity of some patients.
Current DC vaccine preparation method comprises two kinds of methods: antigen load sensitization DC and genetic modification DC.
1) antigen load sensitization DC.The method of antigen load sensitization DC mainly contains tumour cell holoantigen sensitization and tumor-antigen peptide sensitization.Research shows in view of many tumour antigens are not clear and definite yet, the immune response adopting full tumour antigen inducing T cell to produce is wider, multiple different tumour antigen composition stimulates DC simultaneously, induce the specific CTL for different antigenic determinant, hazard boundary is wider, can avoid tumour cell generation immunologic escape, but because full tumour antigen composition is indefinite, may autoimmune response be brought out, there is potential safety hazard.There are synthetic tumor-antigen peptide commodity on the market at present, definite ingredients, security is high, target spot is killed and wounded clear and definite with the CTL that the tumor-antigen peptide load DC of synthetic activates, high specificity, but single antigenic peptide load DC, stimulate the CTL of induction can only express the tumour cell of single antigenic peptide by specific killing, hazard boundary limits to.
2) genetic modification DC.Conventional tumor antigen gene, costimulatory molecules, cytokine, chemokine etc. modify DC at present, and method comprises virus vector and physico-chemical method.Research show physico-chemical method comparatively virus vector not only transfection efficiency is low, and the forfeiture of the dead and surface marker of DC may be caused.And virus vector transfection efficiency is high, and the larger gene fragment of energy load, effect is had for stationary phase and division cells, exogenous gene expression is also more efficient, but though slow-virus transfection efficiency is high, can by allogenic gene Insertion Into Host Cell genome, hazard level is high, adenovirus is comparatively safe, but transfection efficiency is not satisfactory.
3.DC-CTL
Cytotoxic T lymphocyte (Cytotoxic T Lymphocyte, CTL) is effector T cell, can the specific recognition MHC molecule antigen of offering, and then killing tumor cell and the cell etc. by infected by microbes specifically.CTL efficiently and kill and wound target cell specifically, and can not damage healthy tissues.The mechanism of CTL killing tumor cell comprises: 1) release particles enzyme and the direct inducing death of neoplastic cells of pore-forming protein; 2) by Fas/FasL approach inducing apoptosis of tumour cell; 3) the cytokine killing tumor cell such as secretion of gamma-IFN and TNF-α or inhibition tumor cell growth.
With specific tumour antigen load DC, the DC of sensitization is induced a large amount of IL-12 of ripe rear secretion, and then effectively stimulate T cells activation, propagation, the IL-2 secreted after T cell activation stimulates the propagation of self further by the mode of autocrine, the T cell of a large amount of propagation is finally divided into effector T cell, plays targeting anti-tumor effect.The specific for tumour antigen CTL of sensitization DC induction is mainly CD8+T cell, and part is CD4+T cell.Action effect T cell, CTL can kill and wound the tumour cell of expressing particular tumor antigens efficiently.
(2) prior art problem and defect describe
1.DC vaccine preparation technology existing defects:
1) the full tumour antigen in autologous tumor tissue source obtains not easily, and full tumour antigen composition is numerous, may cause autoimmune response, there is security risk;
2) use the single tumor-antigen peptide load DC of synthetic to prepare DC vaccine, definite ingredients, but the tumor-antigen peptide quantity of load is single, result for the treatment of is limited; The CTL that the DC of a certain tumor-antigen peptide of load activates effectively can only kill and wound the tumour cell of expressing this tumour antigen;
3) genetic modification DC method is not perfect, and transfection efficiency is low or safety index is low.
2. when prior art prepares DC vaccine, be generally by antigen and TNF-α and immature DC Dual culture, to induce DC ripe and realize the antigen presentation effect of DC, but the antigen presentation efficiency of DC is not high, and the CTL killing activity of activation is not strong.
3. the ordinary method of preparation CTL goes out CD8+T cell for using flow cytometer or immunological magnetic bead sorting at present, and then use combination of cytokines to carry out the amplification of cell, the conventional cytokine used comprises IL-2, IFN-γ, IL-7 and PHA.But, the mode of selected by flow cytometry apoptosis cell inherently can affect to cytoactive, and flow cytometer will sub-elect the object cell of some amount, positive cell ratio must be high, and not so the time can be long, larger on cytoactive impact, if adopt the method improving cell initial density, one is possible plugging, and two is erroneous judgement and the waste that can increase sorting, the time that cell reads analysis is too much short, and judgement can be affected.Moreover, now domesticly carry out the expensive of selected by flow cytometry apoptosis cell.Immunological magnetic bead sorting cell, getable object cell quantity is larger, little on cell viability impact, purity also can reach 90%, but magnetic micro-beads can not automatically disengage object cell mass, be difficult to complete wash-out and become acellular impurity, certain impact is existed on the follow-up cultivation of object cell.
In addition, existing combination of cytokines cultivates the CTL quantity not sufficient of amplification, and cytoactive is not high, is difficult to meet clinical demand.
Summary of the invention
An object of the present invention is the test kit providing preparation DC-CTL, and the test kit of described preparation DC-CTL comprises: the reagent being separated mononuclearcell;
The reagent of induction DC differentiation and maturation;
The tumor antigen activity epitope peptide of sensitization DC;
The reagent promoting CTL amplification and activate, described promotion CTL increases and the reagent that activates is the combination of one or several and the anti-CD28 monoclonal antibody in CD 3-resisting monoclonal antibody, IFN-γ, IL-1 α, PHA, IL-2, IL-7, anti-ICOS monoclonal antibody.
Further: the reagent of described separation mononuclearcell is Ficoll lymphocyte separation medium, or percoll cellular segregation liquid.
The reagent of induction DC differentiation and maturation: 400-600U/mL IL-4,800-1200U/mL GM-CSF, 800-1200U/mL TNF-α (preferred median).
The tumor antigen activity epitope peptide (tumour antigen expressed by tumour selects the combination of two peptides or multiple peptide) of described sensitization DC: include but not limited to MAGE1:161-169, MAGE-A12:170-178, AIM-2:14-23, TRP-2:180-188, gp100:209-217, HER2:369-377, HER2:342-350, IL-13R α 2:345-354, PSA:248-257, PAP213-221, PAP112-120, PSMA441-450, PSMA624-632, ESO-1:161-180, CEA652-660, survivin96-104, EGFR800-809, MRP3:503-511, SART2:93-101.
Described promotion CTL amplification and the reagent activated: 50-80ng/mL CD 3-resisting monoclonal antibody (preferably 50), 1000-1200U/mL IFN-γ (preferably 1000), 80-150U/mL IL-1 α (preferably 100), 50-100ng/mLPHA (preferably 100), 300-500U/mL IL-2 (preferably 300), 20-30ng/mL IL-7 (preferably 30), the anti-CD28 monoclonal antibody (preferably 30) of 20-50ng/mL, the anti-ICOS monoclonal antibody (preferably 30) of 20-40ng/mL.
Further, the aminoacid sequence of described gp100:209-217 is as shown in SEQ ID NO:1, and described HER2:369-377 aminoacid sequence is as shown in SEQ ID NO:2.
Further: the concentration of described various tumor antigen activity epitope peptide is 10 μ g/ml-20 μ g/ml.
Described DC-CTL test kit also comprises component:
E) PBS damping fluid;
F) serum free medium.
The test kit that another object of the present invention is to the preparation DC-CTL of application of aforementioned prepares DC-CTL, comprises step:
A) reagent being separated mononuclearcell is used to be separated mononuclearcell;
B) the reagent inducing peripheral blood mononuclear cell of induction DC differentiation is used to break up to DC;
C) use tumor antigen activity epitope peptide sensitization DC, then stimulate DC ripe;
D) use the T lymphocyte in the agent amplifies peripheral blood mononuclear cell promoting CTL amplification and activation, and inducer T lymphocyte is to CTL differentiation and propagation.
Further: described step C) in add tumor antigen activity epitope peptide and TNF-α order be: first add tumor antigen activity epitope peptide, after 1-2h, add TNF-α again.
Further: described step D) in add anti-CD28 monoclonal antibody and the order of adding anti-ICOS monoclonal antibody is: add anti-ICOS monoclonal antibody again after adding anti-CD28 monoclonal antibody 1.5-3h (being preferably 2h).
The present invention uses the process of two or more tumor antigen activity epitope peptides sensitization DC for first adding tumor antigen activity epitope peptide, absorb after tumor antigen activity epitope peptide until DC, add inflammatory mediator TNF-α again, to stimulate DC ripe fully, play Antigen-presenting role better, induction stimulates CTL to generate more forcefully.
In test kit of the present invention, promote that CTL amplification and the reagent activated comprise anti-CD28 monoclonal antibody and anti-ICOS monoclonal antibody, T lymphopoiesis can be stimulated, produce cytokine profiles, and be divided into CTL.The present invention adds anti-ICOS monoclonal antibody after adding anti-CD28 monoclonal antibody 1.5-3h (being preferably 2h) again, the costimulatory signal that anti-ICOS monoclonal antibody provides can promote T lymphopoiesis better, regulate the lymphocytic differentiation of T, maintain effect and the function of the rear T lymphocyte (comprising memory T-lymphocyte) of activation, play an important role in immunne response and maintenance effective stage.
The combination of cytokines that the present invention cultivates CTL use can promote T lymphocyte activation and propagation, and improve T lymphocyte clone number, purity is high, and the DC vaccine of preparation can significantly improve the cytotoxicity of the CTL of induction.
The present invention prepares CTL and from peripheral blood mononuclear cell, sub-elects CD8+T cell without the need to using flow cytometer or immunomagnetic beads, and the CTL purity utilizing the inventive method to prepare is high, and proliferative ability is strong, and quantity is many, high to the killing activity of tumour cell.
Accompanying drawing explanation
Fig. 1 is DC flow cytometry result;
Fig. 2 is the situation schematic diagram that DC secretes IL-12;
Fig. 3 is CTL cell counts schematic diagram;
Fig. 4 is CTL flow cytometry result schematic diagram;
Fig. 5 is the situation schematic diagram of CTL emiocytosis IFN-γ;
Fig. 6 is cytotoxic killer experimental result schematic diagram.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
1. tumor antigen activity epitope peptide: adopt the synthesis of standard Fmoc scheme, high performance liquid chromatography carries out purifying and purity check, and mass spectroscopy carries out identifying and molecular weight determination.Result shows the purity of above-mentioned tumor antigen activity epitope peptide higher than 95%, and molecular weight conforms to theoretical value.Above-mentioned tumor antigen activity epitope peptide fully dissolves with aseptic double-distilled water respectively, and concentration is 10ug/mL, and packing is stored in-80 DEG C.
2. separating peripheral blood mononuclear cells
Gather peripheral blood 70-80mL under aseptic condition, blood preparation is delivered to laboratory, carry out the separation of peripheral blood mononuclear cell.By blood transfer to a 2 50mL centrifuge tube, 2000rpm, l0min are centrifugal, and transfer upper strata autologous plasma is in 50mL centrifuge tube, and 56 DEG C of water-bath deactivation 30min, 4 DEG C of refrigerations are for subsequent use.
Remaining blood and 0.01mol/L PBS liquid press 1:1 dilute blow and beat even, lymphocyte separation medium is got with suction pipe, lymphocyte separation medium is 2:1 with the volume ratio of the rear blood of dilution, the top of blood after lymphocyte separation medium is slowly added dilution along centrifugal tube wall, keep liquid interface clear, 2000rpm, 30min.
Take out centrifuge tube, liquid in pipe is divided into 4 layers, is followed successively by from top to bottom: blood plasma, white cloud thin layer (mononuclearcell layer), lymphocyte separation medium, red corpuscle and granulocyte.Draw mononuclearcell layer, add the PBS washed cell of 3-4 times of volume, 1500rpm, 10min, the PBS adding 4-5 times of volume toward cell precipitation washs again, 1000rpm, 10min.
Cell precipitation 30-40mL is resuspended containing the GTT551 serum free medium of 10% autologous plasma, put into 37 DEG C, 5%CO
2incubator leaves standstill 2h, results attached cell and suspension cell, is respectively used to differentiation-inducing one-tenth DC and the cultivation of T lymphocyte.
3. peripheral blood mononuclear cell is to the differentiation-inducing of DC and tumor antigen activity epitope peptide sensitization DC
1) attached cell that step 2 is gathered in the crops is induced to differentiate into DC, concrete grammar is as follows:
In attached cell culturing bottle, add the GTT551 serum free medium of 20mL containing 10% autologous plasma, interpolation final concentration is 500U/mL IL-4,1000U/mL GM-CSF, puts into 37 DEG C, 5%CO
2continue in incubator to cultivate.GM-CSF and IL-4 acting in conjunction can make monocyte directed differentiation be immature DC, and DC now has stronger antigen uptake and working ability, but antigen presentation ability is very weak.
Supplemented the GTT551 serum free medium containing 10% autologous plasma in the 3rd day according to cell growth status, interpolation final concentration is 500U/mL IL-4,1000U/mL GM-CSF, puts into 37 DEG C, 5%CO
2continue in incubator to cultivate, add above-mentioned substratum and cytokine according to cell growth status.
In the 6th day harvested cell, adjustment cell density was 1X10
6/ mL, adds the tumor antigen activity epitope peptide of 10ug/mL respectively.Add 1000U/mL TNF-α after 2h, stimulate DC ripe, play Antigen-presenting role.In the DC vaccine of the 7th day results load tumor antigen activity epitope peptide.
2) phenotypic evaluation of DC
The ripe DC (mature DC, mDC) getting the cultivation immature DC of the 3rd day (immature DC, imDC) and the 7th day respectively carries out the detection of cell surface marker thing.
Add 0.25% trysinization DC, stop digestion rear 1000rpm, 10min, the cell precipitation 0.01mol/L PBS liquid of 4 DEG C of precoolings is washed 2 times, 1000rpm, 10min, by 200ul PBS re-suspended cell precipitation, adjustment cell density is 1X10
6/ ml, lucifuge adds CD83, CD86, CD1a and HLA-DR fluorescence antibody, and 4 DEG C of lucifuges hatch 30min, add 2mL PBS, l000rpm, l0min, washed cell, to remove unconjugated antibody, removes supernatant, cell precipitation PBS liquid is resuspended, 4 DEG C of censorships, and flow cytometer detects.
Flow cytomery result shows the 7th day mDC high expression level CD83, CD86, CD1a and HLA-DR of cultivating, shows successfully to induce DC ripe.
Table 1DC cell surface marker expression (
, %)
*: compared with imDC group, P<0.05.
3) cytokines measurement (IL-12)
Get the mDC that DC cultivates the 3rd day imDC and the 7th day to detect.Take out the enzyme plate of coated antibody, TMB (3,3', 5,5'-Tetra methylbenzidine) blank colour developing hole is set, add the standard substance that 0.1mL dilute by certain multiple and the sample diluted with sample diluting liquid successively.Enzyme plate adds upper cover, 37 DEG C of reaction 90min.Suck the liquid in enzyme plate with automatic washer after reaction.Added successively in (except TMB blank colour developing hole) by every hole 0.1mL by anti-human for vitamin H IL-12 antibody working fluid, 37 DEG C of reaction 60min, 0.01M PBS wash 3 times.Added successively in (except TMB blank colour developing hole) by every hole 0.1mL by ABC working fluid, 37 DEG C of reaction 30min, 0.01M PBS wash 5 times.Add TMB nitrite ion successively by every hole 90 μ L, 37 DEG C of lucifuge reaction 20-25min, add TMB stop buffer successively by every hole 0.1mL, and now blue vertical turn of yellow, measures OD value by microplate reader at 450nm.Sample OD value to deduct after blank well OD value with OD value and standard concentration as XY axle is drawn, and is multiplied by extension rate, calculates IL-12 concentration in sample after typical curve is searched IL-12 concentration.
Result shows that mDC expresses the level of IL-12 apparently higher than the expression level of imDC, has significant difference (P<0.05).
4. peripheral blood mononuclear cell is to CTL proliferative induction
1) suspension cell step 2 gathered in the crops is to CTL proliferative induction, and concrete grammar is as follows:
By the GTT551 serum free medium adjustment suspension cell density containing 10% autologous plasma to 1X10
6/ mL, insert and wrap by the culturing bottle of 50ng/mL CD 3-resisting monoclonal antibody in advance, interpolation final concentration is 100ng/mL PHA, 1000U/mL IFN-γ, puts into 37 DEG C, 5%CO
2continue in incubator to cultivate.
Adding final concentration in the 2nd day is 100U/mL IL-1 α, 300U/mL IL-2,30ng/mL IL-7, puts into 37 DEG C, 5%CO
2continue in incubator to cultivate.
Supplemented the GTT551 serum free medium containing 10% autologous plasma in the 3rd day according to cell growth status, interpolation final concentration is 300U/mL IL-2,30ng/mL IL-7,100ng/mL PHA, puts into 37 DEG C, 5%CO
2continue in incubator to cultivate.
DC vaccine (DC vaccine and the lymphocytic ratio of T are 1:10) Dual culture is added in the 7th day, carry out first round CTL induced amplification, substratum is except 300U/mL IL-2,30ng/mL IL-7,100ng/mL PHA except adding final concentration, also adding final concentration is the anti-CD28 monoclonal antibody of 30ng/mL, adding final concentration after 2h is again the anti-ICOS monoclonal antibody of 30ng/mL, supplement the GTT551 serum free medium containing 10% autologous plasma every other day, add above-mentioned cytokine, to keep the best use of concentration of cytokine, put into 37 DEG C, 5%CO
2continue in incubator to cultivate.
DC vaccine (DC vaccine and the lymphocytic ratio of T are 1:10) Dual culture is again added in the 14th day, differentiation-inducing to CTL of strengthening T lymphocyte, carry out second and take turns CTL induced amplification, it is the anti-CD28 monoclonal antibody of 300U/mLIL-2,30ng/mL IL-7,100ng/mL PHA, 30ng/mL that substratum adds final concentration, adding final concentration after 2h is again the anti-ICOS monoclonal antibody of 30ng/mL, supplement the GTT551 serum free medium containing 10% autologous plasma every other day, add above-mentioned cytokine, put into 37 DEG C, 5%CO
2continue in incubator to cultivate.
In the 21st day results CTL.Set up general culture method group as a control group, general culture method group adopts conventional combination of cytokines to cultivate, and comprises IL-2, IFN-γ, IL-7 and PHA.
The anti-CD28 monoclonal antibody that the present invention adds and anti-ICOS monoclonal antibody can stimulate T lymphopoiesis, produce cytokine profiles, and being divided into CTL, optimal way adds anti-ICOS monoclonal antibody again after adding anti-CD28 about monoclonal antibody 2h (1.5-3h).
The combination of cytokines that the present invention cultivates CTL use can promote T lymphocyte activation and propagation, and improve T lymphocyte clone number, purity is high, and the DC vaccine of preparation can significantly improve the cytotoxicity of the CTL of induction.
2) cell counting
The CTL quantity that general culture method group obtains is (1.2 ± 0.4) × 10
9, the CTL quantity that anti-CD28 monoclonal antibody group (general culture method group+anti-CD28 monoclonal antibody) obtains is (1.9 ± 0.5) × 10
9, the CTL quantity that anti-ICOS monoclonal antibody group (general culture method group+anti-ICOS monoclonal antibody) obtains is (1.6 ± 0.4) × 10
9, the CTL quantity adopting the new cultural method of the present invention to obtain is (2.8 ± 0.9) × 10
9, the CTL quantity that new cultural method obtains, compared with other culture methods, has significant significant difference (P<0.05).
3) phenotypic evaluation of CTL
Get and do not carry out cell phenotype detection with the T lymphocyte of DC vaccine Dual culture and the CTL that cultivates for the 21st day on the 7th day:
The cell precipitation of the centrifugal acquisition 0.0lmol/L PBS liquid of 4 DEG C of precoolings is washed 2 times, 1000rpm, 10min, and by 200ul PBS re-suspended cell precipitation, adjustment cell density is 1 × 10
6/ mL, lucifuge adds CD3, CD4, CD8 antibody, and 4 DEG C of lucifuges hatch 30min, add 2ml PBS, l000rpm, l0min, and washed cell, to remove unconjugated antibody, removes supernatant, and cell precipitation PBS liquid is resuspended, 4 DEG C of censorships, and flow cytometer detects.Set up T cell group, general culture method group and new culture method group, wherein T cell group be not with the T lymphocyte of the DC Dual culture of load tumor antigen activity epitope peptide, general culture method group is the CTL of the DC Dual culture of general culture method preparation and load tumor antigen activity epitope peptide, and new culture method group is the CTL of the DC Dual culture of cultural method of the present invention preparation and load tumor antigen activity epitope peptide.
Result display general culture method group and new culture method group CD3+ cell proportion higher than T cell group, but not statistically significant, and the CD4+ cell proportion of general culture method group and new culture method group declines, CD8+ cell proportion raises, compared with T cell group, there is statistical significance (P<0.05), and new culture method group significantly raises than the CD8+ cell proportion of general culture method group.
Table 2CTL cell surface marker expression (
, %)
4) cytokines measurement (IFN-γ)
Take out the enzyme plate of coated antibody, TMB blank colour developing hole is set, add the standard substance that 0.1mL dilute by certain multiple and the sample diluted with sample diluting liquid successively.Enzyme plate adds upper cover, 37 DEG C of reaction 90min.Suck the liquid in enzyme plate with automatic washer after reaction.Added successively in (except TMB blank colour developing hole) by every hole 0.1mL by anti-human for vitamin H IFN-gamma antibodies working fluid, 37 DEG C of reaction 60min, 0.01M PBS wash 3 times.Added successively in (except TMB blank colour developing hole) by every hole 0.1mL by ABC working fluid, 37 DEG C of reaction 30min, 0.01M PBS wash 5 times.Add TMB nitrite ion successively by every hole 90 μ L, 37 DEG C of lucifuge reaction 20-25min, add TMB stop buffer successively by every hole 0.1mL, and now blue vertical turn of yellow, measures OD value by microplate reader at 450nm.Sample OD value to deduct after blank well OD value with OD value and standard concentration as XY axle is drawn, and is multiplied by extension rate, calculates IFN-γ concentration in sample after typical curve is searched IFN-γ concentration.If control group, general culture method group and new culture method group, often group establishes 3 multiple holes.
Result shows, and new culture method group secretion of gamma-IFN level, considerably beyond the secretion level of general culture method group, has significant difference (P<0.05).
5. cell killing experiment
Adjust CTL (effector cell) respectively and human glioma cells U251 (target cell) density is 1X10
6/ mL and 1X10
7/ mL, add effector cell suspension and target cell suspension than 5:1,10:1,20:1 toward 96 orifice plates by effect target, cumulative volume is 200uL, puts into 37 DEG C, 5%CO
2after cultivating 48h in incubator, every hole adds 20uL CCK-8, and after continuing to hatch 2h, upper microplate reader detects, and reads OD value, kill rate=[1-(experimental group OD value-effector cell organizes OD value)/target cell group OD value] * 100% in 450nm.Experiment is divided into blank group, target cell group, effector cell's group and experimental group (new culture method group, general culture method group), and experimental group prepares CTL by the DC induction of tumor antigen activity epitope peptide gp100:209-217 (SEQ ID NO:1) and HER2:369-377 (SEQ ID NO:2) load.
Result shows, along with effect the increasing of target ratio, new culture method group and the kill rate of general culture method group to human glioma cells U251 raise gradually, and when imitating target than time for 20:1, lethal the strongest to human glioma cells U251, kill rate is the highest.Further, at same effect target than under condition, new culture method group significantly improves than general culture method group kill rate the kill rate of human glioma cells U251, has statistical significance (P<0.05).
Sequence table
Yin Guan bio tech ltd of <110> Shenzhen
<120> prepares test kit and the application thereof of DC-CTL
〈160〉2
〈210〉1
〈211〉9
〈212〉PRT
< 213 > people
〈400〉1
Ile Thr Asp Gln Val Pro Phe Ser Val
1 5
〈210〉2
〈211〉9
〈212〉PRT
< 213 > people
〈400〉2
Lys Ile Phe Gly Ser Leu Ala Phe Leu
1 5 。
Claims (10)
1. prepare a test kit of DC-CTL, comprising:
Be separated the reagent of mononuclearcell;
The reagent of induction DC differentiation and maturation;
The tumor antigen activity epitope peptide of sensitization DC;
The reagent promoting CTL amplification and activate, described promotion CTL increases and the reagent that activates is the combination of one or several and the anti-CD28 monoclonal antibody in CD 3-resisting monoclonal antibody, IFN-γ, IL-1 α, PHA, IL-2, IL-7, anti-ICOS monoclonal antibody.
2. the test kit of preparation DC-CTL as claimed in claim 1, is characterized in that:
The reagent of described separation mononuclearcell is Ficoll lymphocyte separation medium, or percoll cellular segregation liquid.
3. the test kit of preparation DC-CTL as claimed in claim 1, is characterized in that: the reagent of described induction DC differentiation and maturation is one or several in cytokine IL-4, GM-CS, TNF-α; The concentration of IL-4, GM-CSF, TNF-α is respectively 400-600U/mL, 800-1200U/mL, 800-1200U/mL.
4. the test kit of preparation DC-CTL as claimed in claim 1, it is characterized in that: the tumor antigen activity epitope peptide of described sensitization DC is MAGE1:161-169, MAGE-A12:170-178, AIM-2:14-23, TRP-2:180-188, gp100:209-217, HER2:369-377, HER2:342-350, IL-13R α 2:345-354, PSA:248-257, PAP213-221, PAP112-120, PSMA441-450, PSMA624-632, ESO-1:161-180, CEA652-660, survivin96-104, EGFR800-809, MRP3:503-511, one or more combination in SART2:93-101.
5. the test kit of preparation DC-CTL as claimed in claim 1, is characterized in that: in the reagent of described promotion CTL amplification and activation, the concentration of CD 3-resisting monoclonal antibody, IFN-γ, IL-1 α, PHA, IL-2, IL-7 is respectively 50-80ng/mL, 1000-1200U/mL, 80-150U/mL, 50-100ng/mL, 300-500U/mL, 20-30ng/mL; The concentration of described anti-CD28 monoclonal antibody, anti-ICOS monoclonal antibody is respectively 20-50ng/mL, 20-40ng/mL.
6. the test kit of preparation DC-CTL as claimed in claim 4, is characterized in that: the aminoacid sequence of described gp100:209-217 is as shown in SEQ ID NO:1, and described HER2:369-377 aminoacid sequence is as shown in SEQ ID NO:2.
7. the test kit of preparation DC-CTL as claimed in claim 4, is characterized in that: the concentration of described various tumor antigen activity epitope peptide is 10 μ g/ml-20 μ g/ml.
8. the test kit of the preparation DC-CTL as described in any one of claim 1-7, is characterized in that, also comprise component:
E) PBS damping fluid;
F) serum free medium.
9. application rights requires that the test kit of the preparation DC-CTL described in any one of 1-8 prepares the method for DC-CTL, comprises step:
A) reagent being separated mononuclearcell is used to be separated mononuclearcell;
B) the reagent inducing peripheral blood mononuclear cell of induction DC differentiation is used to break up to DC;
C) use tumor antigen activity epitope peptide sensitization DC, then stimulate DC ripe;
D) use the T lymphocyte in the agent amplifies peripheral blood mononuclear cell promoting CTL amplification and activation, and inducer T lymphocyte is to CTL differentiation and propagation.
10. prepare the method for DC-CTL as claimed in claim 9, it is characterized in that: described step C) in DC culturing process, first add tumor antigen activity epitope peptide sensitization, add TNF-α after 1-2h again and facilitate ripe; Described step D) in add anti-CD28 monoclonal antibody and the order of adding anti-ICOS monoclonal antibody is: add anti-ICOS monoclonal antibody again after adding anti-CD28 monoclonal antibody 1.5-3h.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410446761.XA CN104651311B (en) | 2014-09-03 | 2014-09-03 | Prepare DC CTL kit and its application |
| PCT/CN2015/088648 WO2016034094A1 (en) | 2014-09-03 | 2015-08-31 | Kit for preparing dc-ctl and application of kit |
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| CN105112369A (en) * | 2015-08-25 | 2015-12-02 | 北京康爱瑞浩生物科技股份有限公司 | CTL (cytotoxic T lymphocyte) cytomedicine with continuous antitumor activity and preparation method of CTL cytomedicine |
| CN105154400A (en) * | 2015-09-30 | 2015-12-16 | 中国人民解放军第三0二医院 | Amplification method of polyclonal anti-HBV (hepatitis B virus) immune cells |
| WO2016034094A1 (en) * | 2014-09-03 | 2016-03-10 | 深圳市茵冠生物科技有限公司 | Kit for preparing dc-ctl and application of kit |
| CN105695403A (en) * | 2015-12-31 | 2016-06-22 | 深圳市中美康士生物科技有限公司 | Killer immune cell culturing method and application thereof |
| CN106479975A (en) * | 2015-12-30 | 2017-03-08 | 北京昱龙盛世生物科技有限公司 | Panimmunity cell co-cultivation method |
| CN106566806A (en) * | 2016-07-11 | 2017-04-19 | 英威福赛生物技术有限公司 | Method for in-vitro culture and enrichment of CD8+ T cells |
| CN106795492A (en) * | 2015-06-17 | 2017-05-31 | 深圳市达科为生物工程有限公司 | A kind of preparation method of tumor-specific CTL |
| CN109486761A (en) * | 2019-01-17 | 2019-03-19 | 汇麟生物科技(北京)有限公司 | The cultural method of peripheral blood CTL cell |
| CN109825475A (en) * | 2019-01-31 | 2019-05-31 | 苏州明基医院有限公司 | A kind of the induced activation kit and its application method of DC-CTL cell |
| CN113234674A (en) * | 2021-03-15 | 2021-08-10 | 青岛华赛伯曼医学细胞生物有限公司 | T cell activation and amplification method and application thereof |
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| WO2016034094A1 (en) * | 2014-09-03 | 2016-03-10 | 深圳市茵冠生物科技有限公司 | Kit for preparing dc-ctl and application of kit |
| CN106795492A (en) * | 2015-06-17 | 2017-05-31 | 深圳市达科为生物工程有限公司 | A kind of preparation method of tumor-specific CTL |
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| CN106566806B (en) * | 2016-07-11 | 2018-05-04 | 英威福赛生物技术有限公司 | The method that in vitro culture is enriched with CD8+T cells |
| CN109486761A (en) * | 2019-01-17 | 2019-03-19 | 汇麟生物科技(北京)有限公司 | The cultural method of peripheral blood CTL cell |
| CN109825475A (en) * | 2019-01-31 | 2019-05-31 | 苏州明基医院有限公司 | A kind of the induced activation kit and its application method of DC-CTL cell |
| CN113234674A (en) * | 2021-03-15 | 2021-08-10 | 青岛华赛伯曼医学细胞生物有限公司 | T cell activation and amplification method and application thereof |
| CN113234674B (en) * | 2021-03-15 | 2023-02-07 | 青岛华赛伯曼医学细胞生物有限公司 | T cell activation and amplification method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2016034094A1 (en) | 2016-03-10 |
| CN104651311B (en) | 2018-03-13 |
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