CN104651311B - Prepare DC CTL kit and its application - Google Patents
Prepare DC CTL kit and its application Download PDFInfo
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- CN104651311B CN104651311B CN201410446761.XA CN201410446761A CN104651311B CN 104651311 B CN104651311 B CN 104651311B CN 201410446761 A CN201410446761 A CN 201410446761A CN 104651311 B CN104651311 B CN 104651311B
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Abstract
The invention discloses a kind of kit for preparing DC CTL and its application, the kit for preparing DC CTL includes：Separate the reagent of mononuclearcell；Induce the reagent of DC differentiation and maturations；Sensitization DC tumor antigen activity epitope peptide；Promote the reagent of CTL amplifications and activation, the reagent that the promotion CTL is expanded and activated is CD 3-resisting monoclonal antibody, the one or several kinds of and combination of anti-CD28 monoclonal antibodies, anti-ICOS monoclonal antibodies in IFN γ, IL 1 α, PHA, IL 2, IL 7.The present invention prepares CTL and CD8+T cells is sub-elected from PMNC without using flow cytometer or immunomagnetic beads, and the CTL purity prepared using the inventive method is high, and proliferative ability is strong, and quantity is more, high to the killing activity of tumour cell.
Description
Technical field
The invention belongs to oncotherapy kit field, more particularly, to a kind of kit for preparing DC-CTL and its answers
With.
Background technology
(1) prior art describes
1. immune cell therapy
In recent decades, oncotherapy achieves larger progress, but still suffers from some problems：Operative treatment can be effectively
Reduce tumor load, but minimum cancer stove and metastatic carcinoma stove less effective to residual；Chemicotherapy can reduce tumor focus and suppress
The growth of tumour cell, but normal cell and tumour cell can not be effectively distinguished, normal tissue toxic side effect is larger.With
The fast development of cell biology and molecular biology, the immune cell therapy that targeting is strong, toxic side effect is small is by people pole
Big concern.Immune cell therapy points to immunocyte of the input with antitumor activity, direct killing tumour in tumor patient body
Cell or excitating organism anti tumor immune response, so as to reach the purpose for the treatment of tumour.As a kind of new oncotherapy side
Formula, immune cell therapy have turned into the important supplementary means of oncotherapy, have which overcomed the limitation of routine clinical treatment, can
Minimal residual cancer stove is effectively eliminated, suppresses tumor recurrence, improves patient's immunocompetence, is had in clinical therapy of tumor field wide
Wealthy prospect.
2.DC
BMDC (Dendritic Cell, DC) is to be found by American scholar Steinman in 1973, is mesh
Before untill the most strong immunocyte of the antigen presentation function that finds, played a significant role in the induction and regulation and control of immune response.
DC significantly can stimulate T cells to breed, and be the initiator of organism adaptation T cell immune response, exempt from adaptability T cell
" Decision-making Function " of key is played in the induction of epidemic disease response, therefore it is to strengthen antitumor antigens reaction in immunotherapy of tumors
One of ideal treatment means, have received much concern in tumor vaccine field.Since Stanford Univ USA medical center Hsu in 1996
Deng since reporting global first term DC tumor vaccine clinical researches on Nature Medicine, induced tumor antigentic specificity
The DC tumor vaccine correlative studys of effector cell are persistently overheating, and substantial amounts of clinical research is carried out in succession.Sipuleucel-T
(Dendreon, the U.S.), CreaVaxRCC (CreaGene, South Korea) and Hybricell (Genoa Biotechnologia, bar
West) etc. DC tumor vaccines obtain in succession listing approval.DC tumor vaccines move towards clinical via laboratory, its feasibility and safety
Property and the validity of some patientss is confirmed.
DC vaccine preparation methods include two methods at present：Antigen load sensitization DC and genetic modification DC.
1) antigen load sensitization DC.Antigen load sensitization DC method mainly has tumour cell holoantigen sensitization and tumour to resist
Former polypeptide sensitization.Research shows in view of many tumour antigens are still not known, using immune caused by full tumour antigen inducing T cell
Reaction range is wider, and a variety of different tumour antigen compositions stimulate DC simultaneously, induce the specificity for different antigenic determinants
CTL, hazard boundary is wider, can avoid tumour cell that immunologic escape occurs, but because full tumour antigen composition is indefinite, can
Autoimmune response can be induced, potential safety hazard be present.There are artificial synthesized tumor-antigen peptide commodity on the market at present, into clearly demarcated
Really, safe, the CTL killing target spots of the tumor-antigen peptide load DC activation manually synthesized are clear and definite, high specificity, still
Single antigen polypeptide loads DC, stimulate induction CTL can only specific killing express the tumour cell of single antigen polypeptide, killing
Scope is limited to.
2) genetic modification DC.The modification such as conventional tumor antigen gene, costimulatory molecules, cell factor, chemotactic factor (CF) at present
DC, method include viral vector and physico-chemical method.Research shows physico-chemical method, and compared with viral vector, not only transfection efficiency is low, and
The dead forfeitures with surface marker of DC may be caused.And viral vector transfection efficiency is high, and larger genetic fragment can be loaded, for
Resting stage and division cells have effect, and exogenous gene expression is also more efficient, though but slow-virus transfection efficiency is high, can will
Allogenic gene Insertion Into Host Cell genome, degree of danger is high, and 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 specificity knowledge
The antigen that other MHC molecule is offered, and then specifically killing tumor cell and microorganism-infected cell etc..CTL can be efficient
And target cell is specifically killed, without damaging normal structure.The mechanism of CTL killing tumor cells includes：1) discharge granzyme and
The direct inducing death of neoplastic cells of perforin；2) Fas/FasL approach inducing apoptosis of tumour cell is passed through；3) secretion of gamma-IFN and
The cell factor such as TNF-α killing tumor cell suppresses growth of tumour cell.
DC is loaded with specific tumour antigen, the DC of sensitization secretes substantial amounts of IL-12 after being induced maturation, and then effectively
T cells are stimulated to activate, propagation, the IL-2 secreted after T cell activation further stimulates itself by way of autocrine
Propagation, the T cell largely bred finally are divided into effector T cell, play targeting anti-tumor effect.The tumour of sensitization DC inductions resists
Former specific CTL is mainly CD8+T cells, is partly CD4+T cells.As effector T cell, CTL can efficiently kill expression
The tumour cell of particular tumor antigens.
(2) prior art problem and defect description
1.DC vaccine preparation technology existing defects：
1) the full tumour antigen in autologous tumor tissue source is obtained and is not easy, and full tumour antigen composition is numerous, may be triggered
Autoimmune response, security risk be present；
2) DC vaccines, definite ingredients are prepared using artificial synthesized single tumor-antigen peptide load DC, but loaded swollen
Tumor antigen polypeptide quantity is single, and therapeutic effect is limited；Loading the CTL of the DC activation of a certain tumor-antigen peptide can only effectively kill
Express the tumour cell of the tumour antigen；
3) genetic modification DC methods are not perfect, and transfection efficiency is low or safety index is low.
2. prior art prepares DC vaccines, usually antigen and TNF-α and immature DC are co-cultured, with induce DC into
Antigen presentation effect that is ripe and realizing DC, but DC antigen presentation is inefficient, the CTL killing activities of activation are not strong.
3. the conventional method for preparing CTL at present is to go out CD8+T cells using flow cytometer or immunological magnetic bead sorting, then
The amplification of cell is carried out using combination of cytokines, conventional use of cell factor includes IL-2, IFN-γ, IL-7 and PHA.So
And the mode of selected by flow cytometry apoptosis cell can inherently affect to cytoactive, and flow cytometer will
If sub-electing a number of aim cell, positive cell ratio must be high, and not so the time can be long, to cytoactive
Have a great influence, if using the method for improving cell initial density, when possible plugging, second, erroneous judgement and the wave of sorting can be increased
Take, the time that cell reads analysis too much is short, judges to be affected.Furthermore domestic progress selected by flow cytometry apoptosis is thin now
Born of the same parents' is expensive.Immunological magnetic bead sorting cell, getable aim cell quantity is bigger, small on cell viability influence, pure
Degree can also reach 90%, but magnetic micro-beads can not automatically disengage aim cell group, it is difficult to elute completely and become acellular
Impurity, certain influence be present to the follow-up cultivation of aim cell.
In addition, the CTL lazy weights of existing combination of cytokines culture amplification, cytoactive be not high, it is difficult to which satisfaction is faced
Bed demand.
The content of the invention
An object of the present invention is to provide the kit for preparing DC-CTL, the kit bag for preparing DC-CTL
Include：Separate the reagent of mononuclearcell；
Induce the reagent of DC differentiation and maturations；
Sensitization DC tumor antigen activity epitope peptide；
Promote the reagent of CTL amplifications and activation, the reagent of promotions CTL amplification and activation be CD 3-resisting monoclonal antibody,
One or several kinds of and anti-CD28 monoclonal antibodies, anti-ICOS monoclonal antibodies in IFN-γ, IL-1 α, PHA, IL-2, IL-7
Combination.
Further：The reagent of the separation mononuclearcell is Ficoll lymphocyte separation mediums, or percoll thin
Born of the same parents' separating liquid.
Induce the reagent of DC differentiation and maturations：400-600U/mL IL-4、800-1200U/mL GM-CSF、800-1200U/
ML TNF-αs (preferably median).
Tumor antigen activity epitope peptide (the double peptides or a variety of of tumour antigen selection according to expressed by tumour of the sensitization DC
The combination of peptide)：Including 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-13Rα2：345-354、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.
The reagent of the promotion CTL amplifications and activation：50-80ng/mL CD 3-resisting monoclonal antibodies (preferably 50), 1000-
1200U/mL IFN-γs (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 antibodies of 20-50ng/mL
(preferably 30), the anti-ICOS monoclonal antibodies of 20-40ng/mL (preferably 30).
Further, the gp100:209-217 amino acid sequence such as SEQ ID NO：Shown in 1, the HER2:369-
377 amino acid sequences such as SEQ ID NO：Shown in 2.
Further：The concentration of the various tumor antigen activity epitope peptides is 10 μ g/ml-20 μ g/ml.
The DC-CTL kits also include component：
E) PBS；
F) serum free medium.
Another object of the present invention is to the foregoing kit for preparing DC-CTL of application to prepare DC-CTL, including step：
A) using the reagent separation mononuclearcell of separation mononuclearcell；
B) broken up using the reagent inducing peripheral blood mononuclear cell of induction DC differentiation to DC；
C tumor antigen activity epitope peptide sensitization DC) is used, then stimulates DC ripe；
D) using the T lymphocytes in the reagent amplification PMNC for promoting CTL amplifications and activation, and induce
T lymphocytes break up and bred to CTL.
Further：The step C) in addition tumor antigen activity epitope peptide and the order of TNF-α be：First add tumour
Antigen active epitope peptide, TNF-α is added again after 1-2h.
Further：The step D) in the anti-CD28 monoclonal antibodies of addition and the anti-ICOS monoclonal antibodies of addition it is suitable
Sequence is：Anti- ICOS monoclonal antibodies are added again after adding anti-CD28 monoclonal antibodies 1.5-3h (being preferably 2h).
The present invention is lived using two or more tumor antigen activity epitope peptide sensitization DC process first to add tumour antigen
Property epitope peptide, after DC absorbs tumor antigen activity epitope peptide, add inflammatory mediator TNF-α, fully to stimulate DC ripe,
Antigen-presenting role is preferably played, more forcefully induction stimulates CTL to generate.
In kit of the present invention, the reagent of promotion CTL amplifications and activation includes anti-CD28 monoclonal antibodies and resisted
ICOS monoclonal antibodies, T lymphopoiesis can be stimulated, produce cytokine profiles, and be divided into CTL.Present invention addition is anti-
Anti- ICOS monoclonal antibodies are added again after CD28 monoclonal antibodies 1.5-3h (being preferably 2h), what anti-ICOS monoclonal antibodies provided
Costimulatory signal can advantageously promote T lymphopoiesis, adjust the differentiation of T lymphocytes, maintain T lymphocytes after activation
The effect and function of (including memory T-lymphocyte), in immune response and the effective stage is maintained to play an important role.
The combination of cytokines that present invention culture CTL is used can promote T lymphocyte activations and propagation, and it is thin to improve T lymphs
Born of the same parents clone number, and purity is high, and the DC vaccines prepared can significantly improve the CTL of induction cytotoxicity.
The present invention prepares CTL and sub-elected without using flow cytometer or immunomagnetic beads from PMNC
CD8+T cells, the CTL purity prepared using the inventive method is high, and proliferative ability is strong, and quantity is more, to the killing activity of tumour cell
It is high.
Brief description of the drawings
Fig. 1 is DC flow cytometry results；
Fig. 2 is the situation schematic diagram that DC secretes IL-12；
Fig. 3 is CTL cell counts schematic diagrames；
Fig. 4 is CTL flow cytometry result schematic diagrams；
Fig. 5 is the situation schematic diagram of CTL cell secretion of gamma-IFN；
Fig. 6 is cytotoxic killer experimental result schematic diagram.
Embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.
1. tumor antigen activity epitope peptide：Using standard Fmoc schemes synthesize, high performance liquid chromatography carry out purifying and it is pure
Degree analysis, mass spectrography is identified and molecular weight determination.As a result show that the purity of above-mentioned tumor antigen activity epitope peptide is higher than
95%, molecular weight is consistent with theoretical value.Above-mentioned tumor antigen activity epitope peptide is fully dissolved with aseptic double-distilled water respectively, and concentration is
10ug/mL, packing are stored in -80 DEG C.
2. separating peripheral blood mononuclear cells
Peripheral blood 70-80mL is gathered under aseptic condition, blood preparation is delivered into laboratory, carries out PMNC
Separation.Blood is transferred to 2 50mL centrifuge tubes, 2000rpm, l0min centrifugation, transfer upper strata autologous plasma to 50mL centrifuges
In pipe, 56 DEG C of water-baths inactivate 30min, and 4 DEG C of refrigerations are standby.
Remaining blood presses 1 with 0.01mol/L PBS liquid:1 dilution piping and druming is uniform, takes lymphocyte separation medium with suction pipe, drenches
The volume ratio of bar cell separating liquid and blood after dilution is 2：1, centrifugation tube wall in lymphocyte separation medium edge is slowly added to dilute
The top of rear blood is released, keeps liquid interface clear, 2000rpm, 30min.
Centrifuge tube is taken out, liquid in pipe is divided into 4 layers, is followed successively by from top to bottom：Blood plasma, white cloud thin layer (single core
Cellular layer), lymphocyte separation medium, red blood cell and granulocyte.Mononuclearcell layer is drawn, adds the PBS washings of 3-4 times of volume
Cell, 1500rpm, 10min, the PBS that 4-5 times of volume is added toward cell precipitation are washed again, 1000rpm, 10min.
Cell precipitation is resuspended with GTT551 serum free mediums of the 30-40mL containing 10% autologous plasma, is put into 37 DEG C,
5%CO2Incubator stands 2h, harvests attached cell and suspension cell, is respectively used to induce differentiation into the training of DC and T lymphocytes
Support.
3. induction differentiation and tumor antigen activity epitope peptide sensitization DC of the PMNC to DC
1) attached cell for harvesting step 2 is induced to differentiate into DC, and specific method is as follows：
The GTT551 serum free mediums that 20mL contains 10% autologous plasma are added in toward attached cell blake bottle, addition is dense eventually
Spend for 500U/mL IL-4,1000U/mL GM-CSF, be put into 37 DEG C, 5%CO2Continue to cultivate in incubator.GM-CSF and IL-4
Collective effect can make monocyte directed differentiation be immature DC, and DC now has stronger antigen uptake and working ability,
But antigen submission ability is very weak.
In the 3rd day according to GTT551 serum free medium of the cell growth status supplement containing 10% autologous plasma, addition is eventually
Concentration is 500U/mL IL-4,1000U/mL GM-CSF, is put into 37 DEG C, 5%CO2Continue to cultivate in incubator, given birth to according to cell
Long situation adds above-mentioned culture medium and cell factor.
In the 6th day harvesting, adjustment cell density was 1X106/ mL, it is separately added into 10ug/mL tumor antigen activity
Epitope peptide.1000U/mL TNF-αs are added after 2h, stimulates DC ripe, plays Antigen-presenting role.In the 7th day harvest load tumour
The DC vaccines of antigen active epitope peptide.
2) DC phenotypic evaluation
Take respectively culture the 3rd day immature DC (immature DC, imDC) and the ripe DC of the 7th day (mature DC,
MDC the detection of cell surface marker thing) is carried out.
0.25% pancreatin digestion DC is added, terminates 1000rpm, 10min after digestion, by cell precipitation with 4 DEG C of precoolings
0.01mol/L PBS liquid washs 2 times, 1000rpm, 10min, cell precipitation is resuspended with 200ul PBS, adjustment cell density is
1X106/ ml, lucifuge add CD83, CD86, CD1a and HLA-DR fluorescence antibody, and 4 DEG C of lucifuges are incubated 30min, add 2mL PBS,
L000rpm, l0min, cell to be washed to remove uncombined antibody, remove supernatant, cell precipitation is resuspended with PBS liquid, 4 DEG C of censorships,
Flow cytometer detects.
Flow cytomery result shows high expression CD83, CD86, CD1a and the HLA-DR of the mDC of culture in the 7th day, shows
Success induces DC ripe.
Table 1DC cell surface markers expression (, %)
*：Compared with imDC groups, P<0.05.
3) cytokines measurement (IL-12)
The mDC of imDC and the 7th day for taking DC to cultivate the 3rd day is detected.The ELISA Plate of coated antibody is taken out, is set
TMB (3,3', 5,5'-Tetra methylbenzidine) blank colour developing hole, sequentially adds what 0.1mL was diluted by certain multiple
Standard items and the sample diluted with sample diluting liquid.ELISA Plate is plus lid, 37 DEG C of reaction 90min.Automatic washer is used after reaction
Suck the liquid in ELISA Plate.The anti-human IL-12 antibody working solution of biotin is sequentially added into (TMB blank colour developing by every hole 0.1mL
Except hole), 37 DEG C are reacted 60min, and 0.01M PBS are washed 3 times.ABC working solutions are sequentially added into (TMB blank by every hole 0.1mL
Except colour developing hole), 37 DEG C are reacted 30min, and 0.01M PBS are washed 5 times.TMB nitrite ions are sequentially added by every μ L of hole 90,37 DEG C are kept away
Light reaction 20-25min, TMB terminate liquids are sequentially added by every hole 0.1mL, now blueness is vertical turns yellow, with ELIASA in 450nm
Determine OD values.Sample OD values are drawn after subtracting blank well OD values using OD values and standard concentration as XY axles, are looked on standard curve
Extension rate is multiplied by after looking for IL-12 concentration, calculates IL-12 concentration in sample.
As a result show the mDC expression IL-12 horizontal expression apparently higher than imDC, there is significant difference (P<
0.05)。
4. PMNC is to CTL proliferative inductions
1) it is the suspension cell that step 2 harvests is as follows to CTL proliferative inductions, specific method：
Suspension cell density is adjusted to 1X10 with the GTT551 serum free mediums containing 10% autologous plasma6/ mL, insert pre-
First it is coated with the blake bottle of 50ng/mL CD 3-resisting monoclonal antibodies, adds final concentration of 100ng/mL PHA, 1000U/mL IFN-
γ, 37 DEG C are put into, 5%CO2Continue to cultivate in incubator.
Final concentration of 100U/mL IL-1 α, 300U/mL IL-2,30ng/mL IL-7 were added in the 2nd day, is put into 37 DEG C,
5%CO2Continue to cultivate in incubator.
In the 3rd day according to GTT551 serum free medium of the cell growth status supplement containing 10% autologous plasma, addition is eventually
Concentration is 300U/mL IL-2,30ng/mL IL-7,100ng/mL PHA, is put into 37 DEG C, 5%CO2Continue to cultivate in incubator.
DC vaccines were added in the 7th day, and (ratio of DC vaccines and T lymphocytes is 1：10) co-culture, carry out first round CTL
Induced amplification, culture medium also add in addition to final concentration of 300U/mL IL-2,30ng/mL IL-7,100ng/mL PHA is added
The final concentration of anti-CD28 monoclonal antibodies of 30ng/mL, the anti-ICOS monoclonal antibodies of final concentration of 30ng/mL are added again after 2h, every
Its GTT551 serum free mediums of supplement containing 10% autologous plasma, adds above-mentioned cell factor, to keep cell factor most
Good activity, 37 DEG C are put into, 5%CO2Continue to cultivate in incubator.
Adding DC vaccines again in the 14th day, (ratio of DC vaccines and T lymphocytes is 1：10) co-culture, strengthen T lymphs
Induction differentiation of the cell to CTL, carries out the second wheel CTL induced amplifications, and culture medium adds final concentration of 300U/mLIL-2,30ng/
The anti-CD28 monoclonal antibodies of mL IL-7,100ng/mL PHA, 30ng/mL, the anti-ICOS of final concentration of 30ng/mL are added again after 2h
Monoclonal antibody, GTT551 serum free medium of the supplement containing 10% autologous plasma, adds above-mentioned cell factor, is put into 37 every other day
DEG C, 5%CO2Continue to cultivate in incubator.
CTL was harvested in the 21st day.Set up general culture method group as a control group, general culture method group is using conventional cell
Combinations of factors culture, including IL-2, IFN-γ, IL-7 and PHA.
The anti-CD28 monoclonal antibodies and anti-ICOS monoclonal antibodies that the present invention adds can stimulate T lymphopoiesis, production
Raw cytokine profiles, and CTL is divided into, preferred embodiment is to add again after adding anti-CD28 monoclonal antibodies 2h or so (1.5-3h)
Add anti-ICOS monoclonal antibodies.
The combination of cytokines that present invention culture CTL is used can promote T lymphocyte activations and propagation, and it is thin to improve T lymphs
Born of the same parents clone number, and purity is high, and the DC vaccines prepared can significantly improve the CTL of induction cytotoxicity.
2) cell count
The CTL quantity that general culture method group obtains is (1.2 ± 0.4) × 109, anti-CD28 monoclonal antibodies group (conventional training
Support method group+anti-CD28 monoclonal antibodies) the CTL quantity that obtains is (1.9 ± 0.5) × 109, anti-ICOS monoclonal antibodies group is (often
Advise cultivation group+anti-ICOS monoclonal antibodies) the CTL quantity that obtains is (1.6 ± 0.4) × 109, using new culture side of the invention
The CTL quantity that method obtains is (2.8 ± 0.9) × 109, the CTL quantity that new cultural method obtains has compared with other cultivations
Significant significant difference (P<0.05).
3) CTL phenotypic evaluation
The CTL of the 7th day T lymphocyte not co-cultured with DC vaccines and culture in the 21st day is taken to carry out cell phenotype detection：
The cell precipitation for centrifuging acquisition is washed 2 times, 1000rpm, 10min with the 0.0lmol/L PBS liquid of 4 DEG C of precoolings,
Cell precipitation is resuspended with 200ul PBS, adjustment cell density is 1 × 106/ mL, lucifuge add CD3, CD4, CD8 antibody, and 4 DEG C are kept away
Light is incubated 30min, adds 2ml PBS, l000rpm, l0min, and washing cell removes supernatant, cell to remove uncombined antibody
Precipitation is resuspended with PBS liquid, 4 DEG C of censorships, flow cytometer detection.Set up T cell group, general culture method group and new cultivation
Group, wherein T cell group for load tumor antigen activity epitope peptide DC co-culture T lymphocytes, general culture method group
The CTL co-cultured with the DC for loading tumor antigen activity epitope peptide is prepared for general culture method, new cultivation group is trained for the present invention
The method of supporting prepares the CTL co-cultured with the DC of load tumor antigen activity epitope peptide.
As a result show that general culture method group and new cultivation group CD3+ cell proportions are higher than T cell group, but anticipated without statistics
Justice, and the CD4+ cell proportions of general culture method group and new cultivation group decline, the rise of CD8+ cell proportions, with T cell group phase
Than having statistical significance (P<0.05), and new cultivation group than general culture method group the notable rise of CD8+ cell proportions.
Table 2CTL cell surface markers expression (, %)
4) cytokines measurement (IFN-γ)
The ELISA Plate of coated antibody is taken out, TMB blank colour developing hole is set, 0.1mL is sequentially added and is diluted by certain multiple
Standard items and with sample diluting liquid dilute sample.ELISA Plate is plus lid, 37 DEG C of reaction 90min.With automatic board-washing after reaction
Machine sucks the liquid in ELISA Plate.The anti-human IFN-γ antibody working solution of biotin is sequentially added into (TMB blank by every hole 0.1mL
Except colour developing hole), 37 DEG C are reacted 60min, and 0.01M PBS are washed 3 times.ABC working solutions are sequentially added into (TMB by every hole 0.1mL
Except blank colour developing hole), 37 DEG C are reacted 30min, and 0.01M PBS are washed 5 times.TMB nitrite ions are sequentially added by every μ L of hole 90,37
DEG C lucifuge reaction 20-25min, TMB terminate liquids are sequentially added by every hole 0.1mL, and now blueness is vertical turns yellow, is existed with ELIASA
450nm determines OD values.Sample OD values are drawn after subtracting blank well OD values using OD values and standard concentration as XY axles, in standard curve
Extension rate is multiplied by after upper lookup IFN-γ concentration, calculates IFN-γ concentration in sample.If control group, general culture method group and new
Cultivation group, every group sets 3 multiple holes.
As a result show, new cultivation group secretion of gamma-IFN level has considerably beyond the secretion level of general culture method group
Significant difference (P<0.05).
5. cell killing is tested
It is 1X10 to adjust CTL (effector cell) and human glioma cells U251 (target cell) density respectively6/ mL and 1X107/
ML, by effect target than 5：1、10：1、20：1 adds effector cell's suspension and target cell suspension toward 96 orifice plates, cumulative volume 200uL, puts
Enter 37 DEG C, 5%CO220uL CCK-8 are added per hole after 48h is cultivated in incubator, continue to be incubated above ELIASA detection after 2h, in
450nm reading OD values, killing rate=【1- (experimental group OD values-effector cell organizes OD values)/target cell group OD values】* 100%.Experiment
It is divided into blank control group, target cell group, effector cell's group and experimental group (new cultivation group, general culture method group), experimental group is equal
By tumor antigen activity epitope peptide gp100:209-217(SEQ ID NO：And HER2 1):369-377(SEQ ID NO:2) load
DC induction prepare CTL.
As a result show, with effect target than increase, new cultivation group and general culture method group are to human glioma cells U251
Killing rate gradually rise, when effect target ratio be 20:When 1, to the lethal most strong of human glioma cells U251, killing rate is most
It is high.Also, under the conditions of same effect target ratio, killing rate of the new cultivation group to human glioma cells U251 compares general culture method
Group killing rate significantly improves, and has statistical significance (P<0.05).
Sequence table
<110>Yin Guan bio tech ltd of Shenzhen
<120>Prepare DC-CTL kit and its application
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Claims (4)
1. a kind of method that kit for preparing DC-CTL prepares DC-CTL, it is characterised in that the reagent of the DC-CTL
Box, including：
Separate the reagent of mononuclearcell；
Induce the reagent of DC differentiation and maturations；
Sensitization DC tumor antigen activity epitope peptide；
Promote the reagent of CTL amplifications and activation, the reagent of promotions CTL amplification and activation be AntiCD3 McAb monoclonal antibody,
One or several kinds of and anti-CD28 monoclonal antibodies in IFN-γ, IL-1 α, PHA, IL-2, IL-7, anti-ICOS monoclonals
The combination of antibody；
The reagent of the induction DC differentiation and maturations is the one or several kinds in cell factor IL-4, GM-CSF, TNF-α；IL-
4th, GM-CSF, the concentration of TNF-α are respectively 400-600U/mL, 800-1200U/mL, 800-1200U/mL；
CD 3-resisting monoclonal antibody, IFN-γ, IL-1 α, PHA, IL-2, IL-7 in the reagent of the promotion CTL amplifications and activation
Concentration be respectively 50-80ng/mL, 1000-1200U/mL, 80-150U/mL, 50-100ng/mL, 300-500U/mL, 20-
30ng/mL ；The anti-CD28 monoclonal antibodies, the concentration of anti-ICOS monoclonal antibodies are respectively 20-50ng/mL, 20-
40ng/mL, anti-ICOS monoclonal antibodies addition is after anti-CD28 monoclonal antibodies；
It the described method comprises the following steps：
A) using the reagent separation mononuclearcell of separation mononuclearcell；
B) broken up using the reagent inducing peripheral blood mononuclear cell of induction DC differentiation to DC；
C tumor antigen activity epitope peptide sensitization DC) is used, then stimulates DC ripe；
D) using the T lymphocytes in the reagent amplification PMNC for promoting CTL amplifications and activation, and T is induced to drench
Bar cell breaks up and bred to CTL；The reagent of the separation mononuclearcell is Ficoll lymphocyte separation mediums, or
Percoll cell separating liquids；The tumor antigen activity epitope peptide of the sensitization DC is gp100：209-217、HER2 ：369-
377 combination；
The step C) tumor antigen activity epitope peptide sensitization is first added in DC incubations, TNF-α rush is added after 1-2h again
It is ripe；The step D) in the order of the anti-CD28 monoclonal antibodies of addition and the anti-ICOS monoclonal antibodies of addition be：Addition is anti-
Anti- ICOS monoclonal antibodies are added after CD28 monoclonal antibodies 1.5-3h again.
2. the method as claimed in claim 1 for preparing DC-CTL, it is characterized in that：The gp100:209-217 amino acid
Sequence such as SEQ ID NO：Shown in 1, the HER2:369-377 amino acid sequences such as SEQ ID NO：Shown in 2.
3. the method as claimed in claim 2 for preparing DC-CTL, it is characterized in that：The various tumor antigen activity epitope peptides
Concentration be 10 μ g/ml-20 μ g/ml.
4. the method for preparing DC-CTL as described in claim any one of 1-3, it is characterized in that, the kit also includes group
Point：
PBS buffer solutions and serum free medium.
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| PCT/CN2015/088648 WO2016034094A1 (en) | 2014-09-03 | 2015-08-31 | Kit for preparing dc-ctl and application of kit |
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| CN104651311B (en) * | 2014-09-03 | 2018-03-13 | 深圳市茵冠生物科技有限公司 | Prepare DC CTL kit and its application |
| WO2016201658A1 (en) * | 2015-06-17 | 2016-12-22 | 深圳市达科为生物工程有限公司 | Method for preparing tumor specific ctls |
| 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 |
| CN105154400B (en) * | 2015-09-30 | 2021-02-02 | 中国人民解放军第三0二医院 | Amplification method of polyclonal anti-hepatitis B virus immune cells |
| 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 |
| 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 |
| 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 |
| CN113755444A (en) * | 2020-12-29 | 2021-12-07 | 赛元生物科技(杭州)有限公司 | DC cell induction kit and DC cell induction culture method |
| CN112961828A (en) * | 2021-03-09 | 2021-06-15 | 傅松涛 | Method for promoting DNT cell expansion and activation |
| 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 |
| CN113416697A (en) * | 2021-06-30 | 2021-09-21 | 广州先康达生物科技有限公司 | anti-HBV mixed immune cell preparation and preparation method thereof |
| CN113846059B (en) * | 2021-10-12 | 2024-02-27 | 北京中卫医正科技有限公司 | In-vitro amplification culture method for CTL (cytotoxic T lymphocyte) cells activated by induction of 3D tumor holoantigen |
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