CN108379569A - The DC vaccines of efficient load tumour antigen and its method of induced amplification specific for tumour antigen CTL - Google Patents
The DC vaccines of efficient load tumour antigen and its method of induced amplification specific for tumour antigen CTL Download PDFInfo
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- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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Abstract
A kind of method that the present invention provides the DC vaccines and its induced amplification specific for tumour antigen CTL of efficient load tumour antigen.The DC vaccines are prepared by the following method to obtain：(1) tumour antigen is prepared；(2) it acquires, separation and high-efficient culture expand DC cells；(3) tumour antigen and activator sensitization is used to activate DC cells；(4) closing of DC cell surfaces PD L1 molecules.The present invention also provides sequential blood drawings in the method for the external evoked amplification CTL of DC vaccines, and the closing to external evoked 1 molecules of amplification CTL surface PD.The present invention solves the problems, such as that existing tumour antigen immunogenicity is not strong, Antigenic Target is not complete, patient tumour antigen is not easy to obtain, DC and CTL amplification in vitro lazy weights.DC vaccines can effectively induce specific for tumour antigen CTL in vitro and in vivo, and efficiently can generate specific killing to tumour, no obvious toxic-side effects, and potential applicability in clinical practice is wide.
Description
Technical field
The present invention relates to medicine and biotechnology, specifically, it is thin to be related to a kind of low single core of initial amount peripheral blood
Born of the same parents efficiently prepare self load or the DC vaccines of other source tumour antigens and its system of induced amplification specific for tumour antigen CTL
Preparation Method.
Background technology
Tumour is the inevitable outcome of human evolution, and threatens the major disease of human health and life.China is swollen at present
Tumor incidence rises year by year, and traditional operation, chemotherapy, radiotherapy etc. all has larger limitation.Operation can only cut off naked eyes
Finding lump can not thoroughly remove tumour cell, the more vivo environment without changing tumor patient loss of equilibrium；Chemotherapy, radiotherapy can
With fragmentation unit tumour cell, but quite a few entity tumor is insensitive to Radiotherapy chemotherapy, and many side effects are exempted to patient's
Epidemic disease system and quality of life bring series of negative influence, can not also prevent the relapse and metastasis of tumour；Existing molecular targeted medicine
Object is of limited application, and easily generates drug resistance.Therefore, there is an urgent need for open up new therapy approach and means.
Numerous studies show that recovery/reconstruction patients itself antineoplastic immune function is the indispensable strategy of oncotherapy
One of.Immunotherapy of tumors has become the fourth-largest means of oncotherapy in recent years, and tumor-specific immunity treatment is weight
In it is weight, influence tumor-specific immunity treatment several elements include：1) correctly can induce and expand sufficient amount has
The Dendritic Cells (DC) and tumor specific cytotoxicity T cell (CTL) of powerful antigen presentation function；2) best tumour is anti-
It is former；3) in the infiltration to tumor tissues of chemotactic tumor specific cytotoxicity T cell；4) the immunosupress machine of tumor microenvironment is cut off
System.
The sources DC can be different, but are most easily to obtain mononuclearcell from peripheral blood separation.Since DC and CTL is expanded
Relatively difficult, for the cell quantity for meeting needed for treatment, people usually take measures on the cell quantity of culture starting, wrap
It includes：1) 2-3 days injection GM-CSF carry out bone marrow mobilization before mononuclearcell acquisition, to increase the ratio of peripheral mononuclear blood cell
And absolute counting；2) blood cell separator is used, largely mononuclearcell is acquired by the thousands of milliliters of Peripheral Circulation.In this way
Processing strong external intervention actually has been carried out to the marrow of patient and peripheral blood, especially for tumor patient art
Afterwards, the organismic internal environments such as immune system disorderly after Radiotherapy chemotherapy have further resulted in very big interference.Therefore, clinical anxious
A kind of body need to be had not significant impact, started with smaller cell culture initial amount, lived by culture amplification and antigen sensibilization
After change, it can meet the DC vaccines of Treatment need and its effector cell CTL of induced amplification in cell quantity and phenotype.
Other than cell quantity, best tumour antigen is the key that ensure tumor-specific immunity treatment accuracy,
It needs to meet three aspects：1) specificity and stronger immunogenicity of tumour antigen；2) multiple target point；3) individuation.It is so far
Only, it has been found that some are in normal structure low expression and in the tumor associated antigen (TAA) of tumor tissues height expression；Minority exists
Normal structure is not expressed (except testis and placenta), the tumour specific antigen (TSA) only expressed in tumor tissues；Pass through group
Knit the tumour-specific polypeptides of sample storehouse and organization chip and the extensive screening of monoclonal antibody；Believed based on gene sequencing and biology
Cease the neoantigen that credit analysis obtains (for the Neo-Antigen of individuation)；But it for most of tumours, does not still obtain
There is extensive representative tumour specific antigen recognized by many.Tumour antigen at present in existing tumor vaccine is each has something to recommend him：
Full tumour cell tumor seedling and Tumor lysate tumor seedling antigen broad covered area, immunogenicity is strong, but more containing impurity, poor specificity；
Tumor relative polypeptide high specificity, but less immunogenic；Anti-idiotype Antibody Vaccine is often single target spot；Genetic modification swells
Tumor vaccine attempts to improve the immunogenicity of tumour or the activity of antigen presenting cell, but is generally also single target spot, and transgenosis is grasped
Work itself is exactly a kind of damage to antigen presenting cell.Tumour cell discharge vesica antigen (excretion body) can meet multiple target point and
The strong requirement of particulate antigen immunogenicity, but the antigen in cells and supernatant is only collected in preparation process, it has but lost swollen
A large amount of antigens on oncocyte surface.Change in view of the polygenes of tumour cell, the height heterogeneity and tumour of tumor tissues are suffered from
The height of person is personalized, and the specific active immunotherapy of tumour is made to still suffer from great challenge.In addition, tumor vaccine is as a kind of
Biological agent is also contemplated that the efficiency of the obtained tumour antigen of production process from production angle.There is an urgent need to develop target spot covering surfaces
Extensively (multiple target point), specificity is high, the strong tumour antigen preparation method of immunogenicity, to meet the novel DC vaccine antigens of individuation
Load, and can strong induction in vitro and in vivo and amplification multiple target point tumor specific cytotoxicity T cell.
Tumor patient usually has different degrees of immune tolerance, and how break immune is resistant to, and repairs or excitation dendron shape is thin
The powerful antigen submission ability of born of the same parents (DC), it is tumour immunity to make its abundant induced activation tumor specific cytotoxicity T cell (CTL)
The key for the treatment of.Recently, effect of the immune detection point in oncotherapy receives more and more attention, PD-1 antibody and PD-
L1 antibody is currently used to the treatment of tumour.But that people's common concern is the PD-1 that T cell surface is closed with PD-1 antibody
Molecule, with the PD-L1 of PD-L1 antibody blocking tumor cells expressions, to activate or enhance the function of T cell.Actually not only swell
Oncocyte can express PD-L1, and DC can similarly express PD-L1.The ripe DC being especially activated after load antigen can be with
Height expression PD-L1, this is also one kind " brake " mechanism of DC inducing specific immunities, and unclamps this " brake " and effectively answer
In the treatment of clinical disease, using the PD-L1 receptors of closing DC cell surfaces, realize that the antigen of DC is passed to the maximum extent
It is in and activates the ability of CTL, the PD-1 molecules on the surfaces joint closing CTL, the tumor specific cytotoxicity for improving CTL is horizontal, right
It is a kind of approach of worth exploration to break DC tolerances and the immunization therapy of tumour-specific, individuation.
Invention content
The object of the present invention is to provide a kind of DC vaccines of efficient load tumour antigen and its induced amplification tumour antigen are special
The method of anisotropic CTL, the DC vaccines are using low initial amount peripheral blood mononuclear cells come efficiently to prepare load self or other
Source tumour antigen effectively induces, extensive amplification multiple target point tumor specific cytotoxicity T cell in vitro and in vivo
(CTL), it is used for the clinical treatment of tumor patient.
In order to realize the object of the invention, present invention firstly provides a kind of preparations of the DC vaccines of efficient load tumour antigen
Method includes the following steps：
(1) tumour antigen is prepared；
(2) acquisition peripheral blood detaches mononuclearcell, with adherent low initial amount cell directional induction and amplification DC cells；
(3) tumour antigen and activator sensitization is used to activate DC cells；
(4) DC cell surface PD-L1 molecules are closed, DC vaccines are harvested.
In above-mentioned preparation method, tumour antigen and the peripheral blood of step (2) of step (1) come from same individual or different
Body depends on clinical acquisition autologous tumor tissue or the feasibility of cell and the type of tumour antigen.
Step (1) the tumour antigen type is selected from Tumor Tissue Lysates, tumor cell lysate, tumor cell surface
Proteantigen, tumour cell Membrane protein antigen, tumour cell excretion body, other tumours correlation or specific proteins, tomour specific
Property polypeptide, be based on gene sequencing and bioinformatic analysis, predict and prepare one kind or more in the personalized neoantigen of height
Kind.
The tumour antigen both can be used alone, and can also be used in mixed way.The implementation of the preferred version of the present invention
In example, tumour antigen used is that tumor cell surface proteantigen, tumour cell Membrane protein antigen and tumour cell excretion body are pressed
The mixture of mass ratio 1: 1: 2.
Step (1) tumour antigen is obtained by being chemically treated with the following methods of the sequential progress of physical heating：To swollen
After arsenic trioxide culture is added in oncocyte, after tumour cell diminution is rounded, adherent rate reduces, after cytoplasm blebbing, 40-46
DEG C culture cell, to induce the expression of heat shock protein.
Preferably, to it is being detached from patient's tumor tissues or build be culture tumour cell in final concentration of 1umol/ is added
37 DEG C of the arsenic trioxide of L-15umol/L, 5%CO2Culture 4-24 hour, wait for tumour cell diminution be rounded, adherent rate reduction,
After cytoplasm blebbing, 43 DEG C, 5%CO2Culture 1-12 hours.
In the preparation method of above-mentioned DC vaccines provided by the invention, step (2) acquires and the method that is separately cultured DC cells
It is as follows：
1) separation of peripheral blood mononuclear cells：Anticoagulation is extracted from peripheral vein to be added on lymphocyte separation medium, from
The heart detaches mononuclearcell, serum-free DC cell culture fluids is added, cell is resuspended, until cell concentration is 3 × 106-1.5×107A/
ml；
2) separation, directional induction and amplification of DC cells：Mononuclearcell adhere-wall culture 2-6 hours；It is sucked out not adherent thin
Born of the same parents, gained attached cell are DC precursors；The rh-IL-4 and final concentration of final concentration 1000IU are added into DC precursors
The th-GM-CSF of 1000IU, directional induction and amplification DC precursors 7-9 days, gained immature DC cell is for sensitization activation.
The present invention prepares DC vaccine approach key point in step (2) and is, preparing the DC vaccines per person-portion only needs
The peripheries 40-50ml anticoagulation is acquired, with a large amount of DC of mononuclearcell induced amplification of low starting quantity.
One embodiment of the present of invention the step of in (2), the method for acquiring and being separately cultured DC cells is as follows：
1) separation of peripheral blood mononuclear cells：Extract periphery anticoagulation 40-50ml (preferably 45m1), Conventional density gradients
Centrifugal process obtains mononuclearcell, serum-free DC cell culture fluids is added after washing, cell is resuspended, and carries out cell count, and adjustment is thin
Born of the same parents a concentration of 3 × 106-1.5×107A/ml.
The serum-free DC cell culture mediums are special culture media (BYN-PD701), and the lymphocyte separation medium can come
From commercially available commercially produced product, it is preferably purchased from Sigma Products.
2) separation, directional induction and amplification of DC cells：Cell is at 37 DEG C, 5%CO2In incubator, with serum-free DC cells
Culture medium (BYN-PD701) adhere-wall culture 2-6 hours；The cell that non-attached cell can be used for the DC activation of tumour antigen sensitization is sucked out
Malicious T cell (DC-AT) or cytokine induced kill cell (CIK) amplification cultivation, gained attached cell are that DC precursors are thin
Born of the same parents；The rh-IL-4 and final concentration 1000IU of DC cells special culture medias, final concentration 1000IU is added into DC precursors
Rh-GM-CSF, in 37 DEG C of 5%CO2In incubator, directional induction and amplification DC precursors 7-9 days, gained immature DC cell has
Wait for that sensitization activates.According to cell growth and amplification situation, individuation timely and appropriate discovery adds the rh-IL-4 containing final concentration 1000IU
With the serum-free DC cells special culture media (BYN-PD701) of the rh-GM-FCS of final concentration 1000IU.
In the preparation method of the DC vaccines of efficient load tumour antigen provided by the invention, it is thin that step (3) sensitization activates DC
The method of born of the same parents is：Using the antigen in the autologous tumor source from peripheral blood donor, or according to the pathological examination of patient as a result, plus
Enter the mixed rumour antigen of the corresponding tumour to match to pathological examination result, while activator is added, incubation obtains sensitization work
The DC cells of change；The activator is bacterial product, complex immunity globulin, monoclonal antibody, micromolecular compound.
Preferably, the activator is Polyinosinic injection, anti-human CD 40 monoclonal antibody, TNF-d or bacterial product come
The marketed products (injection) in source.In the embodiment of the present invention, the mixed rumour antigen of selection contains tumor cell surface antigen,
Membrane antigen, tumour cell excretion body.It will be appreciated by those skilled in the art that mixed rumour antigen includes but not limited to following
Any two or multiple mixing of tumour antigen：Tumor Tissue Lysates, tumor cell lysate, tumor cell surface albumen are anti-
Original, tumour cell Membrane protein antigen, tumour cell excretion body, other tumours be related or specific proteins, tumour-specific polypeptides,
Height personalization neoantigen based on gene sequencing.
Specifically, the method for step (3) tumour antigen and activator sensitization activation DC cells is as follows：To DC cell concentrations
It is 5 × 106-1.5×107The mixed rumour antigen of 1/10 volume is added in the serum-free DC cell culture fluids of a/ml, and (tumour is thin
Cellular surface proteantigen: tumour cell Membrane protein antigen: tumour cell excretion body mass ratio 1: 1: 2), while final concentration is added
The activator of the Polyinosinic injection and final concentration 2-10 μ g/ml of 0.2-0.8 μ g/ml is incubated overnight, and it is thin to obtain ripe DC
Born of the same parents.
It is highly preferred that the activator is selected from anti-human CD 40 monoclonal antibody.
In the preparation method of the DC vaccines of efficient load tumour antigen provided by the invention, step (4) is activated to sensitization
Anti human PD-L 1 antibody is added in DC cells afterwards, is incubated 0.5-2 hours at 30-40 DEG C, to close PD-L1 points of DC cell surfaces
Son.
Specifically, in step (4), the anti human PD-L 1 antibody of final concentration 1-10ug/ml is added into ripe DC cells,
37 DEG C of 5%CO2Under the conditions of be incubated 0.5-2 hours, close DC cell surface PD-L1 antigen molecules, harvest closing after DC cells,
The DC vaccines of as efficient load tumour antigen, can be directly used for active immunity treatment, i.e., induced amplification CTL in vivo.
On the other hand, the present invention also provides obtained efficient load tumour antigen DC vaccines, induced amplification is swollen in vitro
Purposes in tumor antigen specific CTL.The purposes includes two committed steps：(1) sequential to extract the non-patch of a small amount of peripheral blood separation
Wall mononuclearcell, it is external evoked with DC vaccines, and priority primary culture medium and amplification culture medium culture expand CTL；(2) body
The closing of the surfaces the CTL PD-1 molecules of outer induced amplification.It is specific as follows：
(1) tumour antigen sensitization activates the external evoked amplification tumor-specific CTLs of DC
Tumour antigen sensitization activation DC cell surface height expression CD80, CD83, CD86, HLA-DR (figure prepared by this method
1A, Figure 1B), illustrate that its maturity is higher, also can inducing tumor-specific CTL in vitro, therefore prepared in first time blood drawing swollen
On the tumor antigen sensitization DC harvest same day, 40~50mL of same tumor patient anticoagulation (i.e. sequential blood drawing) is extracted for the second time, separation is single
Not adherent mononuclearcell is sucked out in a nucleus adhere-wall culture, and DC is activated with 3-7: 1 with the tumour antigen sensitization of same day harvest
Ratio mixes, and sets and CTL serum-frees primary culture medium (BYN-PT331) is added in culture bottle, 37 DEG C, 5%CO2Incubator culture, it is secondary
Day anti-human CD3 monoclonal antibodies, anti-human CD28 monoclonal antibodies, rh-IL-2 is added to continue to cultivate.Then a according to cell growth and amplification situation
Body timely and appropriate discovery adds the serum-free amplification culture medium (BYN-PT332) containing rh-IL-2 and carries out CTL amplification cultivations.
(2) closing of the surfaces CTL PD-1 molecules is harvested with CTL
CTL was cultivated to 11-14 days, and when cell log summit of growth, before CTL harvests, anti-human PD-1 Dan Ke are added
Grand antibody 1-10ug/ml (final concentration), the PD-1 molecules on the surfaces closing CTL, 37 DEG C, 5%CO2It is incubated 0.5-2 hours in incubator.
Collect cell suspension simultaneously centrifuge washing 2 times；Leave and take quality-control sample；Gained CTL is resuspended with physiological saline 100mL, and 0.5% people is added
Blood albumin, standby human vein, which instils, to be used；Or it is resuspended using 30mL or 2mL according to clinical demand, standby torso model, intraperitoneal injection
With or Partial tumors local injection.Trypan blu e dyes, and carries out cell count, 95% or more Cell viability；Total number of cells amount 1
×1010Left and right；Flow cytometry cell phenotype shows that CD3+T cells account for 90% or more, and with CD8+ cell (cytotoxicities
T cell) dominant, (Fig. 2A, Fig. 2 B).
The tumour that the DC vaccines of the present invention induce peripheral blood to obtain in vitro is related or specific CTL belongs to guarantor of the invention
Protect range.
Not strong, Antigenic Target is incomplete, tumor patient swollen for existing tumour antigen immunogenicity for the DC vaccines of the present invention
Tumor antigen be not easy to obtain etc. the problem of problem, DC and CTL amplification in vitro lazy weights in terms of tumour antigens preparation and DC and
The closed question of the immune detection point activated in CTL Process of in vitro, it is proposed that total solution, it can be effectively in body
Outer and Immune inducing in vivo specific for tumour antigen CTL, and can specific killing, no obvious toxic-side effects efficiently be generated to tumour.
Beneficial effects of the present invention embody in the following areas：
(1) in terms of preparing tumour antigen, after present invention arsenic trioxide targeted induction tumor stem cell apoptosis, with adding
The method of heat handles tumour cell, both can also induce tumor cells expression heat shock protein with inducing apoptosis of tumour cell.
Heat shock protein can reside in endocytoplasmic reticulum, can also be expressed in cell surface, be important tumour antigen molecule companion
Companion can increase the immunogenicity of tumour antigen.
(2) in terms of the selection of tumour antigen, the available tumour antigen of DC vaccines of the present invention includes not only tumour cell
Surface protein, epicyte protein, intracellular protein, further include in culture solution outside tumour cell after tumor cell culture amplification
The vesica of row.Can full tumor-cell antigen be obtained from tumour cell different level to greatest extent in this way, Antigenic Target covers
Lid is comprehensive, and it is anti-as antigen molecule companion and vesica to have taken into account soluble protein, lipopoteins, its heat shock protein being rich in
Former graininess, increases the immunogenicity of the hybrid antigen；It simultaneously can be from autologous patient excision or biopsy tumor tissues, chest and abdomen
Oedema oncocyte obtains antigen, has expanded the source of autologous tumor antigens, has met the requirement of individuation.
(3) in terms of the acquisition of peripheral blood collection capacity and DC cell concentrations, the present invention utilizes the low single core of initial amount peripheral blood
Cell can harvest comparatively high amts and the DC compared with high maturity, have after culture amplification in 8-10 days and load tumour antigen
Clinical manipulation is simple, and securely and reliably, on the one hand the features such as side reaction is small, high efficiency, it is big due to extracting to alleviate tumor patient
Amount peripheral blood brings painful and to the adverse effect of body；On the other hand it reduces the production cost, ensure that treatment DC
The sufficient dosage of cell.
(4) blocking DC cell surfaces PD-L1 to start t cell proliferation program to inhibit immunology, present invention joint
Break DC immune tolerances using CD40 monoclonal antibodies and PD-L1 monoclonal antibodies, DC is activated with CD40 monoclonal antibodies, DC tables are closed with anti-PD-L1 monoclonal antibodies
Face PD-L1.At present in terms of the immunosuppression mechanism research of PD-1/PDL-1 test points, what those skilled in the art were primarily upon
It is expression programmed cell death molecule (PD-1) after T cell activation, tumour cell then expresses PD-L1 molecules (PD-1's matches in tissue
Body), start the apoptosis of T cell if the two combines, this is one of important immunosuppression mechanism of tumor immune escape.So
And i.e. high expression PD-L1 molecules after often having ignored DC loads tumour antigen and activating, when DC is contacted with T cell, completion tumour
The apoptosis program of T cell may be started during antigen submission and T cell activation.The present invention is closed using anti-PD-L1 monoclonal antibodies
The PD-L1 on the surfaces load tumour antigen DC, blocks this immunosuppression mechanism, immune anti-to activate body antineoplastic specificity
It answers, is internal and external induced amplification tumor specific cytotoxicity T cell, and persistently play T cell effect and lay a good foundation.
(5) enhancing DC the vaccines reaction of Immune inducing in vivo tumor-specific immunity and external evoked amplification tumour-specific again
In terms of CTL, the present invention using when load tumour antigen DC vaccines harvest, acquires same tumor patient for the second time in another way
A small amount of peripheral blood detaches mononuclearcell.It is risen in the zero moment of non-adherent mononuclearcell culture, utilizes DC produced by the present invention
Vaccine is mixed with the pure T cell in higher ratio and mononuclearcell group in vitro, and excluding, tumor patient is peculiar
In the external superior microenvironment of immunosuppressive agent, antigen submission and activating T cell are completed.This method can be from 45ml peripheral bloods
It originates mononuclearcell and obtains CTL total number of cells amount 1 × 1010Left and right, higher than the DC-CIK quantity (1 × 10 of usual method amplification9
Left and right)；95% or more Cell viability；Cell surface can high expression CD3, CD8 molecule.These tumor-specific CTL adoptive transfers
To patient's body can direct killing tumour cell can improve curative effect with DC vaccine use in conjunction.
Description of the drawings
Figure 1A-Figure 1B is that DC cell surfaces height of the present invention expresses CD80, CD83, CD86, HLA-DR typical case's collection of illustrative plates.
Fig. 2A-Fig. 2 B are that CTL cell surfaces height of the present invention expresses CD3, CD8 molecule typical case's collection of illustrative plates.
Specific implementation mode
Following embodiment is used for illustrating the present invention, but is not limited to the scope of the present invention.Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, biomaterial used is commercial goods.
The preparation of the DC vaccines of 1 efficient load tumour antigen of embodiment
1. tumor cell culture
Tumor tissues or tumor tissue biopsy sample of the tumour cell from patient itself operation excision, and have with patient
There is the tumor cell line of same pathological diagnosis feature.Cell line can be the self-built system in laboratory, also be purchased from China national cell guarantor
Tibetan center or U.S. ATCC carry out in vitro culture according to the cellular biology of tumor characteristic respectively.Usually using tire containing 10-15%
The DMEM culture solutions of cow's serum, at 37 DEG C, 5%CO2It is cultivated in incubator, passage amplification.
2. induced apoptosis simultaneously expresses heat shock protein
When waiting for that cell growth to logarithmic phase, 80% is paved with bottom of bottle, drug-treated is carried out, arsenic trioxide (1- is added
15umol/L) continue at 37 DEG C, 5%CO2It is cultivated 4-24 hours in incubator.The diminution of microscopic observation tumour cell is rounded, and is pasted
Wall rate reduces and cytoplasm " blebbing ", and cell is transferred to 43 DEG C of heating, 5%CO2It is cultivated 10 hours in incubator.
3. the preparation of tumour cell excretion body
After by arsenic trioxide and heating sequential induction, apoptosis occurs for tumour cell, and attached cell suspends, and outer row is more
Excretion body.In order to harvest more antigens, can attached cell further be detached with cell scraping, then proceed to following procedure：
1) low-speed centrifugal 300-600g 10-15 minutes, tumour cell precipitation was collected to prepare tumor cell proteins class antigen
(see step 4,5).
2) it collects supernatant high speed centrifugation 10000g 30 minutes, regathers the further ultracentrifugation 100000g of supernatant 60 and divide
Clock collects precipitation, as tumour cell excretion body.
4. the preparation of tumor cell surface proteantigen, using surface protein extracts kit, Mem-PEP Plus Kit,
Purchased from Thermo, Inc.USA.
5. the preparation of tumour cell Membrane protein antigen uses Membrane protein extraction kit, Pierce Cell Surface
Protein lsolation Kit are purchased from Thermo, Inc.USA.It is operated according to commodity service manual, prepares tumor cell membrane
Proteantigen.
6. the preparation of load tumour antigen DC vaccines
1) separation of peripheral blood mononuclear cells
Using it is known in the art that easy peripheral blood mononuclear cells separation method.The present embodiment is used with lower section
Method：Aseptic collection peripheral blood 45ml, is gently added on the lymphocyte separation medium of proportion 1.077, is detached using Density gradient centrifugation
Mononuclearcell.Centrifuge tube middle white cellular layer is drawn, is moved into the centrifuge tube containing physiological saline, cell is washed, abandons
Clearly, serum-free DC cell culture mediums (BYN-PD701) are added, cell is resuspended, carry out cell count.
2) directional induction and amplification of DC
Cell is at 37 DEG C, 5%CO2Adhere-wall culture 2-6 hours in incubator, non-attached cell is sucked out, rh-IL-4, rh- is added
GM-CSF 1000IU (final concentration), 37 DEG C, 5%CO2In incubator, directional induction and amplification DC cells 7-9 days.It is given birth to according to cell
Long and amplification situation, individuation timely and appropriate discovery are added containing rh-IL-4, and the serum-free of rh-GM-CSF 1000IU (final concentration) is trained
Support base (BYN-PD701).
3) the tumour antigen sensitization of DC and activation
DC was cultivated to 7-9 days, using the antigen from autologous patient tumour source, or according to the pathological examination knot of patient
Matching corresponding mixed rumour antigen (the tumor cell surface proteantigen: tumour cell memebrane protein of 1/10 volume is added in fruit
Antigen: tumour cell excretion body mass ratio 1: 1: 2), while Polyinosinic injection (final concentration 0.2-0.8 μ g/ml), anti-human is added
CD40 monoclonal antibodies (whole 2-10 μ g/ml concentration), 37 DEG C, 5%CO2In incubator overnight.
4) closing of the surfaces DC PD-L1 molecules
DC is cultivated and sensitization was to 8-10 day, before harvesting DC, addition anti human PD-L 1 antibody (monarch grow directly from seeds object) 1-
The PD-L1 molecules on the surface 10ug/ml (final concentration), closing DC, 37 DEG C, 5%CO2It is incubated 0.5-2 hours in incubator.
5) harvest of DC cells
After the closing of the surfaces DC PD-L1 molecules, culture solution (not abandoned containing cell) is sucked out, is blown and beaten and is rushed with ice physiological saline
It washes and remains attached cell until falling off, collect simultaneously centrifuge washing 2 times of all cell suspensions；Cell physiological saline 1ml is resuspended standby
Treatment is used.Leave and take quality-control sample, conventional detection microorganism, endotoxin simultaneously；Fluidic cell cell art detects DC cell surfaces
The expression (Figure 1A -1B) of CD80, CD83, CD86, HLA-DR, by Figure 1A -1B visible DC cell surfaces height expression CD80,
CD83、CD86、HLA-DR。
DC vaccines manufactured in the present embodiment detach the low starting in adherent mononuclearcell group from 45 milliliters of periphery anticoagulations
DC precursors are measured, after culture amplification and antigen sensibilization, can finally harvest tumour antigen sensitization DC total quantitys about 1 × 108-2
×108, the needs of dosage and an induced amplification tumor-specific CTL needed for 1-2 DC vaccine therapy can be met simultaneously.It should
Method has clinical application easy to operate, securely and reliably, the features such as side reaction is small, high efficiency, is provided for clinic another excellent
The DC vaccines of choosing and the new way prepared for tumor specific cell.
The preparation of the DC vaccines of 2 efficient load tumour antigen of embodiment
Tumour cell is detached from tumor patient chest, ascites, after culture amplification, disposably from tumour cell and tumour cell
Excretion body in culture solution combines the preparation method for preparing tumour antigen.
1. collecting tumor patient hydrothorax or ascites 500-1000ml under aseptic condition, 300g is centrifuged 8 minutes, abandons supernatant.
2. physiological saline is added to be resuspended to 100ml, cell suspension is made.
3. pair weight density centrifugation：It is from the bottom to top：1st layer of 100% human lymphocyte separating liquid (density 1.077g/
ml)；2nd layer of 75% lymphocyte separation medium (density 1.077g/ml)；3rd confluent monolayer cells suspension.Centrifugation：700g, 20 minutes.
4. the cell for collecting the 1st interface and the 2nd interface, brine 2 times are sucked out respectively.
5. repetitive operation step 3, step 4 are primary.
6. the cell at the 2nd interface is tumour cell.
7. if tumour cell is enough, and desirable tumour cell is directly handled by embodiment 1, and autologous tumor is extracted obtain
Membrane protein antigen.
8. can also be handled after the tumour cell in vitro culture of separation by embodiment 1, except extraction autologous tumor is thin
Membrane-associated protein antigen, while extracting tumour cell vesica.
After prepared by tumour antigen, the method that can refer to embodiment 1 prepares tumour antigen sensitization DC vaccines and CTL.
3 tumour antigen sensitization of embodiment activates the external evoked amplification tumor-specific CTLs of DC
DC vaccines made from embodiment 1 and embodiment 2 are selected respectively.
1, on the day of DC vaccines harvest, second of 40~50 milliliters of heparin anti-coagulating of pumping, according to the 1 of 1 step 6 of embodiment)
With operation 2), the not adherent mononuclearcell of fresh separated is sucked out, with 5: 1 ratio and DC vaccines obtained (with reference to implementation
DC vaccines made from 2 method of example 1 or embodiment) mixing, it sets containing serum-free CTL primary culture mediums (BYN-PT331) culture bottle
Interior overnight, anti-human CD3 monoclonal antibodies, anti-human CD28 monoclonal antibodies, rh-IL-2 is added in next day, at 37 DEG C, 5%CO2It is cultivated in incubator.Thereafter
According to cell growth and amplification situation, individuation timely and appropriate discovery adds the serum-free amplification culture medium (BYN- containing rh-IL-2
PT332 CTL amplification cultivations) are carried out.
2, the harvest of CTL
CTL was cultivated to 11-14 days, and when cell log summit of growth, before CTL harvests, anti-human PD-1 Dan Ke are added
Grand antibody 1-10ug/ml (final concentration), the PD-1 molecules on the surfaces closing CTL, 37 DEG C, 5%CO2It is incubated 0.5-2 hours in incubator.
Collect cell suspension simultaneously centrifuge washing 2 times；Leave and take quality-control sample；CTL is resuspended with physiological saline 100ml, and it is white that 0.5% people's blood is added
Albumen, standby human vein, which instils, to be used；Alternatively, being resuspended using 30ml or 2ml according to clinical demand, standby torso model or intraperitoneal injection
With, or part is for locally injected into tumor.Trypan blu e dyes, and carries out cell count, 95% or more Cell viability；Total number of cells amount
General 1 × 1010Left and right, high person is up to 2 × 1010；Tumor patient CTL cell surfaces can high expression CD3, CD8 molecule (Fig. 2A-
2B)。
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the preparation method of the DC vaccines of efficient load tumour antigen, which is characterized in that include the following steps：
(1) tumour antigen is prepared；
(2) it extracts anticoagulation from peripheral vein and detaches mononuclearcell, with adherent low initial amount cell directional induction and amplification
DC cells；
(3) tumour antigen and activator sensitization is used to activate DC cells；
(4) DC vaccines are harvested after the PD-L1 molecules of closing DC cell surfaces.
2. preparation method according to claim 1, which is characterized in that the periphery of the tumour antigen and step (2) of step (1)
Blood depends on the clinical feasibility and tumour antigen for obtaining autologous tumor tissue or cell from same individual or Different Individual
Type；
Step (1) the tumour antigen type is selected from Tumor Tissue Lysates, tumor cell lysate, tumor cell surface albumen
Antigen, tumour cell Membrane protein antigen, tumour cell excretion body, other tumours are related or specific proteins, tumour-specific are more
It is one or more in peptide, the height personalization neoantigen based on gene sequencing.
3. preparation method according to claim 1, which is characterized in that step (1) described tumour antigen passes through chemical treatment
It is obtained with the following methods of the sequential progress of physical heating：After arsenic trioxide culture is added into tumour cell, tumour cell is waited for
Diminution is rounded, adherent rate reduces, after cytoplasm blebbing, 40-46 DEG C of culture cell, to induce the expression of heat shock protein.
4. preparation method according to claim 1, which is characterized in that prepare the DC vaccines per person-portion only needs in step (2)
40-50ml peripheral vein anticoagulations are extracted, with a large amount of DC of mononuclearcell induced amplification of low starting quantity.
5. according to any preparation methods of claim 1-4, which is characterized in that step (3) sensitization activates the side of DC cells
Method is：Using the antigen in the autologous tumor source from peripheral blood donor, or according to the pathological examination of patient as a result, being added and disease
The mixed rumour antigen for the corresponding tumour that reason testing result matches, while activator is added, it is incubated and obtains the DC of sensitization activation
Cell；
The activator is bacterial product, complex immunity globulin, monoclonal antibody, micromolecular compound.
6. preparation method according to claim 5, which is characterized in that step (4) is that the DC cells after sensitization activation add
Enter anti human PD-L 1 antibody, is incubated 0.5-2 hours at 30-40 DEG C, to close the PD-L1 molecules of DC cell surfaces.
7. the efficient load tumour antigen DC vaccines that any preparation methods of claim 1-6 are prepared, the DC epidemic diseases
Seedling can be directly used for active immunity treatment, can induced amplification specific for tumour antigen CTL in vivo.
8. induced amplification tumour is related in vitro or in vivo or the purposes of specific CTL for DC vaccines described in claim 7.
9. purposes according to claim 8, which is characterized in that induction specific for tumour antigen CTL methods are in patient the
Primary blood drawing prepares DC vaccines and harvests the same day of DC, and the separation of 40~50mL anticoagulation cirumferential bloods is extracted simultaneously second in same individual
Not adherent mononuclearcell is sucked out with DC vaccines with 3-7 in adhere-wall culture:1 ratio mixing, is added CTL serum-free Primary cultures
Base after exo-antigen is offered, is added anti-human CD3 monoclonal antibodies, anti-human CD28 monoclonal antibodies, rh-IL-2 and carries out CTL cultures；
It is changed to amplification culture medium after CTL starts cloning growth and carries out amplification cultivation；CTL is cultivated to cell log summit of growth
When, before CTL harvests, the anti-human PD-1 monoclonal antibodies of final concentration of 1-10ug/ml, the PD-1 on the surfaces closing CTL is added
Molecule is incubated 0.5-2 hours；Collect cell suspension and centrifuge washing；Leave and take quality-control sample；Gained CTL is resuspended with physiological saline,
In case patient is transfused.
10. the DC vaccines described in claim 7 induce the tumour of peripheral blood acquisition related or specific CTL in vitro.
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