CN105832769A - Immune preparation for treating tumor and preparation method thereof - Google Patents
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Abstract
The invention discloses an immune preparation for treating tumor; the immune preparation contains dendritic cells (DC) loaded with a tumor antigen and cytokine-induced killer cells (CIK), the concentration of the DC loaded with the tumor antigen in a culture liquid is 1*10<7> to 1*10<8>, and the concentration of the CIK in the culture liquid is 1*10<9> to 1*10<10>. The invention further provides a preparing method of the immune preparation for treating tumor; the preparing method comprises the steps: collection of mononuclear cells of peripheral blood; isolation and culture of the DC and the CIK; extraction of a tumor antigen gene and viral vector packaging; transfection of the DC with a virus mediated tumor antigen gene; and co-culture of the DC loaded with the tumor antigen and the CIK. The accuracy of positioning of re-transported immune cells in a human body can be improved. The objective defect of a conventional cell immunotherapy technology in the same field is not good in solid tumor treatment effect can be effectively improved; adaptation diseases are wide, and the use is safe and effective.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of for immune formulation treating tumor and preparation method thereof.
Background technology
Cancer is the major disease of serious threat human life and social development, it has also become the major reason of human death.
Within 2015, many countries die from cancer in respect of 9,000,000 people in advance, and the year two thousand thirty, this numeral was up to 11,400,000 people.Cancer
It it is the major disease of serious threat human life, it has also become the major reason of human death.Whole nation tumor Register issues
" 2015 tumor registration annual report " in data show, within 2011, China increases Malignant Tumor Case about 3,370,000 newly,
There are about 2,110,000 people the most dead.Chinese population radix is huge, therefore becomes the country that cancer mortality number is the highest in the world.
National malignant tumor sickness rate is 250.28/10 ten thousand (male 277.77/10 ten thousand, and women 221.37/10 ten thousand)；Dead
Rate is 156.83/10 ten thousand (male 194.88/10 ten thousand, and women 116.81/10 ten thousand).Report is pointed out, 2011 complete
What state's Incidence was the first is pulmonary carcinoma, annual new cases about 650,000, is secondly women with breast cancer, gastric cancer, liver
Cancer and colorectal cancer；What mortality of malignant tumors was the first is pulmonary carcinoma, annual death about 520,000, is secondly hepatocarcinoma, stomach
Cancer, the esophageal carcinoma and colorectal cancer.Cancer Mortality 0-39 year section be in reduced levels, start after 40 years old fast
Speed raises, and peaks during more than 80 age group；Mortality rate was in the age after reduced levels, 45 years old before 45 years old
Group starts quickly to raise.The five year survival rate of population of China major cancers: pulmonary carcinoma 16.1%, hepatocarcinoma 10.1%, the esophageal carcinoma
20.9%, gastric cancer 27.4%, colorectal cancer 47.2%, breast carcinoma 73.0%.
At present, the mechanism of tumor not yet forms generally accepted theory.Researcher generally approve tumor generation,
Develop closely related with the immunologic function of human body.The most under normal circumstances, one it is between tumor and the defensive enginery of body
Plant dynamic balance, and the most this balance is destroyed, it is possible to tumor occurs.The each histiocyte of health is by the external world
The impact of the factors such as factor stimulation or own cells aging variation such as, physics, biology so that some cell internal occurs
Sudden change, becomes precancerous cell；And body's immunity is low or due to dysimmunity, it is impossible to identify in time, kill,
Removing these abnormal cells, mutant cell replicates growth in tissue, destroys organ dysfunction, tumor after reaching some
I.e. occur.Tumor patient, especially malignant tumor patient often immunologic function, particularly cellular immune function are low, because of
And disease in progress, movable, poor prognosis.Therefore, improving immunity of organisms, setting up effective immunne response is that treatment is swollen
The most promising approach of tumor.
Tumor cell immunotherapy is the one in tumor biotherapy method, has significant curative effect, is oncotherapy
The new direction of future development.Science magazine is evaluated 2013 annual ten big sciences and is broken through, and immunotherapy of tumors occupies list
First.This technology is to treat tumor by the principle of " immunocyte of human body self kills tumor cell ", simultaneously can be big
Increase the immunologic function of strong man's body, suppresses growth of tumour cell.It is suitable for each period early, middle and late, to the most postoperative
Close cellular immunization treatment and can not only effectively remove postoperative residue tumor cell, moreover it is possible to improve immunity of organisms, prevent from shifting and multiple
Send out.In for, late tumor patient, can not only attenuation synergistic, accurately kill tumor cell, control tumor and increase,
Can also ease the pain for patient, make patient from the misery of Radiotherapy chemotherapy and toxic and side effects.The method become the operation that continues,
The fifth-largest oncotherapy technology after radiotherapy, chemotherapy, targeted therapies.
The know-why of immunization therapy is:
After the initiating antigen such as tumor antigen stimulate, unsensitized T cell need to be by special antigen presenting cell (antigen
Presenting cells, APC) could activate and function to its submission related antigen.
Dendritic cell (dendritic cell, DC), bone-marrow-derived lymphocyte and mononuclear phagocyte are full-time APC, distribution
In body institute in a organized way and organ.These cells express MHC I and the MHC II factor, possess powerful present antigen
Ability.
Dendritic cell (dendritic cells, DC) is to be currently known the professional antigen presenting cells that function is the strongest.Become
Ripe DC cell can effectively be bred and activation by inducing antigen-specific T cell, is mainly opening of antitumor immunity of organism reaction
Dynamic person and participant.Owing to DC is uniquely can be to the cell of non-sensitized T cell present antigen, therefore DC is antigen presentation function
The strongest APC.Additionally, DC can secrete a large amount of cytokine such as IL-12 etc. after antigen sensibilization, this type cytokines exists
Play an important role during stimulating T cell activation and antitumor.These characteristics of DC make it answer at triggering specific immunity
Play a crucial role in answering, and make DC likely be applied to tumor and the development of Other diseases immunotherapy techniques and improvement.
Cytokine induced kill cell (cytokine-induced killer cells, CIK) is mononuclearcell warp
Malignant cell is had by the class that the cytokine profiles stimulations such as anti-cd 3 antibodies, INF-γ and IL-2 produce highly to be killed
Hindering the T lymphocyte of ability, it is mainly characterized by expressing T cell antigen (such as CD3 and CD8) and NK cell receptor simultaneously
(such as CD56), it has concurrently, and the powerful anti-tumor activity of T cell is restricted with the non-MHC of NK cell kills tumor feature.
Research shows, has cooperative effect between DC and CIK cell, and the two co-cultures, and demonstrates significant antitumor
Effect.As the IL-12 of DC emiocytosis can strengthen the CIK cell lethal effect to tumor, and CIK cell is secreted
IFN-γ can promote DC more effectively present antigen.DC and CIK cell co-culture and constitute an Activated in Vitro and exempt from
Epidemic disease system.The cancerous cell inhibitory action to immunocyte can be reasonably resistant to after feedback, play and more longlasting kill tumor activity,
It is active specific immunotherapy and model that adoptive immunotherapy combines.
In normal human peripheral blood, DC and CIK cell content are the lowest, account for peripheral blood lymphocytes and CD3 respectively+T cell
The 1～5% of total amount, and the above-mentioned two class cell contents of tumor patient are substantially less than normal person.Therefore, the most in a large number
Cultivate DC and CIK cell and feed back and can correct internal immunodeficiency conditions to tumor patient, thus significantly improving
The anti-cancer ability of body.DC-CIK cell is increasingly becoming tumor adoptive cellular immunotherapy because of its efficient Tumor-cytotoxic efiect
One of the best approach.
What the DC of load tumor antigen can improve CIK kills tumor specificity, and CIK can promote DC surface co-stimulatory molecules simultaneously
Express and increase, improve its HLA-II antigen.
The cancer of each patient has unique features.Therefore, it is intended that, maximally effective treatment is that specificity is for cancer
Disease gene expression and assembled state.Gene mutation is the most relevant with human cancer with chromosomal abnormality.Regrettably, by
The generation development causing cancer in gene mutation is typically random event, and each tumour patient has the antigen table of a set of uniqueness
Reach spectrum.This feature of human cancer requires that the immune system of each patient is capable of identify that and target tumor specific antigen.
The challenge brought for the gene expression profile and mutational spectrum of tackling every kind of cancer uniqueness, personalized cancer immunity is controlled
Treatment technology can capture the hereditary information of complete, unique individual tumors, makes the dendritic cell load tumor antigen of patient,
And trigger suitable immunoreation by these dendritic cell modified, can be for expressed by different patients, different tumor
Specific antigen equip immune system, and then identify and resist specific disease.
The treatment of tumor disease experienced by traditional treatment (operation, radiotherapy, chemotherapy) to targeted therapy, then to up-to-date thin
Born of the same parents' immunization therapy, the requirement to its safety and effectiveness and individuation is the most urgent.In following medical domain, cell is exempted from
Epidemic disease therapy as chemotherapy, will become a line method for the treatment of.Along with gene sequencing, Protocols in Molecular Biology, image skill
Art, the fast development such as big data, tumor is precisely treated and also can be obtained significant progress, and will become future tumors Synthetic
The Main Patterns treated.
Tumor-targeting DC-CIK cellular immunotherapy is the disease specific sudden change different for each patient or tumor change
Hapten and the individual therapy that designs, can be described as again tumor accurate cellular immunotherapy technology.Planning commission is defended recently according to country
Message, current China formulates " precisely medical treatment " strategical planning, and this planning maybe will be included into " 13 " weight
Big science and technology is special.
Along with modern industry develops rapidly the acceleration of brought environmental pollution and aged tendency of population, the sickness rate of whole world tumor
By increasing to the 22200000 of the year two thousand thirty from 12,700,000 in 2008, add about 75%, future tumors immunization therapy
The market space is huge.Meanwhile, constantly breaking through and renewal of accurate medical field correlation technique, accurate medical treatment is increasingly
Paid close attention to by market.Wherein, cellular immunotherapy is as an important breakthrough mouth of the accurate medical field of tumor, related industry
It is expected to welcome fast-developing opportunity, and highly important effect will be played in the treatment of tumor disease.
Summary of the invention
For above-mentioned technical problem of the prior art, the invention provides a kind of immune formulation for treating tumor and
Preparation method, described kind is widely used in the immune formulation treating tumor and preparation method thereof prior art to be solved
Nonspecific immunity therapy specific aim strong, the technical problem that usefulness is the highest.
The invention provides a kind of immune formulation for treating tumor, the DC cell containing load tumor antigen and CIK
Cell, the DC cell of described load tumor antigen concentration in culture fluid is 1 × 107-1×108, described CIK
Cell concentration in culture fluid is 1 × 109-1×1010。
Further, the described DC cell of load tumor antigen and the number of CIK cell ratio is for 1:100.
Present invention also offers the preparation method of above-mentioned a kind of immune formulation for treating tumor, comprise the steps:
1) step gathering PERIPHERAL BLOOD MONONUCLEAR CELL；
2) one separates and cultivates DC and the step of CIK cell:
Using DC bed board culture medium culturing PERIPHERAL BLOOD MONONUCLEAR CELL, regulation cell concentration is 6 × 106/ ml,
Insert 37 DEG C, be the CO of 5% containing concentration of volume percent2After incubator hatches 1～3h, non-adherent cell is
CIK precursor, by CIK precursor with CIK culture fluid by 1.5 × 106/ ml density is resuspended, plants into training
Support in device and cultivate；
During cultivating CIK cell, when cultivating beginning, add IFN-γ, final concentration of 3000U/ml；
CD3 monoclonal antibody, IL-1 α and IL-2, the final concentration of 300ng/ml of CD3 monoclonal antibody is added after 24h；IL-1α
Final concentration of 2000U/ml；The final concentration of 1000U/ml of IL-2, is subsequently placed in 37 DEG C, containing body
Long-pending percent concentration is the CO of 5%2Incubator is hatched, and within the 3rd day, starts to change liquid, maintains density in 1-4 × 106
/ml；
Attached cell is DC precursor, DC precursor is added DC bed board culture medium, puts into 37 DEG C,
It is the CO of 5% containing concentration of volume percent2Incubator continues to cultivate, and every 1 day, uses DC to change liquid culture medium
Half amount changes liquid；
3) one is extracted tumor antigen gene and uses the step of viral vector packaging, by tumor sample or pathology mark
This, clean up with physiological saline solution, uses TRIzol method extracting tumor tissues total serum IgE, then
It is packaged into viral vector；
4) step using virus-mediated tumor antigen gene transfection DC cell, the half of the 5th day is cultivated in collection
Adherent immature DC, is adjusted to 5 × 10 with LonZA X-VIVO 15 serum-free medium5/ 0.2ml's
Cell density, is inoculated in culture apparatus, by the virus of the above-mentioned viral vector equipped with tumor antigen gene
Liquid adds, final concentration 2 × 107PFU/ul, virus quantity is 200MOI, 37 DEG C, containing volume basis
Specific concentration is the CO of 5%2Incubator hatches 1～3h, then washs with culture fluid, proceeds to another culture apparatus
In, add DC and change liquid culture medium, add TNF-α, the final concentration of 1000U/ml of TNF-α, continue
Continuous cultivation 40～56h, the load tumor antigen DC cell of the 7th day results maturation；
5) step that load tumor antigen DC with CIK co-cultures, by thin for the load tumor antigen DC of results
Born of the same parents add step 2) CIK cell in co-culture, load tumor antigen DC cell and CIK cell
Quantitative proportion maintains between 1:10～200, cultivates to the 14th day harvesting, and CIK cell quantity reaches
To 1 × 109-1×1010；DC cell quantity reaches 1 × 107-1×108。
Further, a step extracting tumor antigen gene, the volume of tumor tissues is less than TRIzol reagent
The 10% of volume；
Further, described viral vector is adenovirus vector.
Further, described DC bed board culture medium is LonZA X-VIVO 15 serum-free medium, containing quality hundred
Proportion by subtraction concentration is the human serum albumin of 10%, and possibly together with L-Glu, IL-4 and GM-CSF, L-Glu's is final concentration of
The final concentration of 800U/ml of the final concentration of 1000U/ml, GM-CSF of 2mmol/ml, IL-4.
Further, it is LonZA X-VIVO 15 serum-free medium that described DC changes liquid culture medium, containing quality hundred
Proportion by subtraction concentration is the human serum albumin of 10%, and possibly together with L-Glu, IL-4 and GM-CSF, L-Glu's is final concentration of
The final concentration of 1600U/ml of final concentration of 2000U/ml, GM-CSF of 4mmol/ml, IL-4.
Further, described CIK culture fluid is LonZA X-VIVO 15 serum-free medium, containing mass percent
Concentration is the human serum albumin of 10%, possibly together with L-Glu, IL-2, the final concentration of 2mmol/ml, IL-2 of L-Glu
Final concentration of 1000U/ml.
The technical characterstic of the present invention and advantage:
A) targeting interior tumor cell kills, and effectively removes cancerous cell remaining after operation, chemicotherapy and micro-
The recurrence of small lesion, effectively suppression tumor and transfer.
B) strengthen radiation sensitivity, reduce Esophageal carcinoma；The immunosuppressive action of opposing chemotherapeutics, increases
The strong sensitivity to chemotherapeutics, improves the curative effect of chemotherapy.
C) for losing surgical engine meeting or the late tumor patient of cancerous cell Preventive, its clinical condition can be alleviated
Shape, makes body immune system promote, and a part of patient occurs that tumor body reduces even disappearance or long-term band tumor
The therapeutic outcome of existence；And for the invalid patient of chemicotherapy, or chemotherapeutics is produced drug resistance
Patient, can improve sensitivity and produce good curative effect.
D) cellular immunotherapy has immunomodulating and somatic cell repair, can make chemicotherapy side effect symptom
Alleviate or disappear, strengthening patient mental's state and muscle power, be greatly promoted the life quality of tumor patient；
Skin is glossy simultaneously, black speck desalination, varicosis disappear, stop alopecia and grow, poliosis blackening
Send out and wait " rejuvenation " performance.
E) present invention is novel personalized treatment, utilizes genius morbi and the immunity of self that each patient is unique
Cell carrys out early warning and remilitarizes immune system.
F) the disease specific sudden change for different patients and variant antigens are caught.It is by exciting lasting memory T
Cell effect, overcome the immunosuppressant that the disease such as tumor and HIV (human immunodeficiency virus) causes, it is to avoid conventional immunity
The toxic reaction that in Therapeutic Method, adjuvant may cause.
G) this technology is applicable to multiple different cancer and infectious disease, it is intended to overcome obstruction individuation cell to exempt from
Epidemic disease therapy produces and commercial applications facing challenges.
DC-CIK cellular immunotherapy is to kill tumor cell with the immunocyte of human body self, can be greatly enhanced simultaneously
The immunologic function of human body, suppresses growth of tumour cell.Though the present invention can not substitute traditional operation, radiation and chemotherapy completely,
But invent and there is unrivaled advantage:
With routine clinical treatment means odds pair
On the basis of conventional method, tumor antigen RNA is supported on DC cell, allows it identify targetedly swollen
Tumor tissue is also attacked and is killed, accurate positioning, can exciting human autoimmune response accurately, obtain and preferably face
Bed curative effect.
Various immunotherapy odds pair
The present invention also has following feature:
1)Safety is high: utilize human body own cells to kill tumor cell, completely autologous (from patient itself), nontoxic pair
Effect, patient is the most painful；
2)Tumor tissues demand is low: have only to a small amount of tumor specimen, and early and late patient is the most applicable for tumor；
3)With strong points: to target tumor complete set spectrotype, the sudden change of other patients it is different from including individuality, DC cell can lure
Deriving the immunocyte of killing tumor cell, CIK cell has non-specific killing tumor cell；
4)Lasting immunity: start body immune system, recovers body's immunity, lasting killing tumor cell, hinders for a long time
Only cancerous cell diffusion, recurrence；
5)General: rebuild and improve the body's immunity of entire patient, identification comprehensively, search, killing tumor cell,
Effectively prevent recurrence and the transfer of tumor；
6)Completeness: improve body immunity, remaining tumor cells and small metastatic lesion in thorough purged body；
7)Indication is wide: the effectively most entity tumor for the treatment of, and can eliminate and the tumor of transfer insensitive to Radiotherapy chemotherapy
Cell.
Tumor-targeting DC-CIK immunotherapy (tumor targeted DC-CIK immunotherapy) is by extracting
Mononuclearcell in patient self peripheral blood, the middle DC cell induced and load tumor antigen between GMP cleaning sterile,
Expand the most in a large number with after CIK cell co-cultivation, feed back after quality testing is qualified to the therapeutic process of patient.
Main application is the DC cell making load target tumor antigen, has and has specific recognition effect, energy to tumor cell
Enough various entities of popularity identification and neoplastic hematologic disorder；Malignant cell is killed or drops by DC with CIK synergy completely
Low and stable when certain level, can reach clinical cure or the purpose of tumor control of tumor.
The principle of the present invention is: gather a fritter tumor and a small amount of blood sample, uses leucocyte removal method to collect purification and suffers from
The dendritic cell of person self.From patient's specimen (tumor tissues or pathogen), separate a complete set of RNA, use adenovirus to carry
Body loaded equipment is on DC so that it is for disease antigen.DC cell ripe, antigen load is again with CIK cell altogether
Cultivate, can the propagation of effective stimulus CIK cell and immunocompetence.After results, intravenous injection feeds back together with patient's blood plasma,
Body initial CD8+T cell development can be stimulated to become CD8+CD28+ memory T cell, and then cause new cellular immunization.This
Active immunity and passive immunity are effectively combined and are integrated by invention, and tumor tissues is carried out targeting attack, significantly contract
The time that short sufferer immune activation and target kill.Good effect, instant effect.
The present invention compares with prior art, and its technological progress is significant.Owing to the tumor antigen of load can be due to illness different,
The present invention can improve the immunocyte accuracy in human body inner position of feedback.Effectively improve previously same domain cell
The objective deficiency that immunotherapy techniques is not good enough to solid tumor curative effect, indication is extensive, uses safely and effectively.The present invention is not only
Tumor patient specific tumour immunne response can be excited, moreover it is possible to inducing immunological memory, make human body obtain permanent Antineoplastic effect,
Internal residual prevent it from recur after cleaning routine treatment, is one treatment means brand-new, special, effective.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of DC cell.
Fig. 2 is DC-CIK cell form under phase contrast microscope.
Fig. 3 is that DC-CIK cell is being attacked and killing tumor cell.
Fig. 4 is a schematic diagram of the lethal effect of the DC cells against tumor of load tumor specific antigen.
Fig. 5 is a schematic diagram of the lethal effect of the DC cells against tumor of load tumor specific antigen.
Fig. 6 is the photo that tumor cell is supported on DC.
Fig. 7 is the reaction mechanism schematic diagram of the present invention.
Fig. 8 is tumor-targeting DC-CIK immunotherapy schematic flow sheet.
Detailed description of the invention
Embodiment 1
The operating procedure of tumor-targeting DC-CIK immunotherapy:
1) PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) gathers:
Patient venous blood sampling 50ml, uses blood cell separator to separate and enrichment PBMC, Ficoll lymphocyte divides
Chaotropic density－gradient centrifuga－tion method preliminary purification, the ratio separating liquid and blood is 1:1 or 1:1.5,2000rpm, 20
Minute centrifugal, take upper plasma and be placed in another centrifuge tube, 56 degree, within 30 minutes, inactivate, 4 degree save backup.With
Cloud and mist layer (i.e. mononuclearcell layer) slowly drawn by dropper, adds the brine 2 times of 0.9%, 1500rpm,
Within 5-10 minute, it is centrifuged.
2) DC and CIK cell are cultivated
It is 6 × 10 with LonZA X-VIVO 15 serum-free medium (10% human serum albumin) regulation cell concentration6
/ ml, every hole 2ml.Insert 37 DEG C, 5%CO2After incubator hatches 2h, non-adherent cell is CIK precursor,
With CIK culture fluid by 1.5 × 106/ ml density is resuspended, plants into 25cm2In culture bottle, volume about 4-10ml.Patch
Parietal cell is DC precursor, and every hole adds the DC bed board culture medium of 2ml preheating, puts into 37 DEG C, and 5%CO2 is incubated
Case continues to cultivate.Every 1 day, i.e. d2, d4, d6 days use DC that DC cultivates changed liquid culture medium half amount and change liquid.
CIK cultivates d0 days, adds IFN-γ, final concentration of 3000U/ml.CD3 monoclonal antibody is added, eventually after 24h
Concentration is 300ng/ml；IL-1 α, final concentration of 2000U/ml；IL-2, final concentration of 1000U/ml, fill
Dividing to shake up and be placed on 37 DEG C, 5%CO2 incubator is hatched.Within d3 days, start to change liquid, maintain density in 1-4 × 106/ml。
Can carry out expanding bottle according to proliferative conditions to cultivate.
Cell culture fluid formula:
A) DC bed board culture medium: LonZA X-VIVO 15 serum-free medium, containing 10% human serum albumin, L-Glu is eventually
Concentration is the final concentration of 1000U/ml of 2mmol/ml, IL-4, the final concentration of 800U/ml of GM-CSF.
B) DC changes liquid culture medium: LonZA X-VIVO 15 serum-free medium, containing 10% human serum albumin, L-Glu is eventually
Concentration is the final concentration of 2000U/ml of 4mmol/ml, IL-4, the final concentration of 1600U/ml of GM-CSF.
C) CIK culture fluid: LonZA X-VIVO 15 serum-free medium, containing 10% human serum albumin, L-Glu final concentration
For the final concentration of 1000U/ml of 2mmol/ml, IL-2.
3) tumor antigen gene extracts and adenovirus vector is packed
Fresh sample or Pathologic specimen, clean up with physiological saline solution, is cut into 5mm3Fritter, chopping grinding,
Every 50-100mg tissue adds 1ml TRIzol reagent, by homogenizer, piece of tissue is homogeneous.TRIzol method extracting tumor
Total tissue RNA.It is packaged into adenovirus vector Ad-GFP.
Owing to RNA is very easy to be degraded by RNase, so the reagent solution of this part operation and all needing through two with water
Ethyl coke hydrochlorate (DEPC) processes.Use aseptic disposable plastic apparatus special for RNA and automatic pipettor.Experiment
Whole process wears disposable breathing mask and glove.
Pathological tissue and the ratio of TRIzol reagent volume amount: abundant in order to react, tissue volume is not to be exceeded TRIzol
The 10% of reagent volume.RNA precipitate drying time can not long (more than 10 minutes).It is completely dried and will be substantially reduced RNA
The solubility being deposited in subsequent step.
4) adenovirus mediated tumor antigen gene transfection DC cell
The half adherent immature DC cultivating d5 is collected in featheriness, is adjusted to LonZA X-VIVO 15 serum-free medium
5×105The cell density of/0.2ml is inoculated into 24 orifice plates, every hole 0.2ml.Upper step intermediate package there is tumor antigen gene
Adenovirus vector Ad-GFP virus liquid add every hole, final concentration 2 × 107PFU/ul, virus quantity is 200MOI.37
DEG C, 5%CO2 incubator hatches 2h, and period rocks culture plate every 15min, makes virus uniformly transfection DC cell.Thereafter
Wash 2 times with culture fluid, proceed to 6 orifice plates and add and change liquid culture medium, add TNF-α, final concentration of 1000U/ml, continue
The continuous load tumor antigen DC cell cultivating 48h, d7 results maturation.
Viral vector carries tumor antigen gene information transfection DC, can make DC cell continual and steady expressing tumor in human body
A complete set of antigen, it is thus achieved that long lasting immune response.Traditional tumor antigen protein sensitization DC method is due to the antigen often half-life
Shorter, in human body, action time is short, it is therefore desirable to repeatedly immunity (feed back often, the course for the treatment of length) could obtain potent
Cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL), and there is the skill that transduction efficiency is low
Art bottleneck.
The selection of viral vector: we select the adenovirus vector that mammalian cell transfection experiment is conventional.Adenovirus vector
Formed by 5 type adenoviral gene restructuring, owing to its E1 district lacks, virus replication afunction, thus thin for host
Born of the same parents do not result in virus and infect and diffusion.And its unconformity is to host chromosome, insertion mutation will not occur；Transfection effect
The best, can effectively breed, virus titer is high.Carry reporter gene green fluorescent protein (green fluorescent
Protein, GFP) recombinant adenovirus (Ad-GFP) can after transfection fluorescence microscopy Microscopic observation and calculate transfection effect
Rate.
The selection on transfection opportunity: non-ripe dc is relatively strong to picked-up and the working ability of transfected material, and the dc's of maturation is anti-
Former submission ability is stronger.Therefore should transfect before DC maturation so that it is load tumor antigen RNA, after 24 hours again
Ripe with facilitating ripe cytokine TNF-α to promote it.
5) load tumor antigen DC with CIK co-cultures
The load tumor antigen DC cell of results is added CIK cell and co-cultures by d7, need to be by cell quantity scale dimension
Hold at load tumor antigen about DC:CIK=1:100, cultivate to d14 harvesting.
6) quality control: cell cultivate whole process carries out in GMP laboratory, cytoactive, microorganism detection,
Phenotypic examination is carried out according to acceptance criteria.
Acceptance criteria:
A) DC cell cultivate any stage, including the precursor stage, its microorganism detection result, i.e. antibacterial, fungus,
Mycoplasma should be feminine gender, endotoxin ＜ 2EU/ml；DC survival rate ＞ 75%, DC yield ＞ 5%, DC purity ＞
50%；DC Phenotypic examination HLA-DR, CD1a, CD80/86, CD83, CD11c, CD40 express the positive.
B) CIK cell cultivate any stage, including the precursor stage, its microorganism detection result, i.e. antibacterial, fungus,
Mycoplasma should be feminine gender, endotoxin ＜ 2EU/ml；Survival rate ＞ 95%；CIK Phenotypic examination CD3+CD56+ is thin
Born of the same parents 30%, CD4+CD25+ cell ＜ 5%.
C) within the 14th day, collecting cell, CIK cell quantity reaches 1 × 109-1×1010；DC cell quantity reaches 1 × 107-1×
108。
7) cell feeds back scheme
After quality testing is qualified, the centrifuge tube of the cell suspension in culture bottle is collected, 1500r/min, 5min
Centrifuge washing 3 times, is dissolved in normal saline 300ml or the most standby blood plasma containing 0.25% human serum albumin,
It is made into the laggard row vein of cell suspension to feed back.
The clinical condition that cell feeds back:
A) before blood drawing, the routine blood test result medium-sized lymphocyte absolute value ＜ 0.8 of patient can not take blood, and ＞ 0.8 can take blood.
B) DC-CIK cell therapy can after operative treatment 1 week, within before and after Radiotherapy chemotherapy 2 weeks, carry out；Can not be with chemotherapy and whole body
Radiotherapy is carried out simultaneously, in order to avoid the cell fed back is killed destruction and lessens the curative effect.
C) the day hemogram of feedback is normal, without infection fever.Patient does not carry out immuno-suppressive medication.
Single feeds back cell concentration can not be less than 1-2 × 109, feed back continuously or feed back every other day.During cell infusion,
Speed is slightly slow, keep 30-40 drip/minute, infusion overall process about 1.5-2 hour.
Claims (8)
1. the immune formulation being used for treating tumor, it is characterised in that: the DC cell containing load tumor antigen and CIK cell, the DC cell of described load tumor antigen concentration in culture fluid is 1 × 107-1×108, described CIK cell concentration in culture fluid is 1 × 109-1×1010。
A kind of immune formulation for treating tumor the most according to claim 1, it is characterised in that: the described DC cell of load tumor antigen and the number of CIK cell ratio is for 1:100.
3. the preparation method of a kind of immune formulation for treating tumor described in claim 1, it is characterised in that comprise the steps:
One step gathering PERIPHERAL BLOOD MONONUCLEAR CELL；
One separates and cultivates DC and the step of CIK cell:
Using DC bed board culture medium culturing PERIPHERAL BLOOD MONONUCLEAR CELL, regulation cell concentration is 6 × 106/ ml, inserts 37 DEG C, is the CO of 5% containing concentration of volume percent2After incubator hatches 1 ~ 3h, non-adherent cell is CIK precursor, by CIK precursor with CIK culture fluid by 1.5 × 106/ ml density is resuspended, plants in culture apparatus and cultivates；
During cultivating CIK cell, when cultivating beginning, add IFN-γ, final concentration of 3000U/ ml；CD3 monoclonal antibody, IL-1 α and IL-2, the final concentration of 300ng/ml of CD3 monoclonal antibody is added after 24h；The final concentration of 2000U/ ml of IL-1 α；The final concentration of 1000U/ ml of IL-2, is subsequently placed in 37 DEG C, is the CO of 5% containing concentration of volume percent2Incubator is hatched, and within the 3rd day, starts to change liquid, maintains density in 1-4 × 106/ ml；
Attached cell is DC precursor, DC precursor is added DC bed board culture medium, puts into 37 DEG C, be the CO of 5% containing concentration of volume percent2Incubator continues to cultivate, and every 1 day, uses DC to change liquid culture medium half amount and changes liquid；
One is extracted tumor antigen gene and uses the step of viral vector packaging, by tumor sample or Pathologic specimen, cleaning up with physiological saline solution, uses TRIzol method extracting tumor tissues total serum IgE, is then packaged into viral vector；
One step using virus-mediated tumor antigen gene transfection DC cell, collects the half adherent immature DC cultivated the 5th day, is adjusted to 5 × 10 with LonZA X-VIVO 15 serum-free medium5The cell density of/0.2ml, is inoculated in culture apparatus, is added by the virus liquid of the above-mentioned viral vector equipped with tumor antigen gene, final concentration 2 × 107PFU/ul, virus quantity is 200MOI, 37 DEG C, is being the CO of 5% containing concentration of volume percent2Incubator hatches 1 ~ 3
H, then washs with culture fluid, proceeds in another culture apparatus, adds DC and changes liquid culture medium, adds TNF-α, the final concentration of 1000U/ ml of TNF-α, continues cultivation 40 ~ 56h, the load tumor antigen DC cell of the 7th day results maturation；
The step that one load tumor antigen DC with CIK co-cultures, by results load tumor antigen DC cell add step 2) CIK cell in co-culture, the quantitative proportion of load tumor antigen DC cell and CIK cell maintains between 1:10 ~ 200, cultivating to the 14th day harvesting, CIK cell quantity reaches 1 × 109-1×1010；DC cell quantity reaches 1 × 107-1×108。
4. the preparation method of a kind of immune formulation for treating tumor described in claim 1, it is characterised in that: a step extracting tumor antigen gene, the volume of tumor tissues is less than the 10% of TRIzol reagent volume.
5. the preparation method of a kind of immune formulation for treating tumor described in claim 1, it is characterised in that: described viral vector is adenovirus vector.
6. the preparation method of a kind of immune formulation for treating tumor described in claim 1, it is characterized in that: described DC bed board culture medium is LonZA X-VIVO 15 serum-free medium, it is the human serum albumin of 10% containing mass percent concentration, possibly together with L-Glu, IL-4 and GM-CSF, the final concentration of 2mmol/ml of L-Glu, the final concentration of 800U/ml of the final concentration of 1000U/ ml, GM-CSF of IL-4.
7. the preparation method of a kind of immune formulation for treating tumor described in claim 1, it is characterized in that: it is LonZA X-VIVO 15 serum-free medium that described DC changes liquid culture medium, it is the human serum albumin of 10% containing mass percent concentration, possibly together with L-Glu, IL-4 and GM-CSF, the final concentration of 4mmol/ml of L-Glu, the final concentration of 1600U/ml of final concentration of 2000U/ ml, GM-CSF of IL-4.
8. the preparation method of a kind of immune formulation for treating tumor described in claim 1, it is characterized in that: described CIK culture fluid is LonZA X-VIVO 15 serum-free medium, it is the human serum albumin of 10% containing mass percent concentration, possibly together with L-Glu, IL-2, the final concentration of 1000U/ml of the final concentration of 2mmol/ml, IL-2 of L-Glu.
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