CN106834354A

CN106834354A - The preparation method and purposes of a kind of enhanced targeting immunocyte group of modification

Info

Publication number: CN106834354A
Application number: CN201710079331.2A
Authority: CN
Inventors: 不公告发明人
Original assignee: Harbin Xinlian Joint Biological Technology Co Ltd
Current assignee: SHENZHEN HONGSHI KECHUANG BIOLOGICAL TECHNOLOGY DEVELOPMENT Co.,Ltd.
Priority date: 2016-11-22
Filing date: 2017-02-14
Publication date: 2017-06-13

Abstract

Enhanced DC CIK cells targeting immunocyte group the invention discloses a kind of modification and its production and use, wherein the method for preparing DC cells includes：Monocyte is separately cultured, to obtain immature DC cells；Immature DC cell is carried out into tumour antigen to load and carry out induction differentiation culture, to obtain the DC cells of maturation, wherein, the monocyte is to separate to obtain from the PMNC of sample.The method for preparing CIK includes：CIK cell in vitro differentiation culture, obtains immature CIK cell；Modification is prepared to modify CIK cell in vitro with recombinant slow virus；CIK cell and DC co-culture of cells after modification, so as to CIK cell after the modification for obtaining maturation（Triumph‑DC‑CIK）, and then for obtaining more preferable immunotherapy of tumors effect.

Description

The preparation method and purposes of a kind of enhanced targeting immunocyte group of modification

Technical field

The present invention relates to biological technical field, in particular it relates to the enhanced DC-CIK of modification is cell targeted Immunocyte group and its production and use, belongs to cell biology, immunology, therapeutic field of tumor.

Background technology

Malignant tumour is the main fatal disease type of the mankind, as the number one killer for threatening human health.The Ministry of Public Health Recent statistics data, showed, the annual new cancer cases of China about 3,370,000, dead about 211 according to relevant report of cancer in 2012 Ten thousand.Cancer has turned into the big reason of China dead first, and death toll accounts for global number of cancer deaths's a quarter.The world in 2012 Cancer report display, annual cancer new cases about 14,000,000, dead about 8,000,000, this statistics 12,700,000 with 2008 People compares, and number is significantly increased.The same period, the death toll of cancer patient also increased, and increase to from past 7,600,000 people 8200000 people.Report claims, and to the year two thousand thirty, newly-increased cases of cancer will increase by 50%, reach annual 21600000 people.China's new cases are accounted for Global new cases 22%, death toll accounts for 26%, more than a quarter of global number of cancer deaths.Lung cancer morbidity in the middle of male Rate highest, women is breast cancer.

In current clinical therapy of tumor strategy, limitation tumour is controlled with local treatments such as operation and radiotherapies mainly Treat, and then rely primarily on amic therapy method for the MRD after general, metastatic or local treatment and carry out systematicness and control Treat.But therapeutic effect is mostly undesirable, and it is often accompanied by serious side effect.Tumour is multi-step, polygenic mutation work Result, shows to grow, breaks up out of control with apoptosis.The clinically diagnosis of patient has diversity and individual inheritance heterogeneous Property, while often do the appearance and recurrence with tumour DISTANT METASTASES IN focus, make oncotherapy must with the viewpoint of systemic disease, Whole body therapeutic scheme is taken, the tumour of local lesion is not only eliminated, also to control the Preventive of tumour to grow and tumour pair The invasion and attack of important organ, could really effectively improve cure rate and the life cycle of tumour patient.

In recent years, with the development of molecular medicine technology, tumour immunotherapy treatment tumour turns into clinical therapy of tumor Focus, is that tumor patient brings hope.2013《Science》Immunotherapy of tumors is classified as magazine the head of ten big sciences breakthrough Position, cellular immunotherapy method has huge advantage in theory：（1）It does not damage reduction function of immune system, and enhancing on the contrary is exempted from Epidemic disease system.There is fragmentation effect to active breeding and static hiding cancer cell.（2）Can be with broad-spectrum curing kinds cancer, to general It is effective all over crowd.（3）Cancer cell can be suppressed by the immunosurveillance after treatment to evolve, recurrence rate is reduced.Due to tumour Cellular immunotherapy has a wide range of application (can be used for various entity tumors and leukaemia), especially to tumour minimal disease (including Transfer, recurrence stove, the cancer cell in blood) more effectively, have no toxic side effect, and suitable for (such as late period in tumour each stage Radiotherapy chemotherapy is difficult to use), therefore with huge application prospect and the wide market space.

Cellular immunotherapy mainly includes CIK and DC cells：

BMDC（Dendritic cell, DC）It is that the mostly important immunoregulation for being found in recent years is thin with auxiliary Born of the same parents, it is topmost antigen presenting cell, with capture, working process antigen and to T lymphocytes offers antigen molecule Function, and express costimulatory molecules and adhesion molecule；And the Th1 types played a significant role in antitumor immunity of organism can be secreted Cell factor IL-12, induction body produces antigenspecific T lymphocyte, recognition and killing tumor cell.

The killing cell of cytokine profiles induction（Cytokine-induced killer cell, CIK）It is by outside people All blood mononuclear cells use cytokine profiles in vitro（Such as CD 3-resisting monoclonal antibody, IL-2 and IFN-γ）Co-incubation The a group foreign cell obtained after a period of time.Because this kind of cell expresses two kinds of membrane protein molecules of CD3+ and CD56+ simultaneously, therefore Be otherwise known as NK cell sample T lymphocytes, and the non-MHC with the powerful anti-tumor activity of T lymphocytes and NK cells is restricted Kill knurl advantage.Therefore, it is considered as the preferred option of antitumor adoptive cellular immunotherapy of new generation using CIK cell.CIK is thin Effector cell's CD3+ and CD56+ cell in born of the same parents is less in normal human peripheral blood, only 1%-5%.

People have found to add tumor patient autologous DC when in CIK cell culture again after, can interact each other, promote The maturation of both sides' cell, and induce except the cell of proliferation activity more stronger than homologous CIK cell and Geng Gao tumor cytotoxicities activity Group.The present invention carries out early stage modification on the basis of to DC cells and CIK cell propagation, killing characteristic research to CIK cell, And DC is co-cultured with CIK, the CIK cell group being remarkably reinforced can be produced, its propagation efficiency and tumor suppression cytoactive have Significantly increase.

Programmed death acceptor -1（Programmed death 1, PD-1）PD-1 be the type of immunoglobulin superfamily I across Membrane glycoprotein, relative molecular mass is 55KD or so, is made up of cell outskirt, hydrophobicity transmembrane region, cytoplasm district.PD-1 molecules Most it is characterized in significantly that the afterbody of cytoplasm district contains two tyrosine residues, composition immunity receptor tyrosine is participated in respectively and is suppressed Motif (immunoreceptor tyrosine-based inhibitory motifs, ITIM) and immunity receptor tyrosine turn Change motif (immunoreceptor tyrosine-based swith motifs, ITSM) domain.ITIM can make cytoplasm The phosphorylation of section is restored, and plays the function of antagonism antigen receptor, and the tyrosine residue on ITSM is adjusted in the negativity of PD-1 It is required in section.PD-1 parts include PD-L1 and PD-L2, and wherein PD-L1 wide expressions are in T cells, B cells, list Nucleus, macrophage, BMDC, kinds of tumor cells and some non-lymph PD-1/PD-Ls play negative immune Adjustment effect.After the PD-1 and PD-Ls of cell surface are coupled, the Tyr of the ITSM domains of T cell cytoplasmic region can be caused Phosphorylation, then raises phosphatase protein tyrosinase -2 and protein-tyrosine enzyme -1 by the Tyr of phosphorylation, can not only hinder The activation of stagnant extracellular signal-regulated kinase, can also block phosphatidyl-inositol 3-kinase (phosphatidylinositol 3- Hydroxy kinase, PI3K) and serine-threonine protein kinase enzyme (serine/threonine kinase, Akt) Activation, the final secretion for suppressing T lymphopoiesis and relevant cell factor.

Interleukin 2（IL-2）Principal biological function be promote T cells propagation with differentiation, additionally, IL-2 Also B cell proliferation, differentiation and secretory antibody, activation natural can be promoted to kill (NK) cell, the killing of Lymphokine (LAK) cell and CTL (CTL), increase quantity and the increasing of the class of antigen presenting cell surface I and class Ⅱmolecule Strong antigen Presentation etc..Because IL-2 has extensive immunologic function, tumour, infectivity are clinically mainly used at present The treatment of disease, especially curative effect is obvious in terms of kidney and cancerous effusion treatment.Research shows that IL-2 can not direct intervention Tumour cell is killed in the growth of tumour cell, and its antitumor mechanism essentially consists in stimulation, activates substantial amounts of effector cell such as CTL, B cell, NK cells and LAK cells etc..

Therefore this research is by PD-1 of the CIK cell surface overexpression without intracellular functional areas, making tumour table The false acceptor of face PD-L1, and false acceptor is first combined with PD-L1 suppression signals by linker sequences, so as to reduce There is the PD-1 of intracellular functional areas to be combined with the PD-L1 of tumor cell surface on CIK cell surface, reduce tumour to lethal cell Suppression, while secrete IL-2 propagation activation is carried out to cell, greatly enhancing cell-proliferation activity and tumor-killing effect.

The content of the invention

Enhanced DC-CIK cell targeted immunocytes group the invention discloses a kind of modification and preparation method thereof and Purposes, its preparation method is as follows：

1. expression people source PD-1-IRES-IL-2 fragment slow virus is prepared

The SEQ ID NO of composition sequence table:Double chain DNA molecule shown in 1, is connected into commercialization slow virus carrier, with Used as demonstration example, including but not limited to pWPXL carriers construct recombinant plasmid pWPXL-PD-1- IRES- to pWPXL carrier systems IL-2.Virus packaging is completed by following routine operation：

A, using calcium phosphate method by recombinant expression plasmid together with helper plasmid cotransfection HEK293 cells.Inoculation HEK293 cells in In DMEM culture mediums containing 10% hyclone, cultivated under conditions of 37oC and 5% CO2 to exponential phase, collect cell, 5 × 106 cells are inoculated with the Tissue Culture Dish of 10 cm diameters, continue to cultivate 16~24 h.Treat that cell is long to 70~80% IMDM is replaced medium to during density, virus transfection is carried out after being incubated 2~4 h.

, by recombinant plasmid pWPXL-PD-1-IRES-IL-2 and packaging plasmid pMD2.G plasmids and psPAX2 plasmid co-transfections

HEK293 cells（Every 1 × 106 cell about transfects 30-40 μ g recombinant plasmids, and transfection process is by packaging plasmid and phosphoric acid After calcium solution filtering mixing, it is added in culture dish, mixes.After 8-15 h, culture medium is changed into DMEM, after 48~60 h, collect Supernatant.

Plasmid amount such as following table used：

2. viral purification

A. 6 Ultra-clear SW28 centrifuge tubes are taken, with 70% ethanol disinfection after, be placed in superclean bench and open uviol lamp Continue to sterilize 30 minutes.B. the pretreated viral supernatants of about 32ml are added in each Ultra-clear SW28 centrifuge tube Liquid.C. a pipette of 10ml is taken, the sucrose solution of 12 ml 20% is drawn.Pipette is inserted into the bottom of centrifuge tube always Portion, slowly gets 4 ml by sucrose solution.Similarly, the sucrose solution of remaining 8 ml is added separately to another two centrifuge tube In.A clean pipette separately is taken, remaining 3 pipes are equally processed.D. the weight of each pipe is adjusted with PBS, is made corresponding Weight difference between centrifuge tube is no more than 0.1g.E. all 6 centrifuge tubes are put into Beckman SW28 hypervelocity in order In centrifugal head.F. 4 DEG C, 25,000 rpm. (82,700g) are centrifuged 2 hours.G. carefully pipe is taken out from rotary head. Supernatant is outwelled, centrifuge tube is tipped upside down on to be placed on paper handkerchief makes remaining supernatant drain off for 10 minutes.Sop up remaining drop.In ttom of pipe There should be visible precipitation.H. the PBS of 100ml not calcic and magnesium is often added to wash lower precipitation in pipe.I. by SW28 ultracentrifugations Pipe is inserted into 50ml cone bottom centrifuge tubes, is closed the lid.J. dissolved 2 hours at 4 DEG C, gently shaken every 20 minutes.k. 4 DEG C, 500g is centrifuged 1 minute, solution is concentrated on ttom of pipe.L. with 200 μ l pipettors, softly piping and druming makes precipitation resuspended.Avoid producing Raw foam.During liquid in all pipes focused on into a SW28 centrifuge tube.M. the viral suspension after concentrating is distributed into 50 μ l Every part, it is stored in production tube.With being stored in -80 DEG C after broken dry ice quick-frozen.

Virus titer is determined：ELISA method determines totivirus titre：Antigen detection operation is carried out by kit specification.

The preparation of comparison virus

Replacing recombinant plasmid pWPXL-PD-1-IRES-IL-2 with pWPXL plasmids carries out step 1,2, and the solution for obtaining is named as PWPXL virus liquids.

The DC-CIK of modification（Triumph-DC-CIK）Preparation

A. the preparation of PMNC PBMC

Take fresh anticoagulated whole blood, EDTA（Sodium citrate or heparin）Anti-coagulants.It is dilute with isometric PBS or 0.9%NaCl Release whole blood.The separating liquid of certain volume is added in centrifuge tube, by the blood sample tiling after dilution to separating liquid ullage, is kept Two liquid level interface is clears.Separating liquid, the not diluted whole blood of anti-freezing, PBS（Or physiological saline）Volume is 1:1:1., centrifugation 2000rpmx25min, separates to obtain PBMC, then is washed with Hanks liquid cell number is counted under 2 times, mirror, finally uses serum-free medium VIVO-15 regulation cell densities make into 5x106/ml cell suspensions；

B. non-adhering is separated with adherent cell

Cell suspension is moved into 6 orifice plates, 37 DEG C, 5%CO2 cultivates 2h, then gently rinses cell with pipette, and non-adhering is thin Born of the same parents' suspension collection work induction CIK cell in centrifuge tube is standby, and adherent cell is stayed on 6 orifice plates, adds DC nutrient solution 3ml/ holes It is stand-by.

The induction and amplification of DC cells are adhered to

Leaving on 6 orifice plates of adherent cell, per hole, addition DC nutrient solutions 3ml is contained within GM-CSF800U/ml and IL-4500U/ Ml, in 37 DEG C, 5%CO2 cultures are cultivated 4-5 days, every equivalent fluid infusion in 2-3 days once, in the 5d of culture, add restructuring HumanTNF-α (500U/ml), it is standby that induction DC cell maturations collected cell in the 6-7 days；

D. the induction and amplification of non-adhering CIK cell

The non-adherent cell suspension inoculation in centrifuge tube to the 25cm2 trainings containing IFN-γ 1000U/ml VIVO-15 nutrient solutions Bottle is supported, 10-15ml/ bottles, 37 DEG C, in 5%CO2 incubators after 24-36h is placed in, the μ g/ of anti-CD49d McAb 5 prepared with Hanks liquid Ml is coated with 25cm2 blake bottles, while adding final concentration IL-1a100U/mlVIVO-15, IL-2000U/mlVIVO-15 to continue training 48-72h is supported, is counted under mirror, it is 4x105/ml to adjust cell density with CIK nutrient solutions, every 48h by above-mentioned the same terms amplification 1 It is secondary, and add 2%~10% volume autoserum, wherein, the serum origin is in the blood sample.Thus, cultivate Effect is good.Embodiments in accordance with the present invention, the serum free medium is the serum free medium of VIVO-15.According to the present invention Other embodiments, the antibiotic of 0.5 volume %~5 volumes % is added with the serum free medium, preferably celebrate Big mycin.Thereby, it is possible to efficiently prepare targeted immune cell mass.It is standby that culture collected CIK cell to 5-7 days；

Anti-CD49d McAb method for coating：CD3 monoclonal antibodies are added in VIVO-15 nutrient solutions, anti-CD49d McAb concentration is become for 5- The coating buffer of 10ug/ml, adds 5ml coating buffers in 25cm2 blake bottles, and 4C discards whole overnight or after 37 DEG C of incubation 2h Coating buffer；

E. the preparation and load of tumour antigen

Ocal resection sample, it is under aseptic condition, slough and the nonneoplastic tissue removal of cancer side is clean, then use aseptic life Reason salt is washed 3 times；Tumor tissues are shredded with aseptic tissue shear, add the culture mediums of RPMI 1640, be fully ground, 200 mesh without Single cell suspension is collected after bacterium net filtration.With the culture medium re-suspended cells of RPMI 1640 to 1-2 x 107/ml, load 5ml aseptic It is quick-frozen in immersion liquid nitrogen in cryopreservation tube, taken out after 10 min, then be put into 37 DEG C of water-baths 10 min that thaw rapidly, repeatedly 3-5 It is secondary；（Or with -80 DEG C/37 DEG C multigelations 3-5 times）.By in Tumor lysate addition centrifuge tube, 3000rpm is centrifuged 10min, Supernatant is collected, 0.22 μm of membrane filtration is degerming, keep sample detection protein content and bacterium, fungi and mycoplasma, -80 DEG C of preservations are standby With.

Adjustment cell density, will crack tumour cell and is incubated with tumour antigen with the DC cells of culture 7 days, co-cultured, and make As ripe DC cells.

The modification of cell

The CIK cell cultivated 7 days is separately added into 5x108 pWPXL-PD-1-IRES-IL-2 viruses and pWPXL virus liquids, altogether Culture 12 hours.

The preparation of cell and control DC-CIK cells

The DC and CIK1 for modifying:5 mixing are co-cultured produces DC and culture that Triumph-DC-CIK cells will load tumour antigen CIK cell after to the modification of 5-7 days, is counted, and DC and CIK cell are collected in centrifugation respectively, are adjusted with VIVO-15 serum-free mediums Bicelluar density is respectively 2x105 and 1x106, by DC：CIK=1:25cm2 blake bottles 10ml/ is moved into after 5 isometric mixing Bottle, in 37 DEG C, 5%CO2 is cultivated, every equivalent fluid infusion in 2 days once；, every 3 days sub-bottle Amplification Cultures 1 time, expand through 2 times and train The Triumph-DC-CIK cells of 5x109 ~ 1x1010 can be obtained after supporting.The DC-CIK cells prepared with pWPXL virus infection As control.

The preparation of cell preparation

1 ~ 5x109 cells are collected by centrifugation, are washed with 0.9% sodium chloride injection 2 times, be centrifuged, abandon supernatant, cell is moved to

In 250ml infusion bottles, plus 2g human serum albumins and 250ml0.9% sodium chloride injections, it is made cell suspension preparation.

Brief description of the drawings

Fig. 1 PD-1 fragments and IL-2 fragments PCR amplification purpose band electrophoresis detection results；

The real-time PCR testing results of the not all right just first band of immunocyte group PD-1 and IL-2 genes after Fig. 2 modifications；

Inhibitory action of the immunocyte group to human HepG2 cell's propagation after Fig. 3 modifications；

Inhibitory action of the immunocyte group to people U251 cells propagation after Fig. 4 modifications；

Inhibitory action of the immunocyte group to HeLa Cells propagation after Fig. 5 modifications；

Inhibitory action of the immunocyte group to typeⅡ pneumocyte propagation after Fig. 6 modifications；

Inhibitory action of the immunocyte group to MCF-7 Human Breast Cancer Cells propagation after Fig. 7 modifications；

Inhibitory action of the immunocyte group to Human colorectal carcinoma HT29 cells propagation after Fig. 8 modifications；

Inhibitory action of the immunocyte group to human gastric cancer SGC-7901 cells propagation after Fig. 9 modifications；

Specific embodiment：

As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art, Under the premise without departing from the principles of the invention, some improvement and modification can also be made, these improvements and modifications are also considered as this hair Bright protection domain.

Method used is conventional method unless otherwise instructed in following embodiments, and specific steps can be found in：《Molecular Cloning: A Laboratory Manual》（Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor）.

Unless otherwise noted, the carrier and reagent that the embodiment of the present invention is used are all commercial goods, the primer and DNA sequences Row synthesize by Invitrogen companies.

The preparation of the expression people of embodiment 1. source PD-1 fragment slow virus

1. the structure of people source PD-1 fragment slow virus

The SEQ ID NO of composition sequence table:Double chain DNA molecule shown in 4, is connected into commercialization slow virus carrier, with Used as demonstration example, including but not limited to pWPXL carriers construct recombinant plasmid pWPXL-PD-1- IRES- to pWPXL carrier systems IL-2, is identified by PCR, and as a result as shown in figure 1, swimming lane 1 is DL2000 Marker, swimming lane 2 is PD-1 qualification results, Swimming lane 3 is IL-2 qualification results.

Virus packaging is completed by following routine operation：

, by taking pWPXL systems as an example by recombinant plasmid pWPXL-PD-1-IRES-IL-2 and packaging plasmid pMD2.G plasmids and PsPAX2 plasmid co-transfection HEK293 cells（Every 1 × 106 cell about transfects 30-40 μ g recombinant plasmids, and transfection process is to wrap After dress plasmid mixes with calcium phosphate solution filtering, it is added in culture dish, mixes.After 8-15 h, culture medium is changed into DMEM, 48~ After 60 h, supernatant is collected.Plasmid amount such as following table used：

2. viral purification

Virus titer is determined：ELISA method determines totivirus virus titer：Antigen detection operation is entered by kit specification OK.

The preparation of comparison virus

Replacing recombinant plasmid pWPXL-PD-1 with pWPXL plasmids carries out step 1,2, and the solution for obtaining is named as pWPXL virus liquids.

PD-1 detections after embodiment 2, recombinant virus infection CIK cell

To absolutely prove beneficial effects of the present invention, present invention CIK also metainfective to pWPXLd-PD-1-IRES-IL-2 is thin PD-1 and IL-2 transcriptional levels in born of the same parents carry out real-time PCR detections, and step is as follows：

Carry out the extraction of total serum IgE respectively to the cell after pWPXLd-PD-1-IRES-IL-2 infection 4h, 8h and 12h, meanwhile, The CIK cells of infection pWPXLd are set used as control, the CIK cells being uninfected by make blank.Then reverse transcription is into cDNA Whether template, transcribed, with GAPDH as internal reference with the mRNA of the specific primer detection PD-1 and IL-2 for designing. The primer sequence of GAPDH is：

Sense primer:5’-ACCACAGTCCATGCCATCAC-3’

Anti-sense primer:5’-TCCACCACCCTGTTGCTGTA-3’；

The primer sequence of PD-1 is：

Sense primer：5’- GCATGAGCCCCAGCAACCAGACGGACAAGCTG-3’

Anti-sense primer：5’- AGAACACAGGCACGGCTGAGGGGTCCTCCTTC-3’.

Primer sequence be：

Sense primer：5’-CCAGGATGCTCACATTTAAGTTTTAC-3’

Anti-sense primer：5’- GAGGTTTGAGTTCTTCTTCTAGACACTGA-3’

The real-time PCR testing results of the present embodiment are as shown in Fig. 2 wherein, A is that PD-1, B are the testing result of IL-2.

From Fig. 2, relative to the control that blank is the CIK cell for having transfected pWPXLd；The present embodiment is provided The expression quantity of PD-1 and IL-2 fragments of the CIK cell group for having transfected pWPXL-PD-1-IRES-IL-2 slow virus have significantly Rise.It is produced slow in incasing cells HEK293 cells via plasmid packaging system that the result explanation present invention is provided Virus can infect CIK cell, and the cell containing slow virus or slow virus carrier that the present invention is provided can be used to prepare pWPXL- PD-1-IRES-IL-2 slow virus.

Embodiment 3, the DC-CIK of modification（Triumph-DC-CIK）The preparation of cell

8. the preparation of PMNC PBMC

Take fresh anticoagulated whole blood, EDTA（Sodium citrate or heparin）Anti-coagulants.It is dilute with isometric PBS or 0.9% NaCl Release whole blood.The separating liquid of certain volume is added in centrifuge tube, by the blood sample tiling after dilution to separating liquid ullage, is kept Two liquid level interface is clears.Separating liquid, the not diluted whole blood of anti-freezing, PBS（Or physiological saline）Volume is 1:1:1, centrifugation 2000rpmx25min, separates to obtain PBMC, then is washed with Hanks liquid cell number is counted under 2 times, mirror, finally uses serum-free medium VIVO-15 regulation cell densities make into 5x106/ml cell suspensions；

9. non-adhering is separated with adherent cell

Adhere to the induction and amplification of DC cells

Leaving on 6 orifice plates of adherent cell, per hole, addition DC nutrient solutions 3ml is contained within GM-CSF（800U/ml）And IL-4 （500U/ml）, in 37 DEG C, 5%CO2 cultures, in the 6d of culture, add restructuring humanTNF-α (500U/ml), induction DC cells into It is ripe standby in the 5-7 days collection cells；

The induction and amplification of 11. non-adhering CIK cells

The non-adherent cell suspension inoculation in centrifuge tube to the 25cm2 trainings containing IFN-γ 1000U/ml VIVO-15 nutrient solutions Bottle is supported, 10-15ml/ bottles, 37 DEG C, in 5%CO2 incubators after 24-36h is placed in, the μ g/ of anti-CD49d McAb 5 prepared with Hanks liquid Ml is coated with 25cm2 blake bottles, while adding final concentration IL-1a100U/ml VIVO-15, IL-2000U/mlVIVO-15 to continue to train 48-72h is supported, is counted under mirror, it is 4x105/ml to adjust cell density with CIK nutrient solutions, every 48h by above-mentioned the same terms amplification 1 Secondary, it is standby that culture collected CIK cell to 5-7 days；

Anti-CD49d McAb method for coating：CD3 monoclonal antibodies are added in VIVO-15 nutrient solutions, anti-CD49d McAb concentration is become for 5- The coating buffer of 10ug/ml, adds 5ml coating buffers in 25cm2 blake bottles, and 4 DEG C overnight or after 37 DEG C of incubation 2h, discard whole Coating buffer；

The preparation and load of tumour antigen

A, ocal resection sample, it is under aseptic condition, slough and the nonneoplastic tissue removal of cancer side is clean, then with aseptic Physiology salt is washed 3 times；Tumor tissues are shredded with aseptic tissue shear, adds the culture mediums of RPMI 1640, be fully ground, 200 mesh Single cell suspension is collected after aseptic net filtration.With the culture medium re-suspended cells of RPMI 1640 to 1-2 x 107/ml, load 5ml without It is quick-frozen in immersion liquid nitrogen in bacterium cryopreservation tube, taken out after 10 min, then be put into 37 DEG C of water-baths 10 min that thaw rapidly, repeatedly 3- 5 times；（Or with -80 DEG C/37 DEG C, multigelation 3-5 times）.By in Tumor lysate addition centrifuge tube, 3000rpm is centrifuged 10min, collects supernatant, and 0.22 μm of membrane filtration is degerming, and keep sample detection protein content and bacterium, fungi and mycoplasma, -80 DEG C Save backup.

, adjustment cell density, will cracking tumour cell with

The DC cells and tumour antigen that 7 days will be cultivated are incubated, co-culture, and make ripe DC cells.

The modification of cell

With the CIK1 of modification:Triumph-DC-CIK cells are produced in 5 mixing co-cultivations

Cell preparation

1 ~ 5x109 cells are collected by centrifugation, are washed with 0.9% sodium chloride injection 2 times, be centrifuged, abandon supernatant, cell is moved into 250ml In infusion bottle, plus 2g human serum albumins and 250ml0.9% sodium chloride injections, it is made cell suspension preparation.

The detection that the Triumph-DC-CIK of embodiment 4 is acted on inhibiting tumour cells

Testing sample is Triumph-DC-CIK prepared by embodiment 3 and the DC-CIK cells through pWPXL virus modifications.To be in The tumour cells such as the people HepG2 of exponential phase are collected after Trypsin Induced respectively, are prepared into 1x105/mL's Cell suspension, 96 orifice plates are added after piping and druming is uniform, add 200 μ L cancer cell suspensions per hole, in 37 DEG C, 5 % CO2 incubators Continue culture and treat that cell is long to 70-80%, remove culture medium, washed once with PBS, be divided into 4 groups, the 1st, 2 groups of every group of experimental port difference 102,103 Triumph-DC-CIK cells and the μ L of DC-CIK cells 100 is added, 4h, 8h, 12h is cultivated.After culture terminates, often Hole adds 50 μ L MTT solution（5 mg/mL）After being incubated 24h, nutrient solution is discarded, the DMSO of 200 μ L is added per hole, shake 10min Afterwards, with enzyme linked immunological instrument in the OD values that wavelength is 570nm are determined, growth of cancer cells inhibiting rate is by following formula calculating：

Inhibiting rate %=（Control group OD averages-administration group OD values）/ control group OD average × 100 %

Result is shown in Fig. 3.In Fig. 3, A, B figure are respectively HepG2 cells and add 102 and 103 to treat cell, in 4h, the training of 8h, 12h The result that the time of supporting suppresses to cell tumour,

By in same procedure Fig. 4-9, A, B figure are added in being respectively U251, HeLa, A549, MCF-7, HT29, SGC-7901 cell 102 and 103 treatment cells, in 4h, the result that the incubation time of 8h, 12h suppresses to cell tumour.

The present embodiment fully proves that the fragmentation effect of Triumph-DC-CIK cells against tumor is substantially better than unmodified DC- CIK cell.

Understand that the DC-CIK after modification is respectively provided with powerful killing ability to Several Kinds of Malignancy by above-described embodiment, from now on Good prognosis treatment will be provided for tumor patient into clinic, with huge social value and economic worth.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means to combine specific features, structure, material or spy that the embodiment or example are described Point is contained at least one embodiment of the invention or example.In this manual, to the schematic representation of above-mentioned term not Necessarily refer to identical embodiment or example.And, the specific features of description, structure, material or feature can be any One or more embodiments or example in combine in an appropriate manner.

Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not Can these embodiments be carried out with various changes, modification, replacement and modification in the case of departing from principle of the invention and objective, this The scope of invention is limited by claim and its equivalent.

Sequence table

<110>The new joint bio tech ltd in Harbin

<120>The preparation method and purposes of a kind of enhanced DC-CIK targeting immunocyte groups of modification

<210> SEQ ID NO: 1

<211> 621

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> SEQ ID NO: 1

1 ATGCAGATCC CACAGGCGCC CTGGCCAGTC GTCTGGGCGG TGCTACAACT

51 GGGCTGGCGG CCAGGATGGT TCTTAGACTC CCCAGACAGG CCCTGGAACC

101 CCCCCACCTT CTCCCCAGCC CTGCTCGTGG TGACCGAAGG GGACAACGCC

151 ACCTTCACCT GCAGCTTCTC CAACACATCG GAGAGCTTCG TGCTAAACTG

201 GTACCGCATG AGCCCCAGCA ACCAGACGGA CAAGCTGGCC GCCTTCCCCG

251 AGGACCGCAG CCAGCCCGGC CAGGACTGCC GCTTCCGTGT CACACAACTG

301 CCCAACGGGC GTGACTTCCA CATGAGCGTG GTCAGGGCCC GGCGCAATGA

351 CAGCGGCACC TACCTCTGTG GGGCCATCTC CCTGGCCCCC AAGGCGCAGA

401 TCAAAGAGAG CCTGCGGGCA GAGCTCAGGG TGACAGAGAG AAGGGCAGAA

451 GTGCCCACAG CCCACCCCAG CCCCTCACCC AGGCCAGCCG GCCAGTTCCA

501 AACCCTGGTG GTTGGCGGCG GCGGCAGCGG CGGCGGCGGC AGCGGCGGCG

551 GCGGCAGCGG TGTCGTGGGC GGCCTGCTGG GCAGCCTGGT GCTGCTAGTC

601 TGGGTCCTGG CCGTCATCTG A

1-513PD-1 extracellular regions

514-558 linker sequences

559-618 PD-1 transmembrane regions

<210> SEQ ID NO: 2

<211> 462

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> SEQ ID NO: 2

1 ATGTACAGGA TGCAACTCCT GTCTTGCATT GCACTAAGTC TTGCACTTGT

51 CACAAACAGT GCACCTACTT CAAGTTCTAC AAAGAAAACA CAGCTACAAC

101 TGGAGCATTT ACTGCTGGAT TTACAGATGA TTTTGAATGG AATTAATAAT

151 TACAAGAATC CCAAACTCAC CAGGATGCTC ACATTTAAGT TTTACATGCC

201 CAAGAAGGCC ACAGAACTGA AACATCTTCA GTGTCTAGAA GAAGAACTCA

251 AACCTCTGGA GGAAGTGCTA AATTTAGCTC AAAGCAAAAA CTTTCACTTA

301 AGACCCAGGG ACTTAATCAG CAATATCAAC GTAATAGTTC TGGAACTAAA

351 GGGATCTGAA ACAACATTCA TGTGTGAATA TGCTGATGAG ACAGCAACCA

401 TTGTAGAATT TCTGAACAGA TGGATTACCT TTTGTCAAAG CATCATCTCA

451 ACACTGACTT GA

<210> SEQ ID NO: 3

<211> 588

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> SEQ ID NO: 3

1 GCCCCTCTCC CTCCCCCCCC CCTAACGTTA CTGGCCGAAG CCGCTTGGAA

51 TAAGGCCGGT GTGCGTTTGT CTATATGTTA TTTTCCACCA TATTGCCGTC

101 TTTTGGCAAT GTGAGGGCCC GGAAACCTGG CCCTGTCTTC TTGACGAGCA

151 TTCCTAGGGG TCTTTCCCCT CTCGCCAAAG GAATGCAAGG TCTGTTGAAT

201 GTCGTGAAGG AAGCAGTTCC TCTGGAAGCT TCTTGAAGAC AAACAACGTC

251 TGTAGCGACC CTTTGCAGGC AGCGGAACCC CCCACCTGGC GACAGGTGCC

301 TCTGCGGCCA AAAGCCACGT GTATAAGATA CACCTGCAAA GGCGGCACAA

351 CCCCAGTGCC ACGTTGTGAG TTGGATAGTT GTGGAAAGAG TCAAATGGCT

401 CTCCTCAAGC GTATTCAACA AGGGGCTGAA GGATGCCCAG AAGGTACCCC

451 ATTGTATGGG ATCTGATCTG GGGCCTCGGT GCACATGCTT TACATGTGTT

501 TAGTCGAGGT TAAAAAAACG TCTAGGCCCC CCGAACCACG GGGACGTGGT

551 TTTCCTTTGA AAAACACGAT GATAATATGG CCACAACC

<210> SEQ ID NO: 4

<211> 1671

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> SEQ ID NO: 4

1 ATGCAGATCC CACAGGCGCC CTGGCCAGTC GTCTGGGCGG TGCTACAACT

51 GGGCTGGCGG CCAGGATGGT TCTTAGACTC CCCAGACAGG CCCTGGAACC

101 CCCCCACCTT CTCCCCAGCC CTGCTCGTGG TGACCGAAGG GGACAACGCC

151 ACCTTCACCT GCAGCTTCTC CAACACATCG GAGAGCTTCG TGCTAAACTG

201 GTACCGCATG AGCCCCAGCA ACCAGACGGA CAAGCTGGCC GCCTTCCCCG

251 AGGACCGCAG CCAGCCCGGC CAGGACTGCC GCTTCCGTGT CACACAACTG

301 CCCAACGGGC GTGACTTCCA CATGAGCGTG GTCAGGGCCC GGCGCAATGA

351 CAGCGGCACC TACCTCTGTG GGGCCATCTC CCTGGCCCCC AAGGCGCAGA

401 TCAAAGAGAG CCTGCGGGCA GAGCTCAGGG TGACAGAGAG AAGGGCAGAA

451 GTGCCCACAG CCCACCCCAG CCCCTCACCC AGGCCAGCCG GCCAGTTCCA

501 AACCCTGGTG GTTGGCGGCG GCGGCAGCGG CGGCGGCGGC AGCGGCGGCG

551 GCGGCAGCGG TGTCGTGGGC GGCCTGCTGG GCAGCCTGGT GCTGCTAGTC

601 TGGGTCCTGG CCGTCATCTG AGCCCCTCTC CCTCCCCCCC CCCTAACGTT

651 ACTGGCCGAA GCCGCTTGGA ATAAGGCCGG TGTGCGTTTG TCTATATGTT

701 ATTTTCCACC ATATTGCCGT CTTTTGGCAA TGTGAGGGCC CGGAAACCTG

751 GCCCTGTCTT CTTGACGAGC ATTCCTAGGG GTCTTTCCCC TCTCGCCAAA

801 GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC

851 TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC

901 CCCCACCTGG CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT

951 ACACCTGCAA AGGCGGCACA ACCCCAGTGC CACGTTGTGA GTTGGATAGT

1001 TGTGGAAAGA GTCAAATGGC TCTCCTCAAG CGTATTCAAC AAGGGGCTGA

1051 AGGATGCCCA GAAGGTACCC CATTGTATGG GATCTGATCT GGGGCCTCGG

1101 TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAAAC GTCTAGGCCC

1151 CCCGAACCAC GGGGACGTGG TTTTCCTTTG AAAAACACGA TGATAATATG

1201 GCCACAACCA TGTACAGGAT GCAACTCCTG TCTTGCATTG CACTAAGTCT

1251 TGCACTTGTC ACAAACAGTG CACCTACTTC AAGTTCTACA AAGAAAACAC

1301 AGCTACAACT GGAGCATTTA CTGCTGGATT TACAGATGAT TTTGAATGGA

1351 ATTAATAATT ACAAGAATCC CAAACTCACC AGGATGCTCA CATTTAAGTT

1401 TTACATGCCC AAGAAGGCCA CAGAACTGAA ACATCTTCAG TGTCTAGAAG

1451 AAGAACTCAA ACCTCTGGAG GAAGTGCTAA ATTTAGCTCA AAGCAAAAAC

1501 TTTCACTTAA GACCCAGGGA CTTAATCAGC AATATCAACG TAATAGTTCT

1551 GGAACTAAAG GGATCTGAAA CAACATTCAT GTGTGAATAT GCTGATGAGA

1601 CAGCAACCAT TGTAGAATTT CTGAACAGAT GGATTACCTT TTGTCAAAGC

1651 ATCATCTCAA CACTGACTTG A

<210> SEQ ID NO: 5

<211>

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> SEQ ID NO: 5

1 METGlnIleProGlnAlaProTrpProValValTrpAlaValLeuGlnLeuGlyTrpArg

21 ProGlyTrpPheLeuAspSerProAspArgProTrpAsnProProThrPheSerProAla

41 LeuLeuValValThrGluGlyAspAsnAlaThrPheThrCysSerPheSerAsnThrSer

61 GluSerPheValLeuAsnTrpTyrArgMETSerProSerAsnGlnThrAspLysLeuAla

81 AlaPheProGluAspArgSerGlnProGlyGlnAspCysArgPheArgValThrGlnLeu

101 ProAsnGlyArgAspPheHisMETSerValValArgAlaArgArgAsnAspSerGlyThr

121 TyrLeuCysGlyAlaIleSerLeuAlaProLysAlaGlnIleLysGluSerLeuArgAla

141 GluLeuArgValThrGluArgArgAlaGluValProThrAlaHisProSerProSerPro

161 ArgProAlaGlyGlnPheGlnThrLeuValValGlyGlyGlyGlySerGlyGlyGlyGly

181 SerGlyGlyGlyGlySerGlyValValGlyGlyLeuLeuGlySerLeuValLeuLeuVal

201 TrpValLeuAlaValIle

<210> SEQ ID NO: 6

<211>

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> SEQ ID NO: 6

1 METTyrArgMETGlnLeuLeuSerCysIleAlaLeuSerLeuAlaLeuValThrAsnSer

21 AlaProThrSerSerSerThrLysLysThrGlnLeuGlnLeuGluHisLeuLeuLeuAsp

41 LeuGlnMETIleLeuAsnGlyIleAsnAsnTyrLysAsnProLysLeuThrArgMETLeu

61 ThrPheLysPheTyrMETProLysLysAlaThrGluLeuLysHisLeuGlnCysLeuGlu

81 GluGluLeuLysProLeuGluGluValLeuAsnLeuAlaGlnSerLysAsnPheHisLeu

101 ArgProArgAspLeuIleSerAsnIleAsnValIleValLeuGluLeuLysGlySerGlu

121 ThrThrPheMETCysGluTyrAlaAspGluThrAlaThrIleValGluPheLeuAsnArg

141 TrpIleThrPheCysGlnSerIleIleSerThrLeuThr

Claims

1. a kind of slow virus for modifying DC-CIK cells is prepared, it is characterised in that comprising PD-1 extracellular regions and transmembrane region nucleic acid Sequence, is connected between extracellular region and transmembrane region with linker sequences, such as SEQ ID NO：Shown in 1, and interleukin 2 （IL-2）Nucleotide sequence such as SEQ ID NO：Shown in 2, SEQ ID NO：1 and SEQ ID NO：In the middle of 2 with IRES sequences such as SEQ ID NO ：It is attached shown in 3, the complete sequence for being constituted such as SEQ ID NO：Shown in 4, the virus can be thin in infection In cell surface expression PD-1 extracellular regions and IL-2 albumen is secreted after born of the same parents, its protein sequence and SEQ ID NO：5 and SEQ ID NO ：Shown in 6, for the treatment of kinds of tumors.

2. the method according to claim 1, the virus are through the following steps that obtain：Prepare PD-1 extracellular regions and Transmembrane region-IRES-IL-2 nucleotide sequences, commercialization slow virus carrier is connected into by the sequence, by taking pWPXL systems as an example, wherein Including but not limited to the slow virus system, carries out vector construction, by the method for calcium phosphate transfection, carries out viral packaging, harvests It is viral purified rear standby.

3. the method according to claim 1, the DC-CIK cells through the following steps that obtain：It is prepared by one kind The method of DC-CIK cells, it is characterised in that comprise the following steps：Lymphocyte is separated from peripheral blood, then by adherent Treatment makes a distinction, and attached cell partly changes liquid once in 2-3 days by induction differentiation culture is every, to obtain immature DC Cell；4-5 days, harvesting was co-cultured after the tumour antigen inactivation that will be prepared with incubation, traveling one one-step inducing differentiation of going forward side by side Culture, the 2-3 days DC cells to obtain the sensitization of maturation；Not adherent cell is every partly to change within 2-3 days by Fiber differentiation Liquid once, to obtain CIK cell；The CIK cell that will be cultivated 7 days adds 5x10⁸Virus, co-cultures 12 hours；DC and modification CIK1:5 mixing co-cultivations are produced Triumph-DC-CIK cells and will load the DC of tumour antigen and the modification of culture to 5-7 days CIK cell, is counted afterwards, and DC and CIK cell are collected in centrifugation respectively, and bicelluar density point is adjusted with VIVO-15 serum-free mediums Wei not 2x10⁵And 1x10⁶, by DC：CIK=1:25cm is moved into after 5 isometric mixing²Blake bottle 10ml/ bottles, in 37 DEG C, 5%CO₂Training Support, every equivalent fluid infusion in 2 days once；, every 3 days sub-bottle Amplification Cultures 1 time, after 2 Amplification Cultures, you can obtain 5x10⁹~1x10¹⁰Triumph-DC-CIK cells.

4. tumour according to claim 1 includes but is not limited to liver cancer, stomach cancer, breast cancer, colon cancer, cervical carcinoma, nerve Glioma and breast cancer.

5. it includes step in processing individuality：Exempted from the DC-CIK targetings comprising a kind of modification according to claim 1 Epidemic disease cell mass.

6. method according to claim 5, wherein the individuality has received or received or will receive extra Anti-cancer therapies, wherein extra anti-cancer therapies include operation, radiotherapy, chemotherapy, immunization therapy or hormone therapy.