CN108531458A - Treat the genetic engineering natural killer cells product of tumour - Google Patents
Treat the genetic engineering natural killer cells product of tumour Download PDFInfo
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Abstract
The genetic engineering natural killer cells product of tumour is treated, which is to be made of the transduction that people IL 2 and HSV tk genes are stablized to natural killer cells (NK) is interior gene engineering method.The cell products are homogeneous variant cell products, according to having good stability, anti-tumor effect and safety.It can be mass produced under conditions of not adding 2 IL.Using genetic engineering natural killer cells (NK) product of the present invention, it can be prepared into clinical grade gene cell therapeutic preparation, for treating and preventing the various malignant tumours of the mankind.
Description
Technical field
The invention belongs to bio-pharmaceuticals arts, are produced more particularly to the genetic engineering natural killer cells for the treatment of tumour
Product.
Background technology
The biological therapy of tumour is the fourth-largest therapy after operation, radiotherapy, chemotherapy.Due to traditional operation,
The development of chemicotherapy has been enter into plateau, and people throw into sight more and more in the biological therapy of tumour.Gene and cell
Immunotherapy of cancer is the cutting edge technology of tumor biotherapy.Compared with the traditional therapies such as chemotherapy, radiotherapy, have relatively malicious
The advantages such as Small side effects, significant in efficacy.Especially in the past few years, medical profession shows cellular immunotherapy malignant hematologic disease
Prodigious interest, cellular immunotherapy method and chemotherapy and radiation early period have non-crossing clinical effect, and Small side effects.
, it could be observed that in the various blood patients for receiving bone marrow transplantation therapy, after disease relapse, receive the injection of allosome leucocyte
The complete incidence graph [1-4] of the state of an illness can be reached.It, can be into one if handling leucocyte with interleukin 2 (IL-2) before injection
Killing ability and hazard boundary of the step enhancing donor leukocytes to patient's tumour cell.Natural killer cells (NK) be considered as
Donor leukocytes play an important component of decisive role in reacting the killing of patient's tumour cell, be congenital anti-
One important component [5] of infection and malignant tumour immune system.It is different with T cell effect, what they need not be advance
It is immune, so that it may which that, to kill tumour cell, killing activity is not limited by major histocompatibility complex (MHC).NK cells
Tumor cytotoxicity activity be to be realized by number of mechanisms, middle punch esterase approach is most common mechanism.Perforation ester
Enzyme destroys tumor cell membrane, and then apoptosis-induced cell death.Killing activation expressed by NK cells and killerinhibitoryreceptor
Decisive role [6-9] is played to the identification of tumour cell.Killing activated receptor can be thin with tumor cell and virus infection
The mark molecule of cellular surface, and kill Inhibitory receptor and can recognize that MHC I class molecules.When killing activated receptor and mark molecule are nibbled
After conjunction, the signal of one " killing " is sent to NK cells, in normal cell this " kill " signal can be by killerinhibitoryreceptor and I
The inhibition signal that class MHC molecule is sent out after being meshed constrains, to avoid the side effect that occur.Malignant tumour is thin
Born of the same parents are often upper defective in the expression of MHC I classes, no longer interact with killerinhibitoryreceptor, cannot provide inhibition signal, therefore,
It can be killed by NK cells.Regrettably, the NK cell functions of big a variety of malignant tumor patients are by major injury, Wu Fashi
The target of other malignant tumour loses growth in vitro and kills the function [10] of cell.In addition, cancer patient's autologous NK cells
It can be inhibited by the HLA of " self " expression, and then lose the function of killing malignant tumour.It is compared with autologous NK cells, it is of the same race different
Body NK cells will be a more promising cellular immunotherapy product, also be carried for large-scale production cellular immunotherapy product
Possibility is supplied.
NK-92 (ATCC CRL-2407) is one pure, the NK cell strains of allosome activation.It is thin that NK-92 cells lack killing
Born of the same parents inhibit receptor (KIR), blood and solid tumor cell killing ability with wide spectrum.With the LAK (killings of Lymphokine
Cell) cell compares, and NK-92 cells have stronger and wider cancer cell killing ability.But NK-92 cells have IL-2
Dependence can give large-scale production and clinical application to bring many inconvenience.In addition, the NK cells of Clinical practice allosome activation are also taken
With some potential safety problems (such as：Graft versus host disease(GVH disease)).In order to improve the safety of product, before injection in patient body
NK cellular immunity products need to be subjected to micro radiation treatment.Regrettably, while improving safety, micro radiation treatment
Also reduce the immunotherapeutic effects of product.In order to promote NK cells can be mass produced under the conditions of the GMP of specification,
When avoiding clinically using NK cell products, patient injection recombinant il-2 protein product is also given, and the anti-place of graft occurs
The hidden danger of main disease mustn't safely and effectively want the genetic engineering NK cell products of radiation treatment at present it is necessary to develop newly.
Document
1.Porter,D.L.,et al.,Adoptive immunotherapy with donor mononuclear
cell infusions to treat relapse of acute leukemia or myelodysplasia after
allogeneic bone marrow transplantation.Bone Marrow Transplantation,1996.18
(5):p.975-80.
2.Porter,D.L.,et al.,Induction of graft-versus-host disease as
immunotherapy for relapsed chronic myeloid leukemia.New England Journal of
Medicine,1994.330(2):p.100-6.
3.Collins,R.H.,Jr.,et al.,Donor leukocyte infusions in 140patients
with relapsed malignancy after allogeneic bone marrow transplantation.[see
comment].Journal of Clinical Oncology,1997.15(2):p.433-44.
4.Kolb,H.J.,et al.,Graft-versus-leukemia effect of donor lymphocyte
transfusions in marrow grafted patients.European Group for Blood and Marrow
Transplantation Working Party Chronic Leukemia.[see comment].Blood,1995.86
(5):p.2041-50.
5.Zeis,M.,et al.,Allogeneic MHC-mismatched activated natural killer
cells administered after bone marrow transplantation provides a strong graft-
versusleukemia effect in mice.British Journal of Haematology,1997.96(4):
p.757-61.
6.Moretta,A.,et al.,Major histocompatibility complex class I-specific
receptors on human natural killer and T lymphocytes.Immunological Reviews,
1997.155:p.105-17.
7.Lanier,L.L.,NK cell receptors.Annual Review of Immunology,1998.16:
p.359-93.
8.Smyth,M.J.,et al.,New aspects of natural-killer-cell surveillance
and therapy of cancer.Nature Reviews.Cancer,2002.2(11):p.850-61.
9.Chiorean,E.G.and J.S.Miller,The biology of natural killer cells and
implications for therapy of human disease.Journal of Hematotherapy&Stem Cell
Research,2001.10(4):p.451-63.
10.Whiteside,T.L.,N.L.Vujanovic,and R.B.Herberman,Natural killer
cells and tumor therapy.Current Topics in Microbiology&Immunology,1998.230:
p.221-44.
Invention content
The purpose of the present invention is developing the genetic engineering natural killer cells product of completely new treatment tumour, for tumour
Clinical treatment.
The present invention is achieved by the following technical solutions, and genetic engineering natural killer cells (NK) product is
Allow the expression H IL prime factor and the cell suicide factor that natural killer cells stablizes by genetic modification.
Further, H IL prime factor can be human IL-2 or IL-15.
Further, the cell suicide factor can be HSV-tk or cytosine deaminase gene (cytosine
deaminase)。
Further, natural killer cells can be any of people's natural killer cells system.
Further, the genetic modification natural killer cells stablized with the T2A fusions of IL-2 genes and HSV-tk genes.
Further, genetic engineering natural killer cells is used to prepare the gene cell drug production for treating various malignant tumours
Product.
Further, it is produced under gmp conditions using extensive serum free suspension culture technique.
It is possible to further be used to be injected intravenously, intra arterial injection, intratumor injection, hypodermic injection, organ injection, in hydrothorax
Injection in injection or ascites.
Technical scheme of the present invention advantageous effect is：The invention discloses the genetic engineerings of structure treatment tumour to kill naturally
Hinder the method for cell products.Constructed genetic engineering natural killer cells, NK-SBN cells, have good anti-cancer function and
Safety can be tried out in clinical cancer therapy.
Description of the drawings
Fig. 1 is the expression of the IL-2 of genetic engineering NK92 clone cells；
Fig. 2 is the expression of the TK of genetic engineering NK92 clone cells；
Fig. 3 is the TK sensitivity Detection results of genetic engineering NK92 clone cells；
Fig. 4 is the external inhibiting tumor cell effect of NK-SBN cells；
Fig. 5 is the anticarcinogenic effect of NK-SBN cells in animal body；
Fig. 6 is the co-incubation of NK-SBN cells and healthy person peripheral blood mononuclear cells (PBMC).
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
1, the structure of IL-2-T2A-HSV-tk vector genes engineering plasmid carrier and expression
The clone for having synthesized IL-2cDNA and HSV-tk cDNA genetic fragments is ordered from U.S. Life Technologies
Expression plasmid.Expression plasmid is freeze-drying.The IL-2cDNA of synthesis is cloned into pcDNA3.1/Neo (Invitrogen
V79020 IL-2 expression plasmid carriers) have been built up in carrier.The HSV-tk cDNA gene pieces of synthesis are cloned into pcDNA3.1/
Suicide gene plasmid vector has been built up in Hygro (Invitrogen V870-20) carrier.To IL-2cDNA and HSV-tk cDNA
Genetic fragment is sequenced.IL-2cDNA gene orders please refer to sequence table.HSV-tk cDNA gene orders please refer to sequence
Table.Sequencing result confirms that synthesized IL-2 and HSV-tk gene orders and theoretical reference sequence are 100% to comply fully with.
IL-2cDNA pcDNA3.1 (+) carriers and HSV-tk cDNA that will be synthesized from Life Technologies purchases
PcDNA3.1 (+) carrier is transformed into respectively in Stbl3E.coli (Life Technologies, C7373-03), with LB culture mediums
The transformed Stbl3E.coli of genetic engineering plasmid is incubated overnight in 37 DEG C of degree shaking tables, amplification gene engineering plasmid carrier
DNA.With Qiagen plasmid purification columns (QIAprep Spin Miniprep Kit, 27104), the IL-2 of amplification is purified within second day
With HSV-tk genetic engineering plasmid vectors.EcoRI (New England Biolabs, R0101S) and NotI (New is used again
England Biolabs, R0189S) specificity restriction endonuclease cuts HSV-tk gene plasmids simultaneously and pcDNA3.1 (+)-IL-2 is carried
Body reacts 1 hour in 37 DEG C of water-baths, by HSV-tk gene clonings to pcDNA3.1 (+)-IL-2 carriers, builds pcDNA3.1
(+)-IL-2-HSV-tk genetic engineering plasmid vectors.By pcDNA3.1 (+)-IL-2-HSV-tk genetic engineering plasmids of structure,
It is transformed into Stbl3E.coli (Life Technologies, C7373-03), is trained overnight in 37 DEG C of degree shaking tables with LB culture mediums
Support genetic engineering plasmid transformed Stbl3E.coli, the DNA of amplification gene engineering plasmid carrier.Second day with Qiagen matter
Grain purification column (QIAprep Spin Miniprep Kit, 27104), purifies pcDNA3.1 (+)-IL-2-HSV-tk bases of amplification
Because of engineering plasmid carrier.
Using the method for PCR amplification, the T2A double-stranded DNA gene segments from Thoseaasigna virus have been synthesized.
T2A DNA gene orders please refer to sequence table.During PCR amplification, it is added to and Kpn1 at the both ends of T2A DNA sequence dnas
The equitant 14bp sequences of specific inscribe cleavage pcDNA3.1 (+)-IL-2-HSV-tk plasmids.This equitant 14bp sequence
Row are respectively,
5’-AGTTATCAGTCGACGGTACC-3’
3’-CCATGGCCTGTATACGGGCC-5’
Bolded sequence is Kpn1 specificity inscribe cleavage sites.
T2A double-stranded DNA gene segments denaturation by synthesis and renaturation cut synthesis simultaneously with Kpn1 specificity restriction endonuclease
T2A double chain DNA fragments and pcDNA3.1 (+)-IL-2-HSV-tk Plasmid DNA, with In-Fusion HD cloning process
T2A sequences are cloned into pcDNA3.1 (+)-IL-2-HSV-tk carriers by (Clontech, 638909), build pcDNA3.1
(+)-IL-2-T2A-HSV-tk genetic engineering plasmid vectors.
By pcDNA3.1 (+)-IL-2-T2A-HSV-tk genetic engineering plasmids of structure, it is transformed into Stbl3E.coli
(Life Technologies, C7373-03) is incubated overnight the conversion of genetic engineering plasmid with LB culture mediums in 37 DEG C of degree shaking tables
The Stbl3E.coli crossed, the DNA of amplification gene engineering plasmid carrier.Second day with Qiagen plasmid purification columns (QIAprep
Spin Miniprep Kit, 27104), pcDNA3.1 (+)-IL-2-T2A-HSV-tk genetic engineering plasmids for purifying amplification carry
Body.It is spare that the genetic engineering plasmid of purifying is stored in -20 degree refrigerators.
Sequence analysis detection has been done to pcDNA3.1 (+) the IL-2-T2A-HSV-tk genetic engineering plasmid vectors built,
Gene order (7036bp) please refers to sequence table.Sequencing result confirms constructed pcDNA3.1 (+) IL-2-T2A-HSV-tk bases
Because sequence and theoretical reference sequence are 100% to comply fully with.
2, the establishment of genetic engineering NK cells
Using conventional cell culture processes, culture and amplification NK cells, such as NK-92 (ATCC CRL-2407) cell.
With the plamid vector transfection NK cells for expressing IL-2 and HSV-tk suicide genes simultaneously.Plamid vector transfection is according to Life
The recommendation method of the Lipofectamine reagents (#11668-019) of Technology carries out.With containing after cell transfecting
The liquid medium-selection of neomycin stablizes the NK cells of expression IL-2 and HSV-tk suicide genes, establishes the cell mass at initial stage.Gram
Grand purifying cells group finally establishes genetic engineering NK clone cells, and long-term preservation is in liquid nitrogen, for further studying and
Production.
Culture NK-92 (the ATCC in the AMEM culture solutions for being added to 12% cow's serum and the recombinant il-2 of 150U/mL
CRL-2407) cell.With Lipofectamine reagents (Life Technology, #11668-019) while by pcDNA3.1
(+)IL-2-T2A-HSV-tk
Plamid vector transfection is intracellular to NK-92.After transfection 48 hours, culture solution is converted into the training containing neomycin
The NK-92 cells of expression IL-2 and HSV-tk suicide genes are stablized in nutrient solution screening.It is after continuous culture 30 days, survivaling cell is dilute
It releases, and is transferred in 96 orifice plates and continues to cultivate.It is diluted in each hole only there are one cell.
The upgrowth situation for observing cell under the microscope daily, replaces cell culture fluid if necessary.By 3 wheat harvesting periods
Culture expands cell into the hole of 24 orifice plates from the hole of 96 orifice plates, and in each hole plus 400 microlitres of culture solutions continue culture and expand
Increase.It is primary that culture solution was replaced per 3-5 days.NK92 cell growths relatively slowly, after having cultivated 2 wheat harvesting periods, have in 24 orifice plates
A certain amount of cell, for expanding into 6 orifice plates.In each hole plus 2 milliliters of culture solutions continue culture amplification.More per 3-5 days
It is primary to change culture solution.After having cultivated 1 wheat harvesting period, by cell from 6 orifice plates, the T-25 Tissue Culture Flasks containing 5mL are arrived in amplification, with
Just culture is used to analyze detection and liquid nitrogen storage to the cell of sufficient amount.23 T-25 Tissue Culture Flasks are amounted to, each clone is thin
Born of the same parents are individually with a T-25 Tissue Culture Flask.It is primary that culture solution was replaced per 3-5 days as before.After having cultivated 1 wheat harvesting period,
Cell concentration in each T-25 Tissue Culture Flasks has reached 1.1-1.5x106/mL in succession.Out of each T-25 Tissue Culture Flasks
It takes 1mL to be transferred in new T-25 Tissue Culture Flasks to continue to cultivate, is used for cell detection.By remaining cell, 1000rpm from
The heart 5 minutes, obtained cell precipitation is suspended in 2 milliliters of liquid nitrogen storage buffer solution.Liquid nitrogen storage buffer solution is in gene
The DMSO of addition 10% is made into engineering NK92 cell complete growths culture solution.1mL cell suspensions are added to 12 milliliters
Cryovial (cryovial) in, i.e., each genetic engineering NK92 cell clones have 2 cryovials.The cryovial of cell will be filled
It is put into the Mr.Frosty freeze box for having filled alcohol, places into the low-temperature frozen case of -80C overnight, frozen cell.It second day, will
The cell of freezing is put into long term storage in liquid nitrogen storage tank.Amount to the freezing clone cell of 23 genetic engineering NK92.
3, the detection of genetic engineering NK92 cell clones
The structure of foreign gene is imported, number of copies and stability are a highly important matter of genetic engineering NK cells
Figureofmerit.After establishing genetic engineering NK cell clones, is analyzed and studied using conventional molecular biology method and establishing base
Because of the foreign gene structure of engineering NK cell clones.Extract the genomic DNA of cell.According to pcDNA3.1 (+)-IL-2-T2A-
The DNA sequence dnas of HSV-tk plasmids designs PCR primer, IL-2 and HSV-tk genes are detected and determined in the cell with qPCR methods
Copy number.Confirm the integrality for importing foreign gene with analysis of Restriction Endonuclease Profile.In order to confirm that genetic engineering NK is thin
The stability of born of the same parents' foreign gene, culture cell and 30 to 40 generation of continuous passage.Again with above-described molecular biological analysis side
The integrality and copy number in the cell of IL-2 and HSV-tk genes is detected and determined in method, to confirm imports foreign gene
Stability.
The cell of amplification is collected from each T-25 Tissue Culture Flasks.Cell culture fluid is added in centrifuge tube,
1500rpm, 4C, lower centrifugation 5-7 minutes.Supernatant is removed, to the cell of centrifugation bottom of the tube, ice-cold PBS buffer solution is added, is come clear
Wash cell.Again in 1500rpm, 4C, lower centrifugation 5-7 minutes.Supernatant is removed, to the cell of centrifugation bottom of the tube, is added ice-cold
RIPA (radioimmunoprecipitation assay buffer solution) cell pyrolysis liquid.At 4C, stir 30 minutes.Then in 16000g, 4C, under from
The heart 20 minutes.Supernatant is collected, is placed on ice, the concentration of albumen is measured with conventional albumen concentration detection method.For albumen table
It is spare up to detecting.
● the detection of expression of IL-2 albumen
With conventional gel electrophoresis method, protein isolate matter.By the Protein transfer of separation to the film of Western blot
On.The expression of IL-2 is detected with anti-human IL-2 polyclonal antibodies (Thermo Scientific, Cat#710146).Gene work
The expression of the IL-2 of journey NK92 clone cells please refers to Fig.1.All genetic engineering NK92 clone cells are all in different water
IL-2 albumen is expressed on flat.The phenomenon that this can grow with clone cell in the culture solution without IL-2 is consistent.Relatively
For, clone 4,7 and 11 has higher IL-2 expression quantity.
● the expression of HSV-tk albumen is examined
With conventional gel electrophoresis method, protein isolate matter.By the Protein transfer of separation to the film of Western blot
On.The expression of TK is detected with anti-HSV-TK monoclonal antibodies (Thermo Scientific, Cat#MA1-21542).Gene work
The expression of the TK of journey NK92 clone cells please refers to Fig. 2.All genetic engineering NK92 clone cells are all in different level
On express TK albumen.Comparatively, clone 7,13 and 23 has higher TK expression quantity.
● TK sensitivity Detections
According to the expression of the IL-2 and TK of genetic engineering NK92 clone cells, selected cell clone 7, carried out into
The TK sensitivity Detections of one step.As a contrast using ATCC NK-92 cells.7 and ATCC NK-92 cell inoculations will be cloned to phase
In 12 hollow plates answered, 1mL is added per empty, cell concentration 1x105 cells/mL, 12 hollow plates are for cloning 7 cells, and in addition one
A 12 hollow plate is used for ATCC NK-92 cells.The recombination that 150 units per mls are added in the culture solution of ATCC NK-92 cells is white
Cytokine -2.After having cultivated 24 hours, to the intracellular of culture, being separately added into 0,5,10,50,75 and 100 μM, (end is dense
Degree) Ganciclovir (Ganciclovir, GCV, Sigma, Catalog#G2536).There are two empty for each GCV concentration.Continue to train
Support 96 hours.Sampling dyes detection cell activity with trypan blue (trypan blue).Genetic engineering NK92 clone cells
TK sensitivity Detection results please refer to Fig. 3.
Clone 7 cells has apparent sensibility to GCV, and under the conditions of higher than 10 μM of GCV, most cells are 96
With regard to dead after hour.Confirm the activity for the TK genes that gene is transferred to.In contrast, the ATCC NK-92 of TK genes are not contained
Cell does not show GCV apparent sensibility.
● copy number of the IL-2 and HSV-TK genes in cell clone
It is cloned in 7 cells from genetic engineering NK92 and extracts genomic DNA.With quantifying PCR method be detected and determined IL-2 and
HSV-TK suicide gene copy numbers.The standard for using RNase P genes to be calculated as quantitative PCR gene copy number.IL-2 genes are fixed
Measure PCR detection method
Left primer：TGCAACTCCTGTCTTGCATT
Right primer：GCCTTCTTGGGCATGTAAAA
Visit object：GAATCCCAAACTCACCAGGA
TK gene quantification PCR detection methods
Left primer：GCCTTGACCAGGGTGAGATA
Right primer：ATGCTGCCCATAAGGTATCG
Visit object：ATGACAAGCGCCCAGATAAC
PCR cycle method
Denaturation/activation：95 DEG C, 5 minutes, once
Denaturation：95 DEG C, 15 seconds
Annealing/extension:60 DEG C, 1 minute
Cycle 40 times
Copy number of the 1 IL-2 and HSV-TK genes of table in cell clone
Each genetic engineering NK92 clones 7 have 1 about reciprocity copy IL-2 and HSV-TK gene into the cell.Clone cell
It is named as NK-SBN cells.
4, the external anticarcinogenic effect of NK-SBN
Under the conditions of the ratio of different NK-SBN cells and tumour cell, such as 50:1,20:1,10:1,5:1 and 1:1, altogether
With mixed culture NK-SBN cells and tumour cell not of the same race, such as HL-60, K562 and Jurkat cell, using Europium
Cytotoxicity assays (DELFIA, PerkinElmer) measure the cracking strength of NK-SBN cells against tumor cells, to confirm NK-
The antitumor cell effect of SBN cells.The peripheral blood mononuclear cells (PBMC) of the healthy person of IL-2 activation is used in test to make
For control Europium Cytotoxicity assays are calibrated with detergent processing cancer cell (100% cancer cell lysis).
With RPMI culture solution cultures tumour cell (HL60, Raji, K562 and Jurkat cell).PBS is used in combination in centrifuge cell
Clean cell.With DELFIA BATDA reagent label cells, cultivated 30 minutes at 37 DEG C.It is thin after cleaning label with PBS again
Born of the same parents are diluted to 1x105/mL with RPMI culture solutions.Put 5000 cells (50 microlitres) in the sky of 96 hollow plates.It takes in logarithmic growth
The NK-SBN cells of phase, centrifuge cell are used in combination PBS to clean cell.The cell cleaned is suspended in culture solution, concentration 1E6/ millis
It rises.The NK-SBN cells (such as 50 of different proportion are added into 96 hollow plates containing tumour cell:1,20:1,10:1,5:1 He
1:1).100 microlitres of total volume/sky.96 hollow plate is centrifuged at 200g 4 minutes, to ensure that NK-SBN cells and tumour cell have foot
Enough contacts.96 hollow plates are placed on 2 hours of culture in 37 DEG C/5%CO2 cell incubators.It is each with multichannel pipettor mixing
Empty liquid 5-10 times.96 hollow plate is centrifuged at 500g 5 minutes.From it is each it is empty in take out 20 microlitres, be put into one and new be used for
In 96 hollow plates of fluoroscopic examination.200 microlitres of DELFIA Europium are added into each sky and detect liquid.By 96 hollow plate aluminium
Film wraps up, and is placed on orbital shaker and shakes 15 minutes.100 microlitres of Europium standards are added in two empty skies into 96 hollow plates
Liquid.Fluorescence intensity is measured under fluorescence microplate reader (Synergy 2, Biotek) in different times gap.Measure setup condition
For,
Excitation wavelength：330nm
Send out wavelength：615nm
Time lag：400μs
Measurement gap：1000μs
Measure height：365 millimeters
At the processed cancer cell fluorescence intensity/detergent of NK-SBN cell carcinoma killing rates (%)=NK-SBN cells
Managed cancer cell fluorescence intensity.
The external inhibiting tumor cell effect of NK-SBN cells please refers to Fig. 4.NK-SBN genetic engineering NK cells are to kinds of tumors
Cell has apparent killing cracking strength.In contrast, normal human PBMC does not show killing cracking strength then to cancer cell.
5, anticarcinogenic effect in the animal body of NK-SBN
On the basis of demonstrating extracorporeal anti-tumor cell results, further genetic engineering NK cells are verified in animal body
Anti-tumor effect.Since genetic engineering NK cells are people's cell, animal vivo test Reconstruction in Sever Combined Immunodeciency
(SCID) mouse.It is thin that 106 human leukemias of 5x are squeezed into Reconstruction in Sever Combined Immunodeciency (SCID) mouse by abdominal cavity first
Born of the same parents, TA27, after five days, then by the way that 2x 107NK-SBN cells are injected intraperitoneally, every other day injection is primary, 5 times in total.Control group
Mouse receives physiological saline.The health status of mouse is observed, phase and life cycle occur for tumour.Genetic engineering NK cell therapies
Apparent antitumous effect should be shown, the life cycle of mouse is extended.
It is tested using severe combined immunodeficiency (SCID) mouse to do anticarcinogenic effect in NK-SBN cell bodies.First small
The cultured human leukemia cell (cell line TA27) of mouse intraperitoneal inoculation, with RPMI+10% hyclone nutrient solution cultures TA27.
The cell for collecting exponential phase is used for zoopery.It is followed by by NK-SBN cell therapies within four days.In total with 30 mouse.Divide 3
Group, every group of 10 mouse.
1st group：5x 106TA27 cells are injected in first day each mouse peritoneal.TA27 cells are suspended in before injection
In PBS buffer solution.This group of mouse does not receive NK-SBN cell therapies (negative control group), and receiving is physiological saline, often
Injection is primary every two days, 5 times in total.The health status and weight of observation mouse daily.
2nd group：5x 106TA27 cells are injected in first day each mouse peritoneal.TA27 cells are suspended in before injection
In PBS buffer solution.The treatment of injection 2x 107NK-SBN cells in the 5th day, each mouse peritoneal.Every other day inject one
It is secondary, 5 times in total.The health status and weight of observation mouse daily.
3rd group：This group of mouse does not receive the injection of human leukemia cell.
The cell (positive controls) of injection 2x 107NK-SBN in the 5th day, each mouse peritoneal.Every other day note
It penetrates once, 5 times in total, observes the health status and weight of mouse daily.The anticarcinogenic effect of NK-SBN cells in animal body is asked
Refering to Fig. 5.Genetic engineering NK-SBN cell therapies have reached apparent antitumous effect, effectively extend the existence of mouse
Phase.
6, the safety of NK-SBN cells
Using 1:The peripheral blood mononuclear cells (PBMC) of the 1 common healthy person for being mixed NK-SBN cells and activation 4 days,
Then PBMC cell inoculations are cultivated in methylcellulose culture solution, number meter red blood cell colony number, granulocyte and macrophage
Colony number of cell and other colony number of cell, with no peripheral blood mononuclear cells by with NK-SBN cell co-cultures
(PBMC) as a contrast.Experiment uses the peripheral blood mononuclear cells of 3 healthy persons.NK-SBN cells and healthy person peripheral blood list
The co-incubation result of nucleus (PBMC) please refers to Fig. 6.As a result show NK-SBN cells to healthy person peripheral blood mononuclear cells
(PBMC) the no harmful effect of growth.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
Sequence table
<110>Sai Nuo（Shenzhen）Biopharmaceutical Research Inc.
<120>Treat the genetic engineering natural killer cells product of tumour
<160> 7
<210> 1
<211> 462
<212> DNA
<213>It is artificial synthesized
<400> 1
1 ATGTACAGGA TGCAACTCCT GTCTTGCATT GCACTAAGTC TTGCACTTGT CACAAACAGT GCACCTACTT CAAGTTCTAC
81 AAAGAAAACA CAGCTACAAC TGGAGCATTT ACTGCTGGAT TTACAGATGA TTTTGAATGG AATTAATAAT TACAAGAATC
161 CCAAACTCAC CAGGATGCTC ACATTTAAGT TTTACATGCC CAAGAAGGCC ACAGAACTGA AACATCTTCA GTGTCTAGAA
241 GAAGAACTCA AACCTCTGGA GGAAGTGCTA AATTTAGCTC AAAGCAAAAA CTTTCACTTA AGACCCAGGG ACTTAATCAG
321 CAATATCAAC GTAATAGTTC TGGAACTAAA GGGATCTGAA ACAACATTCA TGTGTGAATA TGCTGATGAG ACAGCAACCA
401 TTGTAGAATT TCTGAACAGA TGGATTACCT TTTGTCAAAG CATCATCTCA ACACTAACTT GA
<210> 2
<211> 1191
<212> DNA
<213>It is artificial synthesized
<400> 2
1 ATGGCTTCGT ACCCCGGCCA TCAGCACGCG TCTGCGTTCG ACCAGGCTGC GCGTTCTCGC GGCCATAGCA ACCGACGTAC
81 GGCGTTGCGC CCTCGCCGGC AGCAAGAAGC CACGGAAGTC CGCCCGGAGC AGAAAATGCC CACGCTACTG CGGGTTTATA
161 TAGACGGTCC CCACGGGATG GGGAAAACCA CCACCACGCA ACTGCTGGTG GCCCTGGGTT CGCGCGACGA TATCGTCTAC
241 GTACCCGAGC CGATGACTTA CTGGCGGGTG CTGGGGGCTT CCGAGACAAT CGCGAACATC TACACCACAC AACACCGCCT
321 TGACCAGGGT GAGATATCGG CCGGGGACGC GGCGGTGGTA ATGACAAGCG CCCAGATAAC AATGGGCATG CCTTATGCCG
401 TGACCGACGC CGTTCTGGCT CCTCATATCG GGGGGGAGGC TGGGAGCTCA CATGCCCCGC CCCCGGCCCT CACCCTCATC
481 TTCGACCGCC ATCCCATCGC CGCCCTCCTG TGCTACCCGG CCGCGCGATA CCTTATGGGC AGCATGACCC CCCAGGCCGT
561 GCTGGCGTTC GTGGCCCTCA TCCCGCCGAC CTTGCCCGGC ACAAACATCG TGTTGGGGGC CCTTCCGGAG GACAGACACA
641 TCGACCGCCT GGCCAAACGC CAGCGCCCCG GCGAGCGGCT TGACCTGGCT ATGCTGGCCG CGATTCGCCG CGTTTACGGG
721 CTGCTTGCCA ATACGGTGCG GTATCTGCAG GGCGGCGGGT CGTGGCGGGA GGATTGGGGA CAGCTTTCGG GGACGGCCGT
801 GCCGCCCCAG GGTGCCGAGC CCCAGAGCAA CGCGGGCCCA CGACCCCATA TCGGGGACAC GTTATTTACC CTGTTTCGGG
881 CCCCCGAGTT GCTGGCCCCC AACGGCGACC TGTACAACGT GTTTGCCTGG GCCTTGGACG TCTTGGCCAA ACGCCTCCGT
961 CCCATGCACG TCTTTATCCT GGATTACGAC CAATCGCCCG CCGGCTGCCG GGACGCCCTG CTGCAACTTA CCTCCGGGAT
1041 GATCCAGACC CACGTCACCA CCCCAGGCTC CATACCGACG ATCTGCGACC TGGCGCGCAC GTTTGCCCGG GAGATGGGGG
1121 AGGCTAACTG AAACACGGAA GGAGACAATA CCGGAAGGAA CCCGCGCTAT GACGGCAATA AAAAGACAGA A
<210> 3
<211> 63
<212> DNA
<213>It is artificial synthesized
<400> 3
1 GGAAGCGGAG AGGGCAGAGG AAGTCTGCTA ACATGCGGTG ACGTCGAGGA GAATCCTGGA CCT
<210> 4
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 4
1 AGTTATCAGT CGACGGTACC
<210> 5
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 5
1 CCATGGCCTG TATACGGGCC
<210> 6
<211> 7036
<212> DNA
<213>It is artificial synthesized
<400> 6
1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGTAT
81 CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA
161 CAATTGCATG AAGAATCTGC TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT
241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG CGTTACATAA
321 CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT
401 AACGCCAATA GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT
481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA
561 TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA
641 TGGGCGTGGA TAGCGGTTTG ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC
721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG
801 GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG
881 GGAGACCCAA GCTGGCTAGC GTTTAAACTT AAGCTTATGT ACAGGATGCA ACTCCTGTCT TGCATTGCAC TAAGTCTTGC
961 ACTTGTCACA AACAGTGCAC CTACTTCAAG TTCTACAAAG AAAACACAGC TACAACTGGA GCATTTACTG CTGGATTTAC
1041 AGATGATTTT GAATGGAATT AATAATTACA AGAATCCCAA ACTCACCAGG ATGCTCACAT TTAAGTTTTA CATGCCCAAG
1121 AAGGCCACAG AACTGAAACA TCTTCAGTGT CTAGAAGAAG AACTCAAACC TCTGGAGGAA GTGCTAAATT TAGCTCAAAG
1201 CAAAAACTTT CACTTAAGAC CCAGGGACTT AATCAGCAAT ATCAACGTAA TAGTTCTGGA ACTAAAGGGA TCTGAAACAA
1281 CATTCATGTG TGAATATGCT GATGAGACAG CAACCATTGT AGAATTTCTG AACAGATGGA TTACCTTTTG TCAAAGCATC
1361 ATCTCAACAC TAACTGGATC CGAGGGCAGA GGAAGTCTTC TAACATGCGG TGACGTGGAG GAGAATCCCG GCCCTTTGAA
1441 TTCTATGGCT TCGTACCCCG GCCATCAGCA CGCGTCTGCG TTCGACCAGG CTGCGCGTTC TCGCGGCCAT AGCAACCGAC
1521 GTACGGCGTT GCGCCCTCGC CGGCAGCAAG AAGCCACGGA AGTCCGCCCG GAGCAGAAAA TGCCCACGCT ACTGCGGGTT
1601 TATATAGACG GTCCCCACGG GATGGGGAAA ACCACCACCA CGCAACTGCT GGTGGCCCTG GGTTCGCGCG ACGATATCGT
1681 CTACGTACCC GAGCCGATGA CTTACTGGCG GGTGCTGGGG GCTTCCGAGA CAATCGCGAA CATCTACACC ACACAACACC
1761 GCCTTGACCA GGGTGAGATA TCGGCCGGGG ACGCGGCGGT GGTAATGACA AGCGCCCAGA TAACAATGGG CATGCCTTAT
1841 GCCGTGACCG ACGCCGTTCT GGCTCCTCAT ATCGGGGGGG AGGCTGGGAG CTCACATGCC CCGCCCCCGG CCCTCACCCT
1921 CATCTTCGAC CGCCATCCCA TCGCCGCCCT CCTGTGCTAC CCGGCCGCGC GATACCTTAT GGGCAGCATG ACCCCCCAGG
2001 CCGTGCTGGC GTTCGTGGCC CTCATCCCGC CGACCTTGCC CGGCACAAAC ATCGTGTTGG GGGCCCTTCC GGAGGACAGA
2081 CACATCGACC GCCTGGCCAA ACGCCAGCGC CCCGGCGAGC GGCTTGACCT GGCTATGCTG GCCGCGATTC GCCGCGTTTA
2161 CGGGCTGCTT GCCAATACGG TGCGGTATCT GCAGGGCGGC GGGTCGTGGC GGGAGGATTG GGGACAGCTT TCGGGGACGG
2241 CCGTGCCGCC CCAGGGTGCC GAGCCCCAGA GCAACGCGGG CCCACGACCC CATATCGGGG ACACGTTATT TACCCTGTTT
2321 CGGGCCCCCG AGTTGCTGGC CCCCAACGGC GACCTGTACA ACGTGTTTGC CTGGGCCTTG GACGTCTTGG CCAAACGCCT
2401 CCGTCCCATG CACGTCTTTA TCCTGGATTA CGACCAATCG CCCGCCGGCT GCCGGGACGC CCTGCTGCAA CTTACCTCCG
2481 GGATGATCCA GACCCACGTC ACCACCCCAG GCTCCATACC GACGATCTGC GACCTGGCGC GCACGTTTGC CCGGGAGATG
2561 GGGGAGGCTA ACTGAAACAC GGAAGGAGAC AATACCGGAA GGAACCCGCG CTATGACGGC AATAAAAAGA CAGAAGCGGC
2641 CGCTCGAGTC TAGAGGGCCC GTTTAAACCC GCTGATCAGC CTCGACTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC
2721 CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA TTGCATCGCA
2801 TTGTCTGAGT AGGTGTCATT CTATTCTGGG GGGTGGGGTG GGGCAGGACA GCAAGGGGGA GGATTGGGAA GACAATAGCA
2881 GGCATGCTGG GGATGCGGTG GGCTCTATGG CTTCTGAGGC GGAAAGAACC AGCTGGGGCT CTAGGGGGTA TCCCCACGCG
2961 CCCTGTAGCG GCGCATTAAG CGCGGCGGGT GTGGTGGTTA CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC
3041 CGCTCCTTTC GCTTTCTTCC CTTCCTTTCT CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG GGGCTCCCTT
3121 TAGGGTTCCG ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT TAGGGTGATG GTTCACGTAG TGGGCCATCG
3201 CCCTGATAGA CGGTTTTTCG CCCTTTGACG TTGGAGTCCA CGTTCTTTAA TAGTGGACTC TTGTTCCAAA CTGGAACAAC
3281 ACTCAACCCT ATCTCGGTCT ATTCTTTTGA TTTATAAGGG ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA
3361 TTTAACAAAA ATTTAACGCG AATTAATTCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG
3441 GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT
3521 ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA CTCCGCCCAG
3601 TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC CGCCTCTGCC TCTGAGCTAT
3681 TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT TTGCAAAAAG CTCCCGGGAG CTTGTATATC CATTTTCGGA
3761 TCTGATCAAG AGACAGGATG AGGATCGTTT CGCATGATTG AACAAGATGG ATTGCACGCA GGTTCTCCGG CCGCTTGGGT
3841 GGAGAGGCTA TTCGGCTATG ACTGGGCACA ACAGACAATC GGCTGCTCTG ATGCCGCCGT GTTCCGGCTG TCAGCGCAGG
3921 GGCGCCCGGT TCTTTTTGTC AAGACCGACC TGTCCGGTGC CCTGAATGAA CTGCAGGACG AGGCAGCGCG GCTATCGTGG
4001 CTGGCCACGA CGGGCGTTCC TTGCGCAGCT GTGCTCGACG TTGTCACTGA AGCGGGAAGG GACTGGCTGC TATTGGGCGA
4081 AGTGCCGGGG CAGGATCTCC TGTCATCTCA CCTTGCTCCT GCCGAGAAAG TATCCATCAT GGCTGATGCA ATGCGGCGGC
4161 TGCATACGCT TGATCCGGCT ACCTGCCCAT TCGACCACCA AGCGAAACAT CGCATCGAGC GAGCACGTAC TCGGATGGAA
4241 GCCGGTCTTG TCGATCAGGA TGATCTGGAC GAAGAGCATC AGGGGCTCGC GCCAGCCGAA CTGTTCGCCA GGCTCAAGGC
4321 GCGCATGCCC GACGGCGAGG ATCTCGTCGT GACCCATGGC GATGCCTGCT TGCCGAATAT CATGGTGGAA AATGGCCGCT
4401 TTTCTGGATT CATCGACTGT GGCCGGCTGG GTGTGGCGGA CCGCTATCAG GACATAGCGT TGGCTACCCG TGATATTGCT
4481 GAAGAGCTTG GCGGCGAATG GGCTGACCGC TTCCTCGTGC TTTACGGTAT CGCCGCTCCC GATTCGCAGC GCATCGCCTT
4561 CTATCGCCTT CTTGACGAGT TCTTCTGAGC GGGACTCTGG GGTTCGAAAT GACCGACCAA GCGACGCCCA ACCTGCCATC
4641 ACGAGATTTC GATTCCACCG CCGCCTTCTA TGAAAGGTTG GGCTTCGGAA TCGTTTTCCG GGACGCCGGC TGGATGATCC
4721 TCCAGCGCGG GGATCTCATG CTGGAGTTCT TCGCCCACCC CAACTTGTTT ATTGCAGCTT ATAATGGTTA CAAATAAAGC
4801 AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAG TTGTGGTTTG TCCAAACTCA TCAATGTATC
4881 TTATCATGTC TGTATACCGT CGACCTCTAG CTAGAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT
4961 ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC
5041 ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG
5121 CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC
5201 GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA
5281 GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA
5361 GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA
5441 GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG
5521 CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC
5601 CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG
5681 CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC
5761 GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG
5841 ATCCGGCAAA CAAACCACCG CTGGTAGCGG TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG
5921 AAGATCCTTT GATCTTTTCT ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA
6001 TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA AGTATATATG AGTAAACTTG
6081 GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC
6161 TCCCCGTCGT GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC
6241 TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA CTTTATCCGC
6321 CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA
6401 TTGCTACAGG CATCGTGGTG TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT
6481 ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC AGAAGTAAGT TGGCCGCAGT
6561 GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG
6641 AGTACTCAAC CAAGTCATTC TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAATACG GGATAATACC
6721 GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA CTCTCAAGGA TCTTACCGCT
6801 GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT
6881 GAGCAAAAAC AGGAAGGCAA ACTCTTCCTT TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA
6961 CATATTTGAA TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT GACGTC
<210> 7
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 7
1 TGCAACTCCT GTCTTGCATT
<210> 8
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 8
1 GCCTTCTTGG GCATGTAAAA
<210> 9
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 9
1 GAATCCCAAA CTCACCAGGA
<210> 10
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 10
1 GCCTTGACCA GGGTGAGATA
<210> 11
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 11
1 ATGCTGCCCA TAAGGTATCG
<210> 12
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 12
1 ATGACAAGCG CCCAGATAAC
Claims (8)
1. treating the genetic engineering natural killer cells product of tumour, it is characterised in that, genetic engineering natural killer cells production
Product are to allow the expression H IL prime factor and the cell suicide factor that natural killer cells stablizes by genetic modification.
2. the genetic engineering natural killer cells product for the treatment of tumour as described in claim 1, which is characterized in that the people is white
The cytokine factor is human IL-2 or human IL-15.
3. the genetic engineering natural killer cells product for the treatment of tumour as described in claim 1, which is characterized in that the cell
The factor of committing suiside is HSV-tk or cytosine deaminase gene (cytos ine deaminase).
4. the genetic engineering natural killer cells product for the treatment of tumour as described in claim 1, which is characterized in that the nature
It can be appointing for people's natural killer cells system to kill cell.
5. the genetic engineering natural killer cells product for the treatment of tumour as described in claim 1, which is characterized in that use IL-2 bases
The genetic modification natural killer cells that cause and the T2A fusions of HSV-tk genes are stablized.
6. the genetic engineering natural killer cells product for the treatment of tumour as described in claim 1, which is characterized in that can be used for
Prepare the gene cell drug products for treating various malignant tumours.
7. gene cell drug products as claimed in claim 6, which is characterized in that use under gmp conditions on a large scale without blood
Clear suspension culture techniques production.
8. the genetic engineering natural killer cells product for the treatment of tumour as described in claim 1, which is characterized in that can be used for
Injection in intravenous injection, intra arterial injection, intratumor injection, hypodermic injection, organ injection, the interior injection of hydrothorax or ascites.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810392435.3A CN108531458A (en) | 2018-04-27 | 2018-04-27 | Treat the genetic engineering natural killer cells product of tumour |
| PCT/CN2018/103655 WO2019205403A1 (en) | 2018-04-27 | 2018-08-31 | Genetically engineered natural killer cell product for treating tumor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810392435.3A CN108531458A (en) | 2018-04-27 | 2018-04-27 | Treat the genetic engineering natural killer cells product of tumour |
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| Publication Number | Publication Date |
|---|---|
| CN108531458A true CN108531458A (en) | 2018-09-14 |
Family
ID=63477705
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| CN201810392435.3A Pending CN108531458A (en) | 2018-04-27 | 2018-04-27 | Treat the genetic engineering natural killer cells product of tumour |
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| Country | Link |
|---|---|
| CN (1) | CN108531458A (en) |
| WO (1) | WO2019205403A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114026225A (en) * | 2019-06-27 | 2022-02-08 | 诺和诺德股份有限公司 | Safe immunostealth cells |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11661459B2 (en) | 2020-12-03 | 2023-05-30 | Century Therapeutics, Inc. | Artificial cell death polypeptide for chimeric antigen receptor and uses thereof |
| TW202241935A (en) | 2020-12-18 | 2022-11-01 | 美商世紀治療股份有限公司 | Chimeric antigen receptor system with adaptable receptor specificity |
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| WO2003074692A2 (en) * | 2002-03-05 | 2003-09-12 | Universitätsklinikum Hamburg-Eppendorf Körperschaft des Öffentlichen Rechts | Specific gene expression in colonic cancer cell lines |
| CN1493687A (en) * | 2003-09-05 | 2004-05-05 | 中国科学技术大学 | Interleukin-15 gene modified natural killing cell strain and its preparation method |
| US20140255363A1 (en) * | 2011-09-16 | 2014-09-11 | Baylor College Of Medicine | Targeting the tumor microenvironment using manipulated nkt cells |
| CN107427578A (en) * | 2015-03-27 | 2017-12-01 | 南克维斯特公司 | The cells of NK 92 and monoclonal antibody by genetic modification for treating cancer |
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| US20140369979A1 (en) * | 2012-02-01 | 2014-12-18 | Postech Academy-Industry Foundation | Vector simultaneously expressing dodecameric trail and hsv-tk suicide genes, and anticancer stem cell therapeutic agent using same |
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2018
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- 2018-08-31 WO PCT/CN2018/103655 patent/WO2019205403A1/en active Application Filing
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| WO2003074692A2 (en) * | 2002-03-05 | 2003-09-12 | Universitätsklinikum Hamburg-Eppendorf Körperschaft des Öffentlichen Rechts | Specific gene expression in colonic cancer cell lines |
| CN1493687A (en) * | 2003-09-05 | 2004-05-05 | 中国科学技术大学 | Interleukin-15 gene modified natural killing cell strain and its preparation method |
| US20140255363A1 (en) * | 2011-09-16 | 2014-09-11 | Baylor College Of Medicine | Targeting the tumor microenvironment using manipulated nkt cells |
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| WO2019205403A1 (en) | 2019-10-31 |
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Application publication date: 20180914 |