CN101966335A - Novel HER2/neu gene-modified dendritic cell vaccine - Google Patents
Novel HER2/neu gene-modified dendritic cell vaccine Download PDFInfo
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- CN101966335A CN101966335A CN2009100439747A CN200910043974A CN101966335A CN 101966335 A CN101966335 A CN 101966335A CN 2009100439747 A CN2009100439747 A CN 2009100439747A CN 200910043974 A CN200910043974 A CN 200910043974A CN 101966335 A CN101966335 A CN 101966335A
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Abstract
The invention provides a preparation scheme of a novel HER2/neu gene-modified dendritic cell vaccine, which comprises: 1, constructing a lentivirus vector carrying the HER2/neu gene fragment; 2, preparing recombinant lentiviruses carrying the HER2/neu gene fragment by using the vector; and 3, infecting dendritic cells with the recombinant lentiviruses and obtaining the vaccine. Animal experiment proves the novel HER2/neu gene-modified dendritic cell vaccine can effectively prevent and treat HER2/neu positive tumors.
Description
Technical field
The present invention relates to biology and medical domain, relate more specifically to a kind of preparation of dendritic cell vaccine of new HER2/neu genetic modification.The present invention can be applicable to the prevention and the treatment of HER2/neu positive tumor.
Background technology
HER2/neu is a kind of oncogene, belongs to Epidermal Growth Factor Receptor Family.The expression of crossing of HER2/neu gene frequently betides breast carcinoma, ovarian cancer, carcinoma of endometrium, pulmonary carcinoma and gastroenteric tumor, and it is expressed crossing of breast carcinoma is the correlative factor of prognosis mala.Can effectively reduce the risk of recurrence of the positive breast carcinoma of HER2 at the monoclonal antibody medicine Herceptin of HER2/neu.Up-to-date the expressing excessively of HER2/neu that studies show that can promote the propagation of tumor stem cell and strengthen its aggressive.Above statement of facts HER2/neu is a desirable target of treatment HER2/neu positive tumor.
Dendritic cell (DC) are the most powerful antigen presenting cells of finding so far of function, can stimulate the immunoreation of body immune system generation at specific antigen, these reactions comprise the neutralization of antibody to specific antigen, and the T cell is to direct and indirect lethal effect of carrying the specific antigen cell etc.Therefore, can suppress the proteic signal transduction activity of HER2, kill and wound the antigenic tumor cell of expression HER2, reach the purpose of prevention and treatment HER2 positive tumor behind the HER2/neu gene importing dendritic cell human body being carried out immunity.
At present, the dendritic cell that had several method of gene introduction to be used to prepare the HER2/neu genetic modification (are abbreviated as DC
HER2), mainly comprise recombinant adenovirus, recombinant retrovirus and mRNA electroporation.The dendritic cell that usefulness such as Sakai are carried the recombinant adenovirus infecting mouse derived from bone marrow of neu genetic fragment carry out inoculation to mice then, find the DC that this adenovirus is transformed
HER2Vaccine can significantly stop or postpone the generation of transgenic mouse milk tumor, and two other discovers the DC that adenovirus is transformed
HER2Vaccine can partly or entirely stop the generation of the positive transplantation tumor of mice neu.The dendritic cell that usefulness such as Nabekura are carried the retroviral infection mouse bone marrow cells source of HER2 genetic fragment prepare DC
HER2Vaccine finds that the latter can partly stop the generation of the positive transplantation tumor of mice HER2, can also partly postpone the generation of transgenic mouse milk tumor in addition.Domestic in addition have researcher to adopt the method for mRNA electroporation to prepare DC
HER2Vaccine, experiment in vitro show that this vaccine can activated t cell killing and wounding the neu positive tumor cell.
Though above-mentioned three kinds of method of gene introduction mentioning can both prepare effective DC
HER2Vaccine, but some shortcomings are all arranged separately: behind the adenovirus infection DC genetically modified expression time shorter, the latter may cause the persistency and the insufficient strength of immunological effect; Retrovirus can only infect the cell of division stage, and clinically patient's PERIPHERAL BLOOD MONONUCLEAR CELL induction that adopts becomes DC more, and this process is seldom with cell proliferation, and therefore part has limited its possible clinical practice; There is short problem of antigenic half-life of submission equally in the mRNA electroporation, in addition, there are some researches show that the mRNA electroporation can weaken the function of DC immune stimulatory.
In fact, recombinant slow virus also can efficiently infect dendritic cell, can not weaken the function of DC simultaneously.In addition, the important feature that recombinant slow virus infects is genetically modified long-term expression, these characteristics then might produce lasting and enhanced immunoreation by stimulating immune system for DC, also might cause immunologic tolerance but then, that is to say that the vaccine with its preparation may not have effect.Do not use recombinant slow virus to prepare DC at present
HER2The report of vaccine.
Summary of the invention
The dendritic cell vaccine that the purpose of this invention is to provide a kind of new HER2/neu genetic modification.
Vaccine provided by the invention is that the transfection dendritic cell prepare again by on the slow virus carrier that HER2/neu is recombinated.
On the slow virus transport vehicle of any one commercialization or private exploitation, make up the expressed sequence of a HER2/neu genetic fragment.The slow virus transport vehicle here refers to provide in the slow virus packaging system carrier of genes of interest, plasmid normally, and it also provides the required cis acting element of transcriptional expression of viral entrained nucleic acid in addition.The expressed sequence of HER2/neu genetic fragment comprises the promoter of 5 ' end, can select special promoter of broad-spectrum strong promoter such as cytomegalovirus (CMV) promoter and elongation factor 2 (EF2) promoter etc. or dendritic cell such as CD11c promoter etc. for use, the polyadenylic acid tailing signal of 3 ' end, and the HER2/neu genetic fragment between promoter and tailing signal (removing born of the same parents' inner segment that it has tyrosine kinase activity usually).The concrete condition that may run in the structure comprises: if transport vehicle does not have other genes of interest, directly in position insert the HER2/neu genetic fragment.If transport vehicle has possessed genes of interest, then need replace original genes of interest with the HER2/neu genetic fragment.If carrier does not provide promoter, then need to insert in position the purpose promoter.If this transport vehicle has possessed the purpose promoter, then only need to insert the HER2/neu genetic fragment in the promoter downstream, transport vehicle can provide tailing signal usually.The Protocols in Molecular Biology of using can be with reference to published relevant books, as " molecular cloning ".
1. utilize the carrier package preparation of above-mentioned structure to carry the recombinant slow virus of HER2/neu genetic fragment, be described as follows: adopt the slow virus packaging system packing preparation of any one commercialization or private exploitation to carry the HER2/neu genetic fragment
Recombinant slow virus.Complete slow virus packaging system comprises several plasmids and a package cell line usually, the former is used to provide the required viral structural gene of slow virus packing and the required cis acting element of transcriptional expression of viral entrained nucleic acid, and the latter provides the virus packing required trans acting factor.The packing preparation process can be followed manufacturer's description and document of delivering or relevant books.The recombinant slow virus that carries the HER2/neu genetic fragment of packing gained can be further purified and/or concentrate, but associative operation reference reagent box manufacturer's description and pertinent literature or books.
2. utilize the recombinant slow virus of mentioning in 2 to infect dendritic cell and prepare new DC
HER2Vaccine is described as follows:
The dendritic cell source can comprise bone marrow cells in mice, human PERIPHERAL BLOOD MONONUCLEAR CELL or CD14 positive cell group, hematopoietic stem cell and subgroup thereof, hESC.Usually through the cultivation of a couple of days, these cells with differentiation potential are divided into dendritic cell with major part under effect of cytokines.Not quite alike from the time that different precursor inducing culture dendritic cell are spent, except that the hESC, need 6-10 days usually, 3 days rapid induction method is also arranged.In the inducing culture process of dendritic cell, with the recombinant slow virus that carries the HER2/neu genetic fragment for preparing in due course machine the cell in the cultivating system is infected, mode of infection can comprise multiple, simple coculture infection, perhaps with virus and the centrifugal together centrifugal infection altogether of cell, perhaps repeat even repeatedly infection the perhaps combination of these methods.Can add the factor such as the tumor necrosis factor that stimulates dendritic cell maturation in late stage of culture in cultivating system, phosphoric acid lipopolysaccharide etc. are to stimulate dendritic cell maturation, and reason is that immature dendritic cell might cause immunologic tolerance.Finally can obtain this new DC
HER2Vaccine.Associative operation can be induced and the document of slow virus infection dendritic cell with reference to cultivating about dendritic cell of having delivered, and the specific embodiment in this description will provide this new DC of preparation from bone marrow cells in mice
HER2The operational approach of vaccine.
According to research of the present invention, the recombinant slow virus that above-mentioned usefulness is carried the HER2/neu genetic fragment infects the DC that was obtained in the 4th day that derives from the medullary cell inducing culture.According to the method for routine, generally should infect at the 2nd day.The inventor found, infected having obtained better unexpected effect at the 4th day.
Beneficial effect
In based on the body of mice, find this new DC in the experiment
HER2Vaccine only needs a low dosage (1.5 * 10
5) immunity inoculation just can prevent the positive transplantation tumor of HER2/neu in the intravital growth of mice fully, guard time is at least above 100 days.And the DC that adopts additive method to prepare
HER2Vaccine needs heavy dose (5 * 10 usually
5-1 * 10
6), and just can reach even also do not reach similar effect (table 1) through 2-3 immunity inoculation.The DC of this lentivirus preparation is described
HER2Vaccine is than the DC with the additive method preparation
HER2Vaccine is more effective.Another supports the foundation of above-mentioned conclusion to be, with this new DC
HER2Behind the vaccine low dosage single immunization, can in the mice body, detect the anti-HER2/neu antibody of enough levels, and adopt adenovirus and retrovirus to prepare DC at before this two
HER2In the research of vaccine, the immunity of single higher dosage can only induce generation can't produce antibody on a small quantity or at all in the mice body, and relevant production of antibodies is to estimate an important indicator of vaccine effect in the body.
This new DC
HER2The prophylactic effect of vaccine is further proved long lasting: the HER2/neu antibody in back 100 days mice plasma of immunity still maintain higher level (average 7 times to not inoculating DC
HER2The mice of vaccine), the back 100 days mice of these immunity is carried out the HER2/neu positive tumor cell inoculation second time, tumor does not take place in discovery mice yet after 50 days.Except that prophylactic effect, this new DC
HER2The positive transplantation tumor of HER2/neu that vaccine has taken place to mice also has therapeutic effect, a low dosage (2 * 10
5) subcutaneous injection can significantly suppress growth of tumor.
In this invention, a series of operations well known by persons skilled in the art have been comprised.But creativeness of the present invention be successfully to use known slow virus carrier with the HER2/neu gene transfection to dendritic cell, realized the stability and high efficiency expression, and can be that animal body produces corresponding immunoreation.
The DC of table 1 lentivirus preparation
HER2The DC of vaccine and additive method preparation
HER2The vaccine prevention effect relatively
Be shown as the percentage ratio of healthy no tumor mice when observing persistent period (tumor cell inoculation is counted the 0th day) and last the observation.
Description of drawings
Fig. 1 is the PCR screening of pSin-EF2-neuET-Pur positive colony among the embodiment; Expect that wherein positive colony should amplify size and be the fragment of 2100bp, visible 4 bacterium colonies have all amplified this band, and explanation may be positive colony.The negative contrast of neg, an irrelevant plasmid, the positive contrast of pos pMMTV-neu;
Fig. 2 is that the enzyme action of pSin-EF2-neu-Pur is identified figure; Expect that wherein the single endonuclease digestion fragment is 8500bp, the double digestion fragment is 6400bp, 2100bp.The enzyme action result is consistent with prediction.E+S represents EcoR I and Spe I double digestion;
Fig. 3 is for respectively organizing the no tumor survival curve figure of mice in the prevention experiment; Wherein mice is through DPBS or DC immunity (1.5 * 10
5/ only, and 5/group) back 7 days, with B16neu cell (2 * 10
5/ only) and carry out subcutaneous vaccination, the tumor of mice is respectively organized in lasting observation, and a situation arises.As seen DC
NeuTumor took place on the 100th day in all mices of immune group not yet behind tumor cell inoculation, and DPBS group and DC
CtrlTumor has all taken place respectively in group at the 24th and 31 day;
Fig. 4 is for respectively organizing the tumor growth curve figure of mice in the treatment experiment; At first use 2 * 10
5B16neu carries out tumor inoculation to every mice, treats with DPBS or DC after 7 days or 15 days then.DC dosage is 2 * 10
5/ only, weekly mouse tumor being carried out twice observation and measurement afterwards, the mouse tumor volume uses oval cubature formula to calculate: a * b
2* 0.5236, wherein a and b represent the line of apsides of tumor respectively, and 0.5236 for calculating a constant of oval volume.Left figure and right figure are respectively mice the 7th day and tumor growth curve of receiving treatment in the 15th day behind tumor inoculation.Data show is the average of mean ± standard error among the figure, the time of numeral immunization therapy under the arrow.Every group of 5 mices of treatment experiment in the 7th day, wherein DPBS treatment group has a tumor-bearing mice at unknown cause death in the 17th day, DC
NeuThe treatment group has a no tumor mice unknown cause death in the 25th day, and these two mices all do not participate in statistics since the death time, and the mouse tumor average external volume of respectively organizing in last day observation period (behind the tumor inoculation 25 days) is respectively 2627mm
3(DPBS), 2359mm
3(DC
Ctrl), 374mm
3(DC
Neu); In the treatment experiment in the 15th day, DBPS, DC
CtrlAnd DC
NeuThree groups of mice numbers are respectively 4,4 and 6.The treatment viewing duration does not have dead mouse, respectively organizes the mouse tumor average external volume in last day observation period (behind the tumor inoculation 21 days) and is respectively 1052mm
3, 2306mm
3And 472.6mm
3Respectively organize the mouse tumor average external volume when twice laboratory observation phase finished and carry out the statistical analysis discovery, compare DC
Ctrl(P=0.023) and DPBS (P=0.03), DC
NeuCan significantly suppress the mouse tumor growth, and DPBS group and DC
CtrlThe mouse tumor average external volume of group does not have significant difference (P=0.587).
The specific embodiment
1.Neu the pcr amplification of genetic fragment and purification
Primer sequence upstream 5 '-ggaattccagcctggtccagcctgag-3 '; Downstream 5 '-gactagttcactgtctccttcgtttgatt-3 '.Reaction template is to carry the plasmid pMMTV-neu of neu full-length cDNA, is that our unit makes up by method of the prior art.Amplified reaction uses the super fidelity PCR of the Phusion test kit of NEB company.Reaction system following (volume unit is μ l):
Coamplification two pipes.Program setting is as follows: 98 degree 30s → 95 degree 10s → 62 degree 30s → 72 degree 40s → 72 degree 5min → 4 degree, repeat 30 circulations.The PCR instrument is an eppendorf company product.
The PCR product is run glue, reclaim purification kit with the glue of TaKaRa company product is carried out purification.
2. distinguish enzyme action PCR product and pSin-EF2-SOX2-Pur (the slow virus transport vehicle is available from the Addgene website)
Reaction system following (volume unit is μ l):
Spend the night and 3hr at 37 degree enzyme action respectively.The enzyme action product is run glue, reclaim neu fragment that has sticky end and the big fragment of pSin-EF2-SOX2-Pur carrier of having removed the SOX2 gene respectively.
3. connect and conversion
Coupled reaction system following (volume unit is μ l):
Fragment is mixed the back and is hatched 3min at 65 degree, transfers on ice rapidly again.16 degree reaction 30min.To connect product and be transformed into the super competent cell of stbl3 (Invitrogen company product), bacterium liquid is coated on the LB agar plate that contains the ammonia benzyl, and 37 degree are cultivated.Wait to clone the screening of carrying out positive colony after growing.
4. the enzyme action of screening positive clone and recombinant vector is identified
At first bacterium colony is carried out Preliminary screening with PCR.Picking 4 bacterium colonies altogether from the plate, each bacterium colony all is resuspended in the 10 μ l distilled waters, as template, PCR reaction system following (volume unit is μ l):
Response procedures is set as follows: 95 degree 2min → 95 degree 30s → 62 degree 30s → 72 degree 2min → 72 degree 5min → 4 degree, repeat 30 circulations.Run glue and identify positive colony, the results are shown in Figure 1.The bacterium liquid of positive colony is seeded to 3-4ml LB culture medium, 250rpm jolting 8hr in the 37 degree shaking tables.Plasmid a small amount of extraction agent box with Takara company prepares recombinant vector.Recombinant vector is carried out enzyme action identifies reaction system following (volume unit is μ l):
37 degree reaction 30min.Run the glue qualification result and see Fig. 2.Recombinant vector is named as pSin-EF2-neu-Pur.Embodiment 2 carry the HER2/neu genetic fragment recombinant slow virus (rLVneu) packing preparation and concentrate
1. the preparation of plasmid
Comprise pSin-EF2-neuET-Pur, pCMV8.91 and pMD.G be totally 3 kinds of plasmids.Use a large amount of extraction agent boxes of Invitrogen company to prepare plasmid after will containing the antibacterial large-scale culture of plasmid.
2. the preparation of transfection reagent
0.3M CaCl 2 : 11.025g CaCl
22H
2O dissolves in 240ml Milli-Q water, is settled to 250ml, 0.22 μ m filtration sterilization, and 4 degree are stored.
2 * HBS: add 14ml 5M NaCl solution, 12.5ml 1M HEPES buffer and 250 μ l 1.5M Na among the 200ml Milli-Q water
2HPO
4Solution is adjusted pH value to 7.10 with NaOH or HCl, is settled to 250ml, 0.22 μ m filtration sterilization, and 4 degree are stored.
3.rLVneu preparation (preparative-scales of 10 100mm Tissue Culture Dishs)
(1) thaws 1 pipe 293FT cell (Invitrogen company product) to 100mm ware, overnight incubation from liquid nitrogen container.
(2) add Geneticin, continue to cultivate.
(3) before density surpasses 80%, be passaged to 4 100mm wares, add Geneticin, continue to cultivate.
(4) before density surpasses 80%, be passaged to 10 100mm wares, do not add Geneticin, continue to cultivate.
(5) monitoring cell density, 70-80% begins transfection.
(6) with 10ml 0.3M CaCl
2Add the 50ml centrifuge tube, add pSin-EF2-EGFP-Pur, pCMV8.91 and pMD.G are respectively 90,120 and 30 μ g.
(7) 10ml 2 * HBS adds the 50ml centrifuge tube.
(8) with CaCl
2Dropwise add among 2 * HBS with the DNA mixed liquor.Put upside down or inhale gently and beat mixing.
(9) rapidly with DNA/CaCl
2/ HBS suspension dropwise adds the 100mm ware, and simultaneously rotation is rocked culture dish suspension is dispersed in the culture medium gently.
(10) cell returns incubator and cultivates 6hr.
(11) change liquid.
(12) return incubator and continue to cultivate 48hr.
(13) collect supernatant, 2500g, 10min.
(14) 0.45 μ m Durapore films (ultralow protein adsorption) filter.
(15) supernatant etc. that will contain virus is sub-packed in 3 aseptic 50ml Beckman centrifuge tubes, and every effective DMEM culture medium is filled it up with, and is tight with the lid lid that has sealing ring.
(16) 20000g, the centrifugal 90min precipitation of 4 degree virus.Centrifuge is a Beckman low-temperature and high-speed centrifuge.
(17) abandon supernatant, visible white precipitate drips the aseptic DPBS of 150-200 μ l at place of settling, and 4 degree dissolvings are spent the night.
(18) collect virus, packing ,-80 degree are stored standby.
The complete medium of DC is for containing 10%FBS, 2mM L-glutamine, the RPMI1640 culture medium of 50 μ m beta-mercaptoethanols and 20ng/mlGM-CSF.GM-CSF is a powder, again after the aquation with 100ng/ μ l, the packing of 10 μ l/ pipe is frozen avoids multigelation at-20 degree, faces and uses preceding adding.The nutrient media storage that adds GM-CSF must use up in 7 days at 4 degree.
Except that separating bone, all the other all strictness follow the sterile working.DC cultivates and preferably to use the bacteriology or without the ware/plate of tissue culture treated.
(1) get femur and the tibia of mice, operation in the super-clean bench is brought in alcohol-pickled sterilization into.
(2) RPMI1640 washing by soaking bone cuts off the epiphysis end, with the 0.45mm entry needle medullary cell is gone out from medullary cavity behind the syringe pump 10ml RPMI1640.
(3) the 15ml centrifuge tube is collected medullary cell, the centrifugal 10min of 300g.
(4) during centrifugal, in ware/plate, add fresh complete medium, prepare cell counting.
(5) after centrifugal the finishing, abandon supernatant, the 1ml complete medium is resuspended, the sampling counting.
(6) by 2 * 10
6/ 100mm ware, 4 * 10
5/ 6 orifice bore kinds are gone into.Put in the incubator and cultivate, be designated as the 0th day.
(7) cultivated the 3rd day, add the fresh complete medium of equal-volume.
(8) the centrifugal 10min of 300g collects the 4th day DC.
(9) prepare to infect complex: 120 μ l concentrate rLVneu, 480 μ l RPMI1640, and final concentration is the polybrene of 4 μ g/ml.
(10) after the centrifugal end, collect supernatant, return foramen primum after supplying (4ml/ hole) with fresh culture.
(11) with the infection complex re-suspended cell for preparing, mixing is put and is infected 3hr in the incubator gently.Every during this time 30min-1hr jolting mixing.
(12) after infection finished, 6 orifice plates were returned in 100 μ l/ holes.
(13) collect the 5th day DC and culture medium, add centrifuge tube together, the centrifugal 90min of 800g room temperature.
(14) during centrifugal, every hole adds the 1ml fresh culture and keeps moistening.Prepare to infect complex, the same.
(15) after the centrifugal end, the 3ml supernatant returns every hole, and all the other abandon, and with the infection complex re-suspended cell for preparing, mixing is put and infected 3hr in the incubator gently.Every during this time 30min-1hr jolting mixing.
(16) after infection finished, 6 orifice plates were returned in 100 μ l/ holes.
(17) collect the 6th day DC and culture medium, add centrifuge tube together, the centrifugal 90min of 800g room temperature.
(18) during centrifugal, every hole adds the 2ml fresh culture and keeps moistening, and half amount of finishing is in passing changed the liquid operation.
(19) after the centrifugal end, stay 12ml supernatant re-suspended cell, 2ml returns in supernatant/hole 6 orifice plates.
(20) cultivate the 9th day evening, collect all culture medium, the centrifugal 10min of 300g abandons supernatant, and re-suspended cell behind the complete medium two-fold dilution adds 20ng/ml LPS stimulation and spends the night.
Collected suspension and loose adherent cell in (21) the 10th days, with aseptic DPBS buffer washing 3 times, with 1.5-2 * 10
6/ ml is resuspended among the DPBS, then obtains DC
HER2Vaccine.RLVneu in the 9th and 14 steps of operation is changed and is DPBS, then finally obtain DCctrl, in follow-up embodiment as a negative control.
B16neu is a neu positive tumor cell system, with obtaining behind the stable transduction of the rLVneu B16-F1.B16-F1 is the melanoma cell in strain C57BL/6 mice source.
1.neu positive tumor prevention experiment
(1) get 6-8 C57BL/6 female mice in age in week, be divided into 3 groups at random, every group of 5 mices, with DPBS, DCctrl and DC
HER2Respectively each group mice is carried out immunity.Immunization ways is a subcutaneous injection, and the position is the mice left lower quadrant, and DC dosage is 1.5 * 10
5, volume injected is 100 μ l.Be designated as the 0th day this moment.
(2) the 7th days, tumor cell B16neu was collected in trypsinization.
(3) 10ml DPBS washes 3 times, and each centrifugal condition is 200g5min, with abundant removal serum composition.
(4) remove supernatant, the resuspended counting of DPBS, adjusting concentration is 2 * 10
6/ ml.
(5) 2 * 10
5Tumor cell carries out subcutaneous injection at the bottom right abdomen of each group immune mouse, and volume injected is 100 μ l.
(6) after this observe mice twice weekly, a situation arises for the record mouse tumor, draws no tumor survival curve, sees Fig. 3.
2.neu positive tumor treatment experiment
(1) at first uses 2 * 10
5The B16neu cell carries out subcutaneous vaccination to mice.
(2) 7 days or use DPBS, DCctrl and DC after 15 days
HER2Respectively mice is carried out immunity, dosage is 2 * 10
5
(3) after this observe mice twice weekly,, draw the mouse tumor growth curve, see Fig. 4 with the vernier caliper measurement tumor line of apsides.
Claims (3)
1. the dendritic cell vaccine of a new HER2/neu genetic modification is characterized in that it being that the transfection dendritic cell prepare again by on the slow virus carrier that HER2/neu is recombinated.
2. vaccine according to claim 1 is characterized in that with the recombinant slow virus that carries the HER2/neu genetic fragment dendritic cell being infected.
3. claim 1 or the 2 said vaccines application in preparation treatment or prevention HER2/neu positive tumor medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102993312A (en) * | 2012-12-14 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | GM-CSF-HER2 recombinant protein, method and application thereof |
| CN104928253A (en) * | 2015-05-22 | 2015-09-23 | 北京康爱瑞浩生物科技股份有限公司空港分公司 | Antigenicity-reinforced tumor cell and construction method thereof |
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| US20110027310A1 (en) * | 2007-05-04 | 2011-02-03 | Medin Jeffrey A | Compositions and Methods for Cancer Treatment |
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2009
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102993312A (en) * | 2012-12-14 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | GM-CSF-HER2 recombinant protein, method and application thereof |
| CN104928253A (en) * | 2015-05-22 | 2015-09-23 | 北京康爱瑞浩生物科技股份有限公司空港分公司 | Antigenicity-reinforced tumor cell and construction method thereof |
| CN104928253B (en) * | 2015-05-22 | 2018-08-21 | 北京康爱瑞浩生物科技股份有限公司空港分公司 | A kind of tumour cell and its construction method of antigenicity enhancing |
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