CN101773666A - Therapeutic anticancer vaccine and preparation thereof - Google Patents
Therapeutic anticancer vaccine and preparation thereof Download PDFInfo
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- CN101773666A CN101773666A CN200910168455A CN200910168455A CN101773666A CN 101773666 A CN101773666 A CN 101773666A CN 200910168455 A CN200910168455 A CN 200910168455A CN 200910168455 A CN200910168455 A CN 200910168455A CN 101773666 A CN101773666 A CN 101773666A
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Abstract
The invention relates to a therapeutic anticancer vaccine and preparation thereof, in particular to a preparation method by taking an immune gene-modified tumor cell line as the anticancer vaccine and application of the anticancer vaccine during antineoplaston, wherein the main ingredient of the vaccine is the immune gene-modified tumor cell line, and immune regulatory genes are GM-CSF, TGF-beta, HSP90, TAP-1, IL-4, IL-2, IL-12, B7.1 (CD80), B7.2 (CD86) and the like.
Description
The application is to be December in 2005 30 days the applying date, and application number is 200510135330.2, and denomination of invention is divided an application for the Chinese invention patent application of " anticancer vaccine and preparation thereof ".
Technical field
The present invention relates to anticancer vaccine and preparation thereof, particularly tumor cell line is modified as the preparation method of anti-cancer vaccine and the application in antineoplaston thereof by immunomodulatory gene.
Background technology
Tumor is present serious harm human life's a big disease, though through years of researches and effort, raising by positive tumor prevention and early diagnosis, treatment technology, the generation and the harm of tumor have been reduced to a certain extent, but because the variation of people life style and ambient conditions, the overall morbidity of tumor and mortality are still high, have become first cause of the death in certain areas.
Tumor treatment is usually based on means such as operation, chemotherapy, radiotherapies, though these treatment meanss are especially in the improvement and the improve that all have aspect the chemotherapeutics exploitation more or less, but the malignant tumor patient for late period is comparatively often felt simply helpless, and can't offer help aspect the life cycle and the quality of making the life better prolonging.Along with the overall Study on Mechanism of people to the generation and the development of tumor, from the eighties of last century late nineteen eighties, development has played a kind of new Biotherapeutics mode by biological means tumor is treated gradually, as cytokine therapy, gene therapy etc.The Biotherapeutics mode of tumor is under the situation that routine operation, chemicotherapy all can't carry out, still can carry out killing and wounding to a certain degree to tumor cell, wherein immunization therapy is a kind of relatively effectively oncotherapy means to antineoplastic by transferring immunity of organisms.
The immunization therapy of tumor is divided into passive immunotherapy and active immunity treatment two big classes.In passive immunotherapy, at the antibody of a certain specific tumor-cell antigen after being used for the patient, can with express this antigenic tumor cell and combine, these can be discerned and remove to patient's self immune system by the tumor cell of antibody labeling.In addition, can also be with radiosiotope molecule or lps molecule and antibody coupling, at antibodies direct killing tumor cell behind the tumor cell.The Herceptin that has been used at present clinical treatment breast carcinoma promptly belongs to the tumor passive immunotherapy.
The active immunity treatment of tumor is by importing specific antigen in the tumor patient body, exciting patient's self immunoreation to remove tumor cell.When importing specific antigen, use other composition of enhance immunity reaction toward contact.This specific immunoreation not only can stimulate the antibody (humoral immunization) of B cell generation antigenic specificity, can also the specific T cell of challenging antigen (cellular immunization).The antigen protein of purification, polypeptide, the system based on gene expression, tumor cell or tumor cell lysate etc. all can be used for the active immunity treatment of tumor, i.e. therapeutic tumor vaccine.
Different with chemotherapy, active immunity treatment is the direct killing tumor cell not, but, increase and treat tumor at the immunocyte of tumor cell, the kind and the quantity of antibody by producing a kind of body fluid and/or cell immune response of specific, the targeting at tumor cell.In addition, all right activating immune system of therapeutic tumor vaccine overcomes because the immunosuppressant that tumor growth and progress cause.Effectively tumor vaccine can prolong patient's progression free interval and total life span, and does not have the significant side effects that chemotherapy and other Biotherapeutics cause.
Because the complexity of pathogenesis of cancer mechanism, and the difficulty that obtains the broad-spectrum tumor specific antigen, actively carried out based on the exploitation of the whole-cell vaccines (whole cell vaccine) of tumor cell.This technology is the antigen body with the injection patient with complete cultured tumor cells in vitro, handles the back through body immune system identification and activates specificity antineoplastic mechanism and generation antitumaous effect (Nature Reviews Cancer 4:401-411,2004).In addition, utilize especially dendritic cell (Dendritic cell of immunocyte, DC) In vitro culture and use specific antigen and common stimulation of cytokine and also be the ingredient (NatureReviews Immunology 3,630-641,2003) of this technology as anti-cancer vaccine).
Full cell therapy actively carries out in the U.S. at present with the clinical trial of vaccine, and existing a plurality of clinical trial phases are afterwards carrying out and facing breakthrough (Expert Review Anticancer Therapy 5:635-644,2005).FDA is expected in the audit approval of finishing in the recent period this series products, and (Nature 411,380-384,2001 thereby start the biological product of the antineoplastic new of a class behind the antibody class drug development; Seminars in Oncology 30,1-8,2003).
Present patent application adopts improves the full cell therapy anti-cancer vaccine of technological development.Prepare business development in conjunction with the peculiar phenotype of tumor cell and functional defect and be worth big compound anti-cancer vaccine.But present patent application is utilized the vaccine product of tumor cell line production extensive use, is different from individuality vaccine (autologous orpersonalized vaccine) on the Therapeutic Principle, has the feasibility of suitability for industrialized production.Can be on using in conjunction with other routine clinical treatments or independent the use, having got rid of the individuality vaccine must be in conjunction with the limitation of operative treatment.Therefore, the product of may command quality inspection makes things convenient for clinical treatment to use thereby the employing the art of this patent can be mass-produced.
Combined treatment is to stimulate patient's immune system with opposing tumor development and elimination tumor with the application aim of anti-cancer vaccine, the present patent application main technical points is to adopt the donor of tumor cell line as tumor antigen, utilizes cell inner expression and excretory cytokine to carry out the stimulation and the adjusting of antineoplastic immune.Be fit to the expression of tumor antigen and the condition of offering by building, to improve the dynamics of antitumor immunity of organism reaction.
Summary of the invention
The invention provides a kind of anti-cancer vaccine, the tumor cell line of its main component for modifying through immunomodulatory gene, described immunomodulatory gene is: GM-CSF, TGF-β, HSP90, TAP-1, IL-4, IL-2, IL-12, B7.1 (CD80), B7.2 (CD86), (Nature Reviews Immunology 3,630-641 such as CTLA-4,2003), preferably GM-CSF and TGF-β.Described tumor cell line is all kinds of human tumour cell lines of subculture in vitro separately, at the anticancer type of prepared vaccine and select.Described immunomodulatory gene is all cloned in human chromosomal DNA, the primary cell that described tumor cell line derives from ATCC center (U.S.) or obtains from patient's tumor clone.
Anti-cancer vaccine of the present invention; its preparation method is to be mixed and made into by the present invention being passed through the tumor cell line of immunomodulatory gene modification and containing a certain amount of physiologically acceptable auxiliary element; described auxiliary element can be DMSO; Pentastarch; serum albumin; buffer solution; water, vaccine adjuvant, protective agent; stabilizing agent etc.; preferably: 5%DMSO, 8%Pentastarch, 2% human serum albumin (HSA) and an amount of phosphate buffer; its preparation process can be made the anti-cancer vaccine composite preparation of the injection of unit dosage form according to the conventional production technology of pharmaceutical preparation.
Anti-cancer vaccine of the present invention, the anti-cancer vaccine composite preparation of the injection of described unit dosage form, wherein the amount of the tumor cell line of process immunomodulatory gene modification is 0.1-500x10
6The abundant mixing of cell/mL and helper component forms.
Among the present invention, the selection of tumor cell line is the key of decision vaccine product application, can take different cell lines at different tumour patients.For example hepatocarcinoma patient can be used Hep3B, HepG2 cell line; Cervical cancer patient can use HeLa, ME180 cell line; Lung cancer patient can be used A549 cell line; The clinical history that also can select potentiality to be exploited for use clearly former generation separated strain.Tumor cell line obtains other transgene expression through in-vitro transfection (transfection).Through screening and setting up stable cell lines to be used for the production of anti-cancer vaccine.
Transgene expression and set up stable cell lines and adopt technology such as retrovirus (retroviral vector) DNA in-vitro transfection.Used viral vector makes up and adopts Moloney murine leukaemia virus (MMLV).After having MMLV virus that genetically modified nonautonomy duplicates and producing under given conditions, the exogenous gene that is used for tumor cell line imports.Positive transformant carries out primary election through specific drug resistance labelling earlier, utilizes the connection dyeing of antibody mediated immunity connection or other molecular biology methods to screen again, and the positive cell strain of selecting is used to prepare tumor vaccine.The core of present patent application is that the exogenous gene combination that is imported is played a crucial role by immune identification and expression in vivo for the tumor-cell antigen body.Selected tumor vaccine cell remakes clinical practice behind radioinactivation.
Immunomodulatory gene of the present invention is an exogenous gene, and the applied exogenous gene of the present invention is as follows:
GM-CSF: the huge efficiently regulatory factor of having a liking for cell (macrophage) and DC cell.After the vaccination of tumor-cell antigen body was to the body, entrained specific tumour antigen can carry out antigen presentation (Annual Review of Immunology, 19:47-64,2001) through cross presentation mechanism.GM-CSF plays extremely important adjusting and facilitation in this course.
TGF-β (transforming growth factor β): this is that a class is by the common regulatory factor of most cells.It mainly can suppress function of immune system, especially suppresses tumor antigen and is discerned (Nature Medicine 7:1118-1122,2001) by cell immune system.This gene utilizes the siRNA technology to be suppressed (Cancer Research 64:7596-7603,2004 in the tumor vaccine preparation process; Gene Therapy12:5-11,2005).
TAP-1 (transporter associated with antigen processing): this gene outcome plays the transmitter effect in the antigen presentation process.This gene is the sudden change disappearance in many cancer cell, thereby makes these tumor cells do not discerned (Nature Reviews Cancer 5:263-274,2005) by immune system.
HSP90 (heat shock protein 90): this is that a class is the common gene of most cells, expresses under emergency rating and improves.This genoid expressed products is served as chaperons in cell, help other albumen to form normal configuration with functionating, and auxilin is in intracellular transmission and processing.Therefore, meeting of this class HSP albumen and intracellular various albumen comprise that tumor antigen combines, and are antigen presentation institute essential (International Journal of Hyperthermia 21:717-722,2005).
Present patent application is applied to the preparation of anti-cancer vaccine with above-mentioned exogenous gene, adopts different exogenous genes combinations and improves anti-cancer ability, has synergism.Preferred compound mode is listed in the embodiment of the invention.
Description of drawings
Fig. 1 is the expression of Hep3B-GMCSF/TGF siRNA anti-cancer vaccine cell line GM-CSF and the experiment analysis results that TGF-β suppresses.The A:ELISA method detects the expression of GM-CSF.B:Western blot method detects the expression of TGF-β.
Fig. 2 is the biological effectiveness analysis of the expressed GM-CSF of anti-cancer vaccine cell line.
Fig. 3 is that the anticancer function of anti-cancer vaccine cell line compares.Three groups of (n=10) C57B1/6 mices were accepted different vaccine (2x10 at first day
6Cell through radiation treatment), and at the 10th day immune once more same like cell.Accepted B16 tumor cell (2x10 at the 30th day
5) the SC attack.The tumor of the 50th day three treated animals compares.
Fig. 4 is that the anticancer function of anti-cancer vaccine cell line compares.All animals first with accepted different vaccine (2x10 in ten days
6Cell through radiation treatment), accepted B16 tumor cell (2x10 at the 30th day
5) the SC attack.The animals survived ratio through diagram relatively.
The specific embodiment
Further specify the present invention by the following examples, but not as limitation of the present invention.
Embodiment 1
The vaccine of tumor cells expression GM-CSF and inhibition TGF-β:
This type of tumour-cell vaccine has two kinds of new features simultaneously, and promptly the siRNA technology that efficiently expresses and utilize of GM-CSF has suppressed the generation of TGF-β.This type of vaccine has improved in the full cell of tumor simple expression of GM-CSF and the antitumor immune function that produces, has synergism.
The GM-CSF gene inserts retrovirus transfection carrier rkat43.2 and makes up rkat43.2GM-CSF through the dna clone technology.The description to some extent in the past of the structure of this carrier and feature (Roberts, etc., 1998; J Immunol, 161:375-384.).The GM-CSF gene is via BamHI and ApaI restriction endonuclease and clone into above-mentioned plasmid vector.According to the concrete grammar described in the document, the retroviral vector that has the GM-CSF gene that infective activity arranged by compound transfection MCVecog/p and 6.1CMVamphoenv DNA plasmid and above-mentioned plasmid vector in incasing cells 293T.After the DNA transfection about 4-5 days, cell pyrolysis liquid can be prepared into the retroviral vector that has the GM-CSF gene, and its infective activity is after virus titer is measured and be further used for the preparation of the stable cell lines of GM-CSF gene expression.
The preparation of expression of GM-CSF stable cell lines is by infecting related neoplasms cell line and carrying out cell in vitro cultivation screening and carry out (Cytotechnology 28:31-42,1998).For example prepare anti-Hepatoma Vaccine, at first the Hep3B cell with In vitro culture carries out the retroviral infection that GM-CSF expresses.After one week, the last cell of depositing is done limited dilution and carried out the cell clone cultivation at 96 well culture plates.The monoclonal cell strain of being set up obtains the Hep3B/GM-CSF cell strain of high expressed through the GM-CSF expression screening.
The inhibition of TGF-β is transmitted the siRNA technology by the pSilencer plasmid and is finished.The pSilencer of Ambion company (U.S.)
TM4.1-CMV neo System is used to prepare the siRNA carrier that suppresses the TGF-beta gene expression.With reference to these system's operation instructions, the siRNA oligonucleotide inserts in the plasmid vector, is carried out the synthetic expression of siRNA hairpin structure by the CMV promoter of modified.Be used for the oligonucleotide sequence TGF-β 1/oligoA and the TGF-β 1/oligoB that produce TGF-β siRNA and import plasmid vector DNA by traditional clone technology.
TGF-β1/oligoA
5’-GATCCCCGACTATCGACATGGAGCTGttcaagagaCAGCTCCATGTCGATAGTCTTTTTGGAAATGF-β1/oligoB
5’-TCGATTTCCAAAAAGACTATCGACATGGAGCTGtctcttgaaCAGCTCCATGTCGATAGTCGGG
The plasmid vector that carries TGF-1siRNA imports tumor cell through transfection and suppresses the expression of TGF-β, thereby makes tumor cell produce new biological property.The Hep3B cell of the expression of GM-CSF that has for example set up is imported by TGF-β 1siRNA plamid vector transfection.G418 drug screening through 2-3 week is cultivated, and sets up stable cell line.The stable cell lines that has wherein suppressed TGF-β expression screens via the RT-PCR technology.The stable cell lines that is obtained carries out cell culture and mass production via Cell Factory or bioreactor (Bio-reactor), is used for the clinical trial treatment behind radioinactivation.
Utilize ELISA and Western Blot method, can analyze the expression of GM-CSF and TGF-β effectively.Fig. 1 shows the experimental analysis of hepatocarcinoma Hep3B anti-cancer vaccine cell line, and with comparison shows that of germinal cell system, the new vaccine bebcell that makes up is expression of GM-csf protein, and the expression of TGF-β has simultaneously been suppressed significantly.
The expressed GM-CSF of hepatocarcinoma Hep3B anti-cancer vaccine cell line by experiment confirm for biologic activity is arranged.Fig. 2 is the specific amplified result of the test of TF-1 cell.The cell culture fluid of Hep3B anti-cancer vaccine cell line and various contrast and TF-1 cell co-cultivation, have only the culture fluid of vaccine strain to show the specific amplified result similar, confirm the GM-CSF that the vaccine strain expression has biologic activity to the GM-CSF standard control.
More than test shows that prepared Hep3B-GMCSF/TGF β siRNA anti-cancer vaccine cell line is not only expressed higher GM-CSF, and has suppressed the expression of TGF-β in the cell, has reached the vaccine design requirement.For further proving the vaccine anticancer function, animal experiment also carries out simultaneously in the dependent body.Because the human tumor model is difficult to study in the animal body that immunologic function perfects, mice GM-CSF is used to make up different cell strains with TGF-β 1 gene, thereby has set up the immunology effect that experimental animal model checking GM-CSF expresses and TGF-β 1siRNA suppresses.In the test of Fig. 3, the cell that GM-CSF expresses is arranged and has the cell that GM-CSF expresses and TGF-β 1siRNA suppresses to show the antitumor immune efficacy of vaccines simultaneously, and the latter since the inhibition of TGF-β 1siRNA more shown than having only GM-CSF to express better immunology effect.
According to the above vaccine construction method, all kinds of tumor cell lines of expression of GM-CSF and TGF-β 1siRNA are cloned, comprise lung cancer A549 cell, intestinal cancer LoVo cell, and gastric cancer SNU5 cell, cervical cancer HeLa cell, or the like.
Tumor cells expression GM-CSF and HSP90 also suppress the vaccine of TGF-β:
This type of tumour-cell vaccine has increased the expression of HSP simultaneously except that above feature.Expressing HSP is by add Foot Mouth Disease Virus (FMDV) 2A sequence and follow-up HSP90 gene behind the GM-CSF gene.FMDV 2A sequence for the expression of second gene HSP90 provide essential signal (Donnelly etc., 2001, Journal of General Virology, 82:1027-1041.).The retrovirus transfection carrier rkat43.2GM-CSF/HSP90 of Gou Jianing is used to prepare the retroviral vector that has GM-CSF and HSP90 gene that infective activity is arranged and the stable cell lines that is further used for preparing expression of GM-CSF and HSP90 gene thus.
The stable tumor cell line of new expression of GM-CSF and HSP90 gene imports TGF-β 1siRNA through transfection pSilencer plasmid vector, suppresses the stable cell lines that TGF-β expresses thereby set up.Concrete preparation process is analogous to the preparation of example one described Hep3B/GM-CSF/TGF-β 1siRNA cell.
This type of vaccine can further improve presenting of tumor antigen and immune system recognition.Fig. 4 is used for the in vivo test result of mouse tumor model B16 for several different genes combinations.In this test, the B16 cell makes up B16-GMCSF/TGF-β through in-vitro transfection
-With B16-GMCSF/TGF-β
-/ HSP cell line, three kinds of different cell lines are used for the B16 tumor treatment.At first, test first day, have about 100mm
3The mice of big B16 tumor is divided into three groups (n=8), the anti-cancer vaccine cell line of a kind of radioinactivation of every winding kind.At the 60th day, mice was accepted the B16 injection cell once more.The survival record of three treated animals shows relatively B16-GMCSF/TGF-β through figure
-The prepared vaccine of/HSP cell line shows the strongest anticancer function.
According to the above vaccine construction method, expression of GM-CSF, all kinds of tumor cell lines of HSP and TGF-β 1siRNA are cloned, comprise lung cancer A549 cell, intestinal cancer LoVo cell, gastric cancer SNU5 cell, cervical cancer HeLa cell, or the like.
Tumor cells expression GM-CSF, HSP90 and TAP-1 also suppress the vaccine of TGF-β:
This type of tumour-cell vaccine cell line has multiple new feature to improve presenting of tumor antigen, can become the efficient compound anti-cancer vaccine, has the obvious synergistic effect.
Utilizing the cross presentation mechanism of DC cell to carry out tumor antigen pointedly presents and has shown certain clinical anticancer function at present.Adopt tumor cell to prepare the anticancer biological product that full cell anti-cancer vaccine may become a new generation.Present patent application is scientifically used immunologic mechanism the presenting and discerning with the raising tumor antigen in the antigen presentation, but the full cell anti-cancer vaccine large-scale industrial production of being developed, the tumor antigen of vaccine is presented and immune system recognition is expected to improve greatly, thereby strengthens the anti-cancer ability of vaccine.
The clinical practice of anti-cancer vaccine of the present invention
In clinical practice, at patient's cancer types, related neoplasms cell line (for example pulmonary carcinoma or hepatoma cell line) is used for expression of GM-CSF (and or HSP) and suppresses TGF-β and make up this type of anti-cancer vaccine.The stabilization of vaccines cell line that is obtained is carried out cell culture and mass production via Cell Factory or bioreactor (Bio-reactor), and institute's cell that obtains is through the washing separation and make suspension (0.1～500x10
6Cell/mL/ props up), after the radiation treatment deactivation, be stored in liquid nitrogen or cryogenic refrigerator.Before being used for the clinical trial treatment, after thawing, the anti-cancer vaccine cell preparation of freezing preservation does injection (for example subcutaneous injection) to patient.Generally speaking, each patient need one to six injection be a course of treatment (weekly or every month once, each one), lasted for six to 24 weeks.
Embodiment 5
The preparation of the vaccine of tumor cells expression GM-CSF and inhibition TGF-β:
The vaccine 0.1x10 of embodiment 1 method preparation
6Cell/mL adds 5%DMSO, 8%Pentastarch, and 2% human serum albumin's (HSA) phosphoric acid physiological condition buffer, mix homogeneously is handled through radioinactivation, and is freezing.
Embodiment 6
The preparation of the vaccine of tumor cells expression GM-CSF and inhibition TGF-β:
The vaccine 500x10 of embodiment 1 method preparation
6Cell/mL adds 5%DMSO, 8%Pentastarch, and 2% human serum albumin's (HSA) phosphoric acid physiological condition buffer, mix homogeneously is handled through radioinactivation, and is freezing.
Embodiment 7
Tumor cells expression GM-CSF and HSP90 also suppress the preparation of the vaccine of TGF-β:
The vaccine 100x10 of embodiment 2 methods preparation
6Cell/mL adds 5%DMSO, 8%Pentastarch, and 2% human serum albumin's (HSA) phosphoric acid physiological condition buffer, mix homogeneously is handled through radioinactivation, and is freezing.
Embodiment 8
Tumor cells expression GM-CSF and HSP90 also suppress the preparation of the vaccine of TGF-β:
The vaccine 10x10 of embodiment 2 methods preparation
6Cell/mL adds 5%DMSO, 8%Pentastarch, and 2% human serum albumin's (HSA) phosphoric acid physiological condition buffer, mix homogeneously is handled through radioinactivation, and is freezing.
Claims (10)
1. an anti-cancer vaccine is characterized in that, vaccine composition is expression of GM-CSF and the Hep3B tumor cell line that suppresses TGF β.
2. an anti-cancer vaccine is characterized in that, vaccine composition is expression of GM-CSF and the K-1735 that suppresses TGF β, and preferred cell is a B16 cell line.
3. anti-cancer vaccine, it is characterized in that vaccine composition is expression of GM-CSF and HSP90 and the tumor cell line that suppresses TGF β, preferred tumor cell is B16 cell, lung cancer A549 cell, intestinal cancer LoVo cell, gastric cancer SNU5 cell, cervical cancer HeLa cell a kind of.
4. according to each described anti-cancer vaccine of claim 1-3; described vaccine contains the tumor cell line and the physiologically acceptable auxiliary element through the immunomodulatory gene modification of effective dose; described auxiliary element is selected from DMSO; Pentastarch, serum albumin, buffer solution; water; vaccine adjuvant, protective agent, stabilizing agent.
5. anti-cancer vaccine according to claim 4, the amount of the tumor cell line that the process immunomodulatory gene of described effective dose is modified is 0.1-500x10
6Cell/mL.
6. anti-cancer vaccine according to claim 4, described tumor cell line is handled through radioinactivation, and described auxiliary element is selected from 5%DMSO, 8%Pentastarch, 2% human serum albumin's phosphate buffer.
7. the application of each described anti-cancer vaccine of claim 1-3 in the pharmaceutical preparation of preparation anti-cancer vaccine.
8. the described application of claim 7, cancer wherein are hepatocarcinoma, pulmonary carcinoma, intestinal cancer, gastric cancer, cervical cancer or melanoma.
9. a method for preparing each described anti-cancer vaccine of claim 1-3 is characterized in that, immunomodulatory gene and tumor cell tie up to vitro recombination and make up, the process following steps:
The preparation of the tumor cell line that a modifies through immunomodulatory gene, its step be, imports exogenous gene with virus or plasmid DNA carrier, and be continuous and carry out the stable cell lines screening and obtain;
The tumor cell line that b modifies through immunomodulatory gene is mixed and made into through radioinactivation processing and physiologically acceptable auxiliary element, and described auxiliary element is selected from DMSO, Pentastarch; serum albumin, buffer solution, water; vaccine adjuvant, protective agent, stabilizing agent.
10. method according to claim 9 is characterized in that, the tumor cell line amount of modifying through immunomodulatory gene is 0.1-500x10
6Cell/mL, described auxiliary element is selected from 5%DMSO, 8%Pentastarch,, 2% human serum albumin's phosphate buffer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103961693A (en) * | 2013-01-24 | 2014-08-06 | 熊慧 | Therapeutic vaccine for malignant tumors and composition thereof |
| CN104096238A (en) * | 2013-04-01 | 2014-10-15 | 熊慧 | Malignant tumor therapeutic vaccine and composition thereof |
-
2005
- 2005-12-30 CN CN200910168455A patent/CN101773666A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103961693A (en) * | 2013-01-24 | 2014-08-06 | 熊慧 | Therapeutic vaccine for malignant tumors and composition thereof |
| CN103961693B (en) * | 2013-01-24 | 2016-09-14 | 熊慧 | A kind for the treatment of malignant tumor vaccine and combinations thereof thing |
| CN104096238A (en) * | 2013-04-01 | 2014-10-15 | 熊慧 | Malignant tumor therapeutic vaccine and composition thereof |
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Open date: 20100714 |