CN105031630A - Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof - Google Patents
Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof Download PDFInfo
- Publication number
- CN105031630A CN105031630A CN201510207955.9A CN201510207955A CN105031630A CN 105031630 A CN105031630 A CN 105031630A CN 201510207955 A CN201510207955 A CN 201510207955A CN 105031630 A CN105031630 A CN 105031630A
- Authority
- CN
- China
- Prior art keywords
- csf
- cell
- tumor cell
- neutralizing antibody
- tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses the field of tumor cellular immunotherapy and particularly relates to a tumor cell vaccine which is modified by a PD-1 neutralizing antibody with combination of a GM-CSF factor and a preparation method thereof. In the invention, tumor cellular vaccine therapy is creatively combined with antibody therapy which relieves tumor immune-suppression, so that the prepared tumor cell vaccine simultaneously secreting the PD-1 neutralizing antibody and the GM-CSF factor can not only relieve the immune-suppression status of a tumor micro-environment but also enhance an antitumor immune reaction. The tumor cell vaccine is good in antitumor treatment effect, is excellent in development value and provides a new approach of tumor cellular immunotherapy.
Description
Technical field
The invention belongs to tumor cell immunotherapy field, be specifically related to tumour-cell vaccine of a kind of secretion PD-1 neutralizing antibody and the GM-CSF factor simultaneously and preparation method thereof.
Background technology
Tumor vaccine can human activin self immune system, brings out specific immune response, and improving autoimmunity, is a kind of active immunity treatment method.It has, and untoward reaction is slight, better tolerance, has preventive and therapeutic action concurrently, becomes one of focus of global antitumor research.Programmed death receptor 1 (PD-1) is that T cell regulates the costimulatory molecules conducting inhibition signal in receptor CD28 family, the negativity conditioning signal of mediated immunity reaction, has all played specific immunoregulation effect in tumor generation, viral infection and autoimmune disease.PD-1 wide expression, in the T cell activated, memory t cell and regulatory T cells surface, can lower T cell activity after it is combined with binding partner PD-L1 or PD-L2, is the important molecule producing tumor immune escape.GM-CSF (Granulocytemacrophagecolony-stimulatingfactor) i.e. granulocyte-macrophage colony stimutaing factor can promote that mononuclear cell, macrophage and dendritic cell (DC) are bred, ripe and migration and the lymphocytic amplification of B and T, differentiation carry out the immunoreation that enhancement antigen causes, and conventionally does adjuvant.At present and have no the report of both to be combined for oncotherapy.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of can the anti-tumor cell vaccine jointly modified of PD-1 neutralizing antibody associating GM-CSF gene and preparation method thereof.
This anti-tumor cell vaccine is the tumor cell modified by PD-1 neutralizing antibody and GM-CSF cytokine, is prepared from through x-ray irradiation.
Wherein, the dosage of above-mentioned x-ray irradiation is sublethal dose.
Wherein, the exposure dose of above-mentioned X-ray is total radiation dosage 50-150Gy.
Wherein, the aminoacid sequence of above-mentioned PD-1 neutralizing antibody is for shown in SEQIDNO.8.
Wherein, the aminoacid sequence of above-mentioned GM-CSF is for shown in SEQIDNO.10 or SEQIDNO.12.
Wherein, the nucleotides sequence of the encoding gene of above-mentioned PD-1 neutralizing antibody is classified as shown in SEQIDNO.7, or is its degenerate sequence.
Wherein, the nucleotides sequence of the encoding gene of above-mentioned GM-CSF is classified as shown in SEQIDNO.9 or SEQIDNO.11, or is both respective degenerate sequences.
Wherein, above-mentioned tumor cell is by obtaining the tumor cell expressing the ability of PD-1 neutralizing antibody and GM-CSF after the vector of the encoding gene being loaded with PD-1 neutralizing antibody and GM-CSF.
Wherein, above-mentioned carrier is slow virus carrier.
Wherein, above-mentioned Lentiviral is Ubi-MCS-3FLAG-IRES-puro.
Wherein, the genophore of above-mentioned expression LV-PD-1 neutralizing antibody, LV-mGM-CSF (Mus GM-CSF) or LV-hGM-CSF (human GM-CSF) is prepared by following steps respectively:
The preparation of a, process LAN slow virus carrier: utilize PCR method to increase respectively anti-PD-1, mGM-CSF or hGM-CSF genes of interest from plasmid pcDNA3.1-anti-PD-1, pcDNA3.1-mGM-CSF and the pcDNA3.1-hGM-CSF containing genes of interest, Ubi-MCS-3FLAG-IRES-puro object carrier is carried out BamHI/AgeI enzyme action, digestion products electrophoresis exchanges after reclaiming, its product conversion antibacterial competent cell.First carry out bacterium colony PCR qualification to the clone grown, then the clone positive to PCR qualification checks order and compare of analysis, what comparison was correct is the object plasmid successfully constructed.
B, slow virus packaging detect with titre: by the recombinant virus plasmid of expression genes of interest that builds and two kinds of auxiliary package original paper vector plasmids thereof, each plasmid vector carries out high-purity respectively without endotoxin extracting, transfection 293T cell is carried out by Invitrogen company Lipofectamine2000 operation instruction, after transfection, 8h is replaced by complete medium, after cultivating 48h, collect the cell conditioned medium liquid being rich in lentiviral particle, obtain the slow virus concentrated solution of high titre after concentrated to it, measure in 293T cell and demarcate virus titer.
Further, the upstream and downstream primer sequence of described amplification PD-1 neutralizing antibody, mice GM-CSF gene sequence and GM-CSF Gene sequence is respectively shown in SEQIDNO.1 and 2, shown in SEQIDNO.3 and 4, and shown in SEQIDNO.5 and 6.
Present invention also offers a kind of method preparing above-mentioned anti-tumor cell vaccine, the method comprises the following steps:
The preparation of a, process LAN slow virus carrier: from plasmid pcDNA3.1-anti-PD-1, pcDNA3.1-mGM-CSF or the pcDNA3.1-hGM-CSF containing genes of interest, PCR method is utilized to increase respectively the genes of interest sequence of PD-1 neutralizing antibody, mGM-CSF and hGM-CSF, Ubi-MCS-3FLAG-IRES-puro carrier is carried out BamHI/AgeI enzyme action, digestion products electrophoresis exchanges after reclaiming, its product conversion antibacterial competent cell.First carry out bacterium colony PCR qualification to the clone grown, then the clone positive to PCR qualification checks order and compare of analysis, what comparison was correct is the object plasmid successfully constructed;
B, slow virus packaging detect with titre: by the recombinant virus plasmid of expression genes of interest that builds and two kinds of auxiliary package original paper vector plasmids thereof, three plasmid vector carry out high-purity respectively without endotoxin extracting, transfection 293T cell is carried out by Invitrogen company Lipofectamine2000 operation instruction, complete medium is replaced by after transfection 8h, after cultivating 48h, collect the cell conditioned medium liquid being rich in lentiviral particle, obtain the slow virus concentrated solution of high titre after concentrated to it, measure in 293T cell and demarcate virus titer;
C, Simultaneous Stabilization express the preparation of the tumor cell line of PD-1 neutralizing antibody and GM-CSF, screening and detection of expression:
By slow virus LV-anti-PD-1, LV-GM-CSF infected tumor cell of preparation, the infection multiplicity scope of slow virus is 1-100, grope the best multiplicity of infection of different tumor cell, first LV-anti-PD-1 is infected after determining multiplicity of infection, screen with puromycin, the cell culture supernatant of viral infection is collected, with the expression detecting PD-1 neutralizing antibody after screening; The cell of stable infection LV-anti-PD-1 is infected LV-GM-CSF again, continues screening with puromycin; Collecting cell supernatant, detects GM-CSF and expresses, and confirms that the cell strain of screening can simultaneously high expressed PD-1 neutralizing antibody and GM-CSF;
D, prepare the tumour-cell vaccine of high expressed PD-1 neutralizing antibody and the GM-CSF factor:
Rear collection is counted by filtering out after the stable tumor cell line having infected LV-GM-CSF and LV-anti-PD-1 is cultivated, through the x-ray irradiation of total radiation dosage 60-160Gy, and the tumor cell after predose is counted, morphologic observation, the proliferative conditions of cell after these predoses is detected by CCK-8 cell proliferation experiment, utilize ELISA to detect the level of cellular expression PD-1 neutralizing antibody and GM-CSF albumen after predose, lose multiplication capacity after screening obtains irradiation and can the cell of high expressed PD-1 neutralizing antibody and the GM-CSF factor be tumour-cell vaccine.
Wherein, above-mentioned tumor is colon cancer, melanoma, breast carcinoma or pulmonary carcinoma.
The anti tumor immune response that the present invention brings out to strengthen vaccine, remove the immunosuppressive condition of tumor microenvironment, tumor vaccine is combined with releasing immunosuppressant antibody drug, so both can remove the immunosuppressive condition of tumor microenvironment, anti tumor immune response can be strengthened again, to obtain better antitumous effect.
Beneficial effect of the present invention is: the Antybody therapy that tumour-cell vaccine treatment and releasing tumour immunity suppress creatively combines by the present invention, prepared vaccine so both can remove the immunosuppressive condition of tumor microenvironment, can strengthen anti tumor immune response again.In the universal Tumor models such as CT26 cell, LL/2 cell, B16 cell, 4T1 cell, the propagation of vaccine to tumor cell that this tumor cell of simultaneously expressing PD-1 neutralizing antibody and the GM-CSF factor is prepared through sublethal dose X-ray (being preferably 60 ~ 120Gy) irradiation creates significant depression effect.The multiple vaccine of preparation all achieves significant antitumous effect, wherein for C26 and the B16-F10 vaccine that PD-1 neutralizing antibody and the GM-CSF factor are modified, C26 vaccine therapy results of animal shows, the inhibitory rate 70.6% of coexpression PD-1 neutralizing antibody and GM-CSF group compared with matched group.B16-F10 vaccine therapy results of animal shows, the inhibitory rate 64.9% of coexpression PD-1 neutralizing antibody and GM-CSF group compared with matched group.The adoptive results of animal of C26 vaccine shows, the inhibitory rate 47.2% of coexpression PD-1 neutralizing antibody and GM-CSF group compared with matched group.The adoptive results of animal of B16-F10 vaccine shows, the inhibitory rate 50.8% of coexpression PD-1 neutralizing antibody and GM-CSF group compared with matched group.Express the tumour-cell vaccine of PD-1 neutralizing antibody and the GM-CSF factor while prepared by visible the present invention, antitumor curative effect is good, has good Development volue, and the immune cell therapy for tumor provides new selection.
Accompanying drawing explanation
Fig. 1, Ubi-MCS-3FLAG-IRES-puro carrier structure schematic diagram;
PD-1 neutralization and GM-CSF detection of expression after Fig. 2, C26,4T1, B16-F10 tumour-cell vaccine predose;
Fig. 3, C26 interior therapeutic zoopery tumor growth curve and life cycle.The tumor killing effect of coexpression PD-1 neutralizing antibody and GM-CSF treatment group is 70.6% compared with matched group, the tumor killing effect of PD-1 neutralizing antibody group is 38.5%, the tumor killing effect of GM-CSF group is 30.5%.In addition, the survival time of mice significant prolongation of therapeutic alliance group.
Fig. 4, B16-F10 interior therapeutic zoopery tumor growth curve and life cycle.The tumor killing effect of coexpression PD-1 neutralizing antibody and GM-CSF treatment group is 64.9% compared with matched group, the tumor killing effect of PD-1 neutralizing antibody group is 55.7%, the tumor killing effect of GM-CSF group is 20.9%.In addition, the survival time of mice significant prolongation of therapeutic alliance group.
The adoptive zoopery tumor growth curve of Fig. 5, C26 vaccine and Survival.
The adoptive zoopery tumor growth curve of Fig. 6, B16-F10 vaccine and Survival.
Detailed description of the invention
Below in conjunction with accompanying drawing by the description of detailed description of the invention to describe in detail but not limit the present invention.
Main thought of the present invention will express the slow virus carrier co-infection tumor cell of PD-1 neutralizing antibody and GM-CSF gene respectively, and screening obtains the cell strain of stably express PD-1 neutralizing antibody and GM-CSF gene sequence.By screen simultaneously high expressed PD-1 neutralizing antibody and GM-CSF albumen stable strain cell be prepared from through sublethal dose X-ray (50-150Gy, preferred 60-120Gy) irradiation.Carrier or the host cell that wherein can express PD-1 neutralizing antibody and GM-CSF gene are as main active component.In this area, conventional X-ray prepares tumor vaccine, and usually by making tumor cell lose proliferation activity and Tumor formation after irradiation, but can normal adherent tumor.This state this area is called sub-lethal, and tumor cell can be caused to occur, and the irradiation dose of this state is referred to as sublethal dose.
Collect filtering out stable infection after tumor cell line that LV-GM-CSF, LV-anti-PD-1 and Simultaneous Stabilization infected LV-GM-CSF and LV-anti-PD-1 is cultivated after counting, through the x-ray irradiation of total radiation dosage 60-160Gy, and the tumor cell after predose is counted, morphologic observation, the proliferative conditions of cell after these predoses is detected by CCK-8 cell proliferation experiment, ELISA is utilized to detect the level of cellular expression PD-1 neutralizing antibody and GM-CSF albumen after predose, to optimize sublethal irradiation dosage.
By optimizing, the preferably irradiation dose of various tumor cell is: lung cancer cell line irradiation dose 80-150Gy, preferred 120Gy.Breast cancer cell line irradiation dose 50-100Gy, preferred 60Gy.Melanoma cells B16-F10 irradiation dose 60-120Gy, preferred 100Gy.Colon carcinoma cell line C26 irradiation dose 60-120Gy, preferred 100Gy.
4 kinds of different tumour-cell vaccine have been prepared: lung cancer cell line LL/2 (irradiation dose 80-150Gy in the embodiment of the present invention, preferred 120Gy), breast cancer cell line 4T1 (50-100Gy, preferred 60Gy), Melanoma cells B16-F10 (60-120Gy, preferred 100Gy), colon cancer C26 (60-120Gy, preferred 100Gy).The infection multiplicity scope of slow virus is 1-100, grope the best multiplicity of infection of different tumor cell, first LV-anti-PD-1 is infected after determining multiplicity of infection, screen with puromycin, collect the cell culture supernatant of viral infection after screening, detect the expression of PD-1 neutralizing antibody with indirect ELISA.The cell of stable infection LV-anti-PD-1 is infected LV-GM-CSF, basis of microscopic observation egfp expression again, continues screening with puromycin.Collecting cell supernatant, detects GM-CSF by Elisa method and expresses, and confirms that the cell strain of screening can simultaneously high expressed PD-1 neutralizing antibody and GM-CSF.
It should be noted that, when mouse animal experiment, therefore use mice GM-CSF.Based on substituting in drug study of people and mice, those skilled in the art's experimental result according to the present invention has reason to believe that end user GM-CSF still has close effect.
The acquisition of embodiment one PD-1 neutralizing antibody and GM-CSF gene and the structure of LV-anti-PD-1, LV-GM-CSF slow virus carrier
According to the anti-PD-1 gene order (patentNo:US20030026800A1) that USPO submits, the mGM-CSF gene order (NM_009969.4) that NCBI submits, hGM-CSF gene order (NM_000758.3) has synthesized anti-PD-1 gene respectively, and (nucleotide sequence is as shown in SEQIDNO.7, the aminoacid sequence of the protein of coding is as shown in SEQIDNO.8), (nucleotide sequence is as shown in SEQIDNO.9 for mGM-CSF gene, the aminoacid sequence of the protein of coding is as shown in SEQIDNO.10), (nucleotide sequence is as shown in SEQIDNO.11 for hGM-CSF gene, the aminoacid sequence of the protein of coding is as shown in SEQIDNO.12).And be built into pcDNA3.1-anti-PD-1, pcDNA3.1-mGM-CSF and pcDNA3.1-hGM-CSF plasmid (being synthesized by Nanjing Jin Sirui company).Then PCR method is utilized to increase respectively PD-1 neutralizing antibody, mGM-CSF and hGM-CSF genes of interest, Ubi-MCS-3FLAG-IRES-puro object carrier is carried out BamHI/AgeI enzyme action, digestion products electrophoresis exchanges after reclaiming, its product conversion antibacterial competent cell.First carry out bacterium colony PCR qualification to the clone grown, then the clone positive to PCR qualification checks order and compare of analysis, what comparison was correct is the object plasmid successfully constructed.
The primer sequence that above-mentioned structure mainly uses is as follows:
The forward primer (SEQIDNO.1) of amplification PD-1 neutralizing antibody:
5'-AGGTCGACTCTAGA
gGATCCcGCCACCATGGCTTGGGTGTGGACCTTGC-3 ' (underscore represents the BamHI restriction enzyme site of introducing)
The downstream primer (SEQIDNO.2) of amplification PD-1 neutralizing antibody:
5'-AGTCCATGGTGGCG
aCCGGAcACTCATTCCTGTTGAAGCTC-3 (underscore represents the AgeI restriction enzyme site of introducing)
The forward primer (SEQIDNO.3) of amplification mGM-CSF:
5'-GA
gGATCCcCGGGTACCGGTCGCCACCATGTGGCTGCAGAATTTAC-3 ' (underscore represents the BamHI restriction enzyme site of introducing)
The downstream primer (SEQIDNO.4) of amplification mGM-CSF:
5'-TCACCATGGTGGCG
aCCGGTtTTTGGCCTGGTTTTTTGC-3 (underscore represents the AgeI restriction enzyme site of introducing)
The forward primer (SEQIDNO.5) of amplification hGM-CSF:
5'-GA
gGATCCcAGAGCCTGCTGCTCTTGG-3 ' (underscore represents the BamHI restriction enzyme site of introducing)
The downstream primer (SEQIDNO.6) of amplification hGM-CSF:
5'-TCACCATGGTGGCG
aCCGGTcCAGCAGTCAAAGGGGATGA-3 (underscore represents the AgeI restriction enzyme site of introducing)
By above-mentioned by the recombinant virus plasmid of expression genes of interest that builds and two kinds of auxiliary package original paper vector plasmids thereof, three plasmid vector carry out high-purity respectively without endotoxin extracting, transfection 293T cell is carried out by Invitrogen company Lipofectamine2000 operation instruction, after transfection, 8h is replaced by complete medium, after cultivating 48h, collect the cell conditioned medium liquid being rich in lentiviral particle, obtain the slow virus concentrated solution of high titre after concentrated to it, measure in 293T cell and demarcate virus titer.Obtain LV-anti-PD-1, LV-mGM-CSF, LV-hGM-CSF virus.
After the preparation of embodiment two products C 26 of the present invention, 4T1, B16-F10 tumour-cell vaccine and predose, PD-1 neutralizing antibody and GM-CSF expression detect.
Use slow virus infection colon cancer cell C26 (irradiation dose 100Gy) obtained above, breast cancer cell 4T1 (irradiation dose 100Gy), melanoma cell B16-F10 cell strain (irradiation dose 60Gy), then use x-ray irradiation to lose proliferation activity.Use the same method and also prepared human tumor cells lung cancer cell line A549, breast cancer cell line MCF-7, the cell vaccine of K-1735 A375, colon cancer HCT116.
Colon cancer cell C26, the breast cancer cell 4T1, the melanoma cell B16-F10 cell strain that screen stably express PD-1 neutralizing antibody and the GM-CSF obtained are cultivated, collecting cell supernatant after 48h.Continue the cell conditioned medium cultivating 48h after collecting cell irradiation simultaneously.Elisa detects the expression of PD-1 neutralizing antibody and GM-CSF in cell conditioned medium after predose.Result is see Fig. 2.
The zoopery of embodiment three products C 26 of the present invention vaccine therapy
Often organize 20 Balb/c mices, every right side of mice subcutaneous vaccination 5 × 10
5c26 cell, measures 1 gross tumor volume in every 3 days, and after inoculation C26 cell, 3d, 6d, 9d are with 1 × 10
6tumour-cell vaccine after irradiation treats 3 times continuously.Treatment grouping comprises: the cell irradiation group of the C26 groups of cells after irradiation, the C26 cell irradiation group infecting LV, the C26 cell irradiation group infecting LV-GM-CSF, the C26 cell irradiation group of infection LV-anti-PD-1, simultaneously infection LV-GM-CSF and LV-anti-PD-1, and sets up untreated fish group as blank.Observe the tumor growth of each group of mice and the life cycle of mice, each experiment repeats 3 times, and result is see Fig. 3.Result shows, the inhibitory rate 70.6% of coexpression PD-1 neutralizing antibody and GM-CSF group compared with matched group.Significantly gross tumor volume be can reduce, animal dis motility rate and time-to-live increased.
The zoopery of embodiment four product B 16-F10 vaccine therapy of the present invention
Often organize 20 C57 mices, every right side of mice subcutaneous vaccination 5 × 10
5b16-F10 cell, measures 1 gross tumor volume in every 3 days, and after inoculation B16-F10 cell, 3d, 6d, 9d are with 1 × 10
6tumour-cell vaccine after irradiation treats 3 times continuously.Treatment grouping comprises: the cell irradiation group of the B16-F10 groups of cells after irradiation, the B16-F10 cell irradiation group infecting LV, the B16-F10 cell irradiation group infecting LV-GM-CSF, the B16-F10 cell irradiation group of infection LV-anti-PD-1, simultaneously infection LV-GM-CSF and LV-anti-PD-1, and sets up untreated fish group as blank.Observe the tumor growth of each group of mice and the life cycle of mice, each experiment repeats 3 times, and result is see Fig. 4.Result shows, the inhibitory rate 64.9% of coexpression PD-1 neutralizing antibody and GM-CSF group compared with matched group.Significantly gross tumor volume be can reduce, animal dis motility rate and time-to-live increased.
The adoptive zoopery of embodiment five products C of the present invention 26 vaccine
Often organize 10 Balb/c mices, zoopery is repeated according to therapeutic immunization scheme, immunity terminates rear separation each group of mouse spleen lymphocyte, mouse spleen lymphocyte after being separated to be adopted treatment to tumor-bearing mice by the mode of tail vein injection by 3d, 5d, 7d, 9d after inoculation respectively, often organize 10 mices, 1 × 10
7cells/ is only every.Subcutaneous vaccination 5 × 10 on the right side of every tumor-bearing mice
5c26 cell, measures 1 gross tumor volume in every 3 days.Observe the tumor growth of each group of mice and the life cycle of mice, each experiment repeats 3 times, and result is see Fig. 5.Result shows, the inhibitory rate 47.2% of coexpression PD-1 neutralizing antibody and GM-CSF group compared with matched group.Significantly gross tumor volume be can reduce, animal dis motility rate and time-to-live increased.
The adoptive zoopery of embodiment six product B 16-F10 of the present invention vaccine
Often organize 10 C57 mices, zoopery is repeated according to therapeutic immunization scheme, immunity terminates rear separation each group of mouse spleen lymphocyte, mouse spleen lymphocyte after being separated to be adopted treatment to tumor-bearing mice by the mode of tail vein injection by 3d, 5d, 7d, 9d after inoculation respectively, often organize 10 mices, 1 × 10
7cells/ is only every.Subcutaneous vaccination 5 × 10 on the right side of every tumor-bearing mice
5b16-F10 cell, measures 1 gross tumor volume in every 3 days.Observe the tumor growth of each group of mice and the life cycle of mice, each experiment repeats 3 times, and result is see Fig. 6.Result shows, the inhibitory rate 50.8% of coexpression PD-1 neutralizing antibody and GM-CSF group compared with matched group.Significantly gross tumor volume be can reduce, animal dis motility rate and time-to-live increased.
Claims (14)
1. anti-tumor cell vaccine, is characterized in that: be by the tumor cell of expressing PD-1 neutralizing antibody and the modification of GM-CSF cytokine, be prepared from through x-ray irradiation.
2. anti-tumor cell vaccine according to claim 1, is characterized in that: the dosage of described x-ray irradiation is sublethal dose.
3. anti-tumor cell vaccine according to claim 2, is characterized in that: the exposure dose of described X-ray is total radiation dosage 50-150Gy.
4. the anti-tumor cell vaccine according to any one of claim 1-3, is characterized in that the aminoacid sequence of described PD-1 neutralizing antibody is for shown in SEQIDNO.8.
5. the anti-tumor cell vaccine according to any one of claim 1-3, is characterized in that the aminoacid sequence of described GM-CSF is for shown in SEQIDNO.10 or SEQIDNO.12.
6. anti-tumor cell vaccine according to claim 4, is characterized in that the nucleotides sequence of the encoding gene of described PD-1 neutralizing antibody is classified as shown in SEQIDNO.7.
7. anti-tumor cell vaccine according to claim 5, is characterized in that the nucleotides sequence of the encoding gene of described GM-CSF is classified as shown in SEQIDNO.9 or SEQIDNO.11, or is both respective conservative variant.
8. the anti-tumor cell vaccine according to any one of claim 1-7, is characterized in that: described tumor cell is by obtaining the ability expressing PD-1 neutralizing antibody and GM-CSF after the vector of the encoding gene being loaded with PD-1 neutralizing antibody and GM-CSF.
9. anti-tumor cell vaccine according to claim 8, is characterized in that: described carrier is slow virus carrier.
10. anti-tumor cell vaccine according to claim 9, is characterized in that: described Lentiviral is Ubi-MCS-3FLAG-IRES-puro.
11., according to the anti-tumor cell vaccine of claim according to any one of claim 1-10, is characterized in that: described expression LV-PD-1 neutralizing antibody, the genophore of LV-mGM-CSF or LV-hGM-CSF are prepared by following steps:
The preparation of a, viral vector: utilize PCR method to increase respectively anti-PD-1, mGM-CSF or hGM-CSF genes of interest from plasmid pcDNA3.1-anti-PD-1, pcDNA3.1-mGM-CSF or the pcDNA3.1-hGM-CSF containing genes of interest, Ubi-MCS-3FLAG-IRES-puro object carrier is carried out BamHI/AgeI enzyme action, digestion products electrophoresis exchanges after reclaiming, its product conversion antibacterial competent cell; First carry out bacterium colony PCR qualification to the clone grown, then the clone positive to PCR qualification checks order and compare of analysis, what comparison was correct is the object plasmid successfully constructed;
B, slow virus packaging detect with titre: by the recombinant virus plasmid of expression genes of interest that builds and two kinds of auxiliary package original paper vector plasmids thereof, three plasmid vector carry out high-purity respectively without endotoxin extracting, transfection 293T cell is carried out by Invitrogen company Lipofectamine2000 operation instruction, after transfection, 8h is replaced by complete medium, after cultivating 48h, collect the cell conditioned medium liquid being rich in lentiviral particle, obtain the slow virus concentrated solution of high titre after concentrated to it, measure in 293T cell and demarcate virus titer.
12. the anti-tumor cell vaccine according to any one of claim 1-11, is characterized in that: described tumor is colon cancer, melanoma, breast carcinoma or pulmonary carcinoma.
The method of the anti-tumor cell vaccine described in 13. any one of preparation claim 1-12, is characterized in that comprising the following steps:
The preparation of a, process LAN slow virus carrier: from plasmid pcDNA3.1-anti-PD-1, pcDNA3.1-mGM-CSF or the pcDNA3.1-hGM-CSF containing genes of interest, PCR method is utilized to increase respectively the genes of interest sequence of PD-1 neutralizing antibody, mGM-CSF and hGM-CSF, Ubi-MCS-3FLAG-IRES-puro carrier is carried out BamHI/AgeI enzyme action, digestion products electrophoresis exchanges after reclaiming, its product conversion antibacterial competent cell; First carry out bacterium colony PCR qualification to the clone grown, then the clone positive to PCR qualification checks order and compare of analysis, what comparison was correct is the object plasmid successfully constructed;
B, slow virus packaging detect with titre: by the recombinant virus plasmid of expression genes of interest that builds and two kinds of auxiliary package original paper vector plasmids thereof, three plasmid vector carry out high-purity respectively without endotoxin extracting, transfection 293T cell is carried out by Invitrogen company Lipofectamine2000 operation instruction, complete medium is replaced by after transfection 8h, after cultivating 48h, collect the cell conditioned medium liquid being rich in lentiviral particle, obtain the slow virus concentrated solution of high titre after concentrated to it, measure in 293T cell and demarcate virus titer;
C, Simultaneous Stabilization express tumor cell line screening and the detection of expression of PD-1 neutralizing antibody and GM-CSF:
By slow virus LV-anti-PD-1, LV-GM-CSF infected tumor cell of preparation, the infection multiplicity scope of slow virus is 1-100, grope the best multiplicity of infection of different tumor cell, first LV-anti-PD-1 is infected after determining multiplicity of infection, screen with puromycin, the cell culture supernatant of viral infection is collected, with the expression detecting PD-1 neutralizing antibody after screening; The cell of stable infection LV-anti-PD-1 is infected LV-GM-CSF again, continues screening with puromycin; Collecting cell supernatant, detects GM-CSF and expresses, and confirms that the cell strain of screening can simultaneously high expressed PD-1 neutralizing antibody and GM-CSF;
D, prepare the tumour-cell vaccine of high expressed PD-1 neutralizing antibody and the GM-CSF factor:
To filter out to stablize and infect LV-GM-CSF, the tumor cell line that LV-anti-PD-1 or Simultaneous Stabilization have infected LV-GM-CSF and LV-anti-PD-1 cultivates the rear collection of rear counting, through the x-ray irradiation of total radiation dosage 60-160Gy, and the tumor cell after predose is counted, morphologic observation, the proliferative conditions of cell after these predoses is detected by CCK-8 cell proliferation experiment, ELISA is utilized to detect the level of cellular expression PD-1 neutralizing antibody and GM-CSF albumen after predose, substantially lose multiplication capacity after screening obtains irradiation and can the cell of high expressed PD-1 neutralizing antibody and the GM-CSF factor be tumour-cell vaccine.
14. methods according to claim 13, is characterized in that: described tumor is colon cancer, melanoma, breast carcinoma or pulmonary carcinoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510207955.9A CN105031630A (en) | 2014-04-28 | 2015-04-28 | Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410174315 | 2014-04-28 | ||
| CN201510207955.9A CN105031630A (en) | 2014-04-28 | 2015-04-28 | Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105031630A true CN105031630A (en) | 2015-11-11 |
Family
ID=54438984
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510207955.9A Pending CN105031630A (en) | 2014-04-28 | 2015-04-28 | Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105031630A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109219616A (en) * | 2016-09-19 | 2019-01-15 | 天境生物 | GM-CSF antibodies and uses thereof |
| CN110337305A (en) * | 2017-02-28 | 2019-10-15 | 赛诺菲 | Therapeutic RNA |
| CN111166867A (en) * | 2018-11-09 | 2020-05-19 | 中国科学院分子细胞科学卓越创新中心 | Function and use of PD-1 ubiquitination agonist |
| CN111386042A (en) * | 2017-12-04 | 2020-07-07 | 热生物制品有限公司 | Production of cell-based vaccines |
| CN116510022A (en) * | 2022-11-23 | 2023-08-01 | 武汉滨会生物科技股份有限公司 | Anti-tumor composition, recombinant plasmid composition and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101213297A (en) * | 2005-05-09 | 2008-07-02 | 小野药品工业株式会社 | Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics |
| CN102973922A (en) * | 2012-12-18 | 2013-03-20 | 华东理工大学 | Application of fusion protein |
-
2015
- 2015-04-28 CN CN201510207955.9A patent/CN105031630A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101213297A (en) * | 2005-05-09 | 2008-07-02 | 小野药品工业株式会社 | Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics |
| CN102973922A (en) * | 2012-12-18 | 2013-03-20 | 华东理工大学 | Application of fusion protein |
Non-Patent Citations (1)
| Title |
|---|
| HONGWEI TIAN ET AL: "Cellular immunotherapy using irradiated lung cancer cell vaccine co-expressing GM-CSF and IL-18 can induce significant antitumor effects", 《BMC CANCER》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109219616A (en) * | 2016-09-19 | 2019-01-15 | 天境生物 | GM-CSF antibodies and uses thereof |
| CN109219616B (en) * | 2016-09-19 | 2022-10-21 | 天境生物科技(杭州)有限公司 | GM-CSF antibodies and uses thereof |
| CN110337305A (en) * | 2017-02-28 | 2019-10-15 | 赛诺菲 | Therapeutic RNA |
| US11865159B2 (en) | 2017-02-28 | 2024-01-09 | Sanofi | Therapeutic RNA |
| CN111386042A (en) * | 2017-12-04 | 2020-07-07 | 热生物制品有限公司 | Production of cell-based vaccines |
| CN111166867A (en) * | 2018-11-09 | 2020-05-19 | 中国科学院分子细胞科学卓越创新中心 | Function and use of PD-1 ubiquitination agonist |
| CN111166867B (en) * | 2018-11-09 | 2022-12-20 | 中国科学院分子细胞科学卓越创新中心 | Function and use of PD-1 ubiquitination agonist |
| CN116510022A (en) * | 2022-11-23 | 2023-08-01 | 武汉滨会生物科技股份有限公司 | Anti-tumor composition, recombinant plasmid composition and application thereof |
| CN116510022B (en) * | 2022-11-23 | 2023-12-05 | 武汉滨会生物科技股份有限公司 | Anti-tumor composition, recombinant plasmid composition and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lin et al. | Oncolytic virotherapy: basic principles, recent advances and future directions | |
| JP7295192B2 (en) | modified oncolytic virus | |
| Merlo et al. | The interplay between Epstein-Barr virus and the immune system: a rationale for adoptive cell therapy of EBV-related disorders | |
| CN105031630A (en) | Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof | |
| US10232003B2 (en) | Exogenous tap inhibitor armed oncolytic viruses and therapeutic uses thereof | |
| CN105307671A (en) | Enhanced adoptive cell therapy | |
| TW201835327A (en) | Recombinant herpes simplex virus and use thereof capable of specifically cloning tumor cells in a high standard manner and destroying tumor cells | |
| WO2019062250A1 (en) | Therapeutic agent comprising isolated recombinant oncolytic adenovirus and nk cell, application, kit, and method for treatment of tumor and/or cancer | |
| Sinkovics et al. | Natural and genetically engineered viral agents for oncolysis and gene therapy of human cancers | |
| CN108261426B (en) | Pharmaceutical composition and its application in the drug for the treatment of tumour and/or cancer | |
| CN103614416A (en) | Recombinant oncolytic adenovirus carrying human cell-penetrating peptide p53 and GM-CSF gene, and uses thereof | |
| CN109554353A (en) | Isolated recombination oncolytic poxvirus, pharmaceutical composition and its purposes in the drug for the treatment of tumour and/or cancer | |
| Pourchet et al. | CD8+ T-cell immune evasion enables oncolytic virus immunotherapy | |
| Münz | The role of lytic infection for lymphomagenesis of human γ-herpesviruses | |
| CN105039269A (en) | Novel viral vaccine for treating non-small cell lung cancer and preparation method thereof | |
| Martini et al. | Oncolytic virotherapy: new weapon for breast cancer treatment | |
| WO2023123195A1 (en) | Engineered immune cell target gene of which can be regulated, preparation method therefor, and use thereof | |
| Zhang et al. | Construction of an IL12 and CXCL11 armed oncolytic herpes simplex virus using the CRISPR/Cas9 system for colon cancer treatment | |
| CN107557337A (en) | A kind of immunocyte of safety-type Chimeric antigen receptor modifications of anti-ROR1 and its application | |
| AU2020103637A4 (en) | Recombinant oncolytic herpes simplex virus type ii and its pharmaceutical composition | |
| CN110267671A (en) | For treating the composition and its method of cancer cell | |
| CN116726198B (en) | Antitumor drug and application thereof in brain glioma treatment | |
| CN116966281A (en) | Method and use of engineered T cell loaded oncolytic viruses | |
| CN104434973A (en) | Method for intensifying functions of cytokine-induced killer cells | |
| CN102660579A (en) | HBx and human IL-12 double-gene recombinant vector and liver caner-resistant vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151111 |