WO2019101062A9 - Vaccin recombiné et son application - Google Patents

Vaccin recombiné et son application Download PDF

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WO2019101062A9
WO2019101062A9 PCT/CN2018/116393 CN2018116393W WO2019101062A9 WO 2019101062 A9 WO2019101062 A9 WO 2019101062A9 CN 2018116393 W CN2018116393 W CN 2018116393W WO 2019101062 A9 WO2019101062 A9 WO 2019101062A9
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vaccine
spd1
tumor
cancer
recombinant
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WO2019101062A1 (fr
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孔维
张海红
于湘晖
陆臻桢
刘晨露
徐平
耿飞
谢雨
郭倩倩
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长春百克生物科技股份公司
吉林大学
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Definitions

  • the invention relates to the field of biotechnology drug technology, in particular to a recombinant vaccine and its application.
  • Cancer or malignant tumors are the killers of human health.
  • the causes of tumors are complicated. We have so far lacked a deep understanding of its specific mechanism of formation.
  • Recent studies have shown that there is a cyclical interaction between the body's immune system and tumor cells, that is, the immune system can recognize cancer cells of "non-self" components, and cancer cells can escape the monitoring and attack of the body's immune system through various ways, resulting in patients. Diffusion of tumor cells in vivo.
  • the molecular mechanism of tumor immune escape includes: down-regulation or loss of tumor antigen expression, tumor cells secreting soluble cytokines with immunosuppressive function, tumor microenvironment recruiting immunosuppressive lymphocytes, and tumor cells up-regulating negative synergistic stimulation signals.
  • CTLA4/CD80 CD86
  • PD1/PD-L1 PD-L2
  • BTLA/HVEM TIM3/GAL9
  • Human programmed cell death protein 1 also known as CD279, shares the same immunoglobulin superfamily with two ligands, PD-L1 (B7H1, CD274) and PD-L2 (B7DC, CD273).
  • the type I transmembrane glycoprotein is composed of a cytoplasmic region, a transmembrane anchoring region and an extracellular site binding domain.
  • the key structure that mediates the immunosuppressive function of PD-1 signaling is the immunoreceptortyrosine-based switch motif (ITSM) located in the intracellular signal transduction region.
  • ITMS immunoreceptortyrosine-based switch motif
  • ITSM The motif initiates a tyrosine phosphorylation program to recruit the relevant phosphatase to dephosphorylate the downstream signaling pathway PI3K/AKT (phosphatidylinositol 3 kinase/serine-threonine protein kinase), resulting in disruption of cytokine synthesis And T cell effect is not sensitive.
  • PI3K/AKT phosphatidylinositol 3 kinase/serine-threonine protein kinase
  • T cell effect is not sensitive.
  • the PD-1/PD-L1 signaling pathway is lowly expressed or induced in normal tissues and peripheral lymphoid tissues, which is important for maintaining immune tolerance and preventing the development of autoimmune diseases. Expression accelerates apoptosis of activated T cells.
  • Immune Checkpoint Blockade which uses inhibitors to block the co-suppressive signaling pathway of negative regulatory T cells, deactivates T cells and re-releases their immune activity.
  • the currently developed inhibitors are mainly humanized monoclonal antibodies, which block two important immunological checkpoints, cytotoxic T lymphocyte protein 4 (CTLA4) and PD-1.
  • CTL4 cytotoxic T lymphocyte protein 4
  • PD-1 cytotoxic T lymphocyte protein 4
  • Herbst et al reported a The monoclonal antibody PMDL3280A, which effectively blocked the PD-1 ligand PD-L1, was used to treat 65 patients with bladder cancer who were not treated with chemotherapy.
  • Costimulatory proteins usually exist in two forms: anchored to the cell membrane or secreted extracellularly, and the soluble molecule retains the extracellular ligand-binding domain of the membrane-type molecule and acts in a free manner through the blood circulation to the distal effector molecule. , involved in regulating body immunity.
  • the native human PD-1 molecule comprises the above two protein forms, and both the membrane type and the soluble PD-1 (Soluble PD-1, sPD1) are encoded by the PDCD1 gene, wherein the encoded full-length mRNA
  • the translation product is a membrane-type molecule, and an mRNA splice variant lacking exon 3 is directly translated into sPD1.
  • sPD1 Another way of forming sPD1 may be that membrane-type PD-1 is shed by proteolytic enzymes.
  • the IgV-IgC-like structure of sPD-1 mediates binding to the ligands PD-L1 and PD-L2, but the downstream immunosuppressive signal could not be initiated due to the lack of an intracellular inhibition motif.
  • Wan et al. detected high expression of sPD1 in synovial fluid and peripheral blood of patients with rheumatoid arthritis, accompanied by elevated levels of cytokines associated with T cell activation such as IFN- ⁇ , IL-4 and IL-21.
  • Soluble co-stimulatory molecules are involved in the regulation of immune regulation, including sCD80, sLAG3, sPD1 and sBTLA. These molecules have their own physiological functions in viral infection, tumor and autoimmune diseases.
  • the immunomodulatory effects of sPD1 molecules are manifested in four aspects: 1 is to promote the secretion of some activated cytokines on T lymphocytes (IFN- ⁇ , IL-2, etc.), while reducing the inhibitory factors IL-10 and TGF- ⁇ .
  • 2sPD1 can promote the proliferation of specific CD4-positive and CD8-positive T cells, and the activated T cells up-regulate the expression of Bcl-xl, which attenuates the apoptotic activity of T cells; 3 flow detection of sPD1 molecules to DC cells The effect of CD80, CD86, IL-12 and MHC-II molecular fluorescence intensity was significantly increased, indicating that soluble PD-1 can promote the maturation of DC cells; 4sPD-1 can promote CD8 + by targeting DC The function of T cells.
  • the specific molecular mechanism of activation of the body's immunity by sPD1 molecules remains to be studied, but we believe that its physiological function depends on the antigen-specific cellular immune response, and the sPD1 molecule alone does not have a specific immune effect.
  • Soluble PD1 molecule has a certain preclinical application basis as a form of PD-1 pathway blocker.
  • Shin et al. targeting herpesvirus thymidine kinase (HSVtk), constructed a conditionally replicating adenoviral vector carrying the recombinant HSVtk gene and the recombinant soluble sPD1-Ig gene bicistronic.
  • the double gene was combined with adenovirus for intratumoral injection and gene therapy for colorectal cancer tumor model mice.
  • sPD-1 can stimulate the massive expansion of IFN- ⁇ /TNF- ⁇ Shuangyang T cells and help T cells kill tumors.
  • sPD1-p24 greatly reduced the inhibitory factors in the peripheral phase of the immune system (within six weeks) and in the tumor microenvironment, specifically in the inhibition of myeloid-derived suppressor cells (MDSC) and regulatory T cells. A decrease in the number of sexual T cells.
  • MDSC myeloid-derived suppressor cells
  • the soluble PD1 molecule competes with the PD-L inhibitory pathway to block the PD1/PD-L1 signaling pathway, and has enhanced specific immunity.
  • the preparation process of recombinant sPD-1 vaccine is relatively simple and low cost. We believe that sPD-1 has potential clinical application value as a PD-1 pathway blocker.
  • fusion gene vaccines targeting survivin and mucin 1 have certain application value in anti-tumor immunotherapy.
  • Survivin is a superfamily of apoptosis-inhibiting proteins. Its main function is to resist apoptosis and regulate cell division. Survivin is specifically expressed in embryonic tissues and cancer cells. It is an ideal tumor immunotherapy target; MUC1 is a type I transmembrane glycoprotein.
  • VNTRs tandem repeats
  • the inventors performed MUC1 VNTRs and Survivin fusion expression DNA vaccine epitope design based on two aspects: one is vaccine safety, and the other is vaccine immunogenicity. According to the literature, full-length Survivin exerts anti-apoptotic function in a dimeric form in vivo, and the key site for dimerization is the N-terminal amino acid residues 6, 7, and 10.
  • the inventors used a Survivin deletion splicing (S8) with a 7-amino acid deletion at the N-terminus to design a vaccine, which not only improved the safety of the vaccine, but also ensured the immunogenicity of the Survivin epitope to the greatest extent;
  • Another epitope involved is the MUC1 VNTR tandem repeat, which contains 33 copies of MUC1 VNTRs (abbreviated to 33M), which contain an immunodominant domain PDTRP sequence that mediates efficient MUC1-specific antibody responses. And improve the specific CTL immune effect.
  • the technical problem to be solved by the present invention is to provide a recombinant vaccine and the use thereof, and the present invention further combines sPD1 with a tumor antigen MS to block PD-1/PD involved in antigen presenting DC cells by sPD1.
  • the -L negative signaling pathway allows the vaccine to have better immunogenic and immune effects.
  • the invention provides a molecular combination comprising PD-1, MUC1 and Survivin.
  • the PD-1, the MUCI and the Survivin of the present invention may be a protein molecule or a nucleic acid molecule, which is not limited in the present invention.
  • the sPD-1 protein refers to a soluble PD-1 protein capable of blocking the PD-1 pathway
  • the sPD-1 nucleic acid refers to a DNA molecule capable of encoding the sPD-1 protein.
  • the sPD-1 is a DNA or a protein, which is an extracellular domain of human sPD-1 (denoted as sPD1), and the DNA sequence thereof is derived from a Genbank accession number NM_005018.2.
  • the DNA sequence of sPD-1 used in the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of sPD-1 is shown in SEQ ID NO: 2.
  • the MUC1 protein is derived from a wild MUC1 VNTR protein and/or a mutant thereof and/or a truncated body thereof, and the MUC1 nucleic acid refers to a DNA molecule capable of encoding the MUC1 protein.
  • the DNA sequence of MUCl is as shown in SEQ ID NO: 3; the amino acid sequence thereof is shown in SEQ ID NO: 4.
  • the Survivin protein is derived from a wild Survivin protein and/or a mutant thereof and/or a deletion splicing thereof, preferably a Survivin having a 7-amino acid residue deleted at the N-terminus.
  • the DNA sequence of Survivin is shown in SEQ ID NO: 5; the amino acid sequence thereof is shown in SEQ ID NO: 6.
  • PD-1, MUC1, and Survivin may be fused (denoted as sPD1/MS), or may be used in combination in a non-fused form (denoted as sPD1+MS), which is not limited in the present invention.
  • the experiments of the present invention show that the preparation of the vaccine by the antigen provided by the present invention can have the effect of preventing and treating tumors.
  • the effect of the sPD1/MS vaccine group was significantly better than that of the MS vaccine group (p ⁇ 0.05), and was also significantly better than the sPD1 vaccine (p ⁇ 0.05).
  • the experiments of the present invention prove that the specific cellular immune response induced by the sPD1/MS fusion vaccine is more consistent than the sPD1+MS combined immunization method, indicating that sPD1 and MS act as antigens, and Tumor multi-targets produced effects, while fusion or non-fusion methods had no significant effect on immune response (p>0.05).
  • the tumor which can be controlled by the antigen provided by the present invention is selected from the group consisting of melanoma, colorectal cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, renal cancer, cholangiocarcinoma, gastric cancer, esophageal cancer, bladder cancer, pancreatic cancer, head and neck cancer. , nasopharyngeal carcinoma, oral cancer, cervical cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, squamous cell carcinoma, lymphoma, brain cancer, glioblastoma, medulloblastoma, lymphosarcoma, chorion Epithelial cancer, osteosarcoma, thyroid cancer.
  • the invention adopts breast cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer and melanoma tumor-bearing mice as experimental subjects, and the results show that the vaccine prepared by the antigen provided by the invention can play a good role in inhibiting tumor growth. Moreover, the ability of lymphocytes to secrete IFN- ⁇ specifically in tumor-bearing mice was also significantly increased (P ⁇ 0.01).
  • the tumor control product of the present invention includes a vaccine.
  • the vaccine prepared by the antigen provided by the invention can play a good role in preventing and treating tumors.
  • the vaccine is a DNA vector vaccine, a viral vector vaccine, a protein vaccine, and/or a dendritic cell vaccine.
  • administration of a DNA vector vaccine, a viral vector vaccine, or a protein vaccine alone can both inhibit tumor growth.
  • the data indicates that the DNA vector vaccine is primed and the booster immunization with the recombinant adenovirus vaccine significantly enhances the immune response.
  • Other data show that the anti-tumor drugs can be synergistically promoted while immunizing.
  • the anti-tumor drug is a chemotherapeutic drug, preferably a platinum compound, paclitaxel and/or gemcitabine and/or cisplatin.
  • the chemotherapeutic agent is oxaliplatin.
  • the tumor-preventing product provided by the present invention may include one or a combination of two or more of a DNA vector vaccine, a viral vector vaccine, a protein vaccine, and/or a dendritic cell vaccine.
  • Chemotherapeutic drugs such as oxaliplatin may also be included.
  • the present invention also provides a recombinant vector comprising the DNA sequences of PD-1, MUCl and Survivin.
  • Tissue plasminogen activator (tPA), abbreviated as tPA signal peptide, can effectively promote protein secretion and enhance its ability to induce antibody production.
  • tPA signal peptide Tissue plasminogen activator
  • the present invention modifies the tPA signal peptide sequence at the 5' end of the DNA sequence of PD-1.
  • the DNA sequence in which sPD-1 is linked to the tPA signal peptide sequence is shown in SEQ ID NO: 7.
  • the length of the tPA signal peptide is 69 bp, and the sequence thereof is shown in SEQ ID NO: 11.
  • the DNA sequence (denoted as an MS sequence) to which MUC1 and Survivin are ligated is set forth in SEQ ID NO: 8.
  • recombinant vectors can be constructed with different backbones, but these recombinant vectors should include the PD-1 sequence linked to the tPA signal peptide, the MUC1 sequence and the Survivin sequence, which are described in the present invention.
  • the PD-1 sequence linked to the tPA signal peptide, the order of attachment of the MUC1 sequence and the Survivin sequence is not limited, and the implementation thereof is within the scope of the present invention.
  • the joining sequence of the fusion fragment is: from the 5' end to the 3' end, the tPA signal peptide, the PD-1 sequence, the MUC1 sequence, and the Survivin sequence.
  • the present invention provides a recombinant vector comprising a backbone vector, the DNA sequence shown in SEQ ID NO: 7, the DNA sequence shown in SEQ ID NO: 8, and an adjuvant sequence.
  • the backbone carrier is CpDV.
  • the CpDV vector is constructed by the invention patent of application number 201110086366.1.
  • the adjuvant sequence is an intramolecular adjuvant selected from the group consisting of cytokine adjuvants including, but not limited to, interleukin 2 (IL-2), unmethylated CpG motif of immunostimulatory DNA, and colony stimulating factor GM-CSF.
  • cytokine adjuvants including, but not limited to, interleukin 2 (IL-2), unmethylated CpG motif of immunostimulatory DNA, and colony stimulating factor GM-CSF.
  • IL-2 interleukin 2
  • the DNA sequence thereof is shown in SEQ ID NO: 9.
  • the insertion site of the adjuvant sequence is between the Xba I and BamH I cleavage sites; the DNA sequence set forth in SEQ ID NO: 7 and the insertion site of the DNA sequence set forth in SEQ ID NO: The sites between BglII and EcoR I were digested.
  • the map of the recombinant vector (denoted as CpDV-IL2-sPD1/MS) provided by the present invention is shown in Figure 1-d, which is prepared by Xba I and BamH I cleavage sites of the backbone vector. The adjuvant sequence was inserted, and then the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 were inserted between the Bgl II and EcoR I restriction sites.
  • the tumor control product is a DNA vector vaccine.
  • the present invention also provides a vaccine for preventing and treating tumors, which comprises the recombinant vector provided by the present invention.
  • the recombinant vector is CpDV-IL2-sPD1/MS.
  • the vaccine provided by the present invention is prepared by inserting an adjuvant sequence between the Xba I and BamH I cleavage sites of the backbone vector, and then inserting SEQ ID NO: 7 between the Bgl II and EcoR I cleavage sites.
  • the sequence shown in SEQ ID NO: 8 yielded a recombinant vector.
  • the mouse inoculated with the DNA vector vaccine provided by the present invention can produce a highly effective anti-mucin 1 antibody and anti-survivin antibody, and the antibody titer of both can reach 10000 or more, and the amount of antibody produced is also large, indicating the present invention.
  • the DNA vector vaccine provided is capable of eliciting a strong specific humoral immune response.
  • experiments have shown that the present invention provides that the DNA carrier vaccine can effectively inhibit the growth of tumors, including breast cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, melanoma, and the effect thereof is significantly better (p ⁇ 0.05).
  • a DNA vector vaccine targeting MS or a DNA vector vaccine targeting sPD-1 is used alone.
  • the DNA vector vaccine provided by the present invention is also capable of treating melanoma, colorectal cancer, colorectal cancer, lung cancer, breast cancer, liver cancer, kidney cancer, cholangiocarcinoma, gastric cancer, esophageal cancer, bladder cancer. , pancreatic cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, cervical cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, squamous cell carcinoma, lymphoma, brain cancer, glioblastoma, medulloblastoma Lymphosarcoma, chorionic epithelial cancer, osteosarcoma, thyroid cancer play a good inhibitory role.
  • the present invention also provides a recombinant vector comprising a backbone vector, the DNA sequence shown in SEQ ID NO: 7, and the DNA sequence shown in SEQ ID NO: 8.
  • the fusion protein (sPD1/MS) is required to construct a recombinant protein vaccine.
  • the expression of the fusion protein uses a prokaryotic expression system.
  • the backbone vector provided by the present invention is a pRSET B or PET series vector.
  • the map of the recombinant vector (denoted as pET28a-sPD1/MS) provided by the present invention is shown in Figure 3-b, and is prepared by inserting between the Nde I and Xho I cleavage sites of the backbone vector. sPD1/MS fragment.
  • the present invention provides an expression vector which is produced by transfecting a host cell with a recombinant vector provided by the present invention.
  • the host cell is E. coli BL21.
  • the present invention also provides a fusion protein obtained by transfecting a host vector obtained by the recombinant vector provided by the present invention to obtain an expression vector, and then obtaining the expression.
  • the host cell is E. coli BL21.
  • the purification step is further performed, after the cultured cells are ultrasonically lysed, centrifuged for 30 minutes, and the precipitate is resuspended in the inclusion body dissolution buffer, dissolved, and the supernatant is passed through the nickel column affinity layer. Analysis, obtaining a fusion protein.
  • the tumor control product is a recombinant protein vaccine.
  • the present invention also provides a vaccine for preventing and treating tumors, comprising the fusion protein (sPD1/MS) provided by the present invention.
  • an adjuvant which is Al(OH) 3 .
  • AL(OH) 3 is a common inorganic adjuvant that enhances the immunogenicity of vaccines by inducing T cell differentiation and humoral immune responses.
  • Inoculation of the recombinant protein vaccine provided by the present invention can inhibit the growth of tumor-bearing mouse tumors, and the ability of the spleen lymphocytes of the tumor-bearing mice to specifically secrete IFN- ⁇ is also improved.
  • the tumor is colorectal cancer. Due to the similarity of tumor pathogenesis, the DNA vector vaccine provided by the present invention can also be used for melanoma, colon cancer, lung cancer, breast cancer, liver cancer, kidney cancer, cholangiocarcinoma, stomach cancer, esophageal cancer.
  • bladder cancer pancreatic cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, cervical cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, squamous cell carcinoma, lymphoma, brain cancer, glioblastoma, medulla Maternal tumor, lymphosarcoma, chorionic epithelial cancer, osteosarcoma, thyroid cancer play a good inhibitory role.
  • the present invention provides a recombinant vector comprising a backbone vector, the DNA sequence shown in SEQ ID NO: 7, and the DNA sequence shown in SEQ ID NO: 8, which is pSC11 or pDC316. .
  • pSC11 is a shuttle plasmid commonly used in Modified Vaccinia Ankara (MVA), and the left and right arm (TKL, TKR) of thymidine kinase on the vector can be homologously recombined with MVA, and simultaneously carried out by the lacZ gene on the vector. Blue spot screening of recombinant MVA (rMVA).
  • the map of the recombinant vector (denoted as pSC11-sPD1/MS) provided by the present invention is shown in Figure 4-b, and the recombinant vector is prepared by the SalI cleavage site of the pSC11-MS plasmid.
  • the sPD1 fragment was inserted, and the 5' end of the sPD-1 fragment was ligated with a tPA signal peptide fragment.
  • the pSC11-MS plasmid was constructed from the Chinese invention patent No. 200910252427.X.
  • pDC316 is a commonly used shuttle plasmid for adenovirus.
  • the map of the recombinant vector (denoted as pDC316-sPD1/MS) provided by the present invention is shown in Figure 4-d, and the recombinant vector is prepared by using pDC316-MS.
  • the sPD1 fragment was inserted into the EcoRI restriction site of the plasmid, and the tPA signal peptide fragment was ligated to the 5' end of the sPD-1 fragment.
  • the pDC316-MS plasmid was constructed from the Chinese invention patent No. 200910252427.X.
  • the present invention also provides a recombinant virus which is produced by transfecting a virus with the recombinant vector provided by the present invention.
  • the recombinant vector is pSC11-sPD1/MS and the virus is a poxvirus.
  • the transfection is: the empty MVA virus is infected with the mutant cell of BHK-21 containing the TK gene, and then the shuttle plasmid pSC11-sPD1/MS is co-transfected into BHK cells using lipo2000 reagent, and cultured. After the culture supernatant was infected with BHK cells, the poxvirus transfected with pSC11-sPD1/MS was screened.
  • the recombinant vector is pDC316-sPD1/MS
  • the virus is an adenovirus
  • the transfection is: co-transfection of 293 cells with the AD backbone and the shuttle plasmid pDC316-sPD1/MS, and the adenovirus transfected with pDC316-sPD1/MS is screened.
  • the adenovirus (AD) backbone is pBHGlox ⁇ E1, 3Cre.
  • the present invention also provides a vaccine for preventing and treating tumors, including the recombinant virus provided by the present invention.
  • Inoculation of the recombinant virus vaccine provided by the present invention can significantly inhibit the growth of tumor-bearing mice, wherein the average tumor volume of sPD1/MS recombinant poxvirus-treated mice is reduced by about 25% compared with the control group, and sPD1/MS recombination The average tumor volume of adenovirus treated mice was reduced by approximately 23%.
  • the tumor is melanoma. Due to the similarity of the tumor vaccine mechanism, the viral vector vaccine provided by the present invention is also capable of treating colorectal cancer, colon cancer, lung cancer, breast cancer, liver cancer, kidney cancer, cholangiocarcinoma, gastric cancer, and esophagus.
  • bladder cancer pancreatic cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, cervical cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, squamous cell carcinoma, lymphoma, brain cancer, glioblastoma, Medulloblastoma, lymphosarcoma, chorionic epithelial cancer, osteosarcoma, thyroid cancer play a good inhibitory role.
  • the experiments of the present invention show that vaccination with a DNA vector vaccine and then boosting with a viral vector vaccine can significantly enhance the immune effect. Specifically, the secretion of IFN- ⁇ is increased and the immune response is enhanced.
  • the DNA vector vaccine comprises CpDV-IL2-sPD1/MS, which is a poxvirus vector vaccine (pSC11-sPD1/MS) or an adenoviral vector vaccine (pDC316-sPD1/MS).
  • the present invention provides a tumor control product comprising the DNA vector vaccine, recombinant protein vaccine and/or viral vector vaccine provided by the present invention.
  • the tumor-preventing product provided by the present invention may comprise only one vaccine, or may comprise two vaccines or a combination of two or more vaccines.
  • the experiments of the present invention have shown that administration of a chemotherapeutic drug at the same time as immunization can play a synergistic role.
  • the specific performance is to prolong the survival of tumor-bearing mice.
  • the combination of recombinant sPD1/MS DNA vaccine and oxaliplatin was observed to increase the average survival of mice by 24% (P ⁇ 0.001) compared with PBS group, including recombinant sPD1/MS DNA vaccine.
  • the survival of the combined oxaliplatin-treated group was significantly longer than that of the oxaliplatin-treated group (14% longer) (P ⁇ 0.05).
  • the anti-tumor product provided by the present invention further includes a chemotherapeutic drug.
  • the chemotherapeutic agent is selected from the group consisting of platinum compounds, paclitaxel or gemcitabine.
  • Another tumor-preventing product provided by the present invention includes a vaccine targeting MS and a vaccine targeting sPD-1.
  • the MS-targeted vaccine was constructed by the Chinese invention patent No. 200910252427.X.
  • the vaccine targeting sPD-1 is CpVR-sPD1, which is constructed by inserting a sPD1 fragment between the PstI and BamHI restriction sites of the vector CpVR, and the 5' end of the sPD-1 fragment is linked with a tPA signal. Peptide fragments.
  • the CpVR vector is constructed by the invention patent of the patent number ZL 201110086366.1.
  • the anti-tumor product provided by the present invention further includes a chemotherapeutic drug.
  • the chemotherapeutic agent is selected from the group consisting of platinum compounds, paclitaxel or gemcitabine.
  • the present invention also provides a method for preventing and treating tumors, which comprises administering a tumor-preventing product provided by the present invention.
  • the injection site is stimulated with a live gene importer; the stimulation voltage is 36V; the frequency is 1 Hz; the pulse is 6 times; the pulse width is 20 ms.
  • the method of administering a tumor-preventing product provided by the present invention is: administering a DNA vector vaccine.
  • the strategy of immunization is to use a DNA vector vaccine for priming, and then a DNA vector vaccine for booster immunization.
  • the number of priming is 1 time, and the number of boosting is 2 times.
  • the interval between the priming and booster immunization was 7 days, and the interval between the two booster immunizations was 7 days.
  • the DNA vector vaccine is CpDV-IL2-sPD1/MS.
  • the method of administering a tumor-preventing product provided by the present invention is: administering a recombinant protein vaccine.
  • the strategy of immunization is to use a recombinant protein vaccine for priming, and then use a recombinant protein vaccine for booster immunization.
  • the number of priming is 1 time, and the number of boosting is 2 times.
  • the interval between the priming and booster immunization was 7 days, and the interval between the two booster immunizations was 7 days.
  • the recombinant protein vaccine was prepared from a fusion protein expressed by VR1012-sPD1/MS.
  • the method of administering a tumor-preventing product provided by the present invention is: administering a viral vector vaccine.
  • the strategy of immunization is to use a viral vector vaccine for priming, and then a viral vector vaccine for booster immunization.
  • the number of times of priming is 1 time, and the number of times of boosting is 1 time. There was a 14-day interval between the priming and booster immunization.
  • the viral vector vaccine is a poxvirus vector vaccine or an adenoviral vector vaccine.
  • the method of administering a tumor-preventing product provided by the present invention is: administering a DNA vector vaccine and a viral vector vaccine.
  • the strategy of immunization is to use a DNA vector vaccine for priming, and then a viral vector vaccine for booster immunization.
  • the number of priming is 3, and the number of boosting is 1 time.
  • the interval between the priming and boosting was 2 weeks, and the interval of 3 priming was 2 weeks.
  • the viral vector vaccine is an adenoviral vector vaccine.
  • the method of administering a tumor-preventing product provided by the present invention is: administering a DNA vector vaccine and a viral vector vaccine.
  • the strategy of immunization is to use a DNA vector vaccine for priming, and then a viral vector vaccine for booster immunization.
  • the number of priming is 2, and the number of boosting is 1 time.
  • the interval between the priming and boosting was 2 weeks, and the interval between 2 priming was 2 weeks.
  • the viral vector vaccine is a poxvirus vector vaccine.
  • the method of administering a tumor-preventing product provided by the present invention is: administering a DNA vector vaccine and a viral vector vaccine.
  • the strategy of immunization is to use a DNA vector vaccine for priming, and then a viral vector vaccine for booster immunization.
  • the number of priming is 2, and the number of boosting is 1 time.
  • the interval between the priming and boosting was 2 weeks, and the interval between 2 priming was 2 weeks.
  • the viral vector vaccine is an adenoviral vector vaccine.
  • the method of administering a tumor-preventing product provided by the present invention is: administering a DNA vector vaccine and a viral vector vaccine.
  • the strategy of immunization is to use a combination of a vaccine targeting MS and a vaccine targeting sPD-1 for priming, and then using a viral vector vaccine for boosting.
  • the number of priming is 2, and the number of boosting is 1 time.
  • the interval between the priming and boosting was 2 weeks, and the interval between 2 priming was 2 weeks.
  • the viral vector vaccine is an adenoviral vector vaccine.
  • the method of administering a tumor-preventing product provided by the present invention is: administering a DNA vector vaccine and a viral vector vaccine.
  • the strategy of immunization is to use a DNA vector vaccine for priming, and then a viral vector vaccine for booster immunization. Immunization is given simultaneously with chemotherapy drugs.
  • the number of priming is 2, and the number of boosting is 1 time.
  • the interval between the priming and boosting was 7 days, and the interval between the two primings was 7 days.
  • the first administration of the chemotherapy drug was the next day after the second priming, and the chemotherapy drug was administered every 5 days thereafter for a total of 4 administrations.
  • the viral vector vaccine is a poxvirus vector vaccine.
  • the chemotherapeutic drug is oxaliplatin.
  • the invention provides molecular combinations including PD-1, MUC1 and Survivin.
  • the preparation of the vaccine with the antigen can have good immunogenicity and antitumor activity.
  • the vaccine immunization provided by the present invention produces a specific antibody response against MUC1, Survivin (see Figure 5) and a specific cellular immune response (see Figure 6).
  • CpDV-IL2-sPD1/MS Compared with the DNA vaccine CpDV-IL2-MS, CpDV-IL2-sPD1/MS has a more significant anti-tumor effect in tumor-bearing mice, with a tumor growth inhibition rate of about 53% for melanoma (see Figure 10).
  • the life extension rate of the survival time of melanoma-bearing mice is about 19% (see Fig.
  • the present invention further provides a joint scheme of a therapeutic vaccine and a chemotherapeutic drug.
  • the vaccine provided by the present invention is combined with oxaliplatin to achieve a tumor inhibition rate of 74%, and the tumor growth of the tumor-bearing mouse is significantly inhibited (see FIG. 11); and the vaccine and the present invention provide the vaccine.
  • the average survival time of the mice in the saliplatin-administered group was 62 days, and the life-prolonging effect was statistically significant compared with the positive control oxaliplatin group (see Fig. 11); in addition, the vaccine provided by the present invention was applied to mice. Treatment of breast cancer, lung cancer, liver cancer and gastric cancer models (see Figures 13, 14, 15, and 16), compared with the negative control group, the tumor growth rate of the vaccine group was significantly slowed down.
  • Figure 1 shows the construction strategy of the DNA plasmid vector and the plasmid CpDV-IL2-sPD1/MS; wherein, Figure 1-a shows the recombinant DNA plasmid CpDV-IL2-MS and the plasmid CpDV-IL2-sPD1/MS; Figure 1-b shows the plasmid The construction strategy of T Easy-sPD1/MS; Figure 1-c shows the construction strategy of plasmid T Easy-tPA/sPD1; Figure 1-d shows the construction strategy of vector CpDV-IL2; Figure 1-e shows the recombinant DNA plasmid CpDV-IL2 -sPD1/MS map;
  • Figure 2 shows Western blot analysis of CpDV-IL2-MS, CpDV-IL2-sPD1/MS, CpVR-sPD1 eukaryotic protein expression;
  • Figure 2-a shows the detection of protein expression by anti-MUC1 antibody;
  • -b shows the detection of protein expression by anti-Survivin antibody;
  • Figure 2-c shows the detection of protein expression by anti-PD-1 antibody;
  • Figure 3 shows the construction of the pET28a-sPD1/MS plasmid; wherein, Figure 3-a shows the recombinant plasmid pET28a-sPD1 map; Figure 3-b shows the recombinant plasmid pET28a-sPD1/MS map;
  • Figure 4 shows the construction of a recombinant sPD1/MS viral vector vaccine; wherein, Figure 4-a shows the recombinant shuttle plasmid pSC11-MS map; Figure 4-b shows the recombinant shuttle plasmid pSC11-sPD1/MS map; Figure 4-c shows the recombinant shuttle Plasmid pDC316-MS map; Figure 4-d shows the recombinant shuttle plasmid pDC316-sPD1/MS map;
  • Figure 5 shows the humoral immune response of anti-mucin 1 and survivin in mice immunized with sPD1/MS recombinant DNA vaccine
  • Figure 5-a shows that Balb/c mice were immunized with PBS and recombinant MS, sPD1/MS and sPD1 DNA plasmids, respectively.
  • the serum ELISA detects the anti-mucin 1 antibody
  • Figure 5-b shows the anti-survival antibody level of the immunized mouse
  • Figure 5-c shows the anti-soluble PD1 antibody level in the immunized mouse
  • Figure 5-d shows the mucin 1 in the immunized mouse Serum levels of IgG1 and IgG2a antibodies
  • Figure 5-e shows serum levels of IgG1 and IgG2a antibodies raised against anti-survivin in mice;
  • Figure 6 shows that sPD1/MS recombinant DNA vaccine induces specific cellular immune responses in mice; Balb/c mice are immunized with PBS, and recombinant MS, sPD1/MS and sPD1 DNA plasmids, and spleen lymphocytes are analyzed for mucin 1 and survivin.
  • the level of specific immune response wherein, Figure 6-a shows the number of Elispot spots secreting IFN- ⁇ specific for the MUC1 epitope in the spleen lymphocytes of each group of immunized mice, and Figure 6-b shows the specific secretion of the epitope against Survivin.
  • Figure 6-c shows the CTL activity of spleen lymphocytes of each group of immunized mice, using spleen lymphocytes as effector cells E, and using CT26 cells stably expressing mucin 1 and survivin protein.
  • Target cell T the intensity of CTL reaction was characterized by the percentage of killer target cells labeled by propidium iodide and CFSE double fluorescent dye;
  • Figure 6-d shows that the combination of sPD1 and MS tumor vaccine is specific for mouse spleen lymphocytes Number of Elispot spots secreting IFN- ⁇ ;
  • Figure 7 shows the Prime-Boost immunization strategy for sPD1/MS recombinant DNA vaccine; Balb/c mice were immunized with PBS, recombinant sPD1/MS DNA vaccine, recombinant adenovirus vaccine rAD-MS, and DNA vaccine/rAD-MS vaccine, respectively.
  • Elispot assay was used to detect the specific immune response of murine spleen lymphocytes to mucin 1 and survivin in each group.
  • Figure 7-a shows that the spleen lymphocytes of each group of immunized mice secrete IFN-specific for MUC1 epitope.
  • the number of Elispot spots of ⁇ Figure 7-b shows the number of Elispot spots that specifically secrete IFN- ⁇ for the Survivin epitope;
  • Figure 8 shows the Prime-Boost immunization strategy of sPD1/MS recombinant virus vaccine; Balb/c mice were immunized with PBS, rMVA-sPD1/MS, and DNA vaccine/rMVA-sPD1/MS vaccine respectively, and the spleens of each group were detected by Elispot.
  • the specific immune response activity of lymphocytes to mucin 1 and survivin wherein Figure 8-a shows the number of Elispot spots specifically secreting IFN- ⁇ by spleen lymphocytes of each group against the MUC1 epitope, Figure 8-b Showing the number of Elispot spots that specifically secrete IFN- ⁇ against the Survivin epitope;
  • Figure 9 shows the therapeutic effect of sPD1/MS recombinant DNA vaccine in melanoma model mice; wherein, Figure 9-a shows that after inoculation of MS f + B16 tumor cells subcutaneously in C57BL/6 mice, PBS, MS vaccine and sPD1/MS vaccine, measuring tumor size to the 17th day after inoculation; Figure 9-b shows observation of mouse survival to the 50th day after tumor inoculation;
  • Figure 10 shows the therapeutic effect of sPD1/MS recombinant virus vaccine in melanoma model mice; after inoculation of MS f + B16 tumor cells subcutaneously in C57BL/6 mice, PBS, rMVA-sPD1/MS recombinant poxvirus vaccine and rDA-sPD1/MS recombinant adenovirus was used to measure tumor growth curve on the 12th day after tumor inoculation;
  • Figure 11 shows the therapeutic effect of sPD1/MS recombinant DNA vaccine in colorectal cancer model mice; after inoculation of MS f + CT26 tumor cells subcutaneously in Balb/c mice, PBS, MS vaccine, sPD1/MS vaccine, and Osa Liplatin, MS/oxaliplatin, and sPD1/MS/oxaliplatin; wherein, Figure 11-a shows the measurement of tumor size to the 25th day after inoculation; Figure 11-b shows the small number of executions on the observation deadline. Rats, tumor tissue was removed and tumor weight was measured; Figure 11-c shows the survival of the mice until the 65th day after tumor inoculation;
  • Figure 12 shows the therapeutic effect of sPD1/MS recombinant protein vaccine in mice with colorectal cancer; after inoculation of MS f + CT26 tumor cells subcutaneously in Balb/c mice, PBS, Al(OH)3 adjuvant, and protein were administered separately. /Al(OH)3 treatment, all mice were sacrificed on the 26th day after tumor inoculation; wherein, Figure 12-a shows the tumor weight of each group of mice stripped; Figure 12-b shows the tumor lymphocytes of each group of tumor-bearing mice The ability of cells to specifically secrete IFN- ⁇ against MUC1 and Survivin epitopes;
  • Figure 13 shows the therapeutic effect of sPD1/MS recombinant DNA vaccine in breast cancer model mice; inoculated MS f + 4T1 tumor cells subcutaneously in Balb/c mice, injected with PBS and sPD1/MS vaccine, respectively, from the 11th day after tumor inoculation The mouse tumor volume was measured and the tumor growth curve was recorded.
  • Figure 14 shows the therapeutic effect of sPD1/MS recombinant DNA vaccine in lung cancer model mice; C57BL/6 mice were subcutaneously inoculated with MS f + Lewis tumor cells, injected with PBS and sPD1/MS DNA vaccine, starting from the 8th day after tumor inoculation. Measure and record the mouse tumor growth curve;
  • Figure 15 shows the therapeutic effect of sPD1/MS recombinant DNA vaccine in liver cancer model mice; Balb/c mice were subcutaneously inoculated with MS f + H22 tumor cells, injected with PBS and sPD1/MS vaccines, respectively, starting from the 12th day after tumor inoculation. And record the tumor growth curve of mice;
  • Figure 16 shows the therapeutic effect of sPD1/MS recombinant DNA vaccine in gastric cancer model mice; 615 mice were subcutaneously inoculated with MS f + MFC tumor cells, injected with PBS and sPD1/MS vaccine, respectively, and measured and recorded from the 12th day after tumor inoculation. Mouse tumor growth curve;
  • Figure 17 shows that the cellular immune effect of the vaccine is significantly improved after using the in vivo gene introduction instrument; wherein, Figure 17-a shows the immunization strategy; Figure 17-b shows the level of IFN- ⁇ secreted by the lymphocytes after immunization; Figure 17-c shows CTL level after immunization;
  • Figure 18 shows that after using the in vivo gene introducer, the vaccine is significantly improved in inhibiting tumor growth; wherein, Figure 18-a shows the immunization strategy; Figure 18-b shows the tumor volume after immunization; Figure 18-c shows the post-immunization CTL level; Figure 18-d shows the results of ELISPOT detection after immunization.
  • the invention provides a recombinant vaccine and its application, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application of the present invention may be modified or combined and modified to achieve and apply the present invention without departing from the scope of the present invention. Invention technology.
  • Antigen refers to a substance that stimulates the body to produce a (specific) immune response and binds to immune response product antibodies and sensitized lymphocytes in vitro to produce an immune effect (specific reaction).
  • An antigen is a piece of DNA or DNA fragment capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof.
  • Polypeptide and “protein” are used interchangeably herein to refer to a polymer of contiguous amino acid residues.
  • the terms “nucleic acid” and “nucleotide” are used interchangeably and refer to RNA, DNA, cDNA (complementary DNA) or cRNA (complementary RNA) and derivatives thereof, for example, comprising a modified backbone.
  • Fusion refers to the technique of fusion expression of two or more proteins to form a fusion protein.
  • a fusion protein is obtained by ligating DNA fragments encoding two or more proteins in frame by using recombinant DNA technology and performing protein expression. Fusion use refers to a method in which a sequence of MS and sPD-1 are fused together to prepare a vaccine.
  • Combined use or “co-immunization” refers to the treatment or immunization of a subject with more than one therapeutic agent. "Joint” does not limit the order in which the subject is treated.
  • a first vaccine eg, a vaccine that targets MS
  • a second vaccine eg, a vaccine that targets sPD-1
  • sPD-1 eg, 5 minutes, 15 minutes, 30
  • Minutes 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours before, simultaneously with or after (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 Hours, 6 hours, 12 hours).
  • Control refers to the treatment, prevention or amelioration of a disease or a condition or symptom associated therewith.
  • Vector means a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • “Host cell” means a cell which can be used for introduction into a vector, including but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, a fungal cell such as a yeast cell or an Aspergillus, such as S2 Drosophila cell or Sf9 or the like. Insect cells, or animal cells such as BHK cells, HEK293 cells or human cells.
  • Adjuvant refers to a non-specific immunopotentiator that, when administered together with an antigen or pre-delivered into the body, enhances the body's immune response to the antigen or alters the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials.
  • adjuvant sequence refers to a sequence of an intramolecular adjuvant which is an adjuvant capable of enhancing the immunogenicity of a protein (antigen) to which the fusion is expressed, which is usually a polypeptide fragment.
  • Vaccine refers to a vaccine-based preventive biological product for the prevention and control of the occurrence and prevalence of infectious diseases for human vaccination.
  • DNA vector vaccine refers to a vaccine based on DNA or RNA (eg, a plasmid, such as an expression plasmid), which optionally further comprises an adjuvant.
  • Recombinant protein vaccine is another common form of tumor vaccine. Unlike DNA vaccine or viral vector vaccine, it enters cells in the form of antigenic proteins and directly activates dendritic cells. At the same time, the protein form cannot be integrated into host cells. The genome has a high level of safety.
  • the viral vector described in the "viral vector vaccine” has the ability to replicate as a vector for a foreign gene to maintain its own infectivity.
  • test materials, reagents and instruments used in the present invention are all common commercial products, and are commercially available.
  • the method for constructing a vaccine targeting MS in the present invention refers to the invention patent of application No. 200910252427.X.
  • the human PD-1 extracellular domain was synthesized according to GeneBank No. NM_005018.2 gene, and the 5' end of the gene sequence was ligated with a 69 bp tPA signal peptide sequence (see SEQ ID NO: 10).
  • the purpose of introducing the tPA signal peptide was to increase the fusion protein. Secretory expression ability of sPD1/MS in extracellular.
  • PCR was used to introduce a restriction enzyme site at both ends of the tPA/sPD1 gene, and the PCR reaction system was 50 ⁇ l, including 0.1 ⁇ g of the tPA/sPD1 DNA template described in SEQ ID NO: 1, primers SEQ ID NO: 12 and SEQ ID.
  • Monoclones were picked up in 5 ml of ampicillin-resistant LB medium and incubated at 37 ° C for 220 h at 220 rpm. After harvesting the cultured cells by high-speed centrifugation, the plasmid was extracted from the plasmid extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.), and after digestion with BglII and BamHI, positive clones were identified by 0.8% agarose gel electrophoresis to obtain plasmid T Easy- tPA/sPD1 (see Figure 1-b).
  • the CpVR monocistronic is the inventor's previous transformation of the VR1012 plasmid backbone into the CpG motif (see application number 201110086366.1), and CpVR-sPD1 independently expresses soluble PD-1 protein as a sPD1/MS fusion.
  • a control plasmid format of a nucleic acid vaccine was as follows: the plasmid T Easy-tPA/sPD1 was digested with PstI and BamHI, and the target gene of the linear sPD1 was recombined with the vector CpVR, and the reaction condition was 16 ° C overnight. 10 ⁇ l of the ligation product was transformed into competent E.
  • coli Top 10 kanamycin-resistant plates were coated, and cultured at 37 ° C for 16 h. Monoclones were picked and cultured in 5 ml of Kana-resistant LB medium for 16 h. The cultured cells were harvested at high speed, and the extracted plasmids were digested with PstI and BamHI and identified by 0.8% agarose gel electrophoresis. The positive clone CpVR-sPD1 was obtained by screening.
  • the construction of recombinant DNA vaccine based on sPD1/MS is mainly divided into two steps.
  • the plasmid CpVR-MS containing the gene of interest (see the invention patent No. 200910252427.X) was digested with BglII and BamHI, and the MS fusion tumor antigen gene was inserted into the plasmid T Easy-tPA by T4 DNA ligase.
  • the polyclonal cleavage site of /sPD was mutated at BamHI to obtain plasmid fusion nucleic acid plasmid T Easy-sPD1/MS (see Figure 1-c).
  • a bicistronic CpDV-IL2-sPD1/MS expressing interleukin 2 (IL-2) gene and sPD1/MS fusion nucleic acid The specific technical method is to amplify human interleukin-2 (IL-2) by PCR.
  • IL-2 interleukin-2
  • 0.1 ⁇ g of the template plasmid CpDV-IL2-MS was added to a 50 ⁇ l PCR reaction system, and the primer concentration of SEQ ID was 20 pmol. NO: 14 and SEQ ID NO: 15, other components and reaction conditions are as described above.
  • the PCR product was digested with XbaI and BamHI, and ligated with the vector CpDV (see Patent No. ZL201110086366.1) to prepare the transition plasmid CpDV-IL2.
  • T Easy-sPD1/MS containing sPD 1/MS fusion nucleic acid was digested with BglII and EcoRI, and inserted into the corresponding polyclonal cleavage site of plasmid backbone CpDV-IL2 to prepare the DNA vector vaccine.
  • CpDV-IL2-sPD1/MS ie sPD1/MS vaccine
  • the construction strategy is shown in Figure 1-d.
  • the 293T cell lysis supernatant transfected with the empty vector VR1012 was used as a negative control, and the recombinant plasmids CpDV-IL2-MS (abbreviated as MS), CpDV-IL2-sPD1/MS (referred to as sPD1/MS), and CpVR-sPD1 (referred to as sPD1). 2 ⁇ g were transfected into 293T cells respectively. After 72h, the cell lysate supernatant was collected, separated by SDS-PAGE and transferred to nitrocellulose membrane for Western Blot Western blotting. The recombinant plasmid antigens MUC1, Survivin and protein sPD1 were identified. .
  • Figure 2 shows the MUC1 blot of the MS plasmid, sPD1/MS plasmid and sPD1 plasmid.
  • sPD1 did not show a band as a control plasmid.
  • the expected sizes of plasmid MS and sPD1/MS were 95KD and 120KD, respectively.
  • MS and sPD1/MS actually present a series of gradient protein bands, and the main band size is above 150KD, which may be the glycosylation modification product of MUC1 protein or specific protease.
  • FIG. 2-b shows the Survivin blot of the MS plasmid, sPD1/MS plasmid and sPD1 plasmid. Since Survivin and MUC1 are fusion antigens, the Western blot is similar to the MUC1 result, and the main band position is above 150KD;
  • Figure 2-c shows the PD-1 blot of MS plasmid, sPD1/MS plasmid, sPD1 plasmid and empty vector VR1012. The empty vector did not show a band as a negative control, while the size of sPD1/MS plasmid and sPD1 plasmid were 150KD and 35KD, respectively.
  • the fusion-expressed sPD1/MS protein has no glycosylation modification or hydrolysis of the sPD1 protein, which ensures the structural integrity of sPD1 and can bind to the PD-L1 ligand.
  • the CpDV-IL2-sPD1/MS recombinant gene vaccine of the present invention is an optimized form based on a tumor antigen gene vaccine, and the plasmid structure is shown in Figure 1-a.
  • the tumor antigens MUC1 and Survivin belong to a class of tumor-associated antigens (TAAs), which are derived from their own antigens rather than foreign antigens, which leads to a limited specific T cell immune response.
  • TAAs tumor-associated antigens
  • DNA vaccine designs incorporating tumor antigens with soluble programmed death-1 (PD-1), which blocks immunosuppression, were employed. Fusion expression of soluble PD-1 and tumor antigen enables sPD1-based fusion proteins to target DC cells via receptors, promote DC uptake of antigens and activate B cells and T cells.
  • Protein vaccine is another common form of tumor vaccine. Unlike DNA vaccine or viral vector vaccine, it enters cells in the form of antigenic proteins and directly activates dendritic cells. At the same time, because the protein form cannot be integrated into the host cell genome, Higher security.
  • a prokaryotic expression vector pET28a-sPD1/MS was constructed, and a fusion protein vaccine (sPD1/MS) was induced, and the specific construction process was as follows:
  • the prokaryotic expression plasmid pET28a-sPD1/MS was constructed by homologous recombination method: the pET28a plasmid (see Figure 3-a) was digested with Nde I and Xho I, and the 5289 bp fragment of the vector sequence was recovered by gel. The sequence of sPD1/MS cistron in CpDV-IL2-sPD1/MS was amplified by PCR, and the 5' end of the primer has a sequence homologous to both ends of the cut pET28a vector with corresponding restriction sites (Nde1 and Xho1). ), primer sequence SEQ ID NO. 19
  • the target fragment sPD1/MS was ligated to the vector according to the instructions of the seamless cloning kit-Seamless Assembly Cloning Kit (Cat. No. C5891) to obtain pET28a-sPD1/MS (see Fig. 3-b).
  • the plasmid was transformed into BL21 competent state for amplification, IPTG was added, and the expression was induced at 37 ° C.
  • the cells were collected, and the supernatant was subjected to ultrasonic lysis, and the supernatant was discarded; the precipitate was resuspended by inclusion body Buffer Buffer The mixture was centrifuged overnight at 4 ° C; centrifuged, and the supernatant was subjected to purification on a Ni-NTA affinity chromatography column to carry out an imidazole gradient elution, and finally a purified sPD1/MS fusion protein having a final storage concentration of 1 mg/ml was obtained.
  • MVA modified Vaccinia Ankara
  • the shuttle plasmid of MVA is pSC11, which has a polyclonal cleavage site for insertion of a foreign gene.
  • the sPD1 gene was inserted into the pre-constructed plasmid pSC11-MS (see Patent No. ZL200910252427.X).
  • the pSC11-sPD1/MS was constructed, and the left and right arms (TKL, TKR) of the thymidine kinase on the vector were homologously recombined with MVA, and the blue spot of recombinant MVA (rMVA) was screened by the lacZ gene on the vector.
  • a specific method for constructing a pSC11-sPD1/MS shuttle plasmid is to first perform a PCR reaction by using primers SEQ ID NO: 16 and SEQ ID NO: 17 to obtain a tPA-sPD1 gene into which a restriction enzyme site is introduced, followed by SalI and XhoI. The same enzyme was used for double digestion of tPA/sPD1, and the pSC11-MS plasmid was digested with SalI (Fig. 4-a), and the target gene and vector backbone were recovered by DNA gel and ligated overnight at 16 °C. The ligation product was transformed into Top10 competent state and cultured in ampicillin resistant LB medium to obtain recombinant fusion pSC11-sPD1/MS plasmid (see Figure 4-b).
  • the empty MVA virus with a titer of 0.05 pfu/cell was first infected with the mutant cell of BHK-21 containing the TK gene, and after incubation for 2 hours, the shuttle plasmid pSC11-sPD1/MS was co-transformed using lipo2000 reagent. Dye the BHK cells. After the infected cells were cultured for 72 hours at 37 ° C, 5% CO 2 , the cells were collected. The cells were lysed by sonication and centrifuged at 2000 rpm for 10 min to retain the lysed supernatant.
  • the appropriate amount of supernatant was used as a seed poison, and the poxvirus was sequentially diluted into 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 to infect BHK cells in a six-well plate for 2 hours. Discard the medium, mix with 2% low melting point agarose and 2 ⁇ DMEM-20 medium containing 1% BrdU (inhibited by wild MVA growth) and pre-warmed at 42 °C, add 2 ml medium to each well at room temperature. The mixture was solidified and cultured in an incubator containing 5% CO 2 at 37 ° C for 48 hours. A layer of selective medium was added with 1/120 volume of 4% X-gal more than the lower layer agar.
  • the blue single spot was selected, and the virus was repeatedly cleaved by freezing and thawing for the next round of screening. The steps were the same, so that more than 6 rounds of screening were performed, and finally, a clone containing only the recombinant virus MVA-MS was obtained.
  • AdMax TM Adenovirus Adenovirus vector systems (Microbix Inc.) and a shuttle vector comprising an adenoviral vector.
  • This example uses the shuttle plasmid pDC316-MS (see Patent No. 200910252427.X, Figure 4-c), which was constructed in the previous stage.
  • the sPD1 gene was passed through the PCR method, and the primers SEQ ID NO: 18 and SEQ ID NO: 13 were used. The 5' and 3' ends of the gene were introduced into the EcoRI cleavage site, and the PCR product of the pDC316-MS and sPD1 genes was digested with EcoRI, and ligated overnight at 16 °C.
  • the ligation product was transformed into Top10 competent state and cultured in ampicillin-resistant LB medium at 37 ° C, and subjected to plasmid sequencing to obtain a positive clone pDC316-sPD1/MS (see Fig. 4-d).
  • AD homologous recombination of adenovirus
  • the adenoviral skeleton in this example was pBHGlox ⁇ E1, 3Cre.
  • the AD vector and the shuttle plasmid pDC316-sPD1/MS were co-transfected into 293 cells, and the recombinant adenovirus plaque containing the sPD1/MS fusion nucleic acid was generated by using the E1 protein in the 293 cells over about 10 days.
  • the plaques were identified by PCR, and the reaction conditions were 95 ° C for 30 s, 55 ° C for 30 s, and 72 ° C for 1 min, and the reaction was carried out for 30 cycles, thereby screening the correct plaques for extensive amplification and purification.
  • the ELISA method was used to detect the antibody response of sPD1/MS fusion DNA vaccine against MUC1 and Survivin, and compared with the humoral immunity effect of the MS vaccine (see the invention patent of Patent No. ZL 200910252427.X). 4-6 weeks old Balb/c mice were selected, and sPD1/MS vaccine experimental group, MS vaccine control group, sPD1 plasmid control group and PBS negative control group were set according to Table 1. The preparation of the above DNA plasmid was dissolved in sterile PBS. The final concentration was adjusted to 1 mg/ml.
  • the specific immunization procedures were: vaccination with DNA vector vaccine at week 0 and week 2, and boosting with recombinant adenovirus vaccine at week 4, and 50 ⁇ g of each mouse intramuscular injection vaccine was selected.
  • the mice were subjected to eyelid blood collection two weeks after the last immunization, and the centrifuged serum was used for ELISA antibody detection.
  • mice vaccinated with sPD1/MS vaccine produced highly potent anti-mucin-1 antibodies and anti-survivin antibodies, and the antibody titers of both were up to 10,000 or more.
  • mouse sera diluted 1:125 were prokaryotically expressed and purified, respectively, and the harvested MUCl protein, Survivin protein and soluble sPD1 protein were used as antigen detection specific antibody responses, of which sPD1 Compared with the MS vaccine group, the anti-MUC1 antibody and anti-Survivin antibody absorbance (450nm) readout values were significantly increased in the /MS vaccine group (P ⁇ 0.001), indicating that the optimized sPD1/MS fusion nucleic acid tumor vaccine can initiate strong Specific humoral immune response; and the CpVR-sPD1 plasmid group was negative for the ELISA antibody in the PBS group.
  • ELISA detected sPD1/MS vaccine group and sPD1 plasmid group mouse serum can produce certain anti-PD1 antibody response (see Figure 5-c), however, PD1 antibody titer is low and can not produce a large number of antibodies in vivo to block
  • the PD-1 negative signaling pathway suggests that the sPD1 fusion nucleic acid vaccine may exert anti-tumor effects by activating the mouse's immune system.
  • the sPD1/MS vaccine also showed a more significant advantage than the MS vaccine group in inducing antigen-specific IgG1 (Th2 type) and IgG2a (Th1 type) responses (see Figures 5-d, 5-e).
  • the spleen cells of the MS vaccine group and the sPD1/MS vaccine group secreted IFN- ⁇ spots. Compared with the PBS negative control group, there was a positive reaction. Compared with the number of spots between the sPD1/MS vaccine and the MS vaccine group, the sPD1/MS vaccine was significantly more than MS, and had a statistical significance of P value of 0.0141.
  • the mouse spleen lymphocytes were used as effector cells to stably express CT26 cells fused with the tumor antigen protein MS (designated MS f + CT26, where f is a full abbreviation, representing the full-length survivin protein of the un truncated N-terminal amino acid)
  • MS tumor antigen protein
  • f is a full abbreviation, representing the full-length survivin protein of the un truncated N-terminal amino acid
  • CTL killing activity was measured by propidium iodide (PI)/CFSE two-color fluorescent dye labeling according to a certain target ratio, and the results are shown in Fig. 6-c.
  • the MS vaccine group and the sPD1/MS vaccine group all showed higher CTL killing effects than the PBS group at the effective target ratios of 12.5:1 and 50:1; comparison between the MS vaccine group and the sPD1/MS vaccine group
  • the killing rates of sPD1/MS vaccines for different effective target ratios were 34.76% and 35.56%, respectively, while the MS vaccine kill rates were 21.65% and 30.39%, respectively.
  • the optimized vaccine form CpDV-IL2-sPD1/MS has more CTL killing. active.
  • the preparation of the multi-gene vaccine includes two methods, one is to prepare a fusion nucleic acid vaccine for multi-gene fusion expression, and the other is to jointly immunize two or more genetic vaccines by co-injection.
  • this part of the experiment compared the sPD1/MS recombinant DNA vaccine with the sPD1 combined with the MS vaccine to induce mouse immune activity.
  • Specific experimental groups are shown in Table 2: 4-6 weeks old mice were randomly divided into PBS group, sPD1/MS group and sPD1+MS group, in which PBS group mice were injected with 100 ⁇ l sterilized at 0, 2 and 4 weeks respectively.
  • mice in the sPD1/MS group were injected with recombinant sPD1/MS fusion nucleic acid vaccine at weeks 0 and 2, and boosted with rAD-MS recombinant adenovirus vaccine at week 4; mice in sPD1+MS group at 0th and The previously mixed CpVR-sPD1 plasmid and the CpDV-IL2-MS plasmid (100 ⁇ g each) were injected at 2 weeks, and the recombinant adenovirus vaccine was also boosted at the 4th week.
  • the specific cellular immune response induced by it is at a relatively consistent level, indicating that the sPD1 gene and the MS tumor antigen gene preparation vaccine have a fusion nucleic acid vaccine and a combination.
  • Example 5 Immunization strategy for sPD1/MS tumor vaccine
  • mice DNA immunization-recombinant adenovirus boosting immunization strategy was used to investigate the enhancement of mucin-1 and survivin-specific immune responses induced by recombinant adenovirus vaccine rAD-MS: sPD1/MS vaccine: 4- Six-week-old mice were randomized according to Table 3, 6 in each group, of which group 1 was intramuscularly injected with PBS as a negative control; group 2 was immunized with recombinant adenovirus vaccine rAD-MS alone at week 0 (1 ⁇ 10 8 pfu) /3); Group 3 injected DNA vaccines at weeks -4, -2, and 0; Group 4, DNA vaccines at weeks -6, -4, and -2, and recombinant adenovirus vaccine at week 0. All groups of mice were aseptically taken from the spleen after the neck was sacrificed at the 2nd week, and the immune response level of the immunized mice against the mucin 1 and surviv
  • Fig. 7-a and Fig. 7-b are the results of Elispot detection of spleen lymphocytes of each group of immunized mice corresponding to Table 2.
  • the MUC1 epitope SEQ ID NO: 21
  • the Survivin epitope SEQ ID NO: 22
  • the number of specific secreted IFN- ⁇ spots increased sequentially (P ⁇ 0.01), while the recombinant adenovirus rAD-MS vaccine alone group was negative, indicating that the recombinant adenovirus alone could not induce small
  • Immunization strategies can significantly improve the immune response of the MUC1 epitope (SEQ ID NO: 21) or the Survivin epitope (SEQ ID NO: 22) produced a positive reaction, and
  • mice 4-6 week old C57BL/6 mice were randomly grouped according to Table 4, 5 mice in each group, and the negative control mice were injected with sterile PBS at -4, -2 and 0 weeks, respectively, and the poxvirus was immunized separately. Group mice were immunized with rMVA-sPD1/MS at week 0, and each mouse was injected at a dose of 1 ⁇ 10 8 pfu. In the other experimental group, mice were sPD1/MS recombinant DNA vaccine at -4 and -2 weeks. Exemption, the sPD1/MS recombinant poxvirus vaccine was used for immunization at week 0. All groups of mice were aseptically taken from the spleen after the neck was sacrificed at the 2nd week, and the immune response level of the immunized mice against the mucin 1 and survivin epitopes was evaluated by the Elispot method.
  • mice immunized with recombinant poxvirus alone induced positive mucin 1 and survivin-specific secretion of IFN- ⁇ -Elispot spots (P ⁇ 0.01); mice immunized with sPD1/MS recombinant DNA vaccine priming and recombinant sPD1/MS pox virus booster immunization induced a highly specific cellular immune response in vivo and compared with the rMVA immunized group.
  • the number of Elispot spots produced was significantly increased (P ⁇ 0.01).
  • the above results indicate that the recombinant virus vaccine expressing sPD1/MS has the ability to activate mouse-specific immunity, and the use of Prime-boost immunization strategy is beneficial to the enhancement of this activation.
  • mice 4-6 week old C57BL/6 mice were randomly grouped according to Table 5 (8/group).
  • MS f + B16 1 ⁇ 10 5 B16 cells stably expressing fusion tumor antigen 33M and full-length Survivin protein (hereinafter referred to as MS f + B16) were subcutaneously inoculated into the right proximal end of each group of mice.
  • Vaccination was given for 15 days, in which group 1 was injected with PBS as a negative control, and groups 2 and 3 were injected with MS vaccine and sPD1/MS vaccine, respectively.
  • group 1 was injected with PBS as a negative control
  • groups 2 and 3 were injected with MS vaccine and sPD1/MS vaccine, respectively.
  • the growth of the tumor in the mice was observed. The experiment took the death of the control mice as the observation node.
  • Figure 9-a shows the changes in tumor growth volume of each group of melanoma model mice.
  • MS vaccine and sPD1/MS vaccine all slowed the growth of melanoma, and the sPD1/MS vaccine showed significant antitumor effect (P ⁇ 0.01).
  • the tumor volume inhibition rate reached 52.62% at the end of the observation.
  • the sPD1/MS vaccine was significantly slower from the 15th day of tumor inoculation until the observation deadline (P ⁇ 0.05). It seems that the CpDV-IL2-sPD1/MS DNA vaccine fused with sPD1 can Optimize the anti-tumor effect of vaccine on tumor-bearing mice.
  • the survival of melanoma mice was observed by group 5, and the cut-off time was observed on the 50th day after tumor inoculation.
  • the results are shown in Figure 9-b.
  • the average survival time of the PBS group was 36 days, and the total death limit was 40 days after tumor inoculation.
  • the MS vaccine group had an average survival of 38.5 days and a life extension rate of 6.94%, which showed no life extension compared with the negative control PBS group.
  • the average survival time of the sPD1/MS DNA vaccine group was 43 days, which was 19.44% longer than that of the control group (P value 0.0325). Although the survival of tumor-bearing mice treated with sPD1/MS DNA vaccine was not statistically significant between the MS vaccine-treated group, recombinant sPD1/MS DNA vaccine was superior to the average life expectancy and life-prolonging trend of mice.
  • Example 7 Anti-tumor effect of sPD1/MS recombinant virus vaccine on melanoma model mice
  • mice were randomly divided into PBS negative group, poxvirus vaccine treatment group and adenovirus vaccine treatment group, with 5 mice in each group.
  • Each group of mice was inoculated with 5 ⁇ 10 4 MS f + B16 cells on day 0, and the mice in the treatment group were immunized with sPD1/MS recombinant poxvirus vaccine or recombinant adenovirus vaccine on days 1 and 15 after inoculation.
  • mice were inoculated with an equal volume of sterile PBS as a control. On the 10th day after the tumor attack, the tumor growth of each group of mice was observed. The results are shown in Fig. 10. Compared with the PBS negative control group, the tumors of the rMVA-sPD1/MS vaccine group and the rAD-sPD1/MS vaccine group mice. The growth trend was significantly slowed down (P ⁇ 0.05).
  • the average tumor volume of sPD1/MS recombinant poxvirus treated mice was reduced by about 25% compared with the control group, sPD1
  • the average tumor volume of the /MS recombinant adenovirus treated mice was reduced by about 23%, indicating that the recombinant viral vector vaccine form of the sPD1/MS fusion nucleic acid has an effect of effectively inhibiting tumor growth.
  • Example 8 Anti-tumor effect of sPD1/MS recombinant DNA vaccine on colorectal cancer model mice
  • mice with colorectal cancer were used as a positive control, and the vaccine was used.
  • the combined immunotherapy of the drug oxaliplatin was used to investigate the synergistic effect of the two on tumor therapy in model mice.
  • 4-6 week old Balb/c mice were grouped according to Table 3, 10 mice in each group, and on day 0, 1 ⁇ 10 6 MS f + CT26 tumor cells were inoculated subcutaneously on the right side of the right side of the mouse. On the 1st, 8th, and 15th day, mice in each group were intramuscularly vaccinated.
  • the first group received PBS for the negative control
  • the second and fifth groups received the MS vaccine
  • the third and sixth groups received the sPD1/MS vaccine.
  • Oxaliplatin (Jiangsu Haizheng Pharmaceutical Co., Ltd.) was administered by intraperitoneal injection at a dose of 0.5 mg/kg each time, and administered 5 times at intervals of 5 days.
  • the growth of the tumor in the mice was recorded, and the mice in the PBS control group began to die as the observation time node.
  • To calculate the tumor inhibition rate of each experimental group all the mice were sacrificed at the observation point, and the tumor was excised and the tumor weight was weighed.
  • Figure 11-a is a graph showing tumor growth volume changes in colorectal cancer model mice. It can be seen from the figure that the tumor growth was significantly slower in the sPD1/MS vaccine group compared with the PBS-negative group, and the tumor volume was decreased from the 23rd day (P ⁇ 0.05). At the same time, from the tumor weight calculation rate of each group (see Figure 11-b), the inhibition rate of sPD1/MS vaccine was 30.96%, which was higher than the 17.18% inhibition rate of MS vaccine group. Statistical significance.
  • the combination of vaccine and chemical oxaliplatin showed that the combination of sPD1/MS vaccine and oxaliplatin caused tumor growth to be almost stagnant, the tumor inhibition rate was 74.71%, and the anti-tumor rate of oxaliplatin group was 58.62%.
  • the sPD1/MS vaccine and chemical combination group had a significant effect on inhibiting tumor growth (P ⁇ 0.01).
  • the combined tumor suppressor results of the MS vaccine and the chemical drug were related to the tumor.
  • the saliplatin group was not statistically significant. This indicates that the sPD1/MS vaccine can synergistically anti-tumor with the chemical drug oxaliplatin while significantly inhibiting tumor growth.
  • the survival of colorectal cancer-bearing mice was observed according to the grouping of Table 6.
  • the cut-off time was 65 days after tumor inoculation.
  • the results are shown in Figure 11-c.
  • the average survival time of mice in the recombinant MS vaccine group was 48.5 days.
  • the average survival time of the mice in the recombinant sPD1/MS vaccine group was 52 days, and there was no significant life extension trend in the negative control group (average survival of 50 days).
  • the combination of recombinant MS vaccine or recombinant recombinant sPD1/MS DNA vaccine and oxaliplatin was observed to increase the average survival time of mice by 22% and 24%, respectively. ⁇ 0.001), in which the survival time of the recombinant sPD1/MS DNA vaccine combined with oxaliplatin-treated group was significantly longer than that of the oxaliplatin-treated group (14% longer) (P ⁇ 0.05). .
  • Example 9 Antitumor effect of sPD1/MS recombinant protein vaccine on colorectal cancer-bearing mice
  • Example 4 and Example 5 it can be seen that the vaccine of the sPD1/MS recombinant nucleic acid form can induce strong MUCl and Survivin-specific cellular immunity in mice, and in this embodiment, the sPD1/MS recombinant protein vaccine is used, and the aluminum azole is combined.
  • the strategy of enhancing the immunization of Al(OH)3 is performed in a colorectal cancer model.
  • the specific groupings are shown in Table 7: 4-6 week old Balb/c mice were randomly divided into PBS negative control group, Al(OH)3 adjuvant group and adjuvant combined with recombinant sPD1/MS protein vaccine treatment group, 5 groups in each group. Only mice.
  • mice Each group of mice was inoculated with 1 ⁇ 10 6 MS f + CT26 tumor cells on day 0, and received vaccine treatment on days 1, 8, and 15, wherein the negative group of mice was given a volume of 100 ⁇ l of sterile PBS, Al(OH). 3 adjuvant group mice were injected with an equal volume of 100 ⁇ g dose of Al(OH)3 adjuvant (sigma), and the protein/adjuvant group mice were injected with 100 ⁇ g of each adjuvant and recombinant sPD1/MS protein previously mixed.
  • the mice in the PBS control group began to die as the observation time node, and all the mice were sacrificed by observing the neck of the node, and the tumor was excised and the tumor weight was weighed.
  • the mean tumor weight results of each group of treated tumor-bearing mice are shown in Figure 12-a.
  • the average tumor weight of the mice injected with PBS was 3.45 g, and the average tumor weight of the Al(OH) 3 adjuvant group was 3.19 g.
  • the adjuvant had no antitumor effect, while the sPD1/MS fusion protein was adjuvanted with the adjuvant.
  • the average tumor weight of the immunized mice was 2.62 g, which was about 24% compared with the negative control group, and there was a statistical significance of P ⁇ 0.05.
  • AL(OH)3 is known to be a common inorganic adjuvant, which can enhance the immunogenicity of the vaccine by inducing T cell differentiation and humoral immune response.
  • recombinant sPD1/MS protein can inhibit tumorigenesis.
  • the growth of mouse tumors at the same time, the ability of spleen lymphocytes to secrete IFN- ⁇ specificly in each group of tumor-bearing mice was analyzed (see Figure 12-b).
  • the adjuvant and protein vaccine co-immunization group also had higher Elispot spots.
  • the number (P ⁇ 0.01) reflects the ability of the sPD1/MS recombinant protein vaccine to induce specific anti-tumor immunity in tumor-bearing mice.
  • Example 10 sPD1/MS recombinant DNA vaccine for treating breast cancer model mice
  • mice 4-6 weeks old Balb/c mice were randomly divided into two groups, 10 in each group; mice were vaccinated on day 0 Mouse breast cancer cells (MS f + 4T1) stably expressing mucin 1 and survivin, wherein the experimental group was injected with sPD1/MS DNA vaccine on days 1 and 8, and recombinant adenovirus was administered on day 15. Injections of rAD-MS, another group of mice as a negative control, were given an equal volume of sterile PBS injection.
  • FIG. 13 shows the tumor growth volume curve of breast cancer model mice.
  • the tumor growth rate of the mice in the recombinant sPD1/MS vaccine group was significantly slower than that of the PBS group, and the tumor inhibition rate was about 40%, indicating that sPD1/ MS recombinant DNA vaccine has significant anti-tumor effect on breast cancer (P ⁇ 0.001).
  • Example 11 sPD1/MS recombinant DNA vaccine for treating lung cancer model mice
  • mice 4-6 weeks old C57BL/6 mice were randomly divided into PBS-negative group and sPD1/MS DNA vaccine treatment group according to Table 8. On each day, mice were inoculated with mouse lung cancer cells (MS f + Lewis) stably expressing mucin 1 and survivin at a dose of 1 ⁇ 10 5 / each; Mice were intramuscularly injected with 100 ⁇ l of sterile PBS on days 1, 8, and 15; mice in the DNA vaccine group were injected with sPD1/MS DNA vaccine on days 1, 8, and 15. The tumor growth changes of the mice were observed on the 8th day after the tumor inoculation, and the mice in the PBS control group began to die as the observation time nodes.
  • mice were inoculated with mouse lung cancer cells (MS f + Lewis) stably expressing mucin 1 and survivin at a dose of 1 ⁇ 10 5 / each; Mice were intramuscularly injected with 100 ⁇ l of sterile PBS on days 1, 8, and 15; mice in the DNA vaccine group were
  • Figure 14 is a graph showing the tumor growth volume of lung cancer model mice. The results showed that the tumor growth rate of the mice treated with the immunorecombinant sPD1/MS DNA vaccine alone was significantly slower than that of the PBS-negative mice. Tumor inhibition effect (P ⁇ 0.05). In summary, sPD1/MS recombinant DNA vaccine has an effective anti-tumor therapeutic effect in the treatment of lung cancer.
  • Example 12 sPD1/MS recombinant DNA vaccine for treatment of liver cancer model mice
  • mice 4-6 weeks old Balb/c mice were randomly divided into two groups, PBS negative control group and sPD1/MS DNA vaccine treatment group, 4 in each group; day 0 inoculated with stable transmuxin 1 and survival Mouse hepatoma cell line MS f + H22, in which the experimental group was intramuscularly injected with sPD1/MS DNA vaccine on days 1, 8, and 15, and on day 22, recombinant adenovirus rAD-MS was boosted, and the other group was small. Rats were injected with an equal volume of sterile PBS as a negative control.
  • the tumor growth of the liver cancer model mice treated with the sPD1/MS recombinant DNA vaccine was slowed down (P ⁇ 0.01). It indicated that sPD1/MS recombinant DNA vaccine has significant antitumor effect on liver cancer.
  • Example 13 sPD1/MS recombinant DNA vaccine for treating gastric cancer model mice
  • the former gastric cancer cell was selected to establish a gastric cancer mouse model to investigate the anti-tumor effect of the sPD1/MS DNA vaccine.
  • 615 mice of 4-6 weeks old were randomly divided into two groups of 4 each. Mice in each group were vaccinated with murine murine 1 and survivin in mouse pre-gastric cell line MS f + MFC on day 0, in which the recombinant sPD1/MS vaccine treatment group received intramuscular DNA vaccine on days 1, 8 and 15
  • the recombinant adenovirus rAD-MS was boosted by 22 days, while the other group was injected with an equal volume of sterile PBS as a negative control at the same time. Tumor growth curves of each group of mice were measured on the 12th day after tumor inoculation.
  • the results in Figure 16 showed that the gastric cancer model mice treated with sPD1/MS recombinant DNA vaccine achieved certain tumor suppression effects (P ⁇ 0.05).
  • Example 14 In vivo gene importer promotes immunogenicity and antitumor effect of sPD1/MS recombinant DNA vaccine
  • the in vivo gene introducer promotes the efficiency of plasmid entry into the cell by the action of an electric field at the injection site.
  • the parameters suitable for our vaccine were obtained: voltage: 36V; frequency: 1Hz; pulse number: 6 times; pulse width: 20ms.
  • the immune promoter effect of DNA vaccine was studied in vivo.
  • the results in Figure 17 show that the cellular immune effect of the vaccine is significantly improved after using the in vivo gene introduction instrument. After stimulation with survivin and MUC1 protein, the ability of lymphocytes to secrete IFN- ⁇ and CTL levels were significantly increased.
  • the effect of intramuscular injection of 100ug was achieved with a 20ug dose (P ⁇ 0.05).
  • the use of in vivo gene introduction enhances the immunogenicity of DNA.
  • the results in Figure 18 show that in the inhibition of tumor growth, after using the living gene importer, the lower dose of DNA can be used to achieve the original intramuscular injection of 100 ug, and after maintaining the 100 ug dose using the living gene importer, after the tumor attack 18 At the beginning of the day, the tumor size of the 100 ug group using the in vivo gene introducer was significantly different from that of the intramuscular group (P ⁇ 0.05).
  • the level of immunity in the tumor-bearing mice was examined.
  • the results of CTL and ELISPOT showed that the ability of CTL and IFN- ⁇ secreting in the low-dose group was significantly improved compared with the intramuscular injection group.

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Abstract

La présente invention concerne le domaine technique des médicaments issus des biotechnologies et concerne en particulier un vaccin recombiné et son application. La présente invention concerne un ensemble moléculaire, comprenant PD-1, MUC1 et survivine solubles. Un vaccin préparé par l'ensemble moléculaire peut produire une bonne immunogénicité et une bonne activité antitumorale. L'immunité de vaccin fournie selon la présente invention peut produire des réponses anticorps spécifiques et des réponses immunitaires cellulaires spécifiques pour MUC1 et survivine. Par comparaison avec le vaccin à ADN CpDV-IL2-MS, CpDV-IL2-sPD1/MS présente un effet antitumoral plus significatif chez les souris porteuses de tumeur. L'invention concerne en outre une combinaison de vaccins thérapeutiques et de médicaments chimiothérapeutiques.
PCT/CN2018/116393 2017-11-24 2018-11-20 Vaccin recombiné et son application WO2019101062A1 (fr)

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CN111118063B (zh) * 2019-12-05 2023-04-18 吉林大学 以FAPα和survivin为基础的DNA及其在制备肿瘤疫苗中的应用
CN113577259B (zh) * 2021-08-26 2023-05-26 贵州医科大学 组合物在制备用于抑制或治疗肿瘤的药物中的应用
CN114790248B (zh) * 2022-05-12 2023-11-28 广州医科大学 MUC1-PDL1-IgG1 Fc肿瘤疫苗及其制备方法和应用
CN115976094B (zh) * 2022-12-15 2024-02-20 浙江大学杭州国际科创中心 一种提高内源酶分泌的基因工程菌及其构建方法和应用

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GB0323965D0 (en) * 2003-10-13 2003-11-19 Glaxosmithkline Biolog Sa Immunogenic compositions
CN102665748A (zh) * 2009-05-27 2012-09-12 达娜-法勃肿瘤研究所公司 使用muc1拮抗剂抑制炎症
CN102086453B (zh) * 2009-12-04 2015-01-07 长春百克生物科技股份公司 以粘蛋白1和生存素为靶点的肿瘤dna疫苗及病毒载体疫苗
CN104013973A (zh) * 2009-12-04 2014-09-03 长春百克生物科技股份公司 以粘蛋白1和生存素为靶点的肿瘤dna疫苗及病毒载体疫苗
CN102732543B (zh) * 2011-04-07 2016-04-20 长春百克生物科技股份公司 以粘蛋白1和生存素为靶点的肿瘤基因工程疫苗
ES2792183T3 (es) * 2013-09-13 2020-11-10 Beigene Switzerland Gmbh Anticuerpos anti-PD1 y su uso como productos terapéuticos y de diagnóstico
DK3071697T3 (da) * 2013-11-22 2020-01-27 Dnatrix Inc Adenovirus der udtrykker immuncelle-stimulatorisk(e) receptor agonist(er)
CA3010975A1 (fr) * 2015-01-15 2016-07-21 University Of Copenhagen Pseudo-particule virale a presentation efficace des epitopes

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