WO2019101062A1 - Recombinant vaccine and application thereof - Google Patents

Recombinant vaccine and application thereof Download PDF

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WO2019101062A1
WO2019101062A1 PCT/CN2018/116393 CN2018116393W WO2019101062A1 WO 2019101062 A1 WO2019101062 A1 WO 2019101062A1 CN 2018116393 W CN2018116393 W CN 2018116393W WO 2019101062 A1 WO2019101062 A1 WO 2019101062A1
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vaccine
spd1
tumor
cancer
recombinant
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PCT/CN2018/116393
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Chinese (zh)
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WO2019101062A9 (en
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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.

Abstract

The present invention relates to the technical field of biotech drugs and in particular relates to a recombinant vaccine and an application thereof. The present invention provides a molecular assembly, comprising soluble PD-1、MUC1 and Survivin. A vaccine prepared by the molecular assembly can produce good immunogenicity and anti-tumor activity. The vaccine immunity provided according to the present invention can produce specific antibody responses and specific cellular immune responses for MUC1 and Survivin. Compared with DNA vaccine CpDV-IL2-MS, CpDV-IL2-sPD1/MS has a more significant anti-tumor effect in tumor-bearing mice. The invention further provides a combination of therapeutic vaccines and chemotherapeutic drugs.

Description

重组疫苗及其应用Recombinant vaccine and its application 技术领域Technical field
本发明涉及生物技术药物技术领域，尤其涉及重组疫苗及其应用。The invention relates to the field of biotechnology drug technology, in particular to a recombinant vaccine and its application.
背景技术Background technique
癌症或恶性肿瘤是威胁人类健康的杀手，肿瘤的诱发原因错综复杂，我们至今缺乏对其具体形成机制的深刻理解。最近的研究表明，机体免疫系统与肿瘤细胞间存在循环相互作用，即免疫系统能够识别“非我”成分的癌细胞，癌细胞也能通过多种途径逃避机体免疫系统的监控和攻击，造成患者体内肿瘤细胞的扩散转移。肿瘤免疫逃逸的分子机制包括：肿瘤抗原表达下调或丢失，肿瘤细胞分泌具有免疫抑制功能的可溶性细胞因子，肿瘤微环境招募免疫抑制性淋巴细胞，以及肿瘤细胞上调表达负性协同刺激信号等。其中，在肿瘤局部微环境及干扰素γ等细胞因子的诱导下，活化的T细胞上大量表达负性共刺激分子，这些免疫抑制调控分子将直接影响效应细胞毒性T淋巴细胞(CTL)的抗肿瘤活性，使其杀伤功能减弱甚至丧失。目前发现的抑制性协同刺激分子主要有CTLA4/CD80(CD86)、PD1/PD-L1(PD-L2)、BTLA/HVEM、TIM3/GAL9等。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. Among them, in the local microenvironment of the tumor and the induction of cytokines such as interferon gamma, a large amount of negative costimulatory molecules are expressed on the activated T cells, and these immunosuppressive regulatory molecules will directly affect the anti-effect cytotoxic T lymphocyte (CTL) resistance. Tumor activity reduces or even kills its killing function. The inhibitory costimulatory molecules currently found mainly include CTLA4/CD80 (CD86), PD1/PD-L1 (PD-L2), BTLA/HVEM, TIM3/GAL9 and the like.
人程序性细胞死亡蛋白1(Programmed Death-1，PD-1)又名CD279，它与两个配体PD-L1(B7H1，CD274)和PD-L2(B7DC，CD273)同属免疫球蛋白超家族I型跨膜糖蛋白，由胞浆区、跨膜锚定区和胞外位点结合域三部分构成。介导PD-1信号免疫抑制功能的关键结构，是位于胞内信号转导区的免疫受体酪氨酸转换基序(Immunoreceptortyrosine-based switch motif，ITSM)，当配受体偶联时，ITSM基序启动酪氨酸磷酸化程序以招募相关磷酸酶，使下游的信号通路PI3K/AKT(磷脂酰肌醇3激酶/丝氨酸-苏氨酸蛋白激酶)去磷酸化，从而导致细胞因子合成的中断和T细胞效应不敏感等。PD-1/PD-L1信号通路在正常组织器官及外周淋巴组织低表达或诱导性表达，对维持机体免疫耐受、防止自身免疫病的发生具重要意义；而肿瘤部位PD-1分子的高表达，则加速了活化T细胞的凋亡。Human programmed cell death protein 1 (PD-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. When the receptor is coupled, 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. 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.
2013年，科学杂志授予免疫检查点阻断疗法年度重大科学突破，免疫治疗也真正使人们看到治愈癌症的希望。免疫检查点阻断疗法(Immune Checkpoint Blockade)即应用抑制剂阻断负性调节T细胞的共抑制信号通路，解除T细胞抑制并重新释放其免疫活性。当前开发的抑制剂主要是人源化单克隆抗体，分别阻断两个重要的免疫检查点——细胞毒性T淋巴细胞蛋白4(CTLA4)和PD-1。2014年，Herbst等报导一种能有效阻断PD-1配体PD-L1的单抗药物PMDL3280A治疗65例化药治疗无效的膀胱癌患者，结果30例PD-L1阳性患者中有13例(43％)产生明显的抑瘤肿瘤的效应，其中2人达到病情完全缓解；另外，在PD-L1阴性患者中也有11％的应答率。由此，体现出检查点阻断药物的抗肿瘤免疫应答优势，为癌症免疫治疗提供了新的研究思路。与此同时，单抗类抑制剂也存在一些弊端使得药物临床应用受到限制。例如，部分患者的肿瘤组织免疫组化检测显示没有T淋巴细胞浸润现象，可能导致此部分患者单抗治疗无效；也有一些患者在接受抗CTLA4抗体治疗后，出现致命的自身免疫病相关的不良反应；此外，人源化单克隆抗体制作成本较高，用药费用昂贵。这些问题都说明检查点阻断治疗需建立更加完善的适应症治疗者筛选程序和治疗方案，而目前开发的PD-1抗体形式阻断剂则无法引导抗肿瘤主动免疫，其阻断 剂形式尚待进一步优化。文献资料显示，一种可溶型PD-1(sPD-1)蛋白可与配体PD-L1识别并结合，可作为另一PD-1/PD-L1信号通路阻断剂的形式应用于抗肿瘤免疫。In 2013, the Science Magazine awarded a major scientific breakthrough in the screening of immunological checkpoints. Immunotherapy also really made people see the hope of curing cancer. 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. In 2014, 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. As a result, 13 of the 30 PD-L1 positive patients (43%) developed obvious tumor suppressor tumors. The effect of the two patients reached complete remission; in addition, 11% of the PD-L1 negative patients also responded. Thus, the advantage of the anti-tumor immune response of the checkpoint blocking drug is reflected, which provides a new research idea for cancer immunotherapy. At the same time, there are some drawbacks to the monoclonal antibody inhibitors that limit the clinical application of the drug. For example, immunohistochemical examination of tumor tissue in some patients showed no T lymphocyte infiltration, which may lead to ineffective treatment of this part of the patient; some patients have fatal autoimmune disease-related adverse reactions after receiving anti-CTLA4 antibody treatment. In addition, humanized monoclonal antibodies are costly to manufacture and expensive to use. These problems indicate that checkpoint blockade therapy needs to establish a more complete screening program and treatment plan for the infectors, and the currently developed PD-1 antibody form blocker can not guide anti-tumor active immunity, and its blocker form is still To be further optimized. The literature shows that a soluble PD-1 (sPD-1) protein can be recognized and bound to the ligand PD-L1 and can be used as a blocker of another PD-1/PD-L1 signaling pathway. Tumor immunity.
(1)可溶性PD-1分子的生物学特性(1) Biological characteristics of soluble PD-1 molecules
共刺激蛋白通常存在两种形式：锚定于细胞膜上或分泌到胞外，可溶型分子保留了膜型分子的胞外配受体结合域，以游离方式通过血液循环作用于远端效应分子，参与调节机体免疫。现已证实，天然的人PD-1分子包含上述两种蛋白形式，且膜型与可溶型PD-1(Soluble PD-1，sPD1)皆由PDCD1基因编码，其中，所编码的全长mRNA翻译产物为膜型分子，而一种缺失外显子3的mRNA剪接变异体则直接翻译成sPD1。sPD1形成的另一种方式，可能是膜型PD-1在蛋白水解酶作用下脱落产生。sPD-1的IgV-IgC样结构，介导与配体PD-L1和PD-L2的结合，但由于缺乏胞内抑制基序，无法启动下游免疫抑制信号。Wan等检测类风湿性关节炎患者的关节滑液及外周血中都有sPD1分子的高表达，同时伴随IFN-γ、IL-4、IL-21等T细胞活化相关细胞因子水平升高。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. It has been confirmed that 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. 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-γ, IL-4 and IL-21.
人与小鼠PD-1和PD-L1蛋白的同源性分别达60％和70％，并且胞外结合关键位点的一级序列高度保守。体外试验结果表明重组人sPD1融合蛋白与小鼠的PD-L1、PD-L2有交叉结合作用，说明人源sPD-1也能阻断小鼠PD-1抑制信号通路。The homology between human and mouse PD-1 and PD-L1 proteins was 60% and 70%, respectively, and the primary sequence of extracellular binding critical sites was highly conserved. The results of in vitro experiments indicated that the recombinant human sPD1 fusion protein interacted with PD-L1 and PD-L2 in mice, indicating that human sPD-1 can also block PD-1 inhibition signaling pathway in mice.
(2)可溶型PD-1的生理功能(2) Physiological function of soluble PD-1
可溶型协同刺激分子参与调节机体免疫的相关研究，包括有sCD80、sLAG3、sPD1和sBTLA等，这些分子在病毒感染、肿瘤和自身免疫病等中有各自的生理功能。sPD1分子的免疫调节作用具体表现在四个方面：①是促进T淋巴细胞上一些激活型细胞因子的分泌(IFN-γ、IL-2等)，同时减少抑制型因子IL-10和TGF-β；②sPD1作用能促进特异性CD4阳性和CD8阳性T细胞的增殖，并且活化的T细胞上调抑凋亡基因Bcl-xl的表达，使T细胞凋亡活性减弱；③流式检测sPD1分子对DC细胞的影响，观察到CD80、CD86、IL-12及MHC-II类分子荧光强度均有显著提升，表明可溶性PD-1能够促进DC细胞的成熟；④sPD-1可通过有靶向DC而促进CD8 +T细胞的功能。有关于sPD1分子激活机体免疫的具体分子机制尚待研究，但我们认为其生理功能的发挥有赖于抗原特异性细胞免疫应答，单独的sPD1分子是不具备特异性免疫效应的。 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-γ, IL-2, etc.), while reducing the inhibitory factors IL-10 and TGF-β. 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.
(3)可溶型PD-1在抗肿瘤免疫中的应用(3) Application of soluble PD-1 in anti-tumor immunity
可溶性PD1分子作为一种形式的PD-1通路阻断剂，已有一定的临床前应用基础。Shin等，以疱疹病毒胸苷激酶(HSVtk)为靶点，构建了携载重组HSVtk基因和重组可溶性sPD1-Ig基因双顺反子的条件复制型腺病毒载体。将此双基因联合腺病毒进行瘤内注射，基因治疗结直肠癌肿瘤模型小鼠。结果显示，联合治疗组抑瘤效果达90％，与单一的HSVtk治疗相比有良好的协同效果；而单独的sPD1基因治疗组，则没有任何抗肿瘤作用；当删除小鼠体内CD8阳性T细胞后，sPD1的协同抗瘤作用消失。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. The results showed that the combined treatment group had a tumor inhibition effect of 90%, and had a good synergistic effect compared with the single HSVtk treatment; while the sPD1 gene treatment group alone did not have any anti-tumor effect; when the CD8-positive T cells were removed from the mouse After that, the synergistic anti-tumor effect of sPD1 disappeared.
癌症的免疫治疗中，Chen等考察了模式性sPD-1-p24抗原融合核酸疫苗对表达异种抗原GAG的恶性间皮细胞瘤的抑瘤效果。肌肉接种sPD1-p24 DNA疫苗的荷瘤小鼠，生存期达到100％，八个月后检测小鼠的特异性体液免疫和细胞免疫，结果IgG1/IgG2a及特异性CD8+T细胞数，都依然维持在高水平，体现了sPD-1抗肿瘤免疫应答是长效性。进一步研究发现，sPD-1能激发IFN-γ/TNF-α双阳T细胞的大量扩增，帮助T细胞杀伤肿瘤。另外，sPD1-p24极大地减少了免疫早期(六周以内)外周以及肿瘤微环境中的抑制因素，具体表现 在骨髓来源抑制性细胞(myeloid-derived suppressor cells，MDSC)与调节性T细胞等抑制性T细胞数目的减少。In the immunotherapy of cancer, Chen et al examined the anti-tumor effect of the model sPD-1-p24 antigen fusion nucleic acid vaccine on malignant mesothelioma expressing the heterologous antigen GAG. The tumor-bearing mice inoculated with sPD1-p24 DNA vaccine were 100%. After eight months, the specific humoral and cellular immunity of the mice were detected. The results showed that the number of IgG1/IgG2a and specific CD8+ T cells remained. Maintaining a high level reflects the long-lasting effect of the sPD-1 anti-tumor immune response. Further studies have found that sPD-1 can stimulate the massive expansion of IFN-γ/TNF-α Shuangyang T cells and help T cells kill tumors. In addition, 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.
综上所述，可溶性PD1分子竞争性与PD-L抑制性通路结合而阻断PD1/PD-L1信号通路，同时具有增强特异性免疫效果。与抗体类药物相比，重组sPD-1疫苗的制备过程相对简单、成本较低，我们相信sPD-1用作PD-1途经阻断剂有潜在的临床应用价值。In summary, the soluble PD1 molecule competes with the PD-L inhibitory pathway to block the PD1/PD-L1 signaling pathway, and has enhanced specific immunity. Compared with antibody drugs, 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.
(4)肿瘤基因疫苗(4) Tumor gene vaccine
本发明的发明人前期研究表明，以生存素(Survivin)和粘蛋白1(MUC1)为靶点的融合基因疫苗在抗肿瘤免疫治疗中具有一定的应用价值。Survivin属抑制凋亡蛋白超家族，主要功能是抵抗细胞凋亡并调控细胞分裂，Survivin特异性表达于胚胎组织及癌细胞，是理想的肿瘤免疫治疗靶点；MUC1是I型跨膜糖蛋白，由核心蛋白和多糖支链构成，其中核心蛋白胞外肽段包含数目不等的串联重复序列(VNTRs)，MUC1在肿瘤中的表达因糖基化不完全而暴露多肽核心VNTR区，这些新的多肽表位为T细胞提供了潜在免疫攻击目标。The inventors of the present invention have shown that fusion gene vaccines targeting survivin and mucin 1 (MUC1) 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. Consisting of a core protein and a polysaccharide branch, wherein the core protein extracellular peptide comprises a number of tandem repeats (VNTRs), and the expression of MUC1 in the tumor exposes the polypeptide core VNTR region due to incomplete glycosylation, these new Polypeptide epitopes provide potential immune targets for T cells.
发明人基于两个方面进行MUC1 VNTRs和Survivin融合表达DNA疫苗抗原表位设计：一是疫苗安全性，二是疫苗免疫原性。根据文献，全长Survivin在体内以二聚体形式发挥抗凋亡功能，且二聚形成关键位点是N端第6、第7和第10位氨基酸残基。因此，发明人采用了N端缺失7个氨基酸残基的Survivin缺失剪切体(简称S8)来设计疫苗，如此既提高疫苗安全性，又在最大程度上保证Survivin表位免疫原性；发明中所涉及的另一抗原表位为MUC1 VNTR串联重复序列，该串联重复结构包含33拷贝MUC1 VNTRs(简称为33M)，其中包含免疫显性结构域PDTRP序列，能够介导高效的MUC1特异性抗体反应并提高特异性CTL免疫效应。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. Therefore, 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.
前期研究结果显示，以33M和S8融合核酸为靶点的DNA疫苗(即MS疫苗)能够有效增强疫苗免疫的广谱性和有效性(参见申请号为200910252427.X的申请文件)。另外，MS疫苗构建于经过发明人改造的VR1012双顺反子载体CpDV中，其载体骨架包含一段CpG基序，同时能够独立编码白介素-2(IL-2)蛋白，它们作为免疫佐剂与粘蛋白1/生存素融合肿瘤抗原同步表达，其免疫增强效果已在发明人前期研究工作中开展并验证(参见专利号为ZL200910252427.x和ZL201110086366.1的专利文件)。在此基础上，进一步探究引入sPD-1的后疫苗诱导的的免疫原性和抗肿瘤效果的增强作用。Previous studies have shown that DNA vaccines (ie, MS vaccines) targeting 33M and S8 fusion nucleic acids can effectively enhance the broad spectrum and effectiveness of vaccine immunization (see application number 200910252427.X). In addition, the MS vaccine was constructed in the VR1012 bicistronic vector CpDV modified by the inventors. The vector backbone contains a CpG motif and is capable of independently encoding the interleukin-2 (IL-2) protein as an immunoadjuvant and adhesion. The protein/survivin fusion tumor antigen is synchronously expressed, and its immunopotentiating effect has been carried out and verified in the inventor's preliminary research work (see Patent Documents ZL200910252427.x and ZL201110086366.1). On this basis, the enhancement of the immunogenicity and antitumor effect induced by the post-vaccine introduction of sPD-1 was further explored.
发明内容Summary of the invention
鉴于此，本发明要解决的技术问题在于提供一种重组疫苗及其应用，本发明将sPD1进一步与肿瘤抗原MS进行组合，以sPD1阻断抗原呈递DC细胞过程中所涉及的PD-1/PD-L负性信号通路从而使疫苗具有更好的免疫原性和免疫效应。In view of this, 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.
本发明提供了一种分子组合，包括PD-1、MUC1和Survivin。The invention provides a molecular combination comprising PD-1, MUC1 and Survivin.
本发明所述的PD-1、MUCI和Survivin可为蛋白分子亦可为核酸分子，本发明对此不做限定。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.
sPD-1蛋白指能够阻断PD-1通路的可溶型PD-1蛋白，sPD-1核酸指能够编码sPD-1蛋白的DNA分子。本发明中，所述sPD-1为DNA或蛋白，为人sPD-1胞外段(记为sPD1)，其DNA序列来自Genbank登录号为NM_005018.2的基因。本发明采用的sPD-1的DNA序列如SEQ ID NO：1所示，所述sPD-1的氨基酸序列如SEQ ID NO：2所示。The sPD-1 protein refers to a soluble PD-1 protein capable of blocking the PD-1 pathway, and the sPD-1 nucleic acid refers to a DNA molecule capable of encoding the sPD-1 protein. In the present invention, 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.
MUC1蛋白衍生自野生MUC1 VNTR蛋白和(或)其突变体和(或)其截短体，MUC1核酸指能够编码MUC1蛋白的DNA分子。本发明中，MUC1的DNA序列如SEQ ID NO：3所示；其氨基酸序列如SEQ ID NO：4所示。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. In the present invention, 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.
Survivin蛋白衍生自野生Survivin蛋白和(或)其突变体和(或)其缺失剪接体，优选为N端缺失7个氨基酸残基的Survivin。发明中，Survivin的DNA序列如SEQ ID NO：5所示；其氨基酸序列如SEQ ID NO：6所示。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. In the invention, the DNA sequence of Survivin is shown in SEQ ID NO: 5; the amino acid sequence thereof is shown in SEQ ID NO: 6.
本发明中，PD-1、MUC1和Survivin，三者可融合(记为sPD1/MS)，亦可以非融合的形式联合使用(记为sPD1+MS)，本发明对此不做限定。In the present invention, 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.
本发明所述的分子组合在制备防治肿瘤的产品中的应用。The use of the molecular combination of the present invention in the preparation of a tumor-preventing product.
本发明实验表明，以本发明提供的抗原制备疫苗能够具有防治肿瘤的作用。且sPD1/MS疫苗组的效果显著优于MS疫苗组(p＜0.05)，也显著优于sPD1疫苗(p＜0.05)。并且，本发明实验证明，sPD1/MS融合制得的疫苗与sPD1+MS联合免疫的方式相比，所诱导产生的特异性细胞免疫反应处于较为一致的水平，表明sPD1和MS作为抗原，能够对肿瘤多靶点产生作用，而融合或非融合的方式对免疫效果的影响不显著(p＞0.05)。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). Moreover, 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).
以本发明提供抗原制得疫苗能够防治的肿瘤选自黑色素瘤、结直肠癌、大肠癌、肺癌、乳腺癌、肝癌、肾癌、胆管癌、胃癌、食管癌、膀胱癌、胰腺癌、头颈癌、鼻咽癌、口腔癌、宫颈癌、卵巢癌、子宫癌、前列腺癌、睾丸癌、鳞状细胞癌、淋巴瘤、脑癌、恶性胶质细胞瘤、髓母细胞瘤、淋巴肉瘤、绒毛膜上皮癌、骨肉瘤、甲状腺癌。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.
本发明以乳腺癌、肺癌、肝癌、胃癌、结直肠癌、黑素瘤荷瘤小鼠作为实验对象，结果表明，以本发明提供抗原制得的疫苗能够起到良好的抑制肿瘤生长的作用，且荷瘤小鼠淋巴细胞特异性分泌IFN-γ的能力也得到显著提高(P＜0.01)。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-γ specifically in tumor-bearing mice was also significantly increased (P<0.01).
本发明所述的防治肿瘤的产品中包括疫苗。The tumor control product of the present invention includes a vaccine.
实验表明，本发明提供抗原制得的疫苗能够起到良好的防治肿瘤的作用。所述疫苗为DNA载体疫苗、病毒载体疫苗、蛋白疫苗和/或树突状细胞疫苗。在一些具体实施例中，单独给予DNA载体疫苗、病毒载体疫苗或蛋白疫苗即可既起到抑制肿瘤生长的作用。而在另一些实施例中，数据表明，初免给予DNA载体疫苗，而以重组腺病毒疫苗加强免疫可以显著提升免疫效果。另有数据表明，在免疫的同时给予抗肿瘤的药物，能够起到增效协同的作用。所述抗肿瘤的药物为化疗药物，优选为铂类化合物、紫杉醇和/或吉西他滨和/或顺铂。一些实施例中，化疗药物为奥沙利铂。以奥沙利铂为实验对象，结果表明，重组sPD1/MS疫苗联合奥沙利铂治疗组荷瘤小鼠的生存期与单独的奥沙利铂治疗组荷瘤小鼠(生命延长14％)相比，体现显著的生命延长作用(P＜0.05)。Experiments have shown that 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. In some embodiments, administration of a DNA vector vaccine, a viral vector vaccine, or a protein vaccine alone can both inhibit tumor growth. In still other embodiments, 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. In some embodiments, the chemotherapeutic agent is oxaliplatin. Taking oxaliplatin as the experimental object, the results showed that the survival time of tumor-bearing mice with recombinant sPD1/MS vaccine combined with oxaliplatin treatment group and tumor-bearing mice treated with oxaliplatin alone (life extension 14%) In comparison, significant life extension was demonstrated (P < 0.05).
因此，本发行提供的防治肿瘤的产品中，可包括DNA载体疫苗、病毒载体疫苗、蛋白疫苗和/或树突状细胞疫苗中的一种或两者以上的组合。也可以还包括化疗药物，如奥沙利铂。Therefore, 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.
本发明还提供了包括PD-1、MUC1和Survivin的DNA序列的重组载体。The present invention also provides a recombinant vector comprising the DNA sequences of PD-1, MUCl and Survivin.
组织型纤溶酶原激活因子(tissue plasminogen activator，tPA)，简称tPA信号肽，可有效促进蛋白的分泌，提高其诱导抗体产生的能力。为了能够提高融合蛋白sPD1/MS在胞外的分泌表达能力，本发明在PD-1的DNA序列的5’端修饰tPA信号肽序列。Tissue plasminogen activator (tPA), abbreviated as tPA signal peptide, can effectively promote protein secretion and enhance its ability to induce antibody production. In order to increase the secretory expression ability of the fusion protein sPD1/MS in the extracellular, the present invention modifies the tPA signal peptide sequence at the 5' end of the DNA sequence of PD-1.
本发明中，sPD-1与tPA信号肽序列相连的DNA序列如SEQ ID NO：7所示。In the present invention, the DNA sequence in which sPD-1 is linked to the tPA signal peptide sequence is shown in SEQ ID NO: 7.
其中，tPA信号肽的长度为69bp，其序列如SEQ ID NO：11所示。Wherein, the length of the tPA signal peptide is 69 bp, and the sequence thereof is shown in SEQ ID NO: 11.
本发明一些具体实施例中，MUC1和Survivin连接的DNA序列(记为MS序列)如SEQ ID NO：8所示。In some embodiments of the invention, the DNA sequence (denoted as an MS sequence) to which MUC1 and Survivin are ligated is set forth in SEQ ID NO: 8.
根据所需构建的疫苗形式，可以不同的骨架构建不同的重组载体，但这些重组载体中，都应包括与tPA信号肽相连接的PD-1序列，MUC1序列和Survivin序列，本发明对所述与tPA信号肽相连接的PD-1序列，MUC1序列和Survivin序列的连接顺序不做限定，其实施皆在本发明的保护范围之内。在本发明的实施例中，融合片段的连接顺序为：由5’端至3’端，tPA信号肽、PD-1序列，MUC1序列、Survivin序列。Depending on the form of vaccine to be constructed, different 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. In an embodiment 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.
为了构建DNA载体疫苗，本发明提供了一种重组载体，其包括骨架载体、SEQ ID NO：7所示的DNA序列、SEQ ID NO：8所示的DNA序列和佐剂序列。In order to construct a DNA vector vaccine, 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.
在本发明实施例中，骨架载体为CpDV。CpDV载体由申请号为201110086366.1的发明专利构建。In an embodiment of the invention, the backbone carrier is CpDV. The CpDV vector is constructed by the invention patent of application number 201110086366.1.
所述佐剂序列为分子内佐剂，选自细胞因子类佐剂，包括但不限于白介素2(IL-2)、免疫刺激DNA的非甲基化CpG基序以及集落刺激因子GM-CSF。本发明实施例中，采用的佐剂序列为白介素-2，其DNA序列如SEQ ID NO：9所示。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. In the embodiment of the present invention, the adjuvant sequence used is interleukin-2, and the DNA sequence thereof is shown in SEQ ID NO: 9.
一些实施例中，佐剂序列的插入位点为Xba I和BamH I酶切位点之间；SEQ ID NO：7所示的DNA序列和SEQ ID NO：8所示的DNA序列的插入位点为BglII和EcoR I酶切位点之间。In some embodiments, 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.
一些实施例中，本发明提供的重组载体(记为CpDV-IL2-sPD1/MS)的图谱如图1-d所示，其制备方法为在骨架载体的Xba I和BamH I酶切位点之间插入佐剂序列，然后在Bgl II和EcoR I酶切位点之间插入SEQ ID NO：7和SEQ ID NO：8所示序列。In some embodiments, 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.
本发明提供的重组载体在制备防治肿瘤的产品中的应用。The use of the recombinant vector provided by the invention in the preparation of a tumor-preventing product.
所述防治肿瘤的产品为DNA载体疫苗。The tumor control product is a DNA vector vaccine.
本发明还提供了一种防治肿瘤的疫苗，其中包括本发明提供的重组载体。The present invention also provides a vaccine for preventing and treating tumors, which comprises the recombinant vector provided by the present invention.
优选的，所述重组载体为CpDV-IL2-sPD1/MS。Preferably, the recombinant vector is CpDV-IL2-sPD1/MS.
本发明提供的疫苗的制备方法为，在骨架载体的Xba I和BamH I酶切位点之间插入佐剂序列，然后在Bgl II和EcoR I酶切位点之间插入SEQ ID NO：7和SEQ ID NO：8所示序列，制得重组载体。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.
接种本发明提供的DNA载体疫苗的小鼠能产生高效的抗粘蛋白1抗体和抗生存素抗体，并且两者的抗体滴度可达10000以上，且产生抗体的量也较大，说明本发明提供的DNA载体疫苗能够启动强烈的特异性体液免疫应答。并且，实验表明，本发明提供规定DNA载体疫苗能够有效抑制肿瘤的生长，所述肿瘤包括乳腺癌、肺癌、肝癌、胃癌、结直肠癌、黑素瘤，且其效果显著优于(p＜0.05)单独使用以MS为靶点的DNA载体疫苗，或以sPD-1为靶点的DNA载体疫苗。由于肿瘤免疫治疗机制的相似性，认为本发明提供的DNA载体疫苗也能够对黑色素瘤、结直肠癌、大肠癌、肺癌、乳腺癌、肝癌、肾癌、胆管癌、胃癌、食管癌、膀胱癌、胰腺癌、头颈癌、鼻咽癌、口腔癌、宫颈癌、卵巢癌、子宫癌、前列腺癌、睾丸癌、鳞状细胞癌、淋巴瘤、脑癌、恶性胶质细胞瘤、髓母细胞瘤、淋巴肉瘤、绒毛膜上皮癌、骨肉瘤、甲状腺癌起到良好的抑制作用。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. Moreover, 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. Due to the similarity of tumor immunotherapy mechanisms, 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.
为了构建重组蛋白疫苗，本发明还提供了一种重组载体，包括骨架载体、SEQ ID NO：7 所示的DNA序列、SEQ ID NO：8所示的DNA序列。In order to construct a recombinant protein vaccine, 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.
构建重组蛋白疫苗需获得融合蛋白(sPD1/MS)，本发明中，融合蛋白的表达采用原核表达系统。为了获得原核表达系统表达的融合蛋白，本发明提供的重组载体中骨架载体为pRSET B或PET系列载体。The fusion protein (sPD1/MS) is required to construct a recombinant protein vaccine. In the present invention, the expression of the fusion protein uses a prokaryotic expression system. In order to obtain a fusion protein expressed by a prokaryotic expression system, the backbone vector provided by the present invention is a pRSET B or PET series vector.
一些实施例中，本发明提供的重组载体(记为pET28a-sPD1/MS)的图谱如图3-b所示，其制备方法为在骨架载体的Nde I和Xho I酶切位点之间插入sPD1/MS片段。In some embodiments, 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.
为了制备融合蛋白(sPD1/MS)本发明提供了一种表达载体，由本发明提供的重组载体转染宿主细胞制得。For the preparation of fusion proteins (sPD1/MS) The present invention provides an expression vector which is produced by transfecting a host cell with a recombinant vector provided by the present invention.
所述宿主细胞为大肠杆菌BL21。The host cell is E. coli BL21.
本发明还提供了一种融合蛋白，其由本发明提供的重组载体转染宿主细胞制得表达载体后，表达获得。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.
所述宿主细胞为大肠杆菌BL21。The host cell is E. coli BL21.
所述表达后，还经过纯化的步骤，所述纯化为将培养后的细胞以超声裂解后，离心30min，沉淀以包涵体溶解buffer重悬后、溶解后，上清液经镍柱亲和层析，获得融合蛋白。After the expression, 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.
本发明制得的融合蛋白在制备防治肿瘤的产品中的应用。The use of the fusion protein prepared by the invention in the preparation of a tumor-preventing product.
所述防治肿瘤的产品为重组蛋白疫苗。The tumor control product is a recombinant protein vaccine.
本发明还提供了一种防治肿瘤的疫苗，包括本发明提供的融合蛋白(sPD1/MS)。The present invention also provides a vaccine for preventing and treating tumors, comprising the fusion protein (sPD1/MS) provided by the present invention.
所述疫苗中还包括佐剂，所述佐剂为Al(OH) 3。 Also included in the vaccine is an adjuvant, which is Al(OH) 3 .
AL(OH) 3为常见的无机佐剂，其可通过诱导T细胞分化和体液免疫反应来提高疫苗的免疫原性， AL(OH) 3 is a common inorganic adjuvant that enhances the immunogenicity of vaccines by inducing T cell differentiation and humoral immune responses.
接种本发明提供的重组蛋白疫苗能够抑制荷瘤小鼠肿瘤的生长，且荷瘤小鼠脾淋巴细胞特异性分泌IFN-γ的能力也得到提高。所述肿瘤为结直肠癌，由于肿瘤发病机制的相似性，认为本发明提供的DNA载体疫苗也能够对黑色素瘤、大肠癌、肺癌、乳腺癌、肝癌、肾癌、胆管癌、胃癌、食管癌、膀胱癌、胰腺癌、头颈癌、鼻咽癌、口腔癌、宫颈癌、卵巢癌、子宫癌、前列腺癌、睾丸癌、鳞状细胞癌、淋巴瘤、脑癌、恶性胶质细胞瘤、髓母细胞瘤、淋巴肉瘤、绒毛膜上皮癌、骨肉瘤、甲状腺癌起到良好的抑制作用。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-γ 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.
为了构建病毒载体疫苗，本发明提供了一种重组载体，其中包括骨架载体、SEQ ID NO：7所示的DNA序列、SEQ ID NO：8所示的DNA序列，所述骨架载体为pSC11或pDC316。In order to construct a viral vector vaccine, 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为安卡拉痘病毒(Modified Vaccinia Ankara，MVA)常用的穿梭质粒，该载体上具有的胸苷激酶左臂和右臂(TKL、TKR)可与MVA同源重组，同时通过载体上lacZ基因，进行重组MVA(rMVA)的蓝斑筛选。一些实施例中，本发明提供的重组载体(记为pSC11-sPD1/MS)的图谱如图4-b所示，该重组载体的制备方法为，在pSC11-MS质粒的SalI酶切位点处插入sPD1片段，所述sPD-1片段的5’端连接有tPA信号肽片段。所述pSC11-MS质粒由申请号为200910252427.X的中国发明专利构建。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). In some embodiments, 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为腺病毒常用穿梭质粒，一些实施例中，本发明提供的重组载体(记为pDC316-sPD1/MS)的图谱如图4-d所示，该重组载体的制备方法为，在pDC316-MS质粒的EcoRI酶切位点处插入sPD1片段，所述sPD-1片段的5’端连接有tPA信号肽片段。 所述pDC316-MS质粒由申请号为200910252427.X的中国发明专利构建。pDC316 is a commonly used shuttle plasmid for adenovirus. In some embodiments, 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.
为了构建病毒载体疫苗，本发明还提供了一种重组病毒，其以本发明提供的重组载体转染病毒制得。In order to construct a viral vector vaccine, the present invention also provides a recombinant virus which is produced by transfecting a virus with the recombinant vector provided by the present invention.
在一些实施例中，所述重组载体为pSC11-sPD1/MS，所述病毒为痘病毒。In some embodiments, the recombinant vector is pSC11-sPD1/MS and the virus is a poxvirus.
在此实施例中，所述转染为：将空MVA病毒感染含有TK基因的BHK-21的突变细胞，然后使用lipo2000试剂将穿梭质粒pSC11-sPD1/MS共转染到BHK细胞，培养后以培养上清作种毒感染BHK细胞后，经筛选得到转染pSC11-sPD1/MS的痘病毒。In this embodiment, 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.
在一些实施例中，所述重组载体为pDC316-sPD1/MS，所述病毒为腺病毒。In some embodiments, the recombinant vector is pDC316-sPD1/MS, the virus is an adenovirus.
在此实施例中，所述转染为：将AD骨架和穿梭质粒pDC316-sPD1/MS共转染293细胞，经筛选得到转染pDC316-sPD1/MS的腺病毒。In this embodiment, 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.
所述腺病毒(AD)骨架为pBHGloxΔE1，3Cre。The adenovirus (AD) backbone is pBHGloxΔE1, 3Cre.
本发明提供的重组病毒在制备防治肿瘤的产品中的应用。The use of the recombinant virus provided by the invention in the preparation of a tumor-preventing product.
本发明还提供了一种防治肿瘤的疫苗，包括本发明提供的重组病毒。The present invention also provides a vaccine for preventing and treating tumors, including the recombinant virus provided by the present invention.
接种本发明提供的重组病毒疫苗能够显著抑制荷瘤小鼠肿瘤的生长，其中，sPD1/MS重组痘病毒治疗小鼠的平均肿瘤体积比对照组的肿瘤体积减小约25％，sPD1/MS重组腺病毒治疗小鼠的平均肿瘤体积则减小约23％。所述肿瘤为黑素瘤，由于肿瘤疫苗机制的相似性，认为本发明提供的病毒载体疫苗也能够对结直肠癌、大肠癌、肺癌、乳腺癌、肝癌、肾癌、胆管癌、胃癌、食管癌、膀胱癌、胰腺癌、头颈癌、鼻咽癌、口腔癌、宫颈癌、卵巢癌、子宫癌、前列腺癌、睾丸癌、鳞状细胞癌、淋巴瘤、脑癌、恶性胶质细胞瘤、髓母细胞瘤、淋巴肉瘤、绒毛膜上皮癌、骨肉瘤、甲状腺癌起到良好的抑制作用。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. 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.
另外，本发明实验表明，初免给予DNA载体疫苗，然后以病毒载体疫苗进行加强免疫能够起到显著提升免疫效果的作用。具体表现为，IFN-γ分泌增多，免疫应答增强。所述DNA载体疫苗中包含CpDV-IL2-sPD1/MS，所述病毒载体疫苗为痘病毒载体疫苗(pSC11-sPD1/MS)或腺病毒载体疫苗(pDC316-sPD1/MS)。In addition, 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-γ 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).
本发明提供了一种防治肿瘤的产品，包括本发明提供的DNA载体疫苗、重组蛋白疫苗和/或病毒载体疫苗。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.
本发明提供的防治肿瘤的产品中，可仅包含一种疫苗，也可包含两种疫苗或两者以上的疫苗组合。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.
另外，本发明实验表明，在免疫的同时给予化疗药物能够起到增效的作用。具体表现为，延长荷瘤小鼠的生存期。在疫苗与化药联合治疗组中，观察到重组sPD1/MS DNA疫苗与奥沙利铂的联合使得小鼠平均生存期较PBS组延长24％(P＜0.001)，其中重组sPD1/MS DNA疫苗联合奥沙利铂治疗组的生存期与单独的奥沙利铂治疗组(生命延长14％)相比，体现显著的生命延长作用(P＜0.05)。In addition, 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. In the combination of vaccine and chemical treatment, 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).
本发明提供的防治肿瘤的产品中，还包括化疗药物。所述化疗药物选自铂类化合物、紫杉醇或吉西他滨。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.
另外，研究表明，融合核酸疫苗CpDV-IL2-sPD1/MS以及联合免疫的CpVR-sPD1与CpDV-IL2-MS疫苗都使得T淋巴细胞分泌MS特异性IFN-γ的elispot斑点数目明显增多(P＜0.01)。同时，比较sPD1/MS疫苗与sPD1+MS联合免疫的方式，其所诱导产生的特异性细胞免疫反应处于较为一致的水平，表明sPD1基因与MS肿瘤抗原基因制备的疫 苗，具有融合核酸疫苗和联合共注射疫苗的多种疫苗形式。In addition, studies have shown that the fusion nucleic acid vaccine CpDV-IL2-sPD1/MS and the combined immunization of CpVR-sPD1 and CpDV-IL2-MS vaccines have significantly increased the number of elisapot spots that secrete MS-specific IFN-γ by T lymphocytes (P< 0.01). At the same time, comparing the sPD1/MS vaccine with sPD1+MS combined immunization, 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. A variety of vaccine forms for co-injection of vaccines.
本发明提供的另一种防治肿瘤的产品，包括以MS为靶点的疫苗和以sPD-1为靶点的疫苗。Another tumor-preventing product provided by the present invention includes a vaccine targeting MS and a vaccine targeting sPD-1.
所述以MS为靶点的疫苗由申请号为200910252427.X的中国发明专利构建。The MS-targeted vaccine was constructed by the Chinese invention patent No. 200910252427.X.
所述以sPD-1为靶点的疫苗为CpVR-sPD1，其构建方法为在载体CpVR的PstI和BamHI酶切位点间插入sPD1片段，所述sPD-1片段的5’端连接有tPA信号肽片段。所述CpVR载体由专利号为ZL 201110086366.1的发明专利构建。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.
在疫苗与化药联合治疗组中，观察到重组MS疫苗与奥沙利铂的联合使得小鼠平均生存期较PBS组延长22％(P＜0.001)。In the combination of vaccine and chemical treatment, the combination of recombinant MS vaccine and oxaliplatin was observed to increase the average survival of mice by 22% (P<0.001) compared with PBS.
本发明提供的防治肿瘤的产品中，还包括化疗药物。所述化疗药物选自铂类化合物、紫杉醇或吉西他滨。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.
本发明还提供了一种防治肿瘤的方法，该方法为给予本发明提供的防治肿瘤的产品。The present invention also provides a method for preventing and treating tumors, which comprises administering a tumor-preventing product provided by the present invention.
一些具体实施例中，给予本发明提供的产品后，以活体基因导入仪刺激注射位点；所述刺激的电压为36V；频率1Hz；脉冲6次；脉宽20ms。In some embodiments, after administration of the 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.
一些具体实施例中，给予本发明提供的防治肿瘤的产品的方法为：给予DNA载体疫苗。免疫的策略为采用DNA载体疫苗进行初免，再采用DNA载体疫苗进行加强免疫。所述初免的次数为1次，所述加强免疫的次数为2次。所述初免与加强免疫之间间隔7天，两次加强免疫间的时间间隔为7天。所述DNA载体疫苗为CpDV-IL2-sPD1/MS。In some embodiments, 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.
一些具体实施例中，给予本发明提供的防治肿瘤的产品的方法为：给予重组蛋白疫苗。免疫的策略为采用重组蛋白疫苗进行初免，再采用重组蛋白疫苗进行加强免疫。所述初免的次数为1次，所述加强免疫的次数为2次。所述初免与加强免疫之间间隔7天，两次加强免疫间的时间间隔为7天。所述重组蛋白疫苗由VR1012-sPD1/MS表达的融合蛋白制得。In some embodiments, 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.
一些具体实施例中，给予本发明提供的防治肿瘤的产品的方法为：给予病毒载体疫苗。免疫的策略为采用病毒载体疫苗进行初免，再采用病毒载体疫苗进行加强免疫。所述初免的次数为1次，所述加强免疫的次数为1次。所述初免与加强免疫之间间隔14天。所述病毒载体疫苗为痘病毒载体疫苗或腺病毒载体疫苗。In some embodiments, 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.
一些具体实施例中，给予本发明提供的防治肿瘤的产品的方法为：给予DNA载体疫苗和病毒载体疫苗。免疫的策略为采用DNA载体疫苗进行初免，再采用病毒载体疫苗进行加强免疫。所述初免的次数为3次，加强免疫的次数为1次。所述初免与加强免疫之间间隔2周，3次初免的时间间隔为2周。所述病毒载体疫苗为腺病毒载体疫苗。In some embodiments, 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.
一些具体实施例中，给予本发明提供的防治肿瘤的产品的方法为：给予DNA载体疫苗和病毒载体疫苗。免疫的策略为采用DNA载体疫苗进行初免，再采用病毒载体疫苗进行加强免疫。所述初免的次数为2次，加强免疫的次数为1次。所述初免与加强免疫之间间隔2周，2次初免的时间间隔为2周。所述病毒载体疫苗为痘病毒载体疫苗。In some embodiments, 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.
一些具体实施例中，给予本发明提供的防治肿瘤的产品的方法为：给予DNA载体疫苗和病毒载体疫苗。免疫的策略为采用DNA载体疫苗进行初免，再采用病毒载体疫苗进行加强免疫。所述初免的次数为2次，加强免疫的次数为1次。所述初免与加强免疫之间间隔2周，2次初免的时间间隔为2周。所述病毒载体疫苗为腺病毒载体疫苗。In some embodiments, 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.
一些具体实施例中，给予本发明提供的防治肿瘤的产品的方法为：给予DNA载体疫苗和病毒载体疫苗。免疫的策略为采用以MS为靶点的疫苗和以sPD-1为靶点的疫苗联合进行初免，再采用病毒载体疫苗进行加强免疫。所述初免的次数为2次，加强免疫的次数为1次。所述初免与加强免疫之间间隔2周，2次初免的时间间隔为2周。所述病毒载体疫苗为腺病毒载体疫苗。In some embodiments, 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.
一些具体实施例中，给予本发明提供的防治肿瘤的产品的方法为：给予DNA载体疫苗和病毒载体疫苗。免疫的策略为采用DNA载体疫苗进行初免，再采用病毒载体疫苗进行加强免疫。免疫同时给予化疗药物。所述初免的次数为2次，加强免疫的次数为1次。所述初免与加强免疫之间间隔7天，2次初免的时间间隔为7天。第1次给予化疗药物的时间为第2次初免后的次日，此后每5天给予化疗药物，共给药4次。。所述病毒载体疫苗为痘病毒载体疫苗。所述化疗药物为奥沙利铂。In some embodiments, 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.
本发明提供了分子组合，包括PD-1、MUC1和Survivin。以该抗原制备疫苗，能够具有良好的免疫原性与抗肿瘤活性。本发明提供的疫苗免疫可产生针对MUC1、Survivin的特异性抗体应答(见图5)和特异性细胞免疫应答(见图6)。与DNA疫苗CpDV-IL2-MS相比，CpDV-IL2-sPD1/MS在荷瘤小鼠中具有更显著的抗肿瘤效应，其中对黑素瘤的肿瘤生长抑制率约53％(见图10)，对黑素瘤荷瘤小鼠的生存期的生命延长率约19％(见图10)；本发明进一步提供了治疗性疫苗与化学治疗药物的联合方案。在一个实施案例中，本发明提供的疫苗与奥沙利铂联合给药抑瘤率达到74％，荷瘤小鼠肿瘤生长得到显著性抑制(见图11)；而本本发明提供的疫苗和奥沙利铂给药组小鼠的平均生存期62天，其生命延长效果与阳性对照奥沙利铂组相比具统计意义(见图11)；此外，将本发明提供的疫苗应用于小鼠乳腺癌、肺癌、肝癌和胃癌等模型的治疗(见图13、14、15、16，与阴性对照组相比，疫苗组的肿瘤生长速率都明显减缓。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). 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. 10); the present invention further provides a joint scheme of a therapeutic vaccine and a chemotherapeutic drug. In one embodiment, 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.
附图说明DRAWINGS
图1示DNA质粒载体及质粒CpDV-IL2-sPD1/MS的构建策略；其中，图1-a示重组DNA质粒CpDV-IL2-MS和质粒CpDV-IL2-sPD1/MS；图1-b示质粒T Easy-sPD1/MS的构建策略；图1-c示质粒T Easy-tPA/sPD1的构建策略；图1-d示载体CpDV-IL2的构建策略；图1-e示重组DNA质粒CpDV-IL2-sPD1/MS图谱；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;
图2示Western Blot印迹分析显示CpDV-IL2-MS、CpDV-IL2-sPD1/MS、CpVR-sPD1的真核蛋白表达情况；其中，图2-a示以抗MUC1抗体检测蛋白表达情况；图2-b示以抗Survivin抗体检测蛋白表达情况；图2-c示以抗PD-1抗体检测蛋白表达情况；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;
图3示pET28a-sPD1/MS质粒的构建；其中，图3-a示重组质粒pET28a-sPD1图谱；图3-b示重组质粒pET28a-sPD1/MS图谱；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;
图4示重组sPD1/MS病毒载体疫苗的构建；其中，图4-a示重组穿梭质粒pSC11-MS图谱；图4-b示重组穿梭质粒pSC11-sPD1/MS图谱；图4-c显示重组穿梭质粒pDC316-MS图谱；图4-d显示重组穿梭质粒pDC316-sPD1/MS图谱；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;
图5示sPD1/MS重组DNA疫苗诱导免疫小鼠的抗粘蛋白1和生存素的体液免疫应答；图5-a示Balb/c小鼠分别免疫PBS及重组MS、sPD1/MS和sPD1DNA质粒后的血清ELISA检测抗粘蛋白1抗体，图5-b示免疫小鼠抗生存素抗体水平；图5-c示免疫小鼠抗可溶性PD1抗体水平；图5-d示免疫小鼠中粘蛋白1的IgG1和IgG2a抗体的血清水 平；图5-e示免疫小鼠抗生存素的IgG1和IgG2a抗体的血清水平；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;
图6示sPD1/MS重组DNA疫苗引发小鼠体内特异性细胞免疫应答；Balb/c小鼠分别免疫PBS、及重组MS、sPD1/MS和sPD1DNA质粒，取脾淋巴细胞分析粘蛋白1和生存素特异性免疫应答水平；其中，图6-a示为各组免疫小鼠脾淋巴细胞针对MUC1表位特异性的分泌IFN-γ的Elispot斑点数目，图6-b示针对Survivin表位特异性分泌IFN-γ的Elispot斑点数目；图6-c示各组免疫小鼠的脾淋巴细胞的CTL活性，以脾淋巴细胞作效应细胞E，并选用稳定表达粘蛋白1和生存素蛋白的CT26细胞作靶细胞T，通过碘化丙啶和CFSE双荧光染料染色后所标记的杀伤靶细胞群百分比来表征CTL反应强度；图6-d示sPD1与MS肿瘤疫苗的联合形式对小鼠脾淋巴细胞特异性分泌IFN-γ的Elispot斑点数目；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-γ 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. The number of Elispot spots of IFN-γ; 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-γ;
图7示sPD1/MS重组DNA疫苗的Prime-Boost免疫策略；Balb/c小鼠分别免疫PBS、重组sPD1/MS DNA疫苗、重组腺病毒疫苗rAD-MS，以及DNA疫苗/rAD-MS疫苗，通过Elispot法检测各组免疫小鼠脾淋巴细胞针对粘蛋白1和生存素的特异性免疫应答活性；其中，图7-a示各组免疫小鼠脾淋巴细胞针对MUC1表位特异性的分泌IFN-γ的Elispot斑点数目，图7-b示针对Survivin表位特异性分泌IFN-γ的Elispot斑点数目；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. Among them, 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 γ, Figure 7-b shows the number of Elispot spots that specifically secrete IFN-γ for the Survivin epitope;
图8示sPD1/MS重组病毒疫苗的Prime-Boost免疫策略；Balb/c小鼠分别免疫PBS、rMVA-sPD1/MS，以及DNA疫苗/rMVA-sPD1/MS疫苗，通过Elispot检测各组小鼠脾淋巴细胞针对粘蛋白1和生存素的特异性免疫应答活性，其中，图8-a示各组小鼠脾淋巴细胞针对MUC1表位的特异性分泌IFN-γ的Elispot斑点数目，图8-b示针对Survivin表位特异性分泌IFN-γ的Elispot斑点数目；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-γ by spleen lymphocytes of each group against the MUC1 epitope, Figure 8-b Showing the number of Elispot spots that specifically secrete IFN-γ against the Survivin epitope;
图9示sPD1/MS重组DNA疫苗在黑素瘤模型小鼠的治疗作用；其中，图9-a示在C57BL/6小鼠皮下接种MS f +B16肿瘤细胞后，分别给予PBS、MS疫苗和sPD1/MS疫苗，测量肿瘤大小至接种后第17天的情况；图9-b示观察小鼠生存情况观察至肿瘤接种后第50天； 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;
图10示sPD1/MS重组病毒疫苗在黑素瘤模型小鼠的治疗作用；在C57BL/6小鼠皮下接种MS f +B16肿瘤细胞后，分别给予PBS、rMVA-sPD1/MS重组痘病毒疫苗和rDA-sPD1/MS重组腺病毒医疗，接种肿瘤后第12天测量记录肿瘤生长变化曲线； 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;
图11示sPD1/MS重组DNA疫苗在结直肠癌模型小鼠的治疗作用；在Balb/c小鼠皮下接种MS f +CT26肿瘤细胞后，分别给予PBS、MS疫苗、sPD1/MS疫苗、奥沙利铂、MS/奥沙利铂和sPD1/MS/奥沙利铂；其中，图11-a示测量肿瘤大小至接种后第25天的情况；图11-b示观察截止日当天处死全部小鼠，剥取肿瘤组织并测量瘤重；图11-c示小鼠生存情况观察至肿瘤接种后第65天； 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;
图12示sPD1/MS重组蛋白疫苗在结直肠癌模型小鼠的治疗作用；在Balb/c小鼠皮下接种MS f +CT26肿瘤细胞后，分别给予PBS、Al(OH)3佐剂，以及蛋白/Al(OH)3治疗，接种肿瘤后第26天处死全部小鼠；其中，图12-a示所剥取的各组小鼠肿瘤重量；图12-b示各组荷瘤小鼠脾淋巴细胞针对MUC1和Survivin表位的特异性分泌IFN-γ的能力； 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-γ against MUC1 and Survivin epitopes;
图13示sPD1/MS重组DNA疫苗在乳腺癌模型小鼠的治疗作用；在Balb/c小鼠皮下接种MS f +4T1肿瘤细胞，分别注射PBS和sPD1/MS疫苗，从接种肿瘤后第11天开始测量小鼠肿瘤体积，记录肿瘤生长曲线； 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.
图14示sPD1/MS重组DNA疫苗在肺癌模型小鼠的治疗作用；C57BL/6小鼠皮下接种MS f +Lewis肿瘤细胞，分别注射PBS和sPD1/MS DNA疫苗，从接种肿瘤后第8天开始测量并记录小鼠肿瘤生长曲线； 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;
图15示sPD1/MS重组DNA疫苗在肝癌模型小鼠的治疗作用；Balb/c小鼠皮下接种MS f +H22肿瘤细胞，分别注射PBS和sPD1/MS疫苗，从接种肿瘤后第12天开始测量并记录小鼠肿瘤生长曲线； 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;
图16示sPD1/MS重组DNA疫苗在胃癌模型小鼠的治疗作用；615小鼠皮下接种MS f +MFC肿瘤细胞，分别注射PBS和sPD1/MS疫苗，从接种肿瘤后第12天开始测量并记录小鼠肿瘤生长曲线； 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;
图17示使用活体基因导入仪器后，疫苗的细胞免疫效果得到显著提升；其中，图17-a示免疫策略；图17-b示免疫后淋巴细胞分泌IFN-γ的水平；图17-c示免疫后CTL水平；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-γ secreted by the lymphocytes after immunization; Figure 17-c shows CTL level after immunization;
图18示使用活体基因导入仪后，疫苗在抑制肿瘤生长方面得到显著提升；其中，图18-a示免疫策略；图18-b示免疫后肿瘤体积；图18-c示免疫后CTL水平；图18-d示免疫后ELISPOT检测结果。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.
具体实施方式Detailed ways
本发明提供了重组疫苗及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。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.
本文中所用的细胞培养、分子遗传学、核酸化学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。The cell culture, molecular genetics, nucleic acid chemistry, biochemistry, and immunology laboratory procedures used herein are all routine steps widely used in the corresponding art. Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.
抗原(antigen，Ag)，是指能够刺激机体产生(特异性)免疫应答，并能与免疫应答产物抗体和致敏淋巴细胞在体外结合，发生免疫效应(特异性反应)的物质。抗原为能够在呈递至宿主动物后诱导免疫反应的一段DNA或DNA片段；多肽、表位、半抗原或其任何组合。Antigen (Ag) 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.
“多肽”和“蛋白质”在本文中可互换使用，意指连续氨基酸残基的聚合物。术语“核酸”、“核苷酸”可互换使用，是指RNA、DNA、cDNA(互补DNA)或cRNA(互补RNA)及其衍生物，例如包含经修饰主链的形式。"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.
“融合”是指将两种或更多种蛋白融合表达以形成融合蛋白的技术。通常，通过使用重组DNA技术将编码两种或更多种蛋白的DNA片段符合读框地连接在一起，并进行蛋白质表达来获得融合蛋白。融合使用是指将MS与sPD-1的序列融合在一起，制得疫苗的方法。"Fusion" refers to the technique of fusion expression of two or more proteins to form a fusion protein. Generally, 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.
“联合使用”或“联合免疫”是指使用超过一种的治疗物对受试者进行治疗或免疫。“联合”没有限制对受试者给予治疗的顺序。例如，第一疫苗(例如，以MS为靶点的疫苗)可以在对受试者给予第二疫苗(例如，以sPD-1为靶点的疫苗)之前(例如，5分钟、15分钟、30分钟、45分钟、1小时、2小时、4小时、6小时、12小时之前)、与其同时或在其之后(例如，5分钟、15分钟、30分钟、45分钟、1小时、2小时、4小时、 6小时、12小时)给予。"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. For example, a first vaccine (eg, a vaccine that targets MS) can be administered prior to administering a second vaccine to the subject (eg, a vaccine that targets 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).
“防治”是指治疗、预防或改善疾病或与之相关的病况或症状。"Control" refers to the treatment, prevention or amelioration of a disease or a condition or symptom associated therewith.
“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。"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. In addition, the vector may also contain an origin of replication.
“宿主细胞”是指可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如BHK细胞，HEK293细胞或人细胞等的动物细胞。"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.
“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。"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. There are many 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.
“佐剂序列”是指分子内佐剂的序列，分子内佐剂是能够增强与之融合表达的目的蛋白(抗原)的免疫原性的佐剂，其通常为多肽片段。The "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.
疫苗(vaccine)是指为了预防、控制传染病的发生、流行，用于人体预防接种的疫苗类预防性生物制品。“DNA载体疫苗”是指，基于DNA或RNA(例如质粒，如表达质粒)的疫苗，其任选地还包含佐剂。“重组蛋白疫苗”是肿瘤疫苗的另一常见形式，不同于DNA疫苗或病毒载体疫苗，它是以抗原蛋白形式进入细胞，可直接激活树突状细胞；同时，由于蛋白形式无法整合到宿主细胞基因组，具有较高的安全性。“病毒载体疫苗”中所述病毒载体具有复制能力可作为外源基因的载体而保持自身传染性。Vaccine (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.
本发明采用的试材、试剂、仪器皆为普通市售品，皆可于市场购得。The test materials, reagents and instruments used in the present invention are all common commercial products, and are commercially available.
本发明中涉及以MS为靶点的疫苗其构建方法参照申请号为200910252427.X的发明专利。The method for constructing a vaccine targeting MS in the present invention refers to the invention patent of application No. 200910252427.X.
下面结合实施例，进一步阐述本发明：The present invention is further illustrated below in conjunction with the embodiments:
实施例1：重组DNA载体疫苗sPD1/MS的构建Example 1: Construction of recombinant DNA vector vaccine sPD1/MS
1.1 tPA/sPD1基因合成及T Easy-tPA/sPD1构建1.1 tPA/sPD1 gene synthesis and T Easy-tPA/sPD1 construction
根据GeneBank号NM_005018.2基因合成人PD-1胞外段序列，其基因序列5’端连有69bp tPA信号肽序列(见SEQ ID NO：10)，引入tPA信号肽的目的是为提高融合蛋白sPD1/MS在胞外的分泌表达能力。其后，采用PCR技术在tPA/sPD1基因两端引入酶切位点，PCR反应体系50μl，包括0.1μg SEQ ID NO：1所述的tPA/sPD1 DNA模板，引物SEQ ID NO：12和SEQ ID NO：13终浓度20pmol，5μl Ex Taq Buffer(10×)，4μl dNTP Mixture(各2.5mM)，1.25U Ex Taq酶(Takara公司)；PCR反应条件为98℃10s、55℃30s、72℃30s，共进行35个循环扩增反应。其后将胶回收的PCR产物连接于pGEM T Easy(promega公司)载体上，反应条件为16℃4h。取连接产物10μl，转化入感受态大肠杆菌Top10(Invitrogen公司)，涂布氨苄青霉素抗性平板，37℃培养16h。挑取单克隆于5ml氨苄抗性LB培养基，37℃220rpm震荡培养16h。高速离心收获所培养菌体后，质粒提取试剂盒(北京天根生化科技有限公司)提取质粒，用BglII和BamHI双酶切后，0.8％琼脂糖凝胶电泳鉴定阳性克隆，获得质粒T Easy-tPA/sPD1(见图1-b)。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. Thereafter, PCR was used to introduce a restriction enzyme site at both ends of the tPA/sPD1 gene, and the PCR reaction system was 50 μl, including 0.1 μg of the tPA/sPD1 DNA template described in SEQ ID NO: 1, primers SEQ ID NO: 12 and SEQ ID. NO: 13 final concentration 20 pmol, 5 μl Ex Taq Buffer (10×), 4 μl dNTP Mixture (2.5 mM each), 1.25 U Ex Taq enzyme (Takara); PCR reaction conditions were 98 ° C for 10 s, 55 ° C for 30 s, 72 ° C for 30 s A total of 35 cycles of amplification reaction were performed. Thereafter, the PCR product recovered by the gel was ligated to a pGEM T Easy (promega) vector under the reaction conditions of 16 ° C for 4 h. 10 μl of the ligation product was taken, transformed into competent E. coli Top10 (Invitrogen), and ampicillin-resistant plates were coated and cultured at 37 ° C for 16 h. 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).
1.2 CpVR-sPD1质粒的构建1.2 Construction of CpVR-sPD1 plasmid
CpVR单顺反子是发明人前期在VR1012质粒骨架插入CpG基序改造得来(参见申请号为201110086366.1的发明专利)，CpVR-sPD1独立表达可溶性PD-1蛋白，是作为一种sPD1/MS融合核酸疫苗的对照质粒形式。具体构建方法是：以PstI和BamHI双酶切消化质粒T Easy-tPA/sPD1，胶回收线性sPD1目的基因与载体CpVR相互连接，反应条件是16℃过夜。将连接产物10μl转化入感受态大肠杆菌Top10，涂布卡那霉素抗性平板，37℃培养16h。挑取单克隆于5ml卡那抗性LB培养基震荡培养16h。高速收获培养后的菌体，所提取质粒采用用PstI和BamHI双酶切消化后进行0.8％琼脂糖凝胶电泳的鉴定，筛选获得阳性克隆CpVR-sPD1。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. The specific construction method 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 μl 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.
1.3 CpDV-IL2-sPD1/MS质粒的构建1.3 Construction of CpDV-IL2-sPD1/MS plasmid
基于sPD1/MS重组DNA疫苗的构建主要分为两步。首先，将包含目的基因的质粒CpVR-MS(参见申请号为200910252427.X的发明专利)采用BglII和BamHI双酶切消化，通过T4DNA连接酶作用，将MS融合肿瘤抗原基因插入质粒T Easy-tPA/sPD的多克隆酶切位点BamHI处，从而获得质粒融合核酸质粒T Easy-sPD1/MS(见图1-c)。The construction of recombinant DNA vaccine based on sPD1/MS is mainly divided into two steps. First, 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).
其次，构建表达白介素2(IL-2)基因和sPD1/MS融合核酸的双顺反子CpDV-IL2-sPD1/MS，具体技术方法是：应用PCR技术扩增人白介素-2(IL-2)基因(序列详见SEQ ID NO：9)，在50μl PCR反应体系中，加入0.1μg模板质粒CpDV-IL2-MS(参见专利号为ZL 200910252427.X的发明专利)，终浓度20pmol的引物SEQ ID NO：14和SEQ ID NO：15，其它组分和反应条件如上文所述。PCR产物经过XbaI和BamHI双酶切消化反应，与载体CpDV(参见专利号为ZL201110086366.1的发明专利)相连接制备过渡质粒CpDV-IL2。再将包含sPD 1/MS融合核酸的T Easy-sPD1/MS进行BglII和EcoRI双酶切消化处理，插入到质粒骨架CpDV-IL2的相应多克隆酶切位点处，制成所述DNA载体疫苗CpDV-IL2-sPD1/MS(即sPD1/MS疫苗)，构建策略见图1-d。Secondly, construct 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. For the gene (see SEQ ID NO: 9 for details), 0.1 μg of the template plasmid CpDV-IL2-MS (see Patent No. ZL 200910252427.X) was added to a 50 μl 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. Then, 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.
1.4 重组质粒体外表达肿瘤抗原及sPD1的Western印迹鉴定1.4 Western blot analysis of recombinant plasmid expressing tumor antigen and sPD1 in vitro
以转染空载体VR1012的293T细胞裂解上清为阴性对照，将重组质粒CpDV-IL2-MS(简称MS)、CpDV-IL2-sPD1/MS(简称sPD1/MS)，和CpVR-sPD1(简称sPD1)各2μg分别转染293T细胞，72h后收取细胞裂解上清，经SDS-PAGE分离后转移到硝酸纤维素膜上进行Western Blot蛋白质印迹依次鉴定重组质粒抗原MUC1、Survivin和蛋白sPD1的印迹鉴定情况。结果如图2所示，其中图2-a显示MS质粒、sPD1/MS质粒和sPD1质粒的MUC1印迹，sPD1作为对照质粒没有显示条带，质粒MS和sPD1/MS的预期大小分别是95KD和120KD，为由图中可以看出，MS和sPD1/MS实际呈现系列梯度的蛋白条带，且主条带大小都在150KD以上，这可能是MUC1蛋白的糖基化修饰产物或特异性蛋白酶作用的裂解产物；图2-b显示MS质粒、sPD1/MS质粒和sPD1质粒的Survivin印迹，由于Survivin与MUC1是融合抗原，所以呈现的Western印迹与MUC1结果相似，其主条带位置都在150KD以上；图2-c显示MS质粒、sPD1/MS质粒、sPD1质粒和空载体VR1012的PD-1印迹，空载体作为阴性对照没有显示条带，而sPD1/MS质粒和sPD1质粒的大小分别为150KD和35KD，由该图中可见，融合表达的sPD1/MS蛋白没有关于sPD1蛋白的糖基化修饰或水解作用，保证了sPD1的结构完整性从而能够结合到PD-L1 配体上。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). 2μg 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. . The results are shown in Figure 2, in which Figure 2-a 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. As can be seen from the figure, 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. The cleavage product; Figure 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. As can be seen from the figure, 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.
本发明所述的CpDV-IL2-sPD1/MS重组基因疫苗是一种基于肿瘤抗原基因疫苗的优化形式，质粒结构示意如图1-a所示。肿瘤抗原MUC1和Survivin属于一类肿瘤相关抗原(TAA)，TAA是源于自身的抗原而非外源抗原，这导致其引发的特异性T细胞免疫反应有所限制。为了提高肿瘤基因疫苗的有效性，采用把肿瘤抗原与具有阻断免疫抑制作用的可溶性程序死亡-1(PD-1)相融合的DNA疫苗设计。可溶性PD-1与肿瘤抗原的融合表达，使基于sPD1的融合蛋白能够通过受体介导靶向DC细胞，促进DC对抗原的摄取并激活B细胞和T细胞。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. In order to improve the effectiveness of tumor gene vaccines, 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.
实施例2：重组蛋白疫苗sPD1/MS的制备Example 2: Preparation of recombinant protein vaccine sPD1/MS
蛋白疫苗是肿瘤疫苗的另一常见形式，不同于DNA疫苗或病毒载体疫苗，它是以抗原蛋白形式进入细胞，可直接激活树突状细胞；同时，由于蛋白形式无法整合到宿主细胞基因组，具有较高的安全性。本实施例构建原核表达载体pET28a-sPD1/MS，诱导表达融合蛋白疫苗(sPD1/MS)，具体的构建过程如下：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. In this example, 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:
采用同源重组方法构建原核表达质粒pET28a-sPD1/MS：将pET28a质粒(见图3-a)用Nde I和Xho I双酶切后，胶回收载体序列5289bp片段。PCR扩增CpDV-IL2-sPD1/MS中sPD1/MS顺反子的序列，引物5’端具有与切下来的pET28a载体两端同源的序列并带有相应的酶切位点(Nde1和Xho1)，引物序列SEQ ID NO.19The 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
GATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTAATCCATGGCAGCCAGCTG和SEQ ID NO.20AGCGGCCTGGTGCCGCGCGGCAGCCATATGGATGCAATGAAGAGAGGGC。GATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTAATCCATGGCAGCCAGCTG and SEQ ID NO. 20AGCGGCCTGGTGCCGCGCGGCAGCCATATGGATGCAATGAAGAGAGGGC.
再根据无缝克隆试剂盒-Seamless Assembly Cloning Kit(货号C5891)说明书进行操作将目的片段sPD1/MS与载体连接起来得到了pET28a-sPD1/MS(见图3-b)。将质粒转化至BL21感受态中进行扩增，加入IPTG，37℃诱导表达，4-6小时后收集菌体，对菌体进行超声裂解后弃去上清；将沉淀以包涵体溶解Buffer重悬，4℃磁力搅拌溶解过夜；离心，收上清，进行Ni-NTA亲和层析柱纯化，进行咪唑梯度洗脱，最后获得纯化的贮存终浓度1mg/ml的sPD1/MS融合蛋白。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. After 4-6 hours, 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.
实施例3：重组病毒载体疫苗sPD1/MS的构建Example 3: Construction of recombinant viral vector vaccine sPD1/MS
3.1 重组痘病毒苗rMVA-sPD1/MS的获得3.1 Acquisition of recombinant pox virus vaccine rMVA-sPD1/MS
应用改良的安卡拉痘病毒(Modified Vaccinia Ankara，MVA)协载肿瘤抗原免疫治疗肿瘤，其安全性较高。MVA的穿梭质粒是pSC11，其具有的多克隆酶切位点可以插入外源基因，本实例中将sPD1基因插入前期构建完成的质粒pSC11-MS(参见专利号为ZL200910252427.X的发明专利)，构建pSC11-sPD1/MS，载体上具有的胸苷激酶左臂和右臂(TKL、TKR)可与MVA同源重组，同时通过载体上lacZ基因，进行重组MVA(rMVA)的蓝斑筛选。The use of modified Vaccinia Ankara (MVA) to co-load tumor antigen immunotherapy for tumors is safe. The shuttle plasmid of MVA is pSC11, which has a polyclonal cleavage site for insertion of a foreign gene. In this example, 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.
构建pSC11-sPD1/MS穿梭质粒的具体方法，首先通过引物SEQ ID NO：16和SEQ ID NO：17进行PCR反应，获得两端引入酶切位点的tPA-sPD1基因，其后采用SalI和XhoI同尾酶进行tPA/sPD1的双酶切消化，用SalI单酶切消化pSC11-MS质粒(图4-a)，DNA凝胶回收目的基因和载体骨架，16℃连接过夜。将连接产物转化入Top10感受态，使用氨苄青霉素抗性的LB培养基培养获得重组的融合pSC11-sPD1/MS质粒(见图4-b)。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).
接下来为了获得重组痘病毒，先将滴度0.05pfu/cell的空MVA病毒感染含有TK基因的BHK-21的突变细胞，孵育2小时后，使用lipo2000试剂将穿梭质粒pSC11-sPD1/MS共转染到BHK细胞。被感染的细胞在37℃，5％CO2下培养72小时后，收集细胞。超声波裂解细胞，2000rpm离心10min保留裂解上清。取适量上清作种毒，于六孔板中将痘病毒依次稀释成10 -2、10 -3、10 -4、10 -5、10 -6感染BHK细胞2小时。弃去培养基，用等体积含有1％BrdU(抑制野生MVA生长)且42℃预温的2％低熔点琼脂糖和2×DMEM-20培养基混匀，每孔加入2ml培养基，在室温下凝固，在37℃含5％CO2的培养箱中培养48小时。铺上层选择性培养基，成分比下层琼脂多了1/120体积的4％X-gal。培养过夜，挑选蓝色单斑，反复冻融三次裂解释放病毒，进行下一轮筛选，步骤相同，如此进行6轮以上筛选，最后直至得到只含重组病毒MVA-MS的克隆株。 Next, in order to obtain the recombinant poxvirus, 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. After culturing overnight, 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.
3.2 重组腺病毒疫苗的获得3.2 Acquisition of recombinant adenovirus vaccine
腺病毒AdMax TM Adenovirus载体系统(Microbix公司)包含穿梭载体和腺病毒载体。本实例采用前期构建的穿梭质粒pDC316-MS(参见专利号为200910252427.X的发明专利，图4-c)，将sPD1基因通过PCR方法，由引物SEQ ID NO：18和SEQ ID NO：13在基因的5’和3’端引入EcoRI酶切位点，对pDC316-MS和sPD1基因的PCR产物EcoRI双酶切消化，16℃连接过夜。连接产物转化入Top10感受态，并在37℃氨苄青霉素抗性的LB培养基中培养，经过质粒测序筛选获得阳性克隆pDC316-sPD1/MS(见图4-d)。 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).
其后进行腺病毒(AD)的同源重组，本实例中的腺病毒骨架为pBHGloxΔE1，3Cre。将AD载体和穿梭质粒pDC316-sPD1/MS共转染293细胞，利用293细胞中的E1蛋白，经过10天左右即产生含有sPD1/MS融合核酸的重组腺病毒噬斑。对噬斑进行PCR鉴定，反应条件95℃30s、55℃30s、72℃1min，反应30个循环，从而筛选正确的噬斑进行大量的扩增和纯化。Thereafter, homologous recombination of adenovirus (AD) was carried out, and the adenoviral skeleton in this example was pBHGloxΔE1, 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.
实施例4：sPD1/MS肿瘤疫苗的免疫原性Example 4: Immunogenicity of sPD1/MS tumor vaccine
4.1 sPD1/MS融合核酸疫苗的体液免疫活性4.1 Humoral immunological activity of sPD1/MS fusion nucleic acid vaccine
ELISA方法检测sPD1/MS融合DNA疫苗针对MUC1和Survivin的抗体反应，并与MS疫苗(参见专利号为ZL 200910252427.X的发明专利)的体液免疫效果进行比较。选用4-6周龄Balb/c小鼠，按表1设置sPD1/MS疫苗实验组、MS疫苗对照组、单独sPD1质粒对照组和PBS阴性对照组，上述DNA质粒的制备均以无菌PBS溶解，终浓度调节至1mg/ml。具体的免疫程序是：第0周和第2周以DNA载体疫苗初免，第4周以重组腺病毒疫苗加强免疫，选取小鼠后侧肢肌肉注射疫苗各50μg。最后一次免疫后两周对小鼠进行眼眦采血，离心分离的血清用于ELISA抗体检测。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 μg 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.
表1 sPD1/MS融合核酸疫苗与MS核酸疫苗的免疫原性Table 1 Immunogenicity of sPD1/MS fusion nucleic acid vaccine and MS nucleic acid vaccine
Figure PCTCN2018116393-appb-000001
Figure PCTCN2018116393-appb-000001
结果显示，接种sPD1/MS疫苗的小鼠能产生高效的抗粘蛋白1抗体和抗生存素抗体，并且两者的抗体滴度可达10000以上。如图5-a和5-b所示，按1∶125比例稀释的小鼠血清分别以原核表达并纯化收获的MUC1蛋白、Survivin蛋白和可溶sPD1蛋白作为抗原检测特异性抗体反应，其中sPD1/MS疫苗组与MS疫苗组相比，抗MUC1抗体以及抗Survivin抗体吸光度(450nm)读出值都有显著性提高(P＜0.001)，说明优化的sPD1/MS融合核酸肿瘤疫苗能够启动强烈的特异性体液免疫应答；而接种CpVR-sPD1质粒组与PBS组ELISA抗体反应都呈阴性。另外，ELISA检测到sPD1/MS疫苗组以及sPD1质粒组小鼠血清能产生一定的抗PD1抗体反应(见图5-c)，然而PD1抗体滴度较低无法在体内产生大量的抗体来阻断PD-1负性信号通路，表明sPD1融合核酸疫苗可能是通过激活小鼠的免疫系统发挥抗肿瘤作用的。同时，sPD1/MS疫苗在诱导抗原特异性IgG1(Th2型)和IgG2a(Th1型)应答上也体现比MS疫苗组更为明显的优势(见图5-d、5-e)。The results showed that 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. As shown in Figures 5-a and 5-b, 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. In addition, 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. At the same time, 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).
4.2 sPD1/MS融合核酸疫苗细胞免疫活性检测4.2 sPD1/MS fusion nucleic acid vaccine cell immunoreactivity assay
为了考察重组sPD1/MS DNA疫苗对粘蛋白1和生存素的特异性免疫应答，应用Elispot法测定表1免疫小鼠脾淋巴细胞特异性分泌IFN-γ的斑点数目，同时检测各组免疫小鼠淋巴细胞CTL杀伤活性。Elispot检测结果如图6-a所揭示，经过MUC1蛋白刺激，sPD1质粒组脾细胞对蛋白刺激无应答，显示单独sPD1蛋白无法激活机体特异性细胞免疫效应；MS疫苗组脾细胞分泌的IFN-γ斑点数目较PBS阴性对照组有2倍数值的差异，而sPD1/MS疫苗组脾细胞分泌IFN-γ斑点数目则明显高出PBS阴性组斑点数目数倍，sPD1/MS疫苗与MS疫苗组斑点数目比较没有体现差异。另外，经过Survivin蛋白刺激，结果如图6-b显示，单独的sPD1质粒免疫小鼠同样没有应答反应；而MS疫苗组和sPD1/MS疫苗组免疫小鼠的脾细胞分泌IFN-γ的斑点与PBS阴性对照组相比，均呈现阳性反应，sPD1/MS疫苗与MS疫苗组两组之间的斑点数目相比较，sPD1/MS疫苗显著多于MS，且具P值0.0141的统计学意义。In order to investigate the specific immune response of mucin 1 and survivin by recombinant sPD1/MS DNA vaccine, the number of spots secreting IFN-γ secreted by spleen lymphocytes of mice immunized with Table 1 was determined by Elispot method, and the mice of each group were also tested. Lymphocyte CTL killing activity. The results of Elispot test are shown in Figure 6-a. After stimulation with MUC1 protein, sPD1 plasmid group spleen cells did not respond to protein stimulation, indicating that sPD1 protein alone could not activate the body-specific cellular immune effect; IFN-γ secreted by spleen cells of MS vaccine group The number of spots was twice as large as that of the PBS negative control group, while the number of IFN-γ spots secreted by the sPD1/MS vaccine group was significantly higher than that of the PBS negative group, and the number of spots in the sPD1/MS vaccine and the MS vaccine group. The comparison does not reflect the difference. In addition, after Survivin protein stimulation, the results are shown in Figure 6-b. The mice immunized with the sPD1 plasmid alone did not respond. The spleen cells of the MS vaccine group and the sPD1/MS vaccine group secreted IFN-γ 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.
以免疫小鼠脾淋巴细胞作为效应细胞，以稳定表达融合肿瘤抗原蛋白MS的CT26细胞(命名为MS f +CT26，其中f为full缩写，代表未截短N端氨基酸的全长生存素蛋白)作为靶细胞，按照一定效靶比例混合后采用碘化丙啶(PI)/CFSE双色荧光染料标记法检测CTL杀伤活性，结果见图6-c。由图可知，MS疫苗组和sPD1/MS疫苗组在效靶比12.5∶1和50∶1时，都表征出高于PBS组的CTL杀伤效应；MS疫苗组和sPD1/MS疫苗组之间比较，sPD1/MS疫苗对应不同效靶比的杀伤率分别为34.76％和35.56％，而MS疫苗杀伤率分别为21.65％和30.39％，显然优化的疫苗形式CpDV-IL2-sPD1/MS更具CTL杀伤活性。 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) As target cells, 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. As can be seen from the figure, 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. Obviously, the optimized vaccine form CpDV-IL2-sPD1/MS has more CTL killing. active.
综上，在CpDV-IL2-sPD1/MS疫苗与CpDV-IL2-MS疫苗的免疫原性评价中，优化的疫苗形式sPD1/MS能够诱导更高效的特异性体液免疫应答和细胞免疫应答。In summary, in the immunogenicity evaluation of CpDV-IL2-sPD1/MS vaccine and CpDV-IL2-MS vaccine, the optimized vaccine form sPD1/MS was able to induce a more efficient specific humoral immune response and cellular immune response.
4.3 sPD1与MS肿瘤疫苗的联合形式对小鼠免疫活性的作用4.3 The effect of the combination of sPD1 and MS tumor vaccine on immune activity in mice
通常多基因疫苗的制备方式包括两种，一种是制备多基因融合表达的融合核酸疫苗，另一种则是以共注射的方式将两种或两种以上基因疫苗进行联合免疫。为了探讨sPD1基因联合MS基因所制备疫苗形式的多样性，本部分实验比较sPD1/MS重组DNA疫苗，与sPD1联合MS疫苗所诱导的小鼠免疫活性。具体实验分组如表2所示：4-6周龄小鼠 随机分成PBS组、sPD1/MS组和sPD1+MS组，其中设置PBS组小鼠分别在第0、2和4周注射100μl无菌PBS作为阴性对照；sPD1/MS组小鼠在第0和2周注射重组sPD1/MS融合核酸疫苗，第4周以rAD-MS重组腺病毒疫苗加强免疫；sPD1+MS组小鼠在第0和2周注射事先混匀的CpVR-sPD1质粒和CpDV-IL2-MS质粒(各100μg)，第4周同样采用重组腺病毒苗加强免疫。Generally, 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. In order to investigate the diversity of vaccine forms prepared by the sPD1 gene in combination with the MS gene, 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 μl sterilized at 0, 2 and 4 weeks respectively. PBS was used as a negative control; 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 μg each) were injected at 2 weeks, and the recombinant adenovirus vaccine was also boosted at the 4th week.
表2 sPD1与MS疫苗的联合形式Table 2 Combination of sPD1 and MS vaccine
Figure PCTCN2018116393-appb-000002
Figure PCTCN2018116393-appb-000002
第6周检测各组小鼠脾淋巴细胞特异性分泌IFN-γ的能力，结果如图6-d所示，与PBS阴性对照组相比，融合核酸疫苗CpDV-IL2-sPD1/MS以及联合免疫的CpVR-sPD1与CpDV-IL2-MS疫苗都使得T淋巴细胞分泌MS特异性IFN-γ的elispot斑点数目明显增多(P＜0.01)。同时，比较sPD1/MS疫苗与sPD1+MS联合免疫的方式，其所诱导产生的特异性细胞免疫反应处于较为一致的水平，表明sPD1基因与MS肿瘤抗原基因制备的疫苗，具有融合核酸疫苗和联合共注射疫苗的多种疫苗形式。The ability of spleen lymphocytes to specifically secrete IFN-γ in each group was detected at week 6. The results are shown in Figure 6-d. Compared with the PBS negative control group, the fusion nucleic acid vaccine CpDV-IL2-sPD1/MS and combined immunization Both CpVR-sPD1 and CpDV-IL2-MS vaccines significantly increased the number of elisapot spots secreting MS-specific IFN-γ by T lymphocytes (P<0.01). At the same time, comparing the sPD1/MS vaccine with sPD1+MS combined immunization, 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. A variety of vaccine forms for co-injection of vaccines.
实施例5：sPD1/MS肿瘤疫苗的免疫策略Example 5: Immunization strategy for sPD1/MS tumor vaccine
5.1 sPD1/MS重组DNA疫苗的免疫策略5.1 Immunization strategy for sPD1/MS recombinant DNA vaccine
本部分实验采用DNA初免-重组腺病毒加强免疫的免疫策略，考察重组腺病毒疫苗rAD-MS对sPD1/MS疫苗所诱导的粘蛋白1和生存素特异性免疫应答的加强作用：将4-6周龄小鼠按表3进行随机分组，每组6只，其中第1组肌肉注射PBS作为阴性对照；第2组在第0周单独免疫重组腺病毒疫苗rAD-MS(1×10 8pfu/只)；第3组分别于第-4、-2和0周注射DNA疫苗；第4组则在第-6、-4和-2周注射DNA疫苗，第0周注射重组腺病毒疫苗。所有组别小鼠于第2周拉颈处死后无菌取脾，应用Elispot法评价免疫小鼠针对粘蛋白1和生存素表位的免疫应答水平。 In this part of the experiment, 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 survivin epitopes was evaluated by the Elispot method.
表3 DNA初免-重组腺病毒加强免疫策略Table 3 DNA priming - recombinant adenovirus boosting strategy
Figure PCTCN2018116393-appb-000003
Figure PCTCN2018116393-appb-000003
图7-a和图7-b为表2对应的各组免疫小鼠脾淋巴细胞Elispot检测结果。由图可以看出，经过MUC1表位(SEQ ID NO：21)或者Survivin表位(SEQ ID NO：22)刺激的各组脾淋巴细胞只有第3组和第4组产生了阳性反应，且与PBS阴性对照组相比，其特异 性分泌IFN-γ斑点数目依次增多(依次P＜0.01)，而单独的重组腺病毒rAD-MS疫苗免疫组为阴性结果，说明单独免疫重组腺病毒无法诱导小鼠体内强烈的细胞免疫应答；同时，通过比较DNA疫苗组和DNA/rAD组的结果发现，DNA/rAD组明显增强特异性免疫应答活性(P＜0.05)，说明DNA初免-重组腺病毒加强免疫的免疫策略可以显著提升sPD1/MS疫苗的免疫效果。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. As can be seen from the figure, only the third and fourth groups of spleen lymphocytes stimulated by the MUC1 epitope (SEQ ID NO: 21) or the Survivin epitope (SEQ ID NO: 22) produced a positive reaction, and Compared with the PBS negative control group, the number of specific secreted IFN-γ 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 The strong cellular immune response in the mice; at the same time, by comparing the DNA vaccine group and the DNA/rAD group, the DNA/rAD group significantly enhanced the specific immune response activity (P<0.05), indicating DNA priming-recombinant adenovirus enhancement. Immunization strategies can significantly improve the immune response of the sPD1/MS vaccine.
5.2 sPD1/MS重组病毒疫苗的免疫策略5.2 Immune strategy for sPD1/MS recombinant virus vaccine
本部分实验以sPD1/MS重组痘病毒疫苗作为考察对象。将4-6周龄C57BL/6小鼠按表4随机分组，每组5只小鼠，其中阴性对照组小鼠分别在第-4、-2和0周注射无菌PBS，单独免疫痘病毒组小鼠于第0周免疫rMVA-sPD1/MS，每只小鼠注射剂量为1×10 8pfu，另一实验组小鼠在第-4和-2周采用sPD1/MS重组DNA疫苗进行初免，第0周用sPD1/MS重组痘病毒疫苗进行免疫加强。所有组别小鼠于第2周拉颈处死后无菌取脾，应用Elispot法评价免疫小鼠针对粘蛋白1和生存素表位的免疫应答水平。 This part of the experiment was based on sPD1/MS recombinant poxvirus vaccine. 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.
表4 sPD1/MS重组病毒疫苗的免疫策略Table 4 Immunization strategies for sPD1/MS recombinant virus vaccine
Figure PCTCN2018116393-appb-000004
Figure PCTCN2018116393-appb-000004
结果如图8-a和8-b显示，与PBS阴性对照相比，单独免疫重组痘病毒苗的小鼠诱导产生了阳性的粘蛋白1和生存素特异性分泌IFN-γ的Elispot斑点(P＜0.01)；而采用sPD1/MS重组DNA疫苗初免、重组sPD1/MS痘病毒苗加强免疫的免疫策略的小鼠，其体内诱导产生高效的特异性细胞免疫应答，且与rMVA免疫组相比，所产生Elispot斑点数目显著增多(P＜0.01)。以上结果表明融合表达sPD1/MS的重组病毒疫苗具有激活小鼠特异性免疫的能力，并且应用Prime-boost免疫策略有利于这种激活作用的增强。The results are shown in Figures 8-a and 8-b. Compared to the PBS negative control, mice immunized with recombinant poxvirus alone induced positive mucin 1 and survivin-specific secretion of IFN-γ-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.
实施例6：sPD1/MS重组DNA疫苗对黑素瘤模型小鼠的抗肿瘤作用Example 6: Anti-tumor effect of sPD1/MS recombinant DNA vaccine on melanoma model mice
6.1 疫苗对肿瘤生长的抑制作用6.1 Inhibition of tumor growth by vaccine
肿瘤接种实验检测重组sPD1/MS DNA疫苗对黑素瘤模型小鼠的治疗作用：将4-6周龄C57BL/6小鼠按表5随机分组(8只/组)。第0天，在各组小鼠右侧近尾端背部皮下接种1×10 5个稳定表达融合肿瘤抗原33M和全长Survivin蛋白的B16细胞(以下简称MS f +B16)，第1、8、15天分别给予疫苗注射，其中第1组注射PBS作为阴性对照，第2、3组则分别注射MS疫苗和sPD1/MS疫苗。攻瘤后第7天开始观察肿瘤在小鼠体内的生长情况，实验以对照组小鼠发生死亡为观察结点。 Tumor inoculation experiments were performed to examine the therapeutic effect of recombinant sPD1/MS DNA vaccine on melanoma model mice: 4-6 week old C57BL/6 mice were randomly grouped according to Table 5 (8/group). On day 0, 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. On the 7th day after the tumor attack, the growth of the tumor in the mice was observed. The experiment took the death of the control mice as the observation node.
图9-a为黑素瘤模型小鼠各组肿瘤生长体积变化情况。与阴性对照PBS组相比，MS疫苗与sPD1/MS疫苗都减缓了黑色素瘤的生长，其中sPD1/MS疫苗表现显著抑瘤作用(P＜0.01)，截止观察终点其肿瘤体积抑制率达52.62％。而sPD1/MS疫苗与MS疫苗比较，从接种肿瘤第15天直至观察截止日，肿瘤生长速率减缓明显(P＜0.05)，由此看来，融合sPD1的CpDV-IL2-sPD1/MS DNA疫苗能够优化疫苗对荷瘤小鼠的抑瘤作用。Figure 9-a shows the changes in tumor growth volume of each group of melanoma model mice. Compared with the negative control PBS group, 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. . Compared with the MS vaccine, 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.
表5重组sPD1/MS DNA疫苗治疗黑色素瘤模型小鼠Table 5 Recombinant sPD1/MS DNA vaccine treatment of melanoma model mice
Figure PCTCN2018116393-appb-000005
Figure PCTCN2018116393-appb-000005
6.2 黑色素瘤荷瘤小鼠的生存期研究6.2 Survival study of melanoma-bearing mice
按表5分组进行黑素瘤小鼠生存期的观察，观察截止时间到接种肿瘤后第50天。结果如图9-b所示，PBS组平均生存期为36天，全部死亡截止则为接种肿瘤后第40天。MS疫苗组小鼠平均生存38.5天，生命延长率6.94％，与阴性对照PBS组相比没有生命延长作用。而sPD1/MS DNA疫苗组小鼠平均生存寿命43天，较对照组小鼠生命延长19.44％(P值0.0325)。尽管sPD1/MS DNA疫苗治疗荷瘤小鼠的生存期与MS疫苗治疗组之间不具统计学意义，但从小鼠平均生存寿命和生命延长趋势上，重组sPD1/MS DNA疫苗更具优势。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.
实施例7：sPD1/MS重组病毒疫苗对黑素瘤模型小鼠的抗肿瘤作用Example 7: Anti-tumor effect of sPD1/MS recombinant virus vaccine on melanoma model mice
为了考察sPD1/MS融合核酸疫苗的不同形式对肿瘤的抑制作用，应用rMVA-sPD1/MS和rAD-sPD1/MS重组病毒对黑素瘤荷瘤小鼠进行抗肿瘤免疫治疗。将4-6周龄的C57BL/6小鼠随机分为PBS阴性组、痘病毒疫苗治疗组和腺病毒疫苗治疗组，每组各5只小鼠。各组小鼠在第0天接种5×10 4个MS f +B16细胞，接种后第1和15天对治疗组小鼠分别进行sPD1/MS重组痘病毒疫苗或重组腺病毒疫苗的免疫，阴性组小鼠则接种等体积无菌PBS作为对照。攻瘤后第10天开始观察各组小鼠的肿瘤生长情况，结果如图10显示，与PBS阴性对照组相比，rMVA-sPD1/MS疫苗组和rAD-sPD1/MS疫苗组小鼠的肿瘤生长趋势皆有较明显的减缓(P＜0.05)，截止到攻瘤后第24天为止，sPD1/MS重组痘病毒治疗小鼠的平均肿瘤体积比对照组的肿瘤体积减小约25％，sPD1/MS重组腺病毒治疗小鼠的平均肿瘤体积则减小约23％，表明sPD1/MS融合核酸的重组病毒载体疫苗形式具有有效抑制肿瘤生长的作用。 To investigate the inhibitory effects of different forms of sPD1/MS fusion nucleic acid vaccine on tumors, anti-tumor immunotherapy was performed on melanoma-bearing mice using rMVA-sPD1/MS and rAD-sPD1/MS recombinant viruses. 4-6 week old C57BL/6 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. Group 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). By the 24th day after the tumor attack, 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.
实施例8：sPD1/MS重组DNA疫苗对结直肠癌模型小鼠的抗肿瘤作用Example 8: Anti-tumor effect of sPD1/MS recombinant DNA vaccine on colorectal cancer model mice
8.1 疫苗对肿瘤生长的抑制作用8.1 Inhibition of tumor growth by vaccine
本部分实验目的一是考察疫苗对结直肠癌模型小鼠体内肿瘤生长的抑制作用和对荷瘤小鼠的生命延长作用，另一方面，以化疗药物奥沙利铂作为阳性对照，通过疫苗与化药奥沙利铂的联合免疫治疗来考察二者对模型小鼠肿瘤治疗的协同效果。将4-6周龄Balb/c小鼠按表3进行分组，每组10只，第0天在小鼠右侧近尾端后背皮下接种1×10 6个MS f +CT26肿瘤细胞。第1、8、15天分别对各组小鼠肌肉注射疫苗，其中第1组为阴性对照注射PBS，第2、5组给予MS疫苗，第3、6组给予sPD1/MS疫苗，表6中奥沙利铂(Oxaliplatin，江苏海正药业)采用腹腔注射方式给药，每次注射剂量0.5mg/kg，间隔5天给药一次共5次。攻瘤后第12天开始记录肿瘤在小鼠体内的生长情况，以PBS对照组小鼠开始死亡为观察时间结点。为计算各实验组的肿瘤抑制率，在观察结点拉颈处死全部小鼠，剥离肿瘤并称量瘤重。 The purpose of this part of the experiment is to investigate the inhibitory effect of vaccine on tumor growth in mice with colorectal cancer and the life extension of tumor-bearing mice. On the other hand, the chemotherapy drug oxaliplatin was 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, and 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. On the 12th day after the tumor attack, 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.
图11-a为结直肠癌模型小鼠肿瘤生长体积变化曲线。由图得知，sPD1/MS疫苗组与 PBS阴性组相比肿瘤生长明显减缓，从第23天开始即表现肿瘤体积的减小(P＜0.05)。同时，从瘤重计算的各组肿瘤抑制率(见图11-b)来看，sPD1/MS疫苗的抑瘤率达30.96％，比MS疫苗组17.18％的抑瘤率有提升的趋势但没有统计学意义。疫苗与化药奥沙利铂的联合治疗，结果显示sPD1/MS疫苗与奥沙利铂的联合使得肿瘤生长几乎停滞，抑瘤率74.71％，而奥沙利铂组抑瘤率58.62％。sPD1/MS疫苗与化药联合组与单独的奥沙利铂阳性对照相比，抑制肿瘤生长效果明显(P＜0.01)，相比之下，MS疫苗与化药的联合抑瘤结果则与奥沙利铂组没有统计学意义。说明sPD1/MS疫苗在显著抑制肿瘤生长的同时，能够与化药奥沙利铂发挥协同抗肿瘤作用。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%. Compared with the oxaliplatin-positive control alone, the sPD1/MS vaccine and chemical combination group had a significant effect on inhibiting tumor growth (P<0.01). In contrast, 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.
表6 sPD1/MS DNA疫苗治疗结直肠癌模型小鼠的免疫策略Table 6 Immune strategies of sPD1/MS DNA vaccine in mice with colorectal cancer
Figure PCTCN2018116393-appb-000006
Figure PCTCN2018116393-appb-000006
8.2 结直肠癌荷瘤小鼠的生存期研究8.2 Survival study of colorectal cancer-bearing mice
按表6的分组进行结直肠癌荷瘤小鼠的生存期观察，观察截止时间为接种肿瘤后第65天。结果如图11-c所示，重组MS疫苗组小鼠平均生存48.5天，重组sPD1/MS疫苗组小鼠平均生存期为52天，与阴性对照(平均生存50天)均没有明显生命延长趋势；而在疫苗与化药联合治疗组中，观察到重组MS疫苗或重组重组sPD1/MS DNA疫苗与奥沙利铂的联合使得小鼠平均生存期较PBS组分别延长22％和24％(P＜0.001)，其中重组sPD1/MS DNA疫苗联合奥沙利铂治疗组的生存期与单独的奥沙利铂治疗组(生命延长14％)相比，体现一定的生命延长作用(P＜0.05)。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). In the combination of vaccine and chemical treatment, 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). .
实施例9：sPD1/MS重组蛋白疫苗对结直肠癌荷瘤小鼠的抑瘤作用Example 9: Antitumor effect of sPD1/MS recombinant protein vaccine on colorectal cancer-bearing mice
根据实施例4和实施例5可知sPD1/MS重组核酸形式的疫苗可以诱导小鼠体内产生强烈的MUC1和Survivin特异性细胞免疫，本实施例中则采用sPD1/MS重组蛋白疫苗，同时结合铝佐剂Al(OH)3加强免疫的策略进行结直肠癌模型的治疗。具体分组情况见表7：随机地将4-6周龄Balb/c小鼠分成PBS阴性对照组、Al(OH)3佐剂组以及佐剂联合重组sPD1/MS蛋白疫苗治疗组，每组5只小鼠。各组小鼠于第0天接种1×10 6个MS f +CT26肿瘤细胞，第1、8和15天则接受疫苗治疗，其中阴性组小鼠给予100μl体积的无菌PBS溶液，Al(OH)3佐剂组小鼠注射等体积100μg剂量的Al(OH)3佐剂(sigma公司)，蛋白/佐剂组小鼠注射事先混匀的各100μg佐剂和重组sPD1/MS蛋白。为计算各实验组的肿瘤抑制率，以PBS对照组小鼠开始死亡为观察时间结点，在观察结点拉颈处死全部小鼠，剥离肿瘤并称量瘤重。 According to 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. 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 μl of sterile PBS, Al(OH). 3 adjuvant group mice were injected with an equal volume of 100 μg dose of Al(OH)3 adjuvant (sigma), and the protein/adjuvant group mice were injected with 100 μg of each adjuvant and recombinant sPD1/MS protein previously mixed. In order to calculate the tumor inhibition rate of each experimental group, 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.
表7 sPD1/MS蛋白疫苗治疗结直肠癌模型小鼠的免疫策略Table 7 Immune strategy of sPD1/MS protein vaccine in mice with colorectal cancer
Figure PCTCN2018116393-appb-000007
Figure PCTCN2018116393-appb-000007
各组治疗荷瘤小鼠的平均瘤重结果如图12-a所示。注射PBS的阴性组小鼠平均瘤重3.45g，Al(OH)3佐剂组平均瘤重3.19g，与PBS组相比单独佐剂没有抑瘤效果，而sPD1/MS融合蛋白与佐剂共同免疫的小鼠平均瘤重2.62g，与阴性对照组相比抑瘤率约24％，且有P＜0.05的统计学意义。已知AL(OH)3为常见的无机佐剂，其可通过诱导T细胞分化和体液免疫反应来提高疫苗的免疫原性，在本实例中可以看出重组sPD1/MS蛋白能够抑制荷瘤小鼠肿瘤的生长，同时，分析观察终止的各组荷瘤小鼠脾淋巴细胞特异性分泌IFN-γ的能力(见图12-b)，佐剂与蛋白疫苗共同免疫组也有较高的Elispot斑点数目(P＜0.01)，体现sPD1/MS重组蛋白疫苗诱导荷瘤小鼠体内特异性抗肿瘤免疫的能力。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. Compared with the PBS group, 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. In this example, it can be seen that recombinant sPD1/MS protein can inhibit tumorigenesis. The growth of mouse tumors, at the same time, the ability of spleen lymphocytes to secrete IFN-γ 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.
实施例10：sPD1/MS重组DNA疫苗治疗乳腺癌模型小鼠Example 10: sPD1/MS recombinant DNA vaccine for treating breast cancer model mice
本部分实验考察sPD1/MS融合核酸疫苗对乳腺癌荷瘤小鼠的治疗作用：将4-6周龄的Balb/c小鼠随机分成两组，每组10只；第0天对小鼠接种稳定表达粘蛋白1和生存素的小鼠乳腺癌细胞(MS f +4T1)，其中实验组小鼠在第1天和第8天进行sPD1/MS DNA疫苗的注射，第15天给予重组腺病毒rAD-MS的注射，另组小鼠作为阴性对照，给予等体积的无菌PBS注射。接种肿瘤后第11天左右观察小鼠肿瘤生长变化情况，以PBS对照组小鼠开始死亡为观察时间结点。图13为乳腺癌模型小鼠肿瘤生长体积变化曲线，截至观察终点，重组sPD1/MS疫苗治疗组小鼠肿瘤与PBS组相比其肿瘤生长速率明显减缓，肿瘤抑制率约40％，说明sPD1/MS重组DNA疫苗对乳腺癌具显著性抑瘤作用(P＜0.001)。 This part of the experiment examined the therapeutic effect of sPD1/MS fusion nucleic acid vaccine on breast cancer-bearing 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. The tumor growth changes of the mice were observed about 11 days after the tumor inoculation, and the mice in the PBS control group began to die as the observation time nodes. Figure 13 shows the tumor growth volume curve of breast cancer model mice. As of the end point, 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).
实施例11：sPD1/MS重组DNA疫苗治疗肺癌模型小鼠Example 11: sPD1/MS recombinant DNA vaccine for treating lung cancer model mice
本部分实验考察sPD1/MS融合核酸疫苗对肺癌荷瘤小鼠的治疗作用：将4-6周龄的C57BL/6小鼠按照表8随机地分为PBS阴性组和sPD1/MS DNA疫苗治疗组，每组各15只；第0天对小鼠接种稳定表达粘蛋白1和生存素的小鼠肺癌细胞(MS f +Lewis)，接种剂量为1×10 5个/只；其中PBS阴性组小鼠在第1、8和15天肌肉注射100μl无菌PBS；DNA疫苗组小鼠则在第1、8和15天进行sPD1/MS DNA疫苗的注射。接种肿瘤后第8天开始观察小鼠肿瘤生长变化，以PBS对照组小鼠开始死亡为观察时间结点。 This part of the experiment examined the therapeutic effect of sPD1/MS fusion nucleic acid vaccine on lung cancer-bearing 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 μl 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.
表8 sPD1/MS DNA疫苗治疗结直肠癌模型小鼠的免疫策略Table 8 Immune strategies of sPD1/MS DNA vaccine in mice with colorectal cancer
Figure PCTCN2018116393-appb-000008
Figure PCTCN2018116393-appb-000008
图14为肺癌模型小鼠肿瘤生长体积变化曲线，结果测量单独免疫重组sPD1/MS  DNA疫苗治疗组的小鼠肿瘤，其肿瘤生长速率明显减缓，与PBS阴性组小鼠肿瘤相比，具有显著的抑瘤效果(P＜0.05)。综上表明sPD1/MS重组DNA疫苗治疗肺癌具有有效抗肿瘤治疗作用。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.
实施例12：sPD1/MS重组DNA疫苗治疗肝癌模型小鼠Example 12: sPD1/MS recombinant DNA vaccine for treatment of liver cancer model mice
本部分实施例考察重组sPD1/MS疫苗针对肝癌荷瘤小鼠的治疗作用。将4-6周龄的Balb/c小鼠进行随机分成两组，分别为PBS阴性对照组和sPD1/MS DNA疫苗治疗组，每组各4只；第0天接种稳转粘蛋白1和生存素的小鼠肝癌细胞MS f +H22，其中实验组小鼠在第1、8和15天肌肉注射sPD1/MS DNA疫苗，在第22天注射重组腺病毒rAD-MS加强免疫，另一组小鼠注射等体积无菌PBS作为阴性对照。接种肿瘤后第12天开始观察到小鼠肿瘤的生长并测量肿瘤体积变化，结果如图15所示，sPD1/MS重组DNA疫苗治疗的肝癌模型小鼠肿瘤生长明显趋缓(P＜0.01)，表明sPD1/MS重组DNA疫苗治疗肝癌具有显著抑瘤作用。 In this part of the examples, the therapeutic effect of recombinant sPD1/MS vaccine against liver cancer-bearing mice was examined. 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. On the 12th day after inoculation of the tumor, the growth of the tumor in the mouse was observed and the tumor volume was measured. As a result, as shown in Fig. 15, 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.
实施例13：sPD1/MS重组DNA疫苗治疗胃癌模型小鼠Example 13: sPD1/MS recombinant DNA vaccine for treating gastric cancer model mice
本部分实施例选取前胃癌细胞(MFC)建立胃癌小鼠模型以考察sPD1/MS DNA疫苗的抑瘤作用。将4-6周龄的615小鼠进行随机分两组，每组各4只。各组小鼠在第0天接种稳转粘蛋白1和生存素的小鼠前胃癌细胞MS f +MFC，其中重组sPD1/MS疫苗治疗组在第1、8和15天肌肉注射DNA疫苗，第22天注射重组腺病毒rAD-MS加强免疫，而另一组小鼠在同一时间注射等体积无菌PBS作为阴性对照。接种肿瘤后第12天开始测量各组小鼠的肿瘤生长曲线，图16结果表明sPD1/MS重组DNA疫苗治疗的胃癌模型小鼠，取得一定的抑制肿瘤效果(P＜0.05)。 In this part of the example, the former gastric cancer cell (MFC) 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).
实施例14：活体基因导入仪促进sPD1/MS重组DNA疫苗的免疫原性和抗肿瘤效果Example 14: In vivo gene importer promotes immunogenicity and antitumor effect of sPD1/MS recombinant DNA vaccine
活体基因导入仪在注射位点通过电场的作用促进质粒进入细胞的效率。通过对活体基因导入仪器不同参数的研究，得到了适合我们疫苗的参数：电压：36V；频率：1Hz；脉冲数：6次；脉宽：20ms。本部分实例对活体基因导入仪对DNA疫苗免疫促进效果进行研究。图17结果显示使用活体基因导入仪器后，疫苗的细胞免疫效果得到显著提升。经survivin和MUC1蛋白刺激后，淋巴细胞分泌IFN-γ的能力和CTL水平显著性升高，使用活体基因导入仪时20ug剂量即能达到与肌肉注射100ug的效果(P＜0.05)。即使用活体基因导入增强了DNA的免疫原性。图18结果显示，在抑制肿瘤生长方面，使用活体基因导入仪后，使用较低剂量的DNA即可达到原来肌肉注射100ug的水平，并且当保持100ug剂量使用活体基因导入仪后，攻瘤后18天后开始，使用活体基因导入仪的100ug组小鼠肿瘤大小与肌肉注射组产生显著性差异(P＜0.05)。检测荷瘤鼠体内免疫水平，CTL和ELISPOT结果均表明，使用活体基因导入仪后，即使低剂量组较肌肉注射组的CTL和分泌IFN-γ的能力也均得到显著提升。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. Through the study of different parameters of the living organism gene introduction instrument, the parameters suitable for our vaccine were obtained: voltage: 36V; frequency: 1Hz; pulse number: 6 times; pulse width: 20ms. In this part of the example, 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-γ and CTL levels were significantly increased. The effect of intramuscular injection of 100ug was achieved with a 20ug dose (P<0.05). That is, 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-γ secreting in the low-dose group was significantly improved compared with the intramuscular injection group.
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. These improvements and retouchings should also be considered. It is the scope of protection of the present invention.

Claims (35)

  1. 一种分子组合，其特征在于，包括sPD-1、MUC1和Survivin。A molecular combination characterized by comprising sPD-1, MUC1 and Survivin.
  2. 根据权利要求1所述的分子组合，其特征在于，所述sPD-1的DNA序列如SEQ ID NO：1所示，所述sPD-1的氨基酸序列如SEQ ID NO：2所示。The molecular combination according to claim 1, wherein the DNA sequence of sPD-1 is as shown in SEQ ID NO: 1, and the amino acid sequence of sPD-1 is shown in SEQ ID NO: 2.
  3. 根据权利要求1所述的分子组合，其特征在于，所述MUC1的DNA序列如SEQ ID NO：3所示；其氨基酸序列如SEQ ID NO：4所示。The molecular combination according to claim 1, wherein the DNA sequence of MUCl is as shown in SEQ ID NO: 3; and the amino acid sequence thereof is shown in SEQ ID NO: 4.
  4. 根据权利要求1所述的分子组合，其特征在于，所述Survivin的DNA序列如SEQ ID NO：5所示；其氨基酸序列如SEQ ID NO：6所示。The molecular combination according to claim 1, wherein the DNA sequence of Survivin is as shown in SEQ ID NO: 5; and the amino acid sequence thereof is shown in SEQ ID NO: 6.
  5. 权利要求1～4任一项所述的分子组合在制备防治肿瘤的产品中的应用。Use of the combination of molecules according to any one of claims 1 to 4 for the preparation of a tumor-preventing product.
  6. 根据权利要求5所述的应用，其特征在于，所述肿瘤选自黑色素瘤、结直肠癌、大肠癌、肺癌、乳腺癌、肝癌、肾癌、胆管癌、胃癌、食管癌、膀胱癌、胰腺癌、头颈癌、鼻咽癌、口腔癌、宫颈癌、卵巢癌、子宫癌、前列腺癌、睾丸癌、鳞状细胞癌、淋巴瘤、脑癌、恶性胶质细胞瘤、髓母细胞瘤、淋巴肉瘤、绒毛膜上皮癌、骨肉瘤、甲状腺癌。The use according to claim 5, wherein the tumor is selected from the group consisting of melanoma, colorectal cancer, colon cancer, lung cancer, breast cancer, liver cancer, renal cancer, cholangiocarcinoma, gastric cancer, esophageal cancer, bladder cancer, and pancreas. 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, lymph Sarcoma, chorionic epithelial cancer, osteosarcoma, thyroid cancer.
  7. 根据权利要求5所述的应用，其特征在于，所述产品中包括疫苗。The use according to claim 5, characterized in that the product comprises a vaccine.
  8. 根据权利要求7所述的应用，其特征在于，所述疫苗包括sPD-1、MUC1和Survivin的DNA序列的重组载体。The use according to claim 7, wherein the vaccine comprises a recombinant vector of the DNA sequences of sPD-1, MUCl and Survivin.
  9. 根据权利要求8所述的重组载体，其特征在于，所述sPD-1为sPD-1的DNA序列的5’端修饰tPA信号肽序列。The recombinant vector according to claim 8, wherein the sPD-1 is a 5'-end modified tPA signal peptide sequence of the DNA sequence of sPD-1.
  10. 根据权利要求9所述的重组载体，其特征在于，所述PD-1与tPA信号肽序列相连的DNA序列如SEQ ID NO：7所示。The recombinant vector according to claim 9, wherein the DNA sequence in which the PD-1 is linked to the tPA signal peptide sequence is as shown in SEQ ID NO: 7.
  11. 根据权利要求9所述的重组载体，其特征在于，所述MUC1和Survivin连接的DNA序列如SEQ ID NO：8所示。The recombinant vector according to claim 9, wherein the DNA sequence to which the MUCl and Survivin are ligated is set forth in SEQ ID NO: 8.
  12. 一种重组载体，其特征在于，包括骨架载体、SEQ ID NO：7所示的DNA序列、SEQ ID NO：8所示的DNA序列和佐剂序列。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.
  13. 根据权利要求12所述的重组载体，其特征在于，所述骨架载体为CpDV。The recombinant vector according to claim 12, wherein the backbone vector is CpDV.
  14. 根据权利要求13所述的重组载体，其特征在于，所述佐剂序列为白介素-2，其DNA序列如SEQ ID NO：9所示。The recombinant vector according to claim 13, wherein the adjuvant sequence is interleukin-2, and the DNA sequence thereof is shown in SEQ ID NO: 9.
  15. 权利要求12～14任一项所述重组载体在制备防治肿瘤的产品中的应用。Use of the recombinant vector according to any one of claims 12 to 14 for the preparation of a tumor-preventing product.
  16. 一种防治肿瘤的疫苗，其特征在于，包括权利要求12～14任一项所述的重组载体。A vaccine for preventing and treating tumors, comprising the recombinant vector according to any one of claims 12 to 14.
  17. 根据权利要求16所述的防治肿瘤的疫苗，其特征在于，还包括PBS溶液，权利要求12～14任一项所述重组载体的浓度为0.1mg/mL～10mg/mL。The tumor control vaccine according to claim 16, further comprising a PBS solution, wherein the concentration of the recombinant vector according to any one of claims 12 to 14 is from 0.1 mg/mL to 10 mg/mL.
  18. 权利要求16～17任一项所述疫苗的制备方法为，在骨架载体的Xba I和BamH I酶切位点之间插入佐剂序列，然后在Bgl II和EcoR I酶切位点之间插入SEQ ID NO：7和SEQ ID NO：8所示序列，制得重组载体后，溶解于PBS溶液。The vaccine according to any one of claims 16 to 17, wherein the adjuvant sequence is inserted between the Xba I and BamH I cleavage sites of the backbone vector, and then inserted between the Bgl II and EcoR I cleavage sites. The sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 were prepared by dissolving the recombinant vector in PBS solution.
  19. 一种重组载体，其特征在于，包括骨架载体、SEQ ID NO：7所示的DNA序列和SEQ ID NO：8所示的DNA序列。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.
  20. 根据权利要求19所述的重组载体，其特征在于，所述骨架载体为PET28a。The recombinant vector according to claim 19, wherein the skeleton carrier is PET28a.
  21. 一种表达载体，其特征在于，由权利要求19～20任一项所述重组载体转染宿主细胞制得。An expression vector obtained by transfecting a host cell with the recombinant vector of any one of claims 19 to 20.
  22. 一种融合蛋白，其特征在于，由权利要求21所述的表达载体表达。A fusion protein expressed by the expression vector of claim 21.
  23. 权利要求22所述的融合蛋白在制备防治肿瘤的产品中的应用。Use of the fusion protein of claim 22 for the preparation of a tumor-preventing product.
  24. 一种防治肿瘤的疫苗，其特征在于，包括权利要求23所述的融合蛋白。A vaccine for preventing and treating tumors, comprising the fusion protein of claim 23.
  25. 根据权利要求24所述的疫苗，其特征在于，其中还包括佐剂，所述佐剂为Al(OH) 3、CpG、MPL、QS21、AS02、AS03、Poly-IC及其衍生物等。 The vaccine according to claim 24, further comprising an adjuvant, wherein the adjuvant is Al(OH) 3 , CpG, MPL, QS21, AS02, AS03, Poly-IC and derivatives thereof and the like.
  26. 一种重组载体，其特征在于，包括骨架载体、SEQ ID NO：7所示的DNA序列、SEQ ID NO：8所示的DNA序列，所述载体为pSC11或pDC316。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.
  27. 一种重组病毒，其特征在于，以权利要求28所述的重组载体转染细胞，在细胞内与感染的病毒发生同源重组制得，所述病毒为腺病毒或痘病毒。A recombinant virus obtained by transfecting a cell with the recombinant vector of claim 28, and homologously recombining with an infected virus in a cell, wherein the virus is an adenovirus or a poxvirus.
  28. 权利要求27所述的重组病毒在制备防治肿瘤的产品中的应用。Use of the recombinant virus of claim 27 for the preparation of a tumor-preventing product.
  29. 一种防治肿瘤的疫苗，其特征在于，包括权利要求27所述的重组病毒。A vaccine for preventing and treating tumors, comprising the recombinant virus of claim 27.
  30. 一种防治肿瘤的产品，其特征在于，包括权利要求16～17任一项所述的疫苗、权利要求24～25任一项所述的疫苗和/或权利要求29所述的疫苗。A vaccine for preventing and treating a tumor, comprising the vaccine according to any one of claims 16 to 17, the vaccine according to any one of claims 24 to 25, and/or the vaccine according to claim 29.
  31. 根据权利要求30所述的产品，其特征在于，其中还包括化疗药物。The product of claim 30 further comprising a chemotherapeutic drug.
  32. 一种防治肿瘤的产品，其特征在于，包括以MS为靶点的疫苗并且MS与sPD-1融合表达的疫苗。A tumor-preventing product characterized by comprising a vaccine targeting MS and a vaccine expressed by fusion of MS and sPD-1.
  33. 根据权利要求32所述的产品，其特征在于，其中还包括化疗药物。The product of claim 32 further comprising a chemotherapeutic drug.
  34. 一种防治肿瘤的方法，其特征在于，给予权利要求30～33任一项所述的产品。A method for controlling tumors, which comprises administering the product according to any one of claims 30 to 33.
  35. 根据权利要求34所述的方法，其特征在于，给予权利要求30～33任一项所述的产品后，以活体基因导入仪刺激注射位点。The method according to claim 34, wherein after the product according to any one of claims 30 to 33 is administered, the injection site is stimulated by a living body gene introducing instrument.
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Publication number Priority date Publication date Assignee Title
CN111118063B (en) * 2019-12-05 2023-04-18 吉林大学 FAP alpha and survivin-based DNA and application thereof in preparation of tumor vaccine
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CN114790248B (en) * 2022-05-12 2023-11-28 广州医科大学 MUC1-PDL1-IgG1Fc tumor vaccine and preparation method and application thereof
CN115976094B (en) * 2022-12-15 2024-02-20 浙江大学杭州国际科创中心 Genetically engineered bacterium for improving secretion of endogenous enzyme and construction method and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1889977A (en) * 2003-10-13 2007-01-03 葛兰素史密丝克莱恩生物有限公司 Vaccine compositions comprising an interleukin 18 and saponin adjuvant system
CN102086453A (en) * 2009-12-04 2011-06-08 长春百克生物科技股份公司 Tumor DNA vaccine taking mucin 1 and survivin as targets and viral vector vaccine
CN102665748A (en) * 2009-05-27 2012-09-12 达娜-法勃肿瘤研究所公司 Inhibition 0f inflammation using antagonists of MUC1
CN104013973A (en) * 2009-12-04 2014-09-03 长春百克生物科技股份公司 Tumor DNA (Deoxyribose Nucleic Acid) vaccine and virus vector vaccine taking mucoprotein 1 and surviving as targets
CN105531288A (en) * 2013-09-13 2016-04-27 百济神州有限公司 Anti-PD1 antibodies and their use as therapeutics and diagnostics
WO2016112921A1 (en) * 2015-01-15 2016-07-21 University Of Copenhagen Virus-like particle with efficient epitope display
CN106029889A (en) * 2013-11-22 2016-10-12 德那翠丝有限公司 Adenovirus expressing immune cell stimulatory receptor agonist(s)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102732543B (en) * 2011-04-07 2016-04-20 长春百克生物科技股份公司 With the oncogene engineered vaccine that MUC-1 and survivin are target spot

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1889977A (en) * 2003-10-13 2007-01-03 葛兰素史密丝克莱恩生物有限公司 Vaccine compositions comprising an interleukin 18 and saponin adjuvant system
CN102665748A (en) * 2009-05-27 2012-09-12 达娜-法勃肿瘤研究所公司 Inhibition 0f inflammation using antagonists of MUC1
CN102086453A (en) * 2009-12-04 2011-06-08 长春百克生物科技股份公司 Tumor DNA vaccine taking mucin 1 and survivin as targets and viral vector vaccine
CN104013973A (en) * 2009-12-04 2014-09-03 长春百克生物科技股份公司 Tumor DNA (Deoxyribose Nucleic Acid) vaccine and virus vector vaccine taking mucoprotein 1 and surviving as targets
CN105531288A (en) * 2013-09-13 2016-04-27 百济神州有限公司 Anti-PD1 antibodies and their use as therapeutics and diagnostics
CN106029889A (en) * 2013-11-22 2016-10-12 德那翠丝有限公司 Adenovirus expressing immune cell stimulatory receptor agonist(s)
WO2016112921A1 (en) * 2015-01-15 2016-07-21 University Of Copenhagen Virus-like particle with efficient epitope display

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LU ZHENZHEN: "Study of soluble PD-1 based DNA vaccine targetting MUC1 VNTR and survivin", CNKI, 1 May 2016 (2016-05-01) *
SURVIVIN DNA CNKI, 1 May 2016 (2016-05-01) *

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