WO2019165848A1 - 一种由NF-κB启动的肿瘤细胞特异效应基因表达载体及其表达产物和应用 - Google Patents

一种由NF-κB启动的肿瘤细胞特异效应基因表达载体及其表达产物和应用 Download PDF

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WO2019165848A1
WO2019165848A1 PCT/CN2019/070016 CN2019070016W WO2019165848A1 WO 2019165848 A1 WO2019165848 A1 WO 2019165848A1 CN 2019070016 W CN2019070016 W CN 2019070016W WO 2019165848 A1 WO2019165848 A1 WO 2019165848A1
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gene expression
expression vector
cell
tumor
cells
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王进科
王丹阳
戴薇
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东南大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the invention belongs to the technical field of tumor immunotherapy, and particularly relates to a tumor cell specific effect gene expression vector initiated by the transcription factor NF- ⁇ B, an expression product thereof and an application thereof.
  • the most widely used and most effective tumor immunotherapy is the PD-1/PD-L1 inhibitor (such as PD-1 antibody), but its current overall response rate is only 20-30%; in hematological malignancies
  • Therapeutic effective cell therapy such as CAR-T and TCR-T has not yet made substantial progress in the treatment of solid tumors. Therefore, exploring new cancer treatment technologies will always be the direction of the scientific and medical circles before humans can truly cure cancer.
  • tumor antigens expressed on the surface of cancer cells are inevitably dependent on such tumor antigens (such as the most successful CD19 currently used in CAR-T therapy), that is, targets for tumor immunotherapy. point.
  • tumor antigens such as the most successful CD19 currently used in CAR-T therapy
  • the currently available antigens of this type are extremely limited, and most of them are also expressed in low amounts on normal cells. When applied, they often cause attack on normal cells/organs by CAR-T cells, resulting in autoimmune symptoms. Serious side effects. Therefore, the field is currently focusing on the use of second-generation sequencing technology to find more new antigens.
  • CD123 antigen is highly expressed on acute myeloid leukemia (AML) cells and parental plasmacytoid dendritic cell tumor (BPDCN) cells. Both of these diseases often develop in the bone marrow and can threaten the lives of patients in the short term.
  • AML acute myeloid leukemia
  • BPDCN parental plasmacytoid dendritic cell tumor
  • the universal CAR-T UCART123 can be used to treat immunotherapy for both cancers. It must be pointed out that any treatment plan that separates the patient's cells, cultures in vitro, genetically manipulates, etc., and then returns to the patient, has the risk of in vitro operation, especially genetic manipulation. Therefore, in view of the current problems faced by tumor immunotherapy, new tumor immunotherapy strategies and techniques need to be developed.
  • NF- ⁇ B is an inducible DNA-binding transcription factor, and its role in liver physiology and hepatocellular carcinoma pathology is achieved through its regulated target genes.
  • NF- ⁇ B is an important inflammation-related transcription factor because it regulates the expression of inflammatory mediators.
  • Important inflammatory mediators such as TNF-a, IL-1, IL-6, etc. are direct targets of NF- ⁇ B.
  • Bcl-2 is a well-recognized anti-apoptotic protein, and many tumors express high levels of Bcl-2.
  • the role of NF- ⁇ B in inhibiting tumor cell apoptosis is precisely the direct regulation of the expression of its target gene Bcl-2 by NF- ⁇ B.
  • NF- ⁇ B has been widely activated in almost all tumor cells after extensive scientific research and analysis of clinical case materials. Therefore, NF- ⁇ B is regarded as an excellent target for tumor therapy and drug screening. Therefore, many pharmaceutical companies and scientists are committed to the study of NF- ⁇ B inhibitors, but many of the drugs developed by the researchers have severely toxic side effects in the successful inhibition of NF- ⁇ B and cannot be used as clinical tumor treatment drugs. The reason, the researchers found that although over-activated NF- ⁇ B is critical for cancer cells, its normal level of activation is also important for the normal physiological function of healthy cells. The introduction of NF- ⁇ B inhibitors into cells often leads to excessive inhibition of NF- ⁇ B activity due to the inability to control the number, thereby causing serious side effects.
  • the present invention provides a tumor cell-specific effector gene expression vector initiated by NF- ⁇ B, when the gene expression vector is introduced into a tumor cell with excessive activation of NF- ⁇ B activity, intracellular
  • the over-activated transcription factor NF- ⁇ B activates the vector to express the effector gene on the vector.
  • the gene expression vector can be subjected to tumor immunotherapy based on intracellular NF- ⁇ B activity, and the gene expression vector can specifically express an effector gene in the tumor cell, and the expression product is a cell surface polypeptide or protein, and the cell surface polypeptide or protein It can be used as a new antigenic protein to be recognized by the immune system in the body to produce an immune response, which causes the immune system to kill the tumor cells.
  • the invention also discloses an expression product of a tumor cell specific effect gene expression vector initiated by NF- ⁇ B and its use in preparing a tumor immunotherapy and imaging reagent or medicament.
  • a tumor cell-specific effector gene expression vector initiated by NF- ⁇ B is characterized in that it comprises two sequence elements, a promoter sequence and a promoter sequence regulating gene expression.
  • the NF- ⁇ B response sequence comprises NF- ⁇ B response sequences of various sequences; the NF- ⁇ B response sequence is a DNA sequence which specifically binds to NF- ⁇ B protein, and its main sequence is characterized by a different number Various NF- ⁇ B binding targets.
  • the promoter is an NF- ⁇ B-specific promoter, ie, a promoter that is only NF- ⁇ B-activatable.
  • the minimal promoter includes minimal promoter sequences from various sources, and the minimal promoter sequences of the various sources include natural and artificially screened minimal promoter sequences.
  • HSV-TK herpes simplex virus thymidine kinase
  • the promoter sequence for regulating gene expression is especially the sequence of SEQ ID NO. 1: 5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CTA GAG GGT ATA TAA TGG AAG CTC GAC TTC CAG-3'.
  • NF- ⁇ B response sequence SEQ ID NO. 2: 5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3'
  • minimal promoter sequence SEQ ID NO. 3: 5'-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3').
  • the effector gene is a gene encoding a cell membrane protein, and the protein protruding outside the cell membrane can be used as a new antigen substance to stimulate the body's immune system, causing the immune system of the body to kill the tumor cells.
  • the effector gene is hepatitis B surface antigen encoding gene HBsAg, streptavidin binding peptide encoding gene SBP or calreticulin encoding gene CRT.
  • the gene expression vector is a linear or circular nucleic acid molecule.
  • the nucleic acid molecule is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule, including double-stranded DNA (such as an adenoviral DNA molecule), single-stranded DNA (adeno-associated virus molecule), or a single-stranded RNA molecule (such as lentiviral RNA molecules).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • double-stranded DNA such as an adenoviral DNA molecule
  • single-stranded DNA adeno-associated virus molecule
  • a single-stranded RNA molecule such as lentiviral RNA molecules.
  • the linear nucleic acid molecule includes a common linear DNA molecule (such as a PCR amplified fragment, a restriction fragment), a viral DNA molecule (such as an adenovirus DNA molecule, an adeno-associated virus molecule) or a viral RNA molecule (such as a lentiviral RNA molecule);
  • the circular nucleic acid molecule includes plasmid DNA.
  • the gene expression vector of the gene expression vector of the present invention introduces a gene expression vector into a tumor cell with excessive activation of NF- ⁇ B activity, and an over-activated transcription factor NF- ⁇ B activates the vector to express the vector. Effect gene.
  • the method of introducing the gene expression vector into a cell includes various types of nucleic acid cell introduction methods.
  • the nucleic acid cell introduction method includes an introduction method such as a viral vector, a nanocarrier, a liposome, an electrotransfer or a gene gun.
  • the method for introducing the gene expression vector into a cell is introduction of a nano vector and a viral vector.
  • the method of introducing the gene expression vector into a cell is preferably an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the expression product of the tumor cell-specific effector gene expression vector initiated by NF- ⁇ B which is a polypeptide or protein on the cell surface.
  • the polypeptide or protein on the surface of the cell can be recognized as a new antigenic protein in the body by the immune system to generate an immune response, which causes the immune system to kill the tumor cells.
  • the cell surface polypeptide or protein can be used as a tumor cell artificial marker for in vivo imaging, diagnosis and cell separation of the tumor.
  • the polypeptide or protein on the cell surface includes any polypeptide or protein, and a polypeptide or protein whose expression undergoes glycosylation or the like through the function of the cell itself.
  • the polypeptide or protein is hepatitis B surface antigen (HBsAg), streptavidin binding peptide (SBP), and calreticulin (CRT).
  • HBsAg hepatitis B surface antigen
  • SBP streptavidin binding peptide
  • CRT calreticulin
  • streptavidin-binding peptide can also be used for in vivo imaging and diagnosis of tumors, such as the use of streptavidin-labeled contrast agents such as MRI, CT, PET, near-infrared fluorescence and SBP expressed on the surface of tumor cells. , for tumor imaging, diagnosis, and the like.
  • the gene expression vector of the present invention is a gene expression vector for tumor gene therapy based on intracellular NF- ⁇ B activity, which can be applied to tumor immunotherapy and imaging.
  • the invention relates to a tumor cell specific effect gene expression vector initiated by NF- ⁇ B in the preparation of a tumor immunotherapy and imaging reagent or medicament.
  • NF- ⁇ B is a transcription factor and is in a tumor cell.
  • the activity of both activities is over-activated, and tumor immunotherapy is achieved by a tumor cell-specific effector gene expression vector initiated by NF- ⁇ B.
  • the NF- ⁇ B-initiated tumor cell-specific effector gene expression vector of the present invention is based on a schematic diagram of the principle of intracellular NF- ⁇ B activity-activated effector gene expression in NF- ⁇ B over-activated cells (Fig. 1). 1 reflects the construction of a gene expression vector containing the NF- ⁇ B response sequence, the minimal promoter and the effector gene; when the gene expression vector is introduced into the tumor cell, the tumor cell is overactivated.
  • the transcription factor NF- ⁇ B protein binds to the NF- ⁇ B response sequence on the vector, thereby activating the expression of the effector gene;
  • the expression product of the effector gene is a cell membrane protein, and the protein protruding from the cell membrane can serve as a kind of
  • the new antigenic substance stimulates the body's immune system and causes immune damage to the tumor cells by the body's immune system.
  • the present invention devises a gene expression vector specifically activated by the over-activated transcription factor NF- ⁇ B in tumor cells, which expresses an effector gene specifically in tumor cells; the effector gene is expressed in cells A polypeptide or protein on the surface; a polypeptide or protein on the surface of the cell can be recognized as a new antigenic protein in the body by the immune system, generating an immune response, causing the immune system to kill the tumor cells.
  • the invention provides a new strategy and a new technology for designing a gene expression vector for NF- ⁇ B-specific expression of a gene expression vector, and reforms the principle of tumor-sensitive immunotherapy.
  • the tumor immunotherapy proposed by the present invention breaks through the constraint of the extremely limited number of natural antigens currently present on the surface of tumor cells, and expresses and creates an artificial antigen on the surface of tumor cells by gene therapy technology, thereby triggering strong immunity of the body. Reacts to kill tumor cells in the body. Artificial antigens are not limited by the type and amount.
  • the expression product of the gene expression vector is only expressed on the surface of the tumor cell as an artificial antigen, and is not expressed on the surface of the normal cell, thereby avoiding The challenge of the immune response to normal cells exerts a very specific tumor cell immune attack response. Therefore, immunization of tumor cells by the gene expression vector is a highly specific immunotherapy for tumor cells.
  • the tumor immunotherapy for immunizing tumor cells can pass the gene expression vector through the vein of the nano carrier or the viral vector by means of a nano vector or a viral vector, particularly a highly safe adeno-associated virus vector. It can be completed by administration, and it is a non-invasive gene therapy technology. This avoids the cumbersome, dangerous and damaging treatment process of current tumor treatment, such as surgery, chemotherapy, radiotherapy, CAR-T manufacturing, etc., which is very helpful to improve the quality of life of cancer patients.
  • the NF- ⁇ B-specific activation gene expression vector designed and demonstrated by the present invention is different from the currently used NF- ⁇ B inhibitor, and does not inhibit NF- ⁇ B in the therapeutic principle, but uses NF- ⁇ B to avoid the use of NF- ⁇ B inhibitors have serious side effects in tumor therapy and are therefore a distinctly innovative new strategy for NF- ⁇ B-based tumor therapy.
  • the tumor cell-specific NF- ⁇ B promoter gene expression vector proposed by the present invention can be packaged in an adeno-associated virus (AAV), and the AAV virus is used as an excellent carrier for gene therapy, and is used for preparing a tumor imaging and treatment drug in human body. in.
  • AAV virus vector can be used for single-shot injection for gene therapy of diseases. Therefore, the adeno-associated virus (AAV) is expected to be a simple tumor for treating tumors by carrying the NF- ⁇ B-specific activation gene expression vector designed and demonstrated by the present invention.
  • FIG. 1 is a schematic diagram showing the principle of gene expression of a tumor cell-specific effector gene expression vector initiated by NF- ⁇ B based on an NF- ⁇ B activity-activated effector gene in NF- ⁇ B over-activated cells;
  • gene expression vector is a gene expression vector NF- ⁇ B responsive sequences (NF- ⁇ B binding sequences) are NF- ⁇ B binding sequences (NF- ⁇ B binding sequence); minimal promoter is the minimal promoter; effective gene is the effector gene; transfection is transfection; over activated transcription factor NF - ⁇ B is an over-activated transcription factor NF- ⁇ B; NF- ⁇ B binding and gene expression activation is NF- ⁇ B binding and gene expression activation; effective gene expression is effector gene expression; effective gene products is effect gene product; cell growth arrest/ Apoptosis/dead is cell growth inhibition/apoptosis/death; NF- ⁇ B over-activated cell is NF- ⁇ B over-activated cell;
  • Figure 2 shows the results of fluorescent quantitative PCR detection of NF- ⁇ B expression in different cells; it can be seen that NF- ⁇ B is expressed in tumor cells, but not in normal cells;
  • FIG. 3 shows the DMP-Display-SBP expression vector transfected with Hepa1-6, HepG2, MRC-5, HL7702, and 293T cells, staining the cells with FITC-labeled streptavidin, followed by white field (bright field) And microscopic photographs of cells under green fluorescent (FITC) channels; and superposition of white and green fluorescent channel images, it can be seen that DMP-controlled cell surface display (SBP) is only in tumor cells (Hepa1-6, HepG2) Expression in 293T), no expression in normal cells (MRC-5, HL7702);
  • SBP DMP-controlled cell surface display
  • Figure 4 shows DMP-Display-SBP expression vector transfected with HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7, B16F10, MRC- 5.
  • HL7702 cells After HL7702 cells, the cells were stained with IRDye800CW-labeled streptavidin protein, and then scanned on a near-infrared fluorescence scanner; the fluorescence intensity of each well was quantified to show DMP-controlled cell surface display ( Display) SBP is expressed only in tumor cells (HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7, B16F10), in normal No expression in cells (MRC-5, HL7702);
  • Display DMP-controlled cell surface display
  • Figure 5 shows DMP-Display-SBP expression vector transfected with HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7, B16F10, MRC- 5.
  • the cells were trypsinized and collected, and then stained with IRDye800CW-labeled streptavidin protein; then scanned on a near-infrared fluorescence scanner and photographed on a white field; DMP-controlled cells were observed.
  • SBP Surface-displayed SBP is expressed only in tumor cells (HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7, B16F10) , no expression in normal cells (MRC-5, HL7702);
  • Figure 6 shows that after packaging DMP-Display-SBP into adeno-associated virus (AAV) expression vector (AAV-SBP), 293T, HepG2, Hepa1-6, MRC-5, and HL7702 cells were transfected with AAV-SBP; IRDye800CW-labeled streptavidin protein was stained, and then scanned on a near-infrared fluorescence scanner (Panel A); cells were trypsinized and collected, and stained with IRDye800CW-labeled streptavidin protein.
  • AAV adeno-associated virus
  • the image was scanned on a near-infrared fluorescence scanner and photographed in a white field (panel B); it can be packaged into an AAV-viral vector (AAV-SBP), and the DMP-controlled cell surface-displayed SBP can be efficiently displayed on tumor cells ( HepG2, 293T, Hepa1-6) surface, no expression in normal cells (MRC-5, HL7702);
  • AAV-SBP AAV-viral vector
  • FIG. 7 is a photograph of mouse experiment 1 in which AAV-HBsAg, AAV-SBP, and AAV-CRT viral vectors are transfected into mouse liver cancer cells Hepa1-6 in vitro, and the virus transfected cells and non-transfected cells are harvested. Transplanted into the skin of the mouse (left transplanted virus transfected cells, right transplanted non-transfected cells), the experimental mice were divided into 3 groups of AAV-HBsAg, AAV-SBP and AAV-CRT experimental groups, 10 mice per group, cells After 15 days of transplantation, the mice were observed and photographed;
  • FIG. 8 is a schematic diagram showing the results of tumor size measurement of the experimental mice in FIG. 7; wherein AAV-HBsAg-L is the result of the left tumor size measurement of the HBsAg experimental group in FIG. 8; AAV-HBsAg-R is the right tumor of the HBsAg experimental group in FIG. The results of size determination; AAV-SBP-L is the result of tumor size measurement on the left side of SBP experimental group in Fig. 8; AAV-SBP-R is the result of tumor size measurement on the right side of SBP experimental group in Fig. 8; AAV-CRT-L is Fig.
  • AAV-CRT-R is the result of the right tumor size measurement in the CRT experimental group in Fig. 8; * indicates that the p value is less than 0.05 (significant difference), ** indicates that the p value is less than 0.01 (difference significant);
  • Figure 9 is a photograph of mouse experiment 2; the experimental mice were divided into 4 groups, namely AAV-HBsAg, AAV-SBP, AAV-CRT and AAV-blank (empty virus expressed by MCS) experimental group; empty virus group 11
  • the other 3 groups of 10 mice in each group were subcutaneously transplanted with Hepa 1-6 hepatoma cells in each group, and 7 days after cell transplantation, followed by AAV-HBsAg, AAV-SBP, AAV-CRT and
  • the mice in the AAV-blank experimental group were intravenously injected with AAV-HBsAg, AAV-SBP, AAV-CRT and AAV-blank virus respectively. After the virus injection, they were kept for 7 days, and the mice were observed and photographed.
  • FIG. 10 is a schematic diagram showing the results of tumor size measurement of the experimental mice in FIG. 9; wherein AAV-MCS-L is the result of measuring the left tumor size of the MCS experimental group in FIG. 9; AAV-MCS-R is the right side of the MCS experimental group in FIG. Tumor size determination results; AAV-HBsAg-L is the result of the left tumor size measurement in the HBsAg experimental group in Figure 9; AAV-HBsAg-R is the result of the right tumor size measurement in the HBsAg experimental group in Figure 9; AAV-SBP-L is the graph The results of the left tumor size determination in the SBP experimental group in 9; AAV-SBP-R is the tumor size measurement on the right side of the SBP experimental group in Fig.
  • AAV-CRT-L is the result of the left tumor size measurement in the CRT experimental group in Fig. 9.
  • AAV-CRT-R is the result of tumor size measurement on the right side of the CRT experimental group in Figure 9;
  • AAV-MCS is the combination of rAAV-MCS-L and rAAV-MCS-R;
  • AAV-HBsAg is AAV-HBsAg-L and AAV- The combination of HBsAg-R;
  • AAV-SBP is the combination of AAV-SBP-L and AAV-SBP-R;
  • AAV-CRT is the combination of AAV-CRT-L and AAV-CRT-R, the figure shows AAV-HBsAg,
  • the tumor size of the AAV-SBP and AAV-CRT groups was statistically significant with the tumor size of the AAV-MCS group, respectively, and ** indicates that the p value was less than 0.01 (the difference was extremely significant).
  • HEK-293T human fetal kidney cells
  • HepG2 human liver cancer cells
  • A549 human lung cancer cells
  • HT-29 human colon cancer cells
  • HeLa human cervical cancer cells
  • SKOV3 human ovarian cancer
  • PANC-1 pancreatic cancer cells
  • MDA-MB-453 human breast cancer
  • Hepa 1-6 mouse liver cancer cells
  • mouse macrophages RAW264.7
  • mouse melanoma cells B16F10
  • HL7702 human normal hepatocytes
  • MRC5 human embryonic fibroblasts
  • RNA was extracted with Trizol
  • complementary DNA cDNA
  • the cDNA preparation reaction and procedure were as follows: 10 ⁇ L of the reverse transcription reaction component contained 2 ⁇ L of 5 ⁇ PrimeScript RT Master Mix (Takara), 50 ng of total RNA, and the total volume of the reaction was supplemented to 10 ⁇ L with RNase Free ddH 2 O; and reacted at 37 ° C for 15 minutes. The reaction was heated to 85 ° C for 5 seconds to inactivate the reverse transcriptase, and the reaction solution was stored at 4 ° C. RelA expression was quantitatively analyzed by qPCR.
  • the upper and lower primers for qPCR were 5'-CCT GGA GCA GGC TAT CAG TC-3' (F) and 5'-ATG GGA TGA GAA AGG ACA GG-3' (R).
  • the PCR template was cDNA, and RelA expression was quantitatively analyzed by qPCR.
  • the 10 ⁇ L qPCR reaction contained 5 ⁇ L of Fast SYBR Green Master Mix (ABI), 0.2 ⁇ L of 10 ⁇ M F, 0.2 ⁇ L of 10 ⁇ M R and 1 ⁇ L of cDNA, and the total volume of the reaction was supplemented to 10 ⁇ L with ddH 2 O.
  • the prepared reaction system was amplified by a quantitative PCR instrument (StepOne plus, ABI), and the amplification procedure was set: pre-denaturation at 95 ° C for 10 minutes, and 45 amplification cycles (denaturation at 95 ° C for 15 s, in each Amplification at annealing temperature for 1 minute).
  • the specificity of real-time PCR amplification was determined by the dissolution curve.
  • the relative quantification (RQ) of gene expression was calculated by comparative CT value method.
  • the data was finally expressed as mean ⁇ standard deviation (SD), and the statistical significance was analyzed by t test. to make sure.
  • NF- ⁇ B RelA/p65 was detected in 11 tumor cells and normal cells (HL7702 and MRC-5) by fluorescence quantitative PCR in order to investigate the expression of NF- ⁇ B RelA/p65 gene in various tumor cells and normal cells. Expression ( Figure 2). The results showed that NF- ⁇ B RelA/p65 was expressed in all tumor cell lines, while in normal cells (HL7702 and MRC5), NF- ⁇ B expression was not detected.
  • the NF- ⁇ B activating gene expression vector proposed by the present invention is a gene expression vector specific for NF- ⁇ B over-activation, such as expression of an NF- ⁇ B activating gene expression vector in tumor cells.
  • DMP-Display-SBP An expression vector DMP-Display-SBP was constructed; the vector contains a DMP sequence and a coding sequence for a cell-expressing streptavidin-binding peptide (SBP).
  • DMP comprises the NF- ⁇ B response sequence SEQ ID NO. 2: (5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3') and the minimal promoter sequence SEQ ID NO. :(5'-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3').
  • SBP coding sequence SEQ ID NO.
  • pDisplay TM appears to be cell surface protein or polypeptide fused to the leader sequence Ig ⁇ - chain rats (Ig ⁇ -chain leader sequence) of the N-terminus of the leader sequence may direct The denominator pathway of the protein;
  • the C-terminus of the pDisplay-expressing protein is the platelet derived growth factor receptor (PDGFR) transmembrane region, which anchors the protein to the cell membrane, thereby displaying the protein on the outside of the cell.
  • PDGFR platelet derived growth factor receptor
  • This membrane protein can interact with proteins in cell culture fluids, such as streptavidin in this example and streptavidin-binding peptide (SBP interaction) on the surface of cell membranes.
  • HEK-293T human fetal kidney cells
  • HepG2 human liver cancer cells
  • A549 human lung cancer cells
  • HT-29 human colon cancer cells
  • HeLa human cervical cancer cells
  • SKOV3 human ovarian cancer
  • PANC-1 pancreatic cancer cells
  • MDA-MB-453 human breast cancer
  • Hepa 1-6 mouse liver cancer cells
  • mouse macrophages RAW264.7
  • mouse melanoma cells Cell culture of B16F10
  • HL7702 human normal liver cells
  • MRC5 human embryonic fibroblasts
  • Cell transfection The cell culture medium was changed to serum-free medium for 1 h. The above cells were transfected with DMP-Display-SBP, respectively. Empty lipofected cells were used as transfection controls. The total amount of DNA per cell and the amount of liposome used were determined in accordance with the instructions for liposome products (Lipofectamine 2000; ThermoFisher Scientific). DNA-liposomes were added to serum-free medium for 4 h. Change to serum-containing fresh medium and continue to culture for 20 h.
  • Cell staining Cells were stained with FITC-labeled streptavidin and its IRDye800CW (a near-infrared fluorescent molecule; LiCor)-labeled streptavidin (LiCor). After transfection of the cells, FITC-labeled streptavidin or IRDye800CW-labeled streptavidin (final concentration of 1 ⁇ g/mL) was directly added to the fresh medium. The cells were further cultured for 20 h, the medium was removed, and the cells were washed twice with PBS. The cells were scanned with a fluorescence microscope or a near-infrared fluorescence scanner (Odyssey, LiCor).
  • the inverted cells were observed with an inverted fluorescence microscope (Olympus IX51-DPI71) to observe whether the cell surface produced green fluorescence. At the same time, the cell growth was observed, such as vigorous growth, good adherence, and no pollution. Photographic photographing of multi-field brightfield and green fluorescence observation channels was performed on various treated cells.
  • Hepa1-6, HepG2, MRC-5, HL7702, and 293T cells were transfected with DMP-Display-SBP expression vector; cells were stained with FITC-labeled streptavidin protein, followed by white field (bright field) and green Microscopic photographing of cells under fluorescent (FITC) channels; superposition of white and green fluorescent channel images (Fig. 3). It can be seen that DMP-controlled cell surface display SBP is expressed only in tumor cells (Hepa 1-6, HepG2, 293T) and not in normal cells (MRC-5, HL7702) (Fig. 2).
  • DMP-controlled cell surface display is only in tumor cells (HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7). , expressed in B16F10), was not expressed in normal cells (MRC-5, HL7702) (Fig. 4).
  • DMP-controlled cell surface display is only in tumor cells (HepG2, 293T, HeLa, PANC-1, MDA-MB-453, HT-29, A549, SKOV-3, Hepa1-6, RAW264.7). , expressed in B16F10), was not expressed in normal cells (MRC-5, HL7702) (Fig. 5).
  • AAV-SBP adeno-associated virus expression vector
  • 293T, HepG2, Hepa1-6, MRC-5, and HL7702 cells were transfected with AAV-SBP.
  • the cells were stained with IRDye800CW-labeled streptavidin protein, which was then scanned on a near-infrared fluorescence scanner (Fig. 6A); cells were trypsinized and collected, and then IRDye800CW-labeled streptavidin protein was used. After staining, the image was scanned on a near-infrared fluorescence scanner and photographed on a white field (Fig. 6B).
  • AAV-SBP AAV virus vector
  • DMP-controlled cell surface display expressed SBP can be efficiently displayed on the surface of tumor cells (HepG2, 293T, Hepa1-6) in normal cells (MRC-5, HL7702). No expression ( Figure 6).
  • the pDMP-Display-SBP vector was constructed as in Example 2.
  • the pDMP-Display-HBsAg and pDMP-Display-CRT vectors were prepared according to the construction procedure of the pDMP-Display-SBP vector.
  • HBsAg The coding sequence of HBsAg is shown in SEQ ID NO. 5, and the coding sequence of CRT is shown in SEQ ID NO.
  • rAAV-DMP viral vector construction AAV-Helper-Free System (Stratagene) was used for the experiment. First, three vectors pDMP-Display-SBP, pDMP-Display-HBsAg and pDMP-Display-CRT were constructed. Primers for the CRT and HBsAg genes were designed. The CRT and HBsAg gene sequences were obtained by PCR amplification using human genomic DNA and hepatitis B virus genomic DNA as templates. The CMV promoter in the pAAV-MCS vector was replaced with a DMP promoter to construct a vector named pAAV-DMP.
  • pAAV-DMP-Display-HBsAg, pAAV-DMP-Display-SBP, pAAV-DMP-Display-CRT vector construction "Display - in pDMP-Display-SBP, pDMP-Display-HBsAg and pDMP-Display-CRT vectors
  • the functional gene functional segments, Display-SBP, Display-HBsAg and Display-CRT, were inserted into the pAAV-DMP vector by enzymatic cleavage to construct pAAV-DMP-Display-HBsAg, pAAV-DMP-Display-SBP, pAAV-DMP.
  • - Display-CRT carrier Restriction sites: upstream Bgl II, downstream Pst I.
  • Plasmid pAAV-DMP-Display-SBP was detected with 293T cells: 293T cells were seeded at a density of 1 x 10 5 cells per well in 24-well plates and cultured for 12 hours. Cells were then transfected with pAAV-DMP-Display-SBP for 4 hours using Lipofectamine 2000. After 4 hours of transfection, the medium containing the liposome was discarded, and the fresh medium was incubated with streptavidin-IDy800CW (near-infrared fluorescein IDy800CW-labeled streptavidin) at a final concentration of 1 ⁇ g/mL. Odyssey Infrared Fluorescence Imaging System (LI-COR) test results. Thereafter, the cells were digested with 0.25% (g/mL) trypsin solution and collected by centrifugation, and scanned in a centrifuge tube.
  • streptavidin-IDy800CW near-infrared fluorescein IDy800CW-labeled
  • Virus preparation 293T cells were co-transfected with the two helper plasmids pHelper and pAAV-RC with pAAV-DMP-Display-SBP, pAAV-DMP-Display-HBsAg and pAAV-DMP-Display-CRT, respectively. After 72 hours of transfection, cells and medium were collected and repeatedly thawed 3 times. 1/10 volume of chloroform was added to the cell freeze-thaw, and the mixture was vigorously shaken at 37 ° C for 1 hour. Solid NaCl was added to a final concentration of 1 mol/L, and centrifuged at 12,000 rpm for 5 minutes at 4 ° C.
  • the upper aqueous phase was transferred, and chloroform and precipitate were discarded.
  • PEG 8000 was added to the upper aqueous phase to a final concentration of 1% (w/v), and then the solution was kept in an ice bath for 1 hour. Then, the liquid was centrifuged at 11,000 rpm for 15 minutes, and the supernatant was discarded. The precipitate was washed with a phosphate buffered saline (PBS) solution and suspended. DNase and RNase were added to a final concentration of 1 ⁇ g/mL, and the solution was incubated at room temperature for 30 minutes. Finally, an equal volume of chloroform was added to the liquid to extract the recombinant virus.
  • the obtained viruses are simply referred to as AAV-SBP, AAV-HBsAg and AAV-CRT, respectively.
  • the virus concentration was determined by real-time PCR. Real-time PCR amplification primers are shown in the table below:
  • Mouse experiment 1 (viral transfected cell tumor-implantation experiment): Mouse liver cancer cells were seeded into 24 wells at a density of 1 ⁇ 10 5 cells/well, and cultured for 12 hours.
  • the mouse hepatoma cells Hepa1-6 were transfected in vitro with AAV-HBsAg, AAV-SBP and AAV-CRT viral vectors, respectively.
  • the transfection dose is 5 ⁇ 10 5 vg/cell; vg is the virus genome, ie the viral genome, indicating the unit of the number of viruses.
  • the control cells were non-transfected cells.
  • the virus-transfected cells and non-transfected cells were further cultured for 24 hours, harvested by trypsinization, resuspended in PBS, and then transplanted into the subcutaneous of the mouse (left transplanted virus-transfected cells, right-hand transplanted non-transfected cells). ).
  • the transplant dose was 1 x 10 7 cells/site.
  • the experimental mice were divided into three groups: AAV-HBsAg, AAV-SBP and AAV-CRT experimental group, with 10 rats in each group. After the cells were transplanted for 15 days, the mice were observed and photographed.
  • the experimental mouse strain was BALB/c-Foxn1 nu . All experimental mice were 4 week old females. All experimental mice were purchased from Changzhou Cavans Laboratory Animal Co., Ltd.
  • Mouse experiment 2 (viral blood injection inhibits subcutaneous xenograft experiments in mice): The experimental mice were divided into 4 groups, namely AAV-HBsAg, AAV-SBP, AAV-CRT and AAV-Control experimental groups, 10 in each group.
  • Mouse hepatoma cells Hepa 1-6 were subcutaneously transplanted into each group of mice. The transplant dose was 1 x 10 7 cells/site. After 7 days of cell transplantation, the mice were observed and photographed. Then, AAV-HBsAg, AAV-SBP, AAV-CRT, and AAV-blank experimental mice were intravenously injected with AAV-HBsAg, AAV-SBP, AAV-CRT, and AAV-Control virus, respectively.
  • the injected dose was 1 x 10 9 vg/mouse. After the virus was injected, the animals were kept for 7 days, and the mice were observed and photographed.
  • the experimental mouse strain was BALB/c-Foxn1 nu . All experimental mice were 4 week old females. All experimental mice were purchased from Changzhou Cavans Laboratory Animal Co., Ltd.
  • FIGs. 7 and 8 A picture of experimental mice in mouse experiment 1 is shown in Figure 7. It can be seen that one side of Hepa1-6 cells transfected with AAV-HBsAg, AAV-SBP and AAV-CRT virus in vitro, in AAV-HBsAg and AAV In the -SBP group, 90% of individual tumor growth was significantly inhibited, and no tumor growth was observed. In the AAV-HBsAg group, there were 4 individuals, and not only the tumor on the side of the AAV-HBsAg transfected liver cancer cells disappeared, but also the tumor on the side of the AAV-HBsAg not transfected with the liver cancer cells disappeared.
  • mice experiment 2 The experimental results of mouse experiment 2 are shown in Figs. 9 and 10 .
  • the experimental mouse pictures of mouse experiment 2 are shown in Fig. 9.
  • the experimental mice were divided into 4 groups, which were AAV-HBsAg, AAV-SBP, AAV-CRT and AAV-blank (empty virus expressed by MCS) experimental group;
  • the virus group was 11 mice; the other 3 groups were 10 mice each.
  • Mouse hepatoma cells Hepa 1-6 were subcutaneously transplanted into each group of mice. The cells were reared for 7 days after transplantation.
  • AAV-HBsAg, AAV-SBP, AAV-CRT, and AAV-Control experimental mice were intravenously injected with AAV-HBsAg, AAV-SBP, AAV-CRT, and AAV-Control virus, respectively. After the virus was injected, the animals were kept for 7 days, and the mice were observed and photographed. The tumor size of the four groups of experimental mice was measured and statistically tested. The results are shown in Figure 10. Blood injections of AAV-HBsAg, AAV-SBP and AAV-CRT viruses were reached, and the virus reached tumor tissues and was transfected into tumor cells.
  • the expression of the new antigens HBsAg, SBP and CRT triggers the body's immune response, strongly inhibits tumor growth, and even eliminates tumor cells. It also shows that blood-injected AAV-HBsAg, AAV-SBP and AAV-CRT viruses can quickly reach and transfect tumor cells, and play a role in tumor immunotherapy.

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Abstract

本发明公开了一种由NF-κB启动的肿瘤细胞特异效应基因表达载体及其表达产物和应用。该基因表达载体包含两个序列元件，调控基因表达的启动子序列与启动子序列下游效应基因编码序列；所述启动子序列由一段NF-κB应答序列和最小启动子序列组成。当该基因表达载体导入肿瘤细胞内时，细胞内的序列特异性转录因子NF-κB就会激活该载体，使其表达载体上的效应基因，效应基因表达产物为一种细胞表面的多肽或蛋白质，该细胞表面的多肽或蛋白质不但可以作为新抗原物质激发体内免疫系统对肿瘤细胞的攻击、发挥肿瘤免疫治疗的功效，还可以作为一种肿瘤细胞人工标记，用于肿瘤成像、诊断、细胞分离等。

Description

一种由NF-κB启动的肿瘤细胞特异效应基因表达载体及其表达产物和应用 技术领域
本发明属于肿瘤免疫治疗技术领域，具体涉及一种由转录因子NF-κB启动的肿瘤细胞特异效应基因表达载体及其表达产物和应用。
背景技术
人类对于癌症治疗有最迫切的期望，科学界和医学界为此坚持不懈地付出探索。癌症是目前困扰人类健康威胁人类生命的重大疾病。虽经发病机理的大量基础研究和各种治疗方法的广泛临床实践，但截至目前，科学界和医学界还没有找到一种可治愈癌症的技术。虽然传统的手术切除加放化疗技术已被普遍用癌症治疗，以及最新的肿瘤免疫疗法也已被探索用于癌症治疗，但治疗的效果还与病人对于健康和生命的期望相差甚远。例如，目前应用最广泛且最有效的肿瘤免疫疗法是PD-1/PD-L1抑制剂(如PD-1抗体)，但其目前的总体应答率却只有20-30％；而在血液恶性肿瘤治疗上有效的CAR-T、TCR-T等细胞治疗，在实体瘤治疗方面还没有实质进展。因此，探索新的癌症治疗技术，在人类可真正治愈癌症前，将始终是科学界和医学界努力的方向。
肿瘤治疗最关键和重要的是找到在癌细胞表面表达的特定抗原。目前肿瘤免疫治疗领域的各种细胞疗法(特别是CAR-T疗法)都无法避免地依赖于该类肿瘤抗原(如目前CAR-T治疗中运用的最成功的CD19)，即肿瘤免疫治疗的靶点。但目前发现的可资利用的这类抗原极其有限，且大多数在正常细胞上也低量表达，在应用时往往引起CAR-T细胞对正常细胞/器官的攻击，导致自身免疫症状，会产生严重副作用。因此，目前该领域正在着力运用二代测序技术寻找更多的新抗原。此外，即使找到这类新抗原，CAR-T治疗的个体化细胞生产也因高昂的成本和潜在的新癌化风险严重阻碍其应用，因此不得不诉诸通用型T细胞的创制。但通用型T细胞也并非完全通用，而仅仅是一种可用于罹患某种同一类型抗原型肿瘤患者的T细胞，对于不同抗原型的肿瘤，仍需制备其特异的T细胞。尽管如此，该类通用型CAR-T的研制，仍代表了肿瘤免疫疗法最新的实用化探索。因此，医学界正努力实现通用型T细胞的研制和临床应用，并已取得显著进展。例如，最近(2017年3月)美国FDA已经批准针对肿瘤抗原CD123的通用型CAR-T UCART123(Cellectis)进入临床试验。CD123抗原在急性骨髓性白血病(AML)细胞与母细胞性浆细胞样树突状细胞瘤(BPDCN)细胞上高度表达。这两种疾病都往往在骨髓中发病，且能在短期内威胁到患者的生命。通用型CAR-T UCART123可用于治疗这两种癌症的免疫治疗。必须指出，任何将病人细胞分离出来，进行体外培养、遗传操作等之后，再回输到病人体内的治疗方案，都存在体外操作带来的风险，尤其是遗传操作。因此，鉴于目前肿瘤免疫治疗尚面临的问题，需要开发新的肿瘤免疫治疗策略和技术。
NF-κB是一种可诱导型DNA结合转录因子，其在肝脏生理及肝炎肝癌病理过程中发挥的所有作用都是通过其调控的靶基因来实现的。例如，NF-κB之所以是一种重要的炎症相关转录因子，是因为它能调控炎症介质的表达。重要的炎症介质如TNF-a、IL-1、IL-6等都是NF-κB的直接靶基因。Bcl-2是公认的抗凋亡蛋白，多种肿瘤高表达Bcl-2。NF-κB抑制肿瘤细胞凋亡的作用正是NF-κB通过直接调控其靶基因Bcl-2的表达而实现的。目前，经过大量的科学研究和临床病例材料的检测分析，已经证明NF-κB几乎在所有肿瘤细胞中被广泛激活，因此NF-κB被视为一种优良的肿瘤治疗及药物筛选靶点。因此，许多制药公司和科学 家都致力于研究NF-κB的抑制剂，但研究人员开发的许多药物在成功抑制NF-κB情况却出现了严重毒副作用而不能成为临床肿瘤治疗药物。究其原因，研究人员发现虽然过度激活的NF-κB对于癌细胞非常关键，但其正常的活化水平由于对健康细胞的正常生理功能也很重要。向细胞导入NF-κB抑制剂，因无法控制其数量，往往造成NF-κB活性的过度抑制，从而产生严重的副作用。
如今，基因治疗成为理想的疾病治疗策略，因为人类的绝大部分疾病均是由基因结构变异或是表达异常引起的，所以最理想的疾病治疗策略就是从基因水平上进行矫正。
发明内容
发明目的：针对现有技术存在的问题，本发明提供一种由NF-κB启动的肿瘤细胞特异效应基因表达载体，当该基因表达载体导入NF-κB活性过度活化的肿瘤细胞内时，细胞内过度活化的转录因子NF-κB就会激活该载体，使其表达载体上的效应基因。该基因表达载体基于细胞内NF-κB活性可以进行肿瘤免疫治疗，该基因表达载体可特异性在肿瘤细胞内表达效应基因，其表达产物为细胞表面的多肽或蛋白质，该细胞表面的多肽或蛋白质可作为一种新抗原蛋白，在机体内被免疫系统识别，产生免疫反应，引起免疫系统对肿瘤细胞的免疫杀伤。
本发明还公开了由NF-κB启动的肿瘤细胞特异效应基因表达载体的表达产物及其在制备用于肿瘤免疫治疗及成像试剂或药物中的应用。
技术方案：为了实现上述目的，如本发明所述一种由NF-κB启动的肿瘤细胞特异效应基因表达载体，其特征在于，包含两个序列元件，调控基因表达的启动子序列与启动子序列下游效应基因编码序列；所述启动子序列由一段NF-κB应答序列和最小启动子序列组成。
其中，所述NF-κB应答序列包括各种序列的NF-κB应答序列；所述NF-κB应答序列为一段可与NF-κB蛋白特异性结合的DNA序列，其主要序列特征为含有数量不同的各种NF-κB结合靶点。
所述启动子为一种NF-κB特异性启动子，即只有NF-κB可激活的启动子。
所述最小启动子包括各种来源的最小启动子序列，所述各种来源的最小启动子序列包括天然及人工筛选的最小启动子序列。如纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase，HSV-TK)启动子最小启动子；其主要作用是与基础转录因子及RNA聚合酶II结合，形成通用转录机器，构成基因表达的基本条件。
作为优选，所述调控基因表达的启动子序列尤指序列SEQ ID NO.1:5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CTA GAG GGT ATA TAA TGG AAG CTC GAC TTC CAG-3'。其中NF-κB应答序列(SEQ ID NO.2:5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3')，最小启动子序列(SEQ ID NO.3:5'-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3')。
其中，所述效应基因为编码一种细胞膜蛋白的基因，该蛋白突出在细胞膜外的部分则可以作为一种新抗原物质，刺激机体免疫系统，造成机体免疫系统对肿瘤细胞的免疫杀伤。
作为优选，所述效应基因为乙肝表面抗原编码基因HBsAg、链亲和素结合肽编码基因SBP或者钙网织蛋白编码基因CRT。
其中，基因表达载体为一线性(linear)或环状(circular)核酸分子。
更进一步地，所述核酸分子为脱氧核糖核酸(DNA)或核糖核酸(RNA) 分子，包括双链DNA(如腺病毒DNA分子)、单链DNA(腺相关病毒分子)或单链RNA分子(如慢病毒RNA分子)等。
所述线性核酸分子包括普通线性DNA分子(如PCR扩增片段、酶切片段)、病毒DNA分子(如腺病毒DNA分子、腺相关病毒分子)或病毒RNA分子(如慢病毒RNA分子)等；所述环状核酸分子包括质粒DNA。
本发明所述的基因表达载体的基因表达方法，将基因表达载体导入NF-κB活性过度活化的肿瘤细胞内，细胞内过度活化的转录因子NF-κB就会激活该载体，使其表达载体上的效应基因。
其中，所述基因表达载体导入细胞的方法包括各种类型的核酸细胞导入方法。
作为优选，所述核酸细胞导入方法包括病毒载体、纳米载体、脂质体、电转移或基因枪等导入方式。
进一步地，所述基因表达载体导入细胞的方法为纳米载体及病毒载体导入。
所述基因表达载体导入细胞的方法最佳为腺相关病毒(AAV)载体。
本发明所述由NF-κB启动的肿瘤细胞特异效应基因表达载体的表达产物，所述表达产物为细胞表面的多肽或蛋白质。
其中，所述细胞表面的多肽或蛋白质可作为一种新抗原蛋白，在机体内被免疫系统识别，产生免疫反应，引起免疫系统对肿瘤细胞的免疫杀伤。
其中，所述细胞表面的多肽或蛋白质可作为一种肿瘤细胞人工标记，用于肿瘤的体内成像、诊断、细胞分离。
所述在细胞表面的多肽或蛋白质包括任何多肽或蛋白质，以及表达中经过细胞自身功能发生糖基化等修饰的多肽或蛋白质。
作为优选，所述多肽或蛋白质为乙肝表面抗原(HBsAg)、链亲和素结合肽(SBP)及钙网织蛋白(calreticulin，CRT)。
其中，链亲和素结合肽(SBP)也可用于肿瘤的体内成像及诊断，如利用链亲和素标记的MRI、CT、PET、近红外荧光等造影剂与表达在肿瘤细胞表面的SBP结合，进行肿瘤成像、诊断等。
本发明所述的基因表达载体为一种基于细胞内NF-κB活性进行肿瘤基因治疗的基因表达载体可以应用在肿瘤免疫治疗、成像中。本发明所述由NF-κB启动的肿瘤细胞特异效应基因表达载体在制备用于肿瘤免疫治疗及成像试剂或药物中的应用。
在现有技术中既然向细胞内导入NF-κB抑制剂由于其严重副作用无法形成可用于临床的药物，本发明可反其道而行之，利用NF-κB是一种转录因子且在肿瘤细胞内其活性均过度活化的特性，借助一种由NF-κB启动的肿瘤细胞特异效应基因表达载体实现肿瘤免疫治疗。
本发明的由NF-κB启动的肿瘤细胞特异效应基因表达载体基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达的机理如原理示意图(图1)所示。图1反映本发明构建了一种基因表达载体,该基因表达载体含有NF-κB应答序列、最小启动子及效应基因三种元件；将该基因表达载体导入肿瘤细胞内时，肿瘤细胞内过度活化的转录因子NF-κB蛋白就会结合载体上的NF-κB应答序列，从而激活效应基因的表达；效应基因的表达产物为一种细胞膜蛋白，该蛋白突出在细胞膜外的部分则可以作为一种新抗原物质，刺激机体免疫系统，造成机体免疫系统对肿瘤细胞的免疫杀伤。
有益效果：与现有技术相比，本发明具有如下优点：
1、本发明设计了一种由肿瘤细胞中过度活化的转录因子NF-κB特异性启动表达的基因表达载体，该基因表达载体可特异性在肿瘤细胞内表达效应基因；该效应基因表达在细胞表面的多肽或蛋白质；细胞表面的多肽或蛋白质可作为一种新抗原蛋白，在机体内被免疫系统识别，产生免疫反应，引起免疫系统对肿瘤细胞的免疫杀伤。本发明通过设计的NF-κB特异性启动表达的基因表达载体进行的肿瘤治疗为一种新策略、新技术，革新了肿瘤敏免疫治疗的原理。
2、本发明提出的肿瘤免疫疗法突破了目前依赖肿瘤细胞表面存在的数量极其有限的自然抗原的约束，通过基因治疗技术在肿瘤细胞表面表达和创造一种人工抗原，以此引发机体强烈的免疫反应，杀灭机体内的肿瘤细胞。人工抗原不受种类和数量的限制。
3、由于本发明使用的是一种肿瘤细胞特异的基因表达载体系统，这种基因表达载体表达产物作为人工抗原只会表达在肿瘤细胞表面，而不会表达在正常细胞表面，从而避免了所激发的免疫反应对正常细胞的攻击，发挥非常特异地肿瘤细胞免疫攻击反应。因此，该基因表达载体对于肿瘤细胞进行免疫是一种肿瘤细胞高度特异的免疫疗法。
4、本发明设计的基因表达载体对于肿瘤细胞进行免疫的肿瘤免疫疗法可借助纳米载体或病毒载体，特别是安全性很好的腺相关病毒载体，将基因表达载体通过纳米载体或病毒载体的静脉给药，即可完成治疗，是一种无创基因治疗技术。从而避免了目前肿瘤治疗的手术、化疗、放疗、CAR-T制造等繁琐、危险、损伤性的治疗过程，非常有助于提高肿瘤患者的生活质量。
5、本发明设计和论证的NF-κB特异性激活基因表达载体不同于目前使用的NF-κB抑制剂，在治疗原理上不是抑制NF-κB，而是利用NF-κB，避免了使用NF-κB抑制剂进行肿瘤治疗的严重副作用，因此是一种截然不同的创新性很强的基于NF-κB的肿瘤治疗新策略。
6、本发明提出的肿瘤细胞特异的NF-κB启动基因表达载体可包装在腺相关病毒(AAV)中，利用AAV病毒作为基因治疗优良载体的特性，用于制备人体内肿瘤成像及治疗的药物中。目前AAV病毒载体可单针一次性注射进行疾病的基因治疗，因此，腺相关病毒(AAV)搭载了本发明设计和论证的NF-κB特异性激活基因表达载体后，有望成为治疗肿瘤的简单、无创(一次性静脉滴注)、高效的新型生物药物。这基本上已经确定了新疗法的药物剂型(AAV病毒)、给药方式(静脉滴注)、安全性(AAV载体)等关键成药性环节；为本发明技术的临床应用奠定了基础。
附图说明
图1为本发明由NF-κB启动的肿瘤细胞特异效应基因表达载体基于细胞内NF-κB活性激活效应基因在NF-κB过度活化细胞内的基因表达原理示意图；其中gene expression vector为基因表达载体；NF-κB responsive sequence(NF-κB binding sequences)为NF-κB应答序列(NF-κB结合序列)；minimal promoter为最小启动子；effective gene为效应基因；transfection为转染；over activated transcription factor NF-κB为过度活化的转录因子NF-κB；NF-κB binding and gene expression activation为NF-κB结合及基因表达激活；effective gene expression为效应基因表达；effective gene products为效应基因产物；cell growth arrest/apoptosis/dead为细胞生长抑制/凋亡/死亡；NF-κB over-activated cell为NF-κB过度活化的细胞；
图2为NF-κB在不同细胞中表达的荧光定量PCR检测结果；可见NF-κB 在肿瘤细胞中有表达，但在正常细胞中无表达；
图3为DMP-Display-SBP表达载体转染Hepa1-6、HepG2、MRC-5、HL7702、293T细胞后，对细胞进行FITC标记的链霉亲和素蛋白的染色，之后在白场(bright field)和绿色荧光(FITC)通道下对细胞显微拍照图；再将白场和绿色荧光通道图像的叠加，可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(Hepa1-6、HepG2、293T)中有表达，在正常细胞(MRC-5、HL7702)中无表达；
图4为DMP-Display-SBP表达载体转染HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10、MRC-5、HL7702细胞后，对细胞进行IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像图；再对各孔荧光强度进行量化，可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10)中表达，在正常细胞(MRC-5、HL7702)中无表达；
图5为DMP-Display-SBP表达载体转染HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10、MRC-5、HL7702细胞后，对细胞进行胰酶消化收集，再用IRDye800CW标记的链霉亲和素蛋白的染色；之后在近红外荧光扫描仪上扫描成像并在白场拍照图；可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10)中表达，在正常细胞(MRC-5、HL7702)中无表达；
图6为将DMP-Display-SBP包装成腺相关病毒(AAV)表达载体(AAV-SBP)后，用AAV-SBP转染293T、HepG2、Hepa1-6、MRC-5、HL7702细胞；对细胞进行IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像图(A图)；对细胞进行胰酶消化收集，再用IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像并在白场拍照图(B图)；可见包装成AAV病毒载体(AAV-SBP)，DMP控制的细胞表面展示表达的SBP可以高效地展示在肿瘤细胞(HepG2、293T、Hepa1-6)表面，在正常细胞(MRC-5、HL7702)中无表达；
图7为小鼠实验1的照片；其中AAV-HBsAg、AAV-SBP、AAV-CRT病毒载体在体外转染小鼠肝癌细胞Hepa1-6后，将病毒转染细胞与非转染细胞收获，分左右移植到小鼠皮下(左侧移植病毒转染细胞、右侧移植非转染细胞)，实验小鼠分AAV-HBsAg、AAV-SBP及AAV-CRT实验组3组，每组10只，细胞移植后饲养15日，对小鼠观察并拍照；
图8为图7中实验小鼠肿瘤大小测定结果示意图；其中AAV-HBsAg-L为图8中HBsAg实验组左侧肿瘤大小测定结果；AAV-HBsAg-R为图8中HBsAg实验组右侧肿瘤大小测定结果；AAV-SBP-L为图8中SBP实验组左侧肿瘤大小测定结果；AAV-SBP-R为图8中SBP实验组右侧肿瘤大小测定结果；AAV-CRT-L为图8中CRT实验组左侧肿瘤大小测定结果；AAV-CRT-R为图8中CRT实验组右侧肿瘤大小测定结果；*表示p值小于0.05(差异显著)，**表示p值小于0.01(差异极显著)；
图9为小鼠实验2的照片；其中实验小鼠分4组，分别为AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-blank(空病毒以MCS表示)实验组；空病毒组11只；其他3组每组10只，对每组每只小鼠进行小鼠肝癌细胞Hepa1-6分左右皮 下移植，细胞移植后饲养7日，之后对AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-blank实验组小鼠分别进行AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-blank病毒的静脉注射，病毒注射后继续饲养7日，对小鼠观察并拍照；
图10为图9中实验小鼠肿瘤大小测定结果示意图；其中AAV-MCS-L为图9中MCS实验组左侧肿瘤大小测定结果；AAV-MCS-R为图9中MCS实验组右侧侧肿瘤大小测定结果；AAV-HBsAg-L为图9中HBsAg实验组左侧肿瘤大小测定结果；AAV-HBsAg-R为图9中HBsAg实验组右侧肿瘤大小测定结果；AAV-SBP-L为图9中SBP实验组左侧肿瘤大小测定结果；AAV-SBP-R为图9中SBP实验组右侧侧肿瘤大小测定结果；AAV-CRT-L为图9中CRT实验组左侧肿瘤大小测定结果；AAV-CRT-R为图9中CRT实验组右侧肿瘤大小测定结果；AAV-MCS为rAAV-MCS-L及rAAV-MCS-R的合并；AAV-HBsAg为AAV-HBsAg-L及AAV-HBsAg-R的合并；AAV-SBP为AAV-SBP-L及AAV-SBP-R的合并；AAV-CRT为AAV-CRT-L及AAV-CRT-R的合并，图中显示了AAV-HBsAg、AAV-SBP及AAV-CRT组肿瘤大小分别与AAV-MCS组肿瘤大小的统计学显著性检验结果，**表示p值小于0.01(差异极显著)。
具体实施方式
以下结合实施例和附图对本发明作进一步说明。
实施例1
NF-κB RelA不同细胞表达
实验方法：
细胞培养：HEK-293T(人胎肾细胞)、HepG2(人肝癌细胞)、A549(人肺癌细胞)、HT-29(人结肠癌细胞)、HeLa(人宫颈癌细胞)、SKOV3(人卵巢癌细胞)、PANC-1(胰腺癌细胞)、MDA-MB-453(人乳腺癌)、Hepa1-6(小鼠肝癌细胞)、小鼠巨噬细胞(RAW264.7)、小鼠黑色素瘤细胞(B16F10)、HL7702(人正常肝细胞)及MRC5(人胚胎成纤维细胞)细胞培养，实施例中所用细胞均购自于中科院上海生命科学研究院。细胞培养使用DEME(Hepa1-6、HEK-293T、HepG2、HeLa、PANC-1、MDA-MB-453、RAW264.7、B16F10、MRC-5)或RPMI 1640培养基(A549、HT-29、SKOV-3、HL7702)、10％(v/v)胎牛血清(HyClone)、100units/mL青霉素和100μg/mL链霉素培养；培养环境为含有5％(v/v)CO 2的加湿培养箱中37℃培养。细胞复苏后，按同等密度接种到24孔微孔板(1×10 5/孔)或12孔微孔板(2×10 5/孔)中，培养过夜贴壁后，进行转染。
基因表达检测：收集细胞，用Trizol提取总RNA，逆转录合成互补DNA(cDNA)。cDNA制备反应及程序为：10μL反转录反应组分包含2μL 5×PrimeScript RT Master Mix(Takara)、50ng总RNA，用RNase Free ddH 2O将反应总体积补至10μL；37℃反应15分钟，升温至85℃反应5秒使反转录酶失活，反应液4℃保存。通过qPCR定量分析RelA表达。用于qPCR的上下引物为5′-CCT GGA GCA GGC TAT CAG TC-3′(F)与5′-ATG GGA TGA GAA AGG ACA GG-3′(R)。PCR模板为cDNA，通过qPCR定量分析RelA表达。10μL qPCR反应含有5μL Fast SYBR Green Master Mix(ABI)，0.2μL 10μM F，0.2μL 10μM R和1μL cDNA,用ddH 2O将反应总体积补至10μL。将配制好的反应体系放于荧光定量PCR仪(StepOne plus，ABI)上进行扩增，设置的扩增程序为：95℃预变性10分钟、45个扩增循环(95℃变性15s，在各退火温度下扩增1分钟)。荧光定量PCR扩增的特异性用溶解曲线来分析确定，用比较CT值法来计算基 因表达的相对定量(RQ)，数据最后表示为平均值±标准偏差(SD)，统计显著性用t检验来确定。
实验结果：
为了考察NF-κB RelA/p65基因在各种肿瘤细胞及正常细胞中的表达，用荧光定量PCR检测了NF-κB RelA/p65在11种肿瘤细胞及正常细胞(HL7702及MRC-5)中的表达(图2)。结果表明在所有肿瘤细胞株中NF-κB RelA/p65均有表达，而在正常细胞(HL7702及MRC5)中，检测不得到NF-κB表达。说明用本发明提出的NF-κB激活基因表达载体是一种NF-κB过度活化细胞特异的基因表达载体，如肿瘤细胞中NF-κB激活基因表达载体的表达。
实施例2
DMP-Display-SBP(效应基因细胞表面表达)
实验方法：
载体构建：构建一表达载体DMP-Display-SBP；该载体含有DMP序列及可细胞表达链亲和素结合肽(SBP)的编码序列。其中DMP含有NF-κB应答序列SEQ ID NO.2:(5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC C-3')以及最小启动子序列SEQ ID NO.3:(5'-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3')。SBP编码序列SEQ ID NO.4:ATG GAC GAG AAG ACC ACC GGG TGG CGG GGC GGC CAC GTT GTG GAG GGT CTC GCT GGC GAG CTG GAG CAG CTC AGG GCC CGC TTG GAG CAC CAT CCC CAG GGG CAA CGC GAG CCT ATC GAT TAA。展示表达的骨架序列克隆自载体pDisplay TM(Invitrogen)；pDisplay TM将要展示在细胞膜表面的蛋白或多肽融合在大鼠Igκ-链引导序列(Igκ-chain leader sequence)的N端,该引导序列可指引蛋白的分母途径；pDisplay表达蛋白的C末端为血小板衍生生长因子受体(platelet derived growth factor receptor，PDGFR)跨膜区，该跨膜区将蛋白锚定在细胞膜上，从而将蛋白展示在细胞外侧。这种膜蛋白可以和细胞培养液中的蛋白发生互作，如本实施例中的链霉亲和素与在细胞膜表面的链霉亲和素结合肽(SBP间的互作)。
细胞培养：HEK-293T(人胎肾细胞)、HepG2(人肝癌细胞)、A549(人肺癌细胞)、HT-29(人结肠癌细胞)、HeLa(人宫颈癌细胞)、SKOV3(人卵巢癌细胞)、PANC-1(胰腺癌细胞)、MDA-MB-453(人乳腺癌)、Hepa1-6(小鼠肝癌细胞)、小鼠巨噬细胞(RAW264.7)、小鼠黑色素瘤细胞(B16F10)、HL7702(人正常肝细胞)及MRC5(人胚胎成纤维细胞)细胞培养。细胞培养使用DEME(Hepa1-6、HEK-293T、HepG2、HeLa、PANC-1、MDA-MB-453、RAW264.7、B16F10、MRC-5)或RPMI 1640培养基(A549、HT-29、SKOV-3、HL7702)、10％(v/v)胎牛血清(HyClone)、100units/mL青霉素和100μg/mL链霉素培养；培养环境为含有5％(v/v)CO 2的加湿培养箱中37℃培养。细胞复苏后，按同等密度接种到24孔微孔板(1×10 5/孔)或12孔微孔板(2×10 5/孔)中，培养过夜贴壁后，进行转染。
细胞转染：细胞培养液换为无血清培养基培养1h。分别用DMP-Display-SBP转染上述细胞。空脂质体转染的细胞作为转染对照。每孔细胞的总DNA用量及脂质体用量参考脂质体产品(Lipofectamine 2000；ThermoFisher Scientific)说明书进行。DNA-脂质体加入无血清培养基培养4h。换为含血清新鲜培养基，继续培养20h。
细胞染色：用FITC标记的链亲和素及其IRDye800CW(一种近红外荧光分 子；LiCor公司)标记的链亲和素(LiCor)对细胞染色。细胞转染后再新鲜培养基中直接加入用FITC标记的链亲和素或IRDye800CW标记的链亲和素(终浓度均为1μg/mL)。细胞继续培养20h，去除培养基，PBS洗涤细胞2次。细胞用荧光显微镜或近红外荧光扫描仪(Odyssey，LiCor)扫描成像。细胞观察：用倒置荧光显微镜(Olympus IX51-DPI71)观测拍照各种处理的细胞，主要观察细胞表面是否产生绿色荧光；同时观察细胞生长情况，如生长旺盛、贴壁良好、无污染等。对各种处理的细胞进行多视野明场及绿色荧光观察通道的照相拍摄。
实验结果：
用DMP-Display-SBP表达载体转染Hepa1-6、HepG2、MRC-5、HL7702、293T细胞；对细胞进行FITC标记的链霉亲和素蛋白的染色，之后在白场(bright field)和绿色荧光(FITC)通道下对细胞显微拍照；再将白场和绿色荧光通道图像的叠加(图3)。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(Hepa1-6、HepG2、293T)中有表达，在正常细胞(MRC-5、HL7702)中无表达(图2)。
用DMP-Display-SBP表达载体转染HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10、MRC-5、HL7702细胞后，对细胞进行IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像；再对各孔荧光强度进行量化。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10)中表达，在正常细胞(MRC-5、HL7702)中无表达(图4)。
用DMP-Display-SBP表达载体转染HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10、MRC-5、HL7702细胞后，对细胞进行胰酶消化收集，再用IRDye800CW标记的链霉亲和素蛋白的染色；之后在近红外荧光扫描仪上扫描成像并在白场拍照。可见DMP控制的细胞表面展示(Display)的SBP仅在肿瘤细胞(HepG2、293T、HeLa、PANC-1、MDA-MB-453、HT-29、A549、SKOV-3、Hepa1-6、RAW264.7、B16F10)中表达，在正常细胞(MRC-5、HL7702)中无表达(图5)。
用将DMP-Display-SBP包装成腺相关病毒(AAV)表达载体(AAV-SBP)后，用AAV-SBP转染293T、HepG2、Hepa1-6、MRC-5、HL7702细胞。对细胞进行IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像(图6A)；对细胞进行胰酶消化收集，再用IRDye800CW标记的链霉亲和素蛋白的染色，之后在近红外荧光扫描仪上扫描成像并在白场拍照(图6B)。可见包装成AAV病毒载体(AAV-SBP)，DMP控制的细胞表面展示表达的SBP可以高效地展示在肿瘤细胞(HepG2、293T、Hepa1-6)表面，在正常细胞(MRC-5、HL7702)中无表达(图6)。
实施例3
AAV-HBsAg、AAV-SBP、AAV-CRT免疫治疗小鼠肿瘤
实验方法：
pDMP-Display载体构建：同实施例2构建pDMP-Display-SBP载体。按pDMP-Display-SBP载体的构建程序构建制备pDMP-Display-HBsAg及pDMP-Display-CRT载体。
其中HBsAg的编码序列如SEQ ID NO.5所示，CRT的编码序列如SEQ ID NO.6所示。
rAAV-DMP病毒载体构建：AAV-Helper-Free System(Stratagene)用于实验。首先，构建了三种载体pDMP-Display-SBP，pDMP-Display-HBsAg和pDMP-Display-CRT。设计了CRT和HBsAg基因的引物。以人基因组DNA和乙肝病毒基因组DNA作为模板，通过PCR扩增分别获得获得CRT和HBsAg基因序列。将pAAV-MCS载体中的CMV启动子替换为DMP启动子以构建名为pAAV-DMP的载体。
pAAV-DMP-Display-HBsAg、pAAV-DMP-Display-SBP、pAAV-DMP-Display-CRT载体构建：将pDMP-Display-SBP，pDMP-Display-HBsAg和pDMP-Display-CRT载体中的“展示-效应基因”功能片段，即Display-SBP、Display-HBsAg和Display-CRT，通过酶切割分别插入pAAV-DMP载体中，构建pAAV-DMP-Display-HBsAg、pAAV-DMP-Display-SBP、pAAV-DMP-Display-CRT载体。限制性位点：上游Bgl II，下游Pst I。
用293T细胞检测质粒pAAV-DMP-Display-SBP：将293T细胞以每孔1×10 5个细胞的密度接种在24孔板中并培养12小时。然后使用Lipofectamine 2000将细胞用pAAV-DMP-Display-SBP转染4小时。转染4小时后，丢弃含有脂质体的培养基，并将新鲜培养基与最终浓度为1μg/mL的链亲和素-IDy800CW(近红外荧光素IDy800CW标记的链亲和素)温育。Odyssey红外荧光成像系统(LI-COR)检测结果。之后，用0.25％(g/mL)胰蛋白酶溶液消化细胞并通过离心收集，在离心管中扫描。
病毒制备：用pAAV-DMP-Display-SBP、pAAV-DMP-Display-HBsAg和pAAV-DMP-Display-CRT分别与两种辅助质粒pHelper和pAAV-RC共转染293T细胞。转染72小时后，收集细胞和培养基，反复冻融3次。向细胞冻融液中加入1/10体积的氯仿，37℃剧烈振荡1小时。加入终浓度为1mol/L的固体NaCl，4℃下12000转/分钟离心5分钟，转移上层水相，弃去氯仿和沉淀。在上层水相中加入PEG8000至终浓度达1％(w/v)，然后将溶液保持在冰浴中1小时。然后，液体以11000转/分钟离心15分钟，弃上清。沉淀用磷酸缓冲盐(PBS)溶液洗涤并悬浮。加入DNase和RNase至终浓度为1μg/mL，然后将溶液在室温下保温30分钟。最后，将等体积的氯仿加入到液体中以提取重组病毒。所获得的病毒分别简称为：AAV-SBP、AAV-HBsAg和AAV-CRT。通过实时PCR确定病毒浓度。实时PCR扩增引物见下表：
Figure PCTCN2019070016-appb-000001
小鼠实验1(病毒转染细胞植瘤实验)：将小鼠肝癌细胞以1×10 5细胞/孔的密度接种到24孔中，培养12小时。用AAV-HBsAg、AAV-SBP及AAV-CRT病毒载体分别转染在体外转染小鼠肝癌细胞Hepa1-6。转染剂量为5×10 5vg/细胞；vg为virus genome，即病毒基因组，表示病毒个数的单位。对照组细胞为非转染细胞。将病毒转染细胞与非转染细胞继续培养24小时后，胰酶消化收获，重悬 与PBS后，分左右移植到小鼠皮下(左侧移植病毒转染细胞、右侧移植非转染细胞)。移植剂量为1×10 7细胞/处。实验小鼠分AAV-HBsAg、AAV-SBP及AAV-CRT实验组3组，每组10只。细胞移植后饲养15日，对小鼠观察并拍照。实验小鼠品系为BALB/c-Foxn1 nu。所有实验小鼠均为4周龄雌鼠。所有实验小鼠均购自常州卡文斯实验动物有限公司。
小鼠实验2(病毒血液注射抑制小鼠皮下移植瘤实验)：实验小鼠分4组，分别为AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-Control实验组，每组10只。对每组每只小鼠进行小鼠肝癌细胞Hepa1-6分左右皮下移植。移植剂量为1×10 7细胞/处。细胞移植后饲养7日，对小鼠观察并拍照。之后对AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-blank实验组小鼠分别进行AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-Control病毒的静脉注射。注射剂量为1×10 9vg/只小鼠。病毒注射后继续饲养7日，对小鼠观察并拍照。实验小鼠品系为BALB/c-Foxn1 nu。所有实验小鼠均为4周龄雌鼠。所有实验小鼠均购自常州卡文斯实验动物有限公司。
实验结果：
小鼠实验1的实验结果如图7、图8所示。小鼠实验1的实验小鼠图片如图7所示，可见接种了AAV-HBsAg、AAV-SBP、AAV-CRT病毒体外转染小鼠肝癌细胞Hepa1-6的一侧，在AAV-HBsAg和AAV-SBP组中，90％的个体肿瘤生长被显著抑制，未见肿瘤生长。在AAV-HBsAg组中有4只个体，不但接种AAV-HBsAg转染肝癌细胞一侧的肿瘤消失，而且接种AAV-HBsAg未转染肝癌细胞一侧的肿瘤也消失了。在AAV-SBP组中有3只个体，不但接种AAV-SBP转染肝癌细胞一侧的肿瘤消失，而且接种AAV-SBP未转染肝癌细胞一侧的肿瘤也消失了。在AAV-CRT组中，70％的个体接种了AAV-CRT转染肝癌细胞一侧的肿瘤消失了；其中1只接种AAV-SBP未转染肝癌细胞一侧的肿瘤也消失了。可见该实验取得非常理想的治疗效果。对3组实验小鼠的肿瘤大小进行测定并进行统计学检验，结果如图8所示，表明AAV-HBsAg、AAV-SBP及AAV-CRT病毒的转染显著抑制了皮下移植瘤的生长，说明这些病毒载体的转染是细胞表面产生新生抗原HBsAg、SBP及CRT，引发机体免疫反应，强烈抑制了肿瘤生长，甚至消除肿瘤细胞。
小鼠实验2的实验结果如图9、图10所示。小鼠实验2的实验小鼠图片如图9所示，实验小鼠分4组，分别为AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-blank(空病毒以MCS表示)实验组；空病毒组为11只小鼠；其他3组每组10只。对每组每只小鼠进行小鼠肝癌细胞Hepa1-6分左右皮下移植。细胞移植后饲养7日。之后对AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-Control实验组小鼠分别进行AAV-HBsAg、AAV-SBP、AAV-CRT及AAV-Control病毒的静脉注射。病毒注射后继续饲养7日，对小鼠观察并拍照。对4组实验小鼠的肿瘤大小进行测定并进行统计学检验，结果如图10所示，表明AAV-HBsAg、AAV-SBP及AAV-CRT病毒的血液注射，病毒到达肿瘤组织并转染肿瘤细胞，表达产生新生抗原HBsAg、SBP及CRT，引发机体免疫反应，强烈抑制了肿瘤生长，甚至消除肿瘤细胞。同时也说明血液注射的AAV-HBsAg、AAV-SBP及AAV-CRT病毒可快速到达并转染肿瘤细胞，发挥肿瘤免疫治疗作用。
Figure PCTCN2019070016-appb-000002
Figure PCTCN2019070016-appb-000003
Figure PCTCN2019070016-appb-000004
Figure PCTCN2019070016-appb-000005

Claims (13)

  1. 一种由NF-κB启动的肿瘤细胞特异效应基因表达载体，其特征在于，包含两个序列元件，调控基因表达的启动子序列与启动子序列下游效应基因编码序列；所述启动子序列由一段NF-κB应答序列和最小启动子序列组成。
  2. 根据权利要求1所述的基因表达载体，其特征在于，所述NF-κB应答序列包括各种序列的NF-κB应答序列；所述NF-κB应答序列为一段可与NF-κB蛋白特异性结合的DNA序列，其主要序列特征为含有数量不同的各种NF-κB结合靶点。
  3. 根据权利要求1所述的基因表达载体，其特征在于，所述调控基因表达的启动子为一种NF-κB特异启动子，即只有NF-κB可激活的启动子。
  4. 根据权利要求1所述的基因表达载体，其特征在于，所述最小启动子包括各种来源的最小启动子序列，所述各种来源的最小启动子序列包括天然及人工筛选的最小启动子序列。
  5. 根据权利要求1所述的基因表达载体，其特征在于，所述效应基因为乙肝表面抗原编码基因HBsAg、链亲和素结合肽编码基因SBP或者钙网织蛋白编码基因CRT。
  6. 根据权利要求1所述的基因表达载体，其特征在于，所述基因表达载体为一线性或环状核酸分子。
  7. 根据权利要求6所述的基因表达载体，其特征在于，所述线性核酸分子包括普通线性DNA分子、病毒DNA分子或病毒RNA分子；所述环状核酸分子包括质粒DNA。
  8. 一种利用权利要求1所述的基因表达载体的基因表达方法，其特征在于，将基因表达载体导入NF-κB活性过度活化的肿瘤细胞内，细胞内过度活化的转录因子NF-κB就会激活该载体，使其表达载体上的效应基因。
  9. 根据权利要求8所述的基因表达方法，其特征在于，所述基因表达载体导入细胞的方法包括病毒载体、纳米载体、脂质体、电转移或基因枪导入方式。
  10. 一种权利要求1所述由NF-κB启动的肿瘤细胞特异效应基因表达载体激活效应基因的表达产物，其特征在于，所述表达产物为细胞表面的多肽或蛋白质。
  11. 根据权利要求10所述的表达产物，其特征在于，所述细胞表面的多肽或蛋白质作为一种抗原蛋白，在机体内被免疫系统识别，产生免疫反应，引起免疫系统对肿瘤细胞的免疫杀伤。
  12. 根据权利要求10所述的表达产物，其特征在于，所述细胞表面的多肽或蛋白质作为一种肿瘤细胞人工标记，用于肿瘤的体内成像、诊断、细胞分离。
  13. 一种权利要求1所述由NF-κB启动的肿瘤细胞特异效应基因表达载体在制备用于肿瘤免疫治疗及成像试剂或药物中的应用。
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