WO2021031551A1 - 一种调控脂质代谢的复制型溶瘤腺病毒及其应用 - Google Patents

一种调控脂质代谢的复制型溶瘤腺病毒及其应用 Download PDF

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WO2021031551A1
WO2021031551A1 PCT/CN2020/078360 CN2020078360W WO2021031551A1 WO 2021031551 A1 WO2021031551 A1 WO 2021031551A1 CN 2020078360 W CN2020078360 W CN 2020078360W WO 2021031551 A1 WO2021031551 A1 WO 2021031551A1
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tumor
apoa1
cancer
oncolytic adenovirus
cells
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French (fr)
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魏继武
董杰
徐天成
孔令凯
沃冠群
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南京诺惟生物科技有限公司
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Priority to JP2022511351A priority Critical patent/JP7316711B2/ja
Priority to EP20855578.9A priority patent/EP4019641A4/en
Publication of WO2021031551A1 publication Critical patent/WO2021031551A1/zh
Priority to US17/676,152 priority patent/US11629362B2/en
Priority to US18/295,174 priority patent/US20230365993A1/en

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Definitions

  • the invention relates to the field of tumor biotherapy, and relates to a replication-type oncolytic adenovirus that effectively inhibits tumor growth, invasion and metastasis, and applications thereof.
  • oncolytic virus itself can replicate in tumor cells and exert an oncolytic effect. At the same time, it can activate immune cells, cause immune cell infiltration in the tumor area, and induce an anti-tumor immune response.
  • oncolytic viruses can carry foreign genes, express foreign proteins locally on tumors, and directly exert the anti-tumor effects of protein drugs.
  • T-Vec the first oncolytic virus drug
  • the recombinant herpes simplex virus-1 can express GM-CSF to activate DC to exert anti-tumor activity.
  • the development of drugs for oncolytic viruses is mainly by constructing recombinant expression vectors to release immune activating factors or immune checkpoint antibodies to activate the patient’s immune function and exert anti-tumor effects, such as a kind of human penetrating peptide p53 and GM- Recombinant oncolytic adenovirus with CSF gene (CN201310460980), and a Newcastle disease oncolytic virus (CN201811560794) expressing PD-L1 single-chain antibody.
  • the current existing technology cannot effectively control tumor metastasis.
  • Apolipoprotein ApoA1 is the most abundant component in the ApoA family. It is synthesized in the liver and small intestine and is a single polypeptide composed of 243 amino acid residues.
  • ApoA1 is an activator of lecithin cholesterol acyltransferase, mainly present in high-density lipoprotein (HDL), and is the main apolipoprotein of HDL.
  • the main function of ApoA1 is to mediate the reverse transport of cholesterol, that is, transport cholesterol from peripheral cells to the liver.
  • ApoA1 binds to the specific receptor adenosine triphosphate binding cassette transporter A1 (ABCA1) on the cell surface to take up cell free cholesterol and phospholipids to generate new HDL.
  • ABCA1 specific receptor adenosine triphosphate binding cassette transporter A1
  • Lecithin cholesterol lipid acyltransferase converts free cholesterol in HDL particles into cholesterol lipids, which are then transported to the liver for metabolism.
  • LCAT Lecithin cholesterol lipid acyltransferase
  • the document's plan is to construct in vitro dimyristoylphosphatidylcholine (DMPC), free cholesterol (FC), ApoA1 composition ratio of 200:9:1 PL, and then form a complex with oncolytic adenovirus The way it works.
  • This method not only has a complicated preparation process, but also has a short duration of drug effect.
  • the purpose of the present invention is to provide a replicating oncolytic adenovirus capable of inhibiting tumor growth and tumor invasion and metastasis and its application.
  • the construction scheme of the replicating oncolytic adenovirus vector includes: an experimental grade tagged adenovirus vector and a pharmaceutical grade unlabeled adenovirus vector.
  • the experimental-grade adenovirus replication element E1A is controlled by the first constitutive promoter CMV.
  • the GFP sequence is connected upstream of E1A and the A2 connection sequence is connected in the middle.
  • the foreign target gene ApoA1 inserted into the E1 region is controlled by the second constitutive promoter CMV.
  • ApoA1 has a signal peptide sequence at the 5'end, and a His tag sequence and a PA sequence at the 3'end.
  • the recombinant oncolytic adenovirus constructed by this scheme can replicate in tumor cells to kill tumor cells and inhibit tumor growth. And it can express and secrete ApoA1 protein, which is a transport protein involved in lipid metabolism. We found that the recombinant fusion ApoA1 oncolytic adenovirus can achieve the effect of inhibiting the invasion and migration of tumor cells.
  • One aspect of the present invention relates to the construction of a replicating oncolytic adenovirus AD5-ApoA1 vector, which can be operably inserted or contains the following foreign genes:
  • the target gene sequence of ApoA1 or its degenerate sequence, and its nucleotide sequence is shown in SEQ ID NO:1. Specifically, its 5'end includes a signal peptide recognition sequence.
  • ApoA1 is located in the E1 region of adenovirus, preferentially located in the E1B region.
  • the vector construction of the replicating oncolytic adenovirus AD5-ApoA1 can be operably inserted or contains the following foreign genes:
  • Experimental level the first promoter, GFP sequence, 2A linking sequence, E1A early activation replication original, insulating sequence, second promoter, target gene sequence or its degenerate sequence, His tag sequence, PA sequence are connected in sequence.
  • the first promoter, the GFP sequence, the 2A linking sequence, and the E1A early activation copy original are in the same expression frame;
  • the second promoter, the target gene sequence or its degenerate sequence, His tag sequence, and PA sequence are in the same expression frame;
  • the promoter can be a constitutive promoter, an inducible promoter, or a specific promoter; preferably, the first The first promoter is a constitutive promoter, an inducible promoter, a specific promoter, and the second promoter is a constitutive promoter.
  • the constitutive promoter is preferably CMV, SV40 or EF1a. More preferably, it is a CMV promoter, and the nucleic acid sequence is shown in SEQ ID NO: 2.
  • the first promoter, E1A early activation of the original copy, the insulating sequence, the second promoter, the target gene sequence or its degenerate sequence, and the PA sequence are connected in sequence.
  • the protein amino acid sequence of the expressed ApoA1 gene is shown in SEQ ID NO: 3.
  • the vector construction of replicating oncolytic adenovirus AD5-ApoA1 according to any one of the present invention is characterized in that the oncolytic adenovirus includes A, B, C, D, E and F subclasses, preferably C Subclass adenovirus. More preferably, the human adenovirus type 5 AD5.
  • Another aspect of the present invention relates to a replicating oncolytic adenovirus, which contains the recombinant oncolytic adenovirus vector according to any one of the present invention, including E1A replication elements, and ApoA1 independent expression cassette; specifically The recombinant oncolytic virus vector was recombined in 293T cells.
  • Another aspect of the present invention relates to any replicating oncolytic adenovirus vector or any replicating oncolytic adenovirus of the present invention in anti-tumor invasion and metastasis, alleviating tumor cachexia, anti-tumor inflammation, and anti-tumor inflammation.
  • the cancer or tumor is liver cancer, breast cancer, lung cancer, colon cancer, gastric cancer, pancreatic cancer, cervical cancer, melanoma, prostate cancer, ovarian cancer, lymphoma, gallbladder cancer, esophageal cancer , Kidney cancer, nasopharyngeal cancer, laryngeal cancer, thyroid tumor, mediastinal tumor or glioma; preferably liver cancer, breast cancer, lung cancer, colon cancer.
  • Tumor invasion and metastasis are the root causes of death of cancer patients. At present, there is no anti-tumor drug that can effectively control tumor invasion and metastasis, and there is no definite oncolytic virus drug that can resist tumor invasion and metastasis.
  • the recombinant replicating oncolytic adenovirus AD5-ApoA1 described in the present invention is the first oncolytic virus capable of inhibiting the invasion and metastasis of malignant tumors.
  • the probability of occurrence of cachexia in advanced tumors is about 60% or more, which is also an important contributing factor leading to the death of advanced cancer patients.
  • the most significant sign of cachexia is progressive wasting (weight loss). There is no clinically effective delay in tumor disease. Liquid medicine or method.
  • the recombinant replicating oncolytic adenovirus AD5-ApoA1 of the present invention has an unexpected effect of resisting the progression of cachexia, can effectively maintain the body weight of colon cancer mice, and significantly extend the survival time.
  • the replicating oncolytic adenovirus can also effectively up-regulate the specific receptor ABCA1 that infects tumor cells with high expression of ApoA1, and can greatly enhance the cholesterol transport effect of AD5ApoA1, resulting in unexpected synergistic effects.
  • AD5ApoA1 can also effectively control tumor-promoting inflammation pathways, inhibit the key enzyme IDO-1 that mediates tumor immune escape, and can effectively restore the body's immune surveillance of tumors.
  • the recombinant replicating oncolytic adenovirus AD5 ApoA1 of the present invention has multiple anti-tumor effects at the same time: inhibiting tumor invasion and metastasis, delaying the cachexia process of malignant tumors, and upregulating the high expression of ApoA1 in infected tumor cells
  • the specific receptor ABCA1 cooperates with anti-tumor, inhibits IDO-1 and promotes tumor inflammation and restores anti-tumor immune surveillance.
  • the above effects cooperate with each other to produce excellent anti-tumor effects, which can be used to prepare anti-tumor drugs.
  • AD5 ApoA1 The construction scheme of replicating oncolytic adenovirus AD5 ApoA1. After AD5 ApoA1 infects cells, the cells can express and secrete apolipoprotein ApoA1;
  • Apolipoprotein ApoA1 significantly inhibits the invasion and migration of breast cancer cells
  • the replicating AD5 ApoA1 significantly up-regulates the APOA1 specific receptor ABCA1 (enhancing the ability of APOA1 to transport cholesterol);
  • apolipoprotein ApoA1 significantly inhibits the activation of STAT3, a key molecule that promotes tumor inflammation;
  • apolipoprotein ApoA1 significantly reduces IDO-1, a key enzyme that inhibits anti-tumor immunity.
  • ApoA1 has gene ID: 335 in NCBI.
  • This gene encodes apolipoprotein A-I, which is the main protein component of high-density lipoprotein (HDL) in plasma.
  • the encoded precursor protein is processed by proteolysis to produce mature protein, which promotes the excretion of cholesterol from tissues to the liver. It is a cofactor for lecithin cholesterol acyltransferase (LCAT), which is responsible for the formation of most plasma cholesterol esters.
  • LCAT lecithin cholesterol acyltransferase
  • Enzyme This gene is closely related to the other two apolipoprotein genes on chromosome 11. Defects of this gene are related to high-density lipoprotein deficiency, including Tangier disease, and systemic non-neurological amyloidosis. Alternative splicing results in multiple transcript variants, at least one of which encodes a preprotein.
  • NM_000039.2/NP_000030.1 cDNA sequence/protein sequence
  • NM_001318017.2/NP_001304946.1 NM_001318018.2/NP_001304947.1
  • NM_001318021.1/NP_001304950.1 NM_000039.2/NP_000030.1
  • E1A virus E1 region genes can be further divided into E1A and E1B.
  • E1A is mainly composed of two components, 289R (or 13S) and 243R (or 12S).
  • the cell transcription factor After the adenovirus genome enters the cell nucleus, the cell transcription factor first binds to the enhancer upstream of the E1A region to express the E1A protein. The function of this protein is to regulate cell metabolism and make viral DNA easier to replicate in the cell.
  • the E1A protein can also activate the promoters of other early genes (E1B, E2A, E2B, E3, and E4).
  • E2B drives three other early gene transcription unit terminal protein precursors (pTP), which are related to virus replication, Expression of single-stranded DNA binding protein (ssDBP, single-stranded DNA binding proteins) and DNA polymerase (DNA pol, DNA polymerase), the expression products of these three genes are tightly combined into a complex with at least three cellular proteins The interaction initiates the replication of the viral genome.
  • pTP early gene transcription unit terminal protein precursors
  • ssDBP single-stranded DNA binding protein
  • DNA pol DNA polymerase
  • GFP and E1A are connected by a 2A linking sequence.
  • 2A itself as the connecting sequence has only post-translational protein cleavage sites, and the 2A peptide will remain in the protein before and after 2A.
  • adenovirus serotypes of which 52 human adenoviruses are divided into six subgroups (A, B, C, D, E, and F). They The tropism, tumorigenicity and disease history of host cells are different. The type 2 and type 5 adenoviruses commonly used in gene therapy belong to the C subgroup in serology, with 95% homology in DNA sequence. The process of adenovirus infection of cells starts from the adhesion of the scalp region of adenovirus cilia to specific receptors on the cell surface.
  • CAR coxsackie/adenovirus receptor
  • the drugs of the present invention can be manufactured by methods well known in the art, such as conventional biologically active preparations, including inactivated vaccines or vaccine viruses.
  • the biological agent is selected as an attenuated live vaccine virus.
  • the application of the medicine of the present invention includes three aspects: preventive products, therapeutic products, and diagnostic products.
  • the application is a therapeutic product.
  • Treatment is single treatment, adjuvant treatment or combination treatment.
  • the drug administration routes of the present invention include but are not limited to oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, Intraperitoneal, intramuscular, subcutaneous, intravenous, and tumor in situ administration.
  • the preferred route of administration is intravenous injection and tumor in situ administration.
  • FIG. 1 is a schematic diagram of the gene structure of the recombinant oncolytic adenovirus AD5ApoA1 expressing soluble ApoA1 of the present invention.
  • B Hepa1-6 mouse liver cancer cells were respectively infected with AD5 CON and AD5 ApoA1. After 72 hours of different multiplicity of infection (MOI), the supernatant of the infected cells was collected, and the secretion of the fusion protein ApoA1 was detected by the dot blot method . Data represents three independent replicate experiments. GFP, green fluorescent protein; E1A, virus early region 1 replication element (early region 1); ApoA1, free fusion protein ApoA1; His, histidine tag.
  • GFP green fluorescent protein
  • E1A virus early region 1 replication element (early region 1)
  • ApoA1 free fusion protein ApoA1
  • His histidine tag.
  • Figure 2 shows the replication and oncolysis of the recombinant oncolytic adenovirus AD5 ApoA1 of the present invention
  • FIG. 3 ApoA1 inhibits the migration and invasion of breast cancer cells 4T1 in vitro.
  • A Mouse breast cancer cells 4T1 were transiently transfected to express ApoA1 non-replicating adenovirus or negative control. After 72 hours, the migration of cells to the scratch was observed under a microscope.
  • B Construction of a stable transgenic 4T1 strain that stably expresses ApoA1. 4T1 cells wild-type (4T1-wt) and 4T1 cells (4T1-ApoA1) with high expression of ApoA1 were cultured for 24 hours, and the supernatant was collected. Western blot was used to detect the secretion level of ApoA1.
  • Figure 4 shows the anti-tumor effect of the recombinant oncolytic adenovirus AD5 ApoA1 in vivo (4T1 breast cancer solid tumor model).
  • the anti-tumor effect of AD5 ApoA1 was evaluated in the 4T1 subcutaneous tumor model of breast cancer.
  • the experimental scheme is shown in Figure (A). Show.
  • (B) Balb/c's right side was subcutaneously inoculated with 1 ⁇ 10 5 4T1 mouse breast cancer cells, and 2.5 ⁇ 10 8 pfu (plaque forming unit) AD5 con or AD5 ApoA1 was injected intratumorally to monitor tumor size in real time.
  • C The mice were sacrificed on the 10th day after the treatment, and the tumors were separated to detect the size and weight.
  • Figure 5 shows the anti-tumor effect of the recombinant oncolytic adenovirus AD5 ApoA1 in vivo (4T1 carcinoma in situ model).
  • the anti-tumor effect of AD5 ApoA1 was evaluated in the 4T1 carcinoma in situ model.
  • the experimental scheme is shown in Figure (A).
  • Balb/c mice were inoculated with 1 ⁇ 10 5 4T1 cells in the right mammary gland, and 2.5 ⁇ 10 8 pfu AD5 CON and AD5 ApoA1 were injected into the tumor. The tumor size was monitored in real time.
  • B Successfully established a mouse orthotopic breast cancer metastasis model. Untreated tumor-bearing mice will invade the peritoneum and metastasize to the lungs.
  • C Orthotopic tumor volume in mice
  • D Survival status of mice. The data represents three independent repeated trials. *, p ⁇ 0.05; **, p ⁇ 0.01.
  • Figure 6 shows the anti-tumor effect in vivo of the recombinant oncolytic adenovirus AD5 ApoA1 (mouse lung cancer LLC subcutaneous tumor model). The anti-tumor effect of AD5 ApoA1 was evaluated in the LLC subcutaneous tumor model.
  • the experimental scheme is shown in Figure (A).
  • (B) C57/BL6 mice were subcutaneously inoculated with 5 ⁇ 10 6 LLC mouse lung cancer cells. After the mice developed tumors, 1 ⁇ 10 8 pfu AD5 CON or AD5 ApoA1 was injected intratumorally to monitor the tumor size in real time.
  • C Body weight of mice. *p ⁇ 0.05.
  • Figure 7 shows the in vivo anti-tumor effect of the recombinant oncolytic adenovirus AD5 ApoA1 of the present invention (a colon cancer cell C26 subcutaneous tumor model that can cause cachexia).
  • the anti-tumor effect of AD5 ApoA1 was evaluated in the C26 subcutaneous tumor model.
  • the experimental scheme is shown in Figure (A).
  • (B) Balb/c mice were subcutaneously inoculated with 1 ⁇ 10 6 C26 mouse colon cancer cells. After the mice developed tumors, 2.5 ⁇ 10 8 pfu AD5 CON or AD5 ApoA1 were injected intratumorally to monitor the tumor size in real time.
  • C Weight of mice (the degree of weight loss reflects the level of progression of cachexia symptoms). *p ⁇ 0.05.
  • Fig. 8 shows the anti-tumor effect of the recombinant oncolytic adenovirus AD5 ApoA1 in vivo (H22 subcutaneous tumor model).
  • the anti-tumor effect of AD5 ApoA1 was evaluated in the H22 subcutaneous tumor model.
  • the experimental plan is shown in Figure (A).
  • (B) Balb/c mice were subcutaneously inoculated with 1 ⁇ 10 6 H22 mouse liver cancer cells. After the mice developed tumors, 2.5 ⁇ 10 8 pfu AD5 CON or AD5 ApoA1 were injected intratumorally to monitor the tumor size in real time.
  • C Tumor growth of each mouse.
  • D Survival of mice. **p ⁇ 0.01.
  • Figure 9 shows the anti-tumor (LM3 subcutaneous tumor) study of the recombinant oncolytic adenovirus AD5 ApoA1 of the present invention in a humanized mouse model.
  • NCG NOD-Prkdc scid Il2rg null mice were subcutaneously inoculated with 5 ⁇ 10 6 LM3 human hepatoma cells, tumors appeared after mice via the tail vein 2 ⁇ 10 6 human peripheral blood mononuclear cells (PBMC). After that, 2x10 8 pfu AD5 CON or AD5 ApoA1 was injected intratumorally.
  • B Detection of tumor size. The data represents the results of three independent repeated experiments. *p ⁇ 0.05.
  • FIG. 10 ApoA1 protein inhibits the expression of breast cancer invasion-related proteins.
  • A 4T1-wt cells and 4T1-ApoA1 cells were cultured for 24h, and mRNA was extracted for RNAseq. The figure shows the relative expression level. The relative expression level is the relative expression level of 4T1-wt being 1, 4T1-ApoA1 expression level than the value of 4T1-wt being 4T1-ApoA1.
  • B 4T1-wt cells and 4T1-ApoA1 cells were cultured for 24h, and the expression level of Krt14 was detected by QPCR. Krt14, keratin 14; Cdh3, cadherin 3; Smn1, Survival Of Motor Neuron 1, Telomeric; Mki67, cell proliferation marker ki-67. **p ⁇ 0.01.
  • Figure 11 Adenovirus up-regulates the expression level of ApoA1 receptor molecule Abca1 in tumor tissues.
  • Balb/c mice were inoculated with 4T1 tumor cells. After tumor formation, 2.5x10 8 pfu AD5 con or AD5 ApoA1 virus was injected into the tumor. After 48 hours, tumor tissue RNA was extracted for RNAseq detection.
  • the figure shows the relative expression level. The relative expression level is based on the PBS group as 1, and the ratio of other groups to the PBS group is the relative expression level of this group.
  • FIG. 12 shows that the recombinant replicating oncolytic adenovirus AD5 ApoA1 of the present invention inhibits the activation of stat1, stat3 and NFkB signaling pathways and the secretion of TNF-alpha, IL-6 and IL-1beta.
  • AD5 ApoA1 or AD5 CON were infected with human mononuclear macrophages THP-1 or human liver cancer cells HCC-LM3 for 24 hours at a MOI of 5 at a multiplicity of infection. Or add TNF-a to stimulate for 30 minutes, and detect the phosphorylation level of stat1, stat3 and IkBa in the cells.
  • FIG 13 shows that the recombinant replicating oncolytic adenovirus AD5 ApoA1 inhibits the expression of IDO1.
  • AD5 ApoA1 or AD5 CON were infected with human hepatocellular carcinoma cells HCC-LM3 and SMMC-7721 for 24 hours at a MOI of 5 at a multiplicity of infection. After 30 minutes of stimulation with IL-6, the expression of IDO1 in the cells was detected. The data represents the results of three independent repeated experiments.
  • the experimental device materials and reagents involved in the present invention are as follows:
  • Biological safety cabinet advance, Class II Biological Safety Cabinet, The Baker Company
  • carbon dioxide incubator (FORMA SERIES II WATER JACKET CO 2 incubator, Thermo), cryogenic centrifuge (HERAEUS MEGAFUGE 1.0R, Thermo), vertical electrophoresis tank (BIO-RAD), Electrophoresis instrument (BIO-RAD), semi-dry transfer-transfer instrument (BIO-RAD), western blot exposure system (Alpha Innotech)
  • PCR instrument PCR Thermal Cycler Dice, TaKaRa
  • real-time quantitative PCR instrument and analysis software (ABI384) , Sequence Detection Software, Version 1.3.1), microplate reader (VERSA max microplate reader), complete set of pipettes (eppendorf and RAININ), cell counter (Countstar Automated cell counter, Inno-Alliance Biotech Inc., Wilmington, USA ), flow cytometer (FACSCalibur, Becton, Dickinson and Company, USA), FlowJo software (Version 7.6.5, Tree
  • the primers are all synthesized by GenScript.
  • the DMEM high glucose medium, double antibodies, and serum required for tumor cell culture were purchased from Invitrogen (Shanghai).
  • Quantitative RT-PCR reagent Faststart Universal SYBR Green Master (Roche, 04913914001).
  • Example 1 AD5-ApoA1 virus plasmid construction, virus rescue and amplification
  • the constructed shuttle vector AD5-pShuttle-ApoA1 was linearized with PmeI and transferred to competent pAdEasy-BJ5183, and screened using LB plates containing 50ug/ml kanamycin, and positive clones were selected for culture identification, and the correct ones were identified
  • the cloned plasmid was re-transformed into DH5a competence for secondary screening and identification. After the identification was correct, the plasmid was extracted to obtain the full length AD5 ApoA1 plasmid.
  • the full-length AD5-ApoA1 plasmid was linearized with Pad. After purification, 1ug/well was transfected into 293T cells in a 6-well plate, cultured in 5% CO2, 37°C, after 2 days, the cells were digested and transferred to a 10cm plate, and the medium was changed in 2-3 days When 80% of the cells appear diseased, use 10ml of culture medium to blow down the cells and collect them into a 15ml centrifuge tube. Repeated freezing and thawing twice, centrifuging at 3000rpm/min for 15min, collecting the virus supernatant and storing at -80°C as the virus seed.
  • the virus was collected by the above method, and the virus was purified by cesium chloride density gradient centrifugation; the titer was determined by the TCID50 method.
  • Example 2 Determination of the titer of AD5-ApoA1 virus
  • Virus gradient dilution prepare EP tubes, add 1170 ⁇ l DMEM containing fetal bovine serum to each EP tube; add 130 ⁇ l virus solution to the first EP tube, mix well, and mark as 10-1; from the first one Pipette 50 ⁇ l from the EP tube into the second EP tube, mix well, mark as 10-2; and so on, until the dilution reaches the desired gradient.
  • V the initial volume of cell culture medium per well (ml/well)
  • AD5 ApoA1 and AD5 CON viruses infect tumor cells at the same MOI.
  • the cells are harvested at different times, and the same amount of virus suspension is obtained after repeated freezing and thawing and centrifugation. 293T cells are used for virus titer determination; virus replication ability changes are analyzed.
  • AD5 ApoA1 and AD5 CON viruses were used to infect tumor cells with an MOI of 1 to 100 viruses. After 72 hours, MTT was used to detect cell activity and evaluate the tumor-killing effect of AD5 ApoA1.
  • the cell clone is seeded in a 96-well plate coated with type I collagen, and the morphology of the cell clone is constantly monitored.
  • mice choose 6-8 week-old Balb/c mice to establish a subcutaneous tumor model in the right axilla or subcutaneously. 4-6 days later, measure the tumor size to 200mm 3 , and divide the mice into 3 groups randomly, namely: no treatment group, control AD5 CON virus treatment group, AD5 ApoA1 virus treatment group; according to the group, use the corresponding intratumoral injection of virus, each injected with a virus amount of 2.5 ⁇ 10 8 pfu, and track the tumor volume and body weight. After the natural death of the mouse, the survival time of the mouse is recorded.
  • Glue preparation and electrophoresis SDS-PAGE separation gel and concentrated gel of different concentrations are prepared according to different requirements. According to the calculation results of protein quantification, the loading amount of each sample is adjusted to 30 ⁇ g. Electrophoresis conditions: concentrated gel 80V 30min, separation gel 120V, about 80min, provided that the bands are separated and will not run out.
  • Transfer membrane Prepare the filter paper and PVDF membrane, soak the PVDF membrane with methanol first, and then soak the filter paper in the transfer buffer for use. Carefully remove the glue from the glass plate, soak it in the transfer buffer solution, and place it in the order of the negative electrode-filter paper-PVDF membrane-glue-filter paper-positive electrode sandwich to drive out bubbles, according to the required strip size, constant flow 110mA transfer film 60-70min.
  • Primary antibody incubation Incubate the primary antibody overnight at 4°C.
  • Secondary antibody incubation Wash the strips with washing buffer for 10 minutes each time, a total of three times; then incubate with the corresponding HPR-labeled secondary antibody at room temperature for 1 hour.
  • Exposure Wash the strips with washing buffer for 10 minutes each time, a total of three times; Expose the strips with a chemiluminescent solution on a WB exposure instrument, and obtain strip images.
  • the 10 ⁇ l system composition of real-time quantitative PCR 2.6 ⁇ l PCR water, 0.2 ⁇ l upstream and downstream primers, 2 ⁇ l template and 5 ⁇ l SYBR Green fluorescent dye. After the samples are mixed, they are amplified on the ABI 384 PCR machine.
  • the results in Figure 2 show that compared with the control virus, the constructed replicating oncolytic adenovirus AD5 ApoA1 is in human liver cancer cell lines HCC-LM3, SMMC-7721, human renal clear cell cancer cell line 786-O, and human liver cancer cell lines.
  • the strains HuH-7, HepG2, human bladder cancer cell line T24, and human lung cancer cell line H1299 have stronger replication ability. And it showed stronger oncolytic ability in Huh-7 and T24.
  • the viral load of the intratumoral replicating AD5 ApoA1 was significantly higher than that of AD5con. It shows that the expression of ApoA1 can promote adenovirus replication and oncolysis.
  • Fig. 6 show that the tumor volume of the mouse lung cancer LLC subcutaneous tumor injected with AD5 ApoA1 adenovirus is smaller than that of the control virus and PBS treatment group, indicating that AD5 ApoA1 adenovirus significantly inhibits the growth of mouse lung cancer in mice.
  • Fig. 11 show that the replicating adenovirus can up-regulate the expression of Abca1, a specific receptor molecule of ApoA1, thereby further enhancing the ability of ApoA1 to transport cholesterol.
  • AD5 ApoA1 adenovirus can inhibit the expression of indoleamine 2,3 dioxygenase 1 (IDO1) of liver cancer cell lines HCC-LM3 and SMMC-7721 in vitro, inhibit immune tolerance, and enhance anti-tumor immunity.
  • IDO1 indoleamine 2,3 dioxygenase 1
  • the present invention provides for the first time a replicating oncolytic adenovirus AD5 ApoA1 that can inhibit tumor growth and block tumor invasion and metastasis.
  • the virus not only has stronger replication and oncolytic ability in tumor cells than Ad5con virus, but also can highly express apolipoprotein ApoA1, which can be secreted outside the cell, and up-regulate the specificity of infected tumor cells to express ApoA1.
  • the receptor ABCA1 can greatly enhance the role of AD5 and ApoA1 in transporting cholesterol, produce unexpected synergistic effects, and exert a variety of anti-tumor effects; inhibit the invasion and migration of malignant tumor cells, block the pro-tumor inflammation pathway, and inhibit the mediation of tumor immune escape
  • the key enzyme IDO-1 can effectively restore the body's immune surveillance of tumors.
  • the recombinant replicating oncolytic adenovirus AD5-ApoA1 of the present invention has an unexpected effect against the progression of cachexia, can effectively maintain the body weight of colon cancer mice, and significantly extend the survival time.
  • the recombinant replicating oncolytic adenovirus AD5 ApoA1 of the present invention has multiple anti-tumor effects at the same time: inhibiting tumor invasion and metastasis, delaying the cachexia process of malignant tumors, and upregulating the high expression of ApoA1 in infected tumor cells.
  • the specific receptor ABCA1 cooperates with anti-tumor, inhibits IDO-1 and promotes tumor inflammation and restores anti-tumor immune surveillance.
  • a virus integrates a variety of unique roles and mechanisms in tumor treatment at the same time, which complement each other and have unexpected effects.

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Abstract

提供了一种抑制肿瘤生长和转移的复制型溶瘤腺病毒AD5-ApoA1、其构建方法及其在制备抗肿瘤药物中的应用。该病毒可以在肿瘤细胞内快速复制并产生溶瘤作用。感染该病毒的肿瘤细胞能够高表达载脂蛋白ApoA1，该蛋白分子能够大量分泌到胞外，显著抑制肿瘤细胞的侵袭和迁移，抑制促肿瘤炎症通路，显著降低导致肿瘤免疫逃逸的关键分子IDO-1。该病毒在肝癌、乳腺癌、结肠癌和肺癌的小鼠中，能显著抑制肿瘤生长、抑制肿瘤的侵袭、延缓恶液质的进展和延长荷瘤小鼠生存时间。

Description

一种调控脂质代谢的复制型溶瘤腺病毒及其应用 技术领域
本发明涉及肿瘤生物治疗领域，涉及一种有效抑制肿瘤生长和侵袭转移的复制型溶瘤腺病毒及其应用。
背景技术
我国每年新增的癌症患者约400万人，每年有近300万人死于癌症。传统的手术、放疗、化疗等方案在一定程度上抑制了肿瘤的进展，但难以控制肿瘤的侵袭、转移和复发。
近年来，肿瘤的生物治疗手段成为不可或缺的一部分。溶瘤病毒本身能够在肿瘤细胞中复制并发挥溶瘤作用，同时能够激活免疫细胞，导致肿瘤局部的免疫细胞浸润，并诱发抗肿瘤免疫应答。另外，溶瘤病毒可以携带外源基因，在肿瘤局部表达外源蛋白，直接发挥蛋白药物的抗肿瘤作用。2015年，第一个溶瘤病毒药物(T-Vec，Agmen公司)被FDA批准上市，重组的单纯疱疹病毒-1能够表达GM-CSF激活DC发挥抗肿瘤活性。现有技术中对溶瘤病毒的药物开发主要是通过构建重组表达载体使病毒释放免疫激活因子或免疫检查点抗体来激活患者免疫功能发挥抗肿瘤作用，如一种携带人穿膜肽p53与GM-CSF基因的重组溶瘤腺病毒(CN201310460980)，以及一种表达PD-L1单链抗体的新城疫溶瘤病毒(CN201811560794)。但目前现有的技术对肿瘤转移并不能进行有效控制。
载脂蛋白ApoA1是ApoA家族中最多的一种组分，在肝脏和小 肠中合成，是由243个氨基酸残基所组成的单一多肽。ApoA1是卵磷脂胆固醇酰基转移酶的活化剂，主要存在于高密度脂蛋白(HDL)中，是HDL的主要载脂蛋白。ApoA1主要功能是介导胆固醇的逆向转运，即将胆固醇由外周细胞转运到肝脏。ApoA1通过与细胞表面的特异性受体三磷酸腺苷结合盒转运体A1(ABCA1)结合，摄取细胞的游离胆固醇和磷脂，生成新生的HDL。通过卵磷脂胆固醇脂酰基转移酶(LCAT)，将HDL颗粒中的游离胆固醇转变为胆固醇脂，然后运输到肝脏进行代谢。现有文献报道，将载有ApoA1野生型(WT)或突变型(V156V)的脂蛋白体(PL)和溶瘤腺病毒耦联形成复合体，可以有效促进溶瘤病毒的对Hep3B细胞的抗肿瘤活性(Mol Cells.2012；34:143-148)。但该技术方案对肿瘤的侵袭，转移和复发未给出是否具有相应的技术效果。此外，该文献的方案是先通过体外构建二肉豆蔻酰磷脂酰胆碱(DMPC)，游离胆固醇(FC)，ApoA1组成比为200:9:1的PL，再和溶瘤腺病毒形成复合物的方式发挥作用。这种方式不仅制备过程复杂，而且药物效应持续的时间较短暂。
因此目前尚缺乏一种能够表达脂质代谢相关的基因的复制型溶瘤病毒载体和溶瘤病毒，使其有效抑制肿瘤的生长，并能达到控制转移复发的效果。
发明内容
本发明的目的在于提供一种既能抑制肿瘤生长又能抑制肿瘤侵袭转移的复制型溶瘤腺病毒及其应用。
为实现上述发明目的，本发明采用了下列技术方案：
一种能够分泌载脂蛋白ApoA1的复制型溶瘤腺病毒载体和重组溶瘤病毒。具体地，该复制型溶瘤腺病毒载体的构建方案包括：实验级带标签的腺病毒载体和药用级无标签的腺病毒载体。实验级腺病毒复制元件E1A由第一组成型启动子CMV控制，在E1A上游连接GFP序列，中间由A2连接序列连接。E1区插入的外源目的基因ApoA1由第二组成型启动子CMV控制，ApoA1的5’端具有信号肽序列，3’端含有His标签序列及PA序列。
此方案构建重组溶瘤腺病毒能够在肿瘤细胞内复制杀伤肿瘤细胞，抑制肿瘤生长。并且其能够表达分泌ApoA1蛋白，它是一种参与脂质代谢的转运蛋白。我们发现重组融合ApoA1溶瘤腺病毒能够达到抑制肿瘤细胞的侵袭和迁移效果。
由此提供了下述发明：
(1)本发明的一个方面涉及一种复制型溶瘤腺病毒AD5-ApoA1的载体构建，其可操作的插入或者包含下述外源基因：
ApoA1目的基因序列或其简并序列，其核苷酸序列如SEQ ID NO：1所示。具体地，其5’端包括信号肽识别序列。
其中ApoA1位于腺病毒E1区，优先地位于E1B区。
根据本发明任一项所述的复制型溶瘤腺病毒AD5-ApoA1的载体构建，其可操作的插入或者包含下述外源基因：
实验级：第一启动子、GFP序列、2A连接序列、E1A早期激活复制原件、绝缘序列、第二启动子，目的基因序列或其简并序列、His标签序列、PA序列依次连接。
具体的，第一启动子、GFP序列、2A连接序列、E1A早期激活复制原件同一个表达框内；
第二启动子，目的基因序列或其简并序列、His标签序列、PA序列在同一个表达框内；启动子可以为组成型启动子，诱导型启动子，特异型启动子；优选地，第一启动子以为组成型启动子，诱导型启动子，特异型启动子，第二启动子同为组成型启动子。组成型启动子，优选地为CMV、SV40或EF1a。更优选的为CMV启动子，核酸序列为SEQ ID NO：2所示。
药用级：第一启动子、E1A早期激活复制原件、绝缘序列、第二启动子，目的基因序列或其简并序列、PA序列依次连接。
其表达的ApoA1基因的蛋白氨基酸序列如SEQ ID NO：3所示。
根据本发明任一项所述的复制型溶瘤腺病毒AD5-ApoA1的载体构建，其特征在于所述的溶瘤腺病毒包括A、B、C、D、E和F亚类，优选的C亚类腺病毒。更优选地，人5型腺病毒AD5。
(2)本发明的另一个方面涉及一种复制型溶瘤腺病毒，其含有本发明中任一项所述的重组溶瘤腺病毒载体，包括了E1A复制元件，以及ApoA1独立表达框；具体的重组溶瘤病毒载体在293T细胞中重组得到。
(3)本发明的再一个方面涉及任一项复制型溶瘤腺病毒载体或者本发明中任一项复制型溶瘤腺病毒在抗肿瘤侵袭转移，缓解肿瘤恶液质，抗肿瘤炎症，抗肿瘤免疫药物的用途；具体地，所述癌症或者肿瘤为肝癌、乳腺癌、肺癌、结肠癌、胃癌、胰腺癌、宫颈癌、黑色素瘤、前列腺癌、卵巢癌、淋巴瘤、胆囊癌、食管癌、肾癌、鼻咽癌、 喉癌、甲状腺肿瘤、纵隔肿瘤或神经胶质瘤；优选地为肝癌、乳腺癌、肺癌、结肠癌。
肿瘤的侵袭和转移是导致肿瘤患者死亡的根本原因。目前还没有能够有效控制肿瘤侵袭与转移的抗肿瘤药物，也没有确切的能够抗肿瘤侵袭转移的溶瘤病毒药物。本发明所描述的重组复制型溶瘤腺病毒AD5-ApoA1，是迄今为止的第一个具有确切抑制恶性肿瘤侵袭转移的溶瘤病毒。
另外，晚期肿瘤发生恶液质的几率约60％以上，也是导致晚期癌症患者死亡的重要促进因素恶液质最显著的体征就是进行性消瘦(体重下降)，临床上没有任何有效的延缓肿瘤恶液质的药物或方法。本发明所述的重组复制型溶瘤腺病毒AD5-ApoA1具有预料不到的对抗恶液质进展的作用，能够有效地维持结肠癌小鼠的体重，并显著延长生存时间。
复制型溶瘤腺病毒，还能够有效地上调感染肿瘤细胞高表达ApoA1的特异性受体ABCA1，能够大大增强AD5ApoA1的转运胆固醇的作用，产生意料不到的协同作用。最后，AD5ApoA1还能够有效控制促肿瘤的炎症通路，抑制介导肿瘤免疫逃逸的关键酶IDO-1，能够有效地恢复机体对肿瘤的免疫监视。综述所述，本发明所述的重组复制型溶瘤腺病毒AD5 ApoA1同时兼具多种抗肿瘤的作用：抑制肿瘤的侵袭转移、延缓恶性肿瘤的恶液质进程、上调感染肿瘤细胞高表达ApoA1的特异性受体ABCA1协同抗肿瘤、抑制IDO-1及促肿瘤炎症并恢复抗肿瘤免疫监视。以上作用相互协作，产生极好的抗肿瘤作用，可以用来制备抗肿瘤药物。
具体地，本发明：
1)复制型溶瘤腺病毒AD5 ApoA1的构建方案。AD5 ApoA1感染细胞后，细胞能够表达和分泌载脂蛋白ApoA1；
2)与对照病毒相比，复制型AD5 ApoA1在肿瘤细胞中有更强的复制和溶瘤能力；
3)载脂蛋白ApoA1显著抑制乳腺癌细胞的侵袭和迁移；
4)动物实验中复制型AD5 ApoA1显著抑制小鼠乳腺癌皮下瘤的生长；
5)动物实验中复制型AD5 ApoA1显著抑制小鼠原位乳腺的侵袭和转移，显著延长小鼠生存时间；
6)动物实验中复制型AD5 ApoA1显著抑制小鼠肺癌的生长；
7)动物实验中复制型AD5 ApoA1显著抑制小鼠结肠癌的生长，能显著缓解小鼠恶液质进展；
8)动物实验中复制型AD5 ApoA1显著抑制小鼠肝癌的生长并显著延长小鼠生存时间；
9)人源化小鼠模型中，复制型AD5 ApoA1显著抑制肝癌的生长并显著延长小鼠生存时间；
10)在乳腺癌细胞中，复制型ApoA1显著抑制促肿瘤侵袭的关键蛋白K14；
11)在癌细胞中，复制型AD5 ApoA1显著上调APOA1的特异性受体ABCA1(增强APOA1转运胆固醇的能力)；
12)在巨噬细胞和肝癌细胞中，载脂蛋白ApoA1显著抑制促肿瘤炎症的关键分子STAT3的活化；
13)在巨噬细胞和肝癌细胞中，载脂蛋白ApoA1显著降低抑制抗肿瘤免疫的关键酶IDO-1。
发明详述
术语“ApoA1”在NCBI中基因ID：335。该基因编码载脂蛋白A-I，是血浆中高密度脂蛋白(HDL)的主要蛋白组分。编码的前体蛋白经蛋白水解处理，生成成熟蛋白，促进胆固醇从组织外排到肝脏排泄，是卵磷脂胆固醇酰基转移酶(LCAT)的辅助因子，LCAT是一种负责大多数血浆胆固醇酯形成的酶。该基因与11号染色体上的另外两个载脂蛋白基因密切相关。该基因的缺陷与高密度脂蛋白缺乏有关，包括丹吉尔病，以及系统性非神经性淀粉样变性。选择性剪接导致多个转录本变异，其中至少有一个编码一个前蛋白。
ApoA1基因有多个转录本，分别为NM_000039.2/NP_000030.1(cDNA序列/蛋白序列)，NM_001318017.2/NP_001304946.1，NM_001318018.2/NP_001304947.1，NM_001318021.1/NP_001304950.1。
术语“E1A”病毒的E1区基因可以进一步分为E1A和E1B。E1A主要由两种成分构成，分别为289R(或13S)和243R(或12S)。腺病毒基因组进入细胞核后，细胞转录因子首先与E1A区上游的增强子结合，表达E1A蛋白，该蛋白的作用是调节细胞代谢，使病毒DNA更易于在细胞中复制。E1A蛋白还可以激活其他早期基因(E1B、E2A、E2B、E3和E4)的启动子，其中E2B驱动另外三个与病毒复制有关 的早期基因转录单位末端蛋白前体(pTP,precursor terminal protein)、单链DNA结合蛋白(ssDBP,single-stranded DNA binding proteins)以及DNA聚合酶(DNA pol,DNA polymerase)的表达，这三个基因的表达产物紧密地结合成一个复合物，与至少三种细胞蛋白相互作用，启动病毒基因组的复制。本发明中E1A前包含一个独立的CMV启动子。GFP和E1A中间由2A连接序列连接。
不拘于理论限制，作为连接序列的2A本身只有翻译后蛋白剪切位点，2A肽段将保留在2A前后的蛋白。使用2A连接GFP和E1A，使得早期复制元件和GFP标签蛋白分离。
不拘于理论限制，在腺病毒血清型选择方面已发现100余个血清型，其中人腺病毒有52种，分为A、B、C、D、E和F六个亚群(subgroup)，它们对宿主细胞的亲嗜性、致瘤性及疾病史各不相同。基因治疗常用人的2型及5型腺病毒在血清学上均属于C亚群，在DNA序列上有95％的同源性。腺病毒感染细胞的过程是从腺病毒纤毛的头节区粘附到细胞表面的特异性受体开始的。因为人腺病毒主要与柯萨奇B病毒共用一种受体，因此这种受体被称为柯萨奇/腺病毒受体即CAR(coxsackie/adenovirus receptor)。低水平表达CAR的无疑制约腺病毒的转导效率。然而，由于C亚群腺病毒载体已经被成功应用临床，其安全性已在人体中广泛测试，作为载体安全性高。本发明重组腺病毒能在肿瘤细胞内复制。
本发明所述的药物，可以采用本领域熟知的方法制造，如常规的生物活性制剂，包括灭活疫苗或或疫苗病毒等。优选地，生物制剂选择为减毒的活疫苗病毒。
本发明所述的药物，应用包括预防制品，治疗制品，诊断制品三 个方面。优选地，应用为治疗制品。治疗为单独治疗，辅助治疗或者联合治疗。
本发明所述的药物的给药途径，包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内、肿瘤原位给药。优选的给药途径是静脉注射、肿瘤原位给药。
附图说明
图1(A)为本发明的表达可溶性ApoA1的重组溶瘤腺病毒AD5ApoA1的构建的基因结构原理图。(B)Hepa1-6小鼠肝癌细胞分别感染AD5 CON和AD5 ApoA1，在不同的感染复数(MOI)72h后，被感染细胞的上清被收集起来，通过dot blot的方法检测融合蛋白ApoA1的分泌。数据代表三次独立性重复实验。GFP，绿色荧光蛋白；E1A，病毒早期区域1复制元件(early region 1)；ApoA1，游离融合蛋白ApoA1；His，组氨酸标签。
图2为本发明的重组溶瘤腺病毒AD5 ApoA1的复制与溶瘤(A)人肝癌细胞株HCC-LM3、SMMC-7721，人肾透明细胞癌细胞株786-O,人肝癌细胞株HuH-7、HepG2，人膀胱癌细胞株T24，人肺癌细胞株H1299分别感染AD5 CON和AD5 ApoA1(MOI＝0.1)，分别在相应时间点收取细胞，提取病毒基因组DNA，通过Q-PCR检测AD5的拷贝数。P值均小于0.01。(B)人肾透明细胞癌细胞株786-O或人膀胱癌细胞株T24，在病毒不同MOI值感染下，24后通过荧光显微镜或流式细胞术检测病毒感染的细胞数量。绿色荧光蛋白表示病毒的感染。(C)人肝癌细胞株HuH-7在不同MOI值的AD5 CON和AD5 ApoA1病毒感染72h后，显微镜检测细胞形态。(D)人膀胱癌细胞 株T24,不同MOI值的AD5 con和AD5 ApoA1病毒感染72h后，CCK8检测细胞活率。(E)Balb/c小鼠接种4T1肿瘤细胞，待肿瘤形成后，瘤内注射2.5x10 8pfu AD5 con或AD5 ApoA1病毒，48h后QPCR检测病毒E1A基因表达量，可反映病毒基因拷贝数。*p<0.05，**p<0.01，数据代表三次独立性重复实验。
图3ApoA1在体外抑制乳腺癌细胞4T1的迁移和侵袭。(A)小鼠乳腺癌细胞4T1瞬时转染表达ApoA1非复制型腺病毒或阴性对照。72小时后显微镜观察细胞向划痕的迁移。(B)构建4T1稳定表达ApoA1的稳转株。4T1细胞野生型(4T1-wt)和高表达ApoA1的4T1细胞(4T1-ApoA1)培养24h后收集上清，Western blot检测ApoA1的分泌水平。
(C)先用悬滴培养4T1-wt或4T1-ApoA1细胞获取细胞克隆，然后将细胞克隆种植于I型胶原包被的96孔细胞培养板。在相应时间点进行监测。(D)Transwell小室实验检测4T1-wt或4T1-ApoA1的侵袭能力。结果代表三次独立重复实验。
图4为本发明的重组溶瘤腺病毒AD5 ApoA1的体内抗肿瘤作用(4T1乳腺癌实体瘤模型)在乳腺癌4T1皮下瘤模型中评估AD5 ApoA1的抗肿瘤效果，实验方案如图(A)所示。(B)Balb/c右侧皮下接种1×10 5 4T1小鼠乳腺癌细胞，瘤内注射2.5×10 8pfu(空斑形成单位)AD5 con或AD5 ApoA1，实时监测肿瘤大小。(C)治疗后第10天处死小鼠，分离肿瘤检测大小和重量。(D)治疗后小鼠的体重。数据代表三次独立性重复试验。*p<0.05。
图5为本发明的重组溶瘤腺病毒AD5 ApoA1的体内抗肿瘤作用(4T1原位癌模型)。在4T1原位癌模型中评估AD5 ApoA1的抗肿瘤 效果，实验方案如图(A)所示。Balb/c小鼠右侧乳腺接种1×10 54T1细胞，瘤内注射2.5×10 8pfu AD5 CON和AD5 ApoA1，肿瘤大小被实时监测。(B)成功建立小鼠原位乳腺癌转移模型。未经治疗的荷瘤小鼠会发生侵袭腹膜的转移以及肺部转移。(C)小鼠原位肿瘤体积(D)小鼠的生存情况。数据代表三次独立性重复试验。*，p<0.05；**，p<0.01。
图6为本发明的重组溶瘤腺病毒AD5 ApoA1的体内抗肿瘤作用(小鼠肺癌LLC皮下瘤模型)。在LLC皮下瘤模型中评估AD5 ApoA1的抗肿瘤效果，实验方案如图(A)所示。(B)C57/BL6小鼠皮下接种5×10 6LLC小鼠肺癌细胞，小鼠出现肿瘤后，瘤内注射1×10 8pfu AD5 CON或AD5 ApoA1，实时监测肿瘤大小。(C)小鼠的体重。*p<0.05。
图7为本发明的重组溶瘤腺病毒AD5 ApoA1的体内抗肿瘤作用(能引起恶液质的结肠癌细胞C26皮下瘤模型)。在C26皮下瘤模型中评估AD5 ApoA1的抗肿瘤效果，实验方案如图(A)所示。(B)Balb/c小鼠皮下接种1×10 6C26小鼠结肠癌细胞，小鼠出现肿瘤后，瘤内注射2.5×10 8pfu AD5 CON或AD5 ApoA1，实时监测肿瘤大小。(C)小鼠体重(体重的减轻程度反映恶液质症状进展水平)。*p<0.05。
图8为本发明的重组溶瘤腺病毒AD5 ApoA1的体内抗肿瘤作用(H22皮下瘤模型)。在H22皮下瘤模型中评估AD5 ApoA1的抗肿瘤效果，实验方案图如图(A)所示。(B)Balb/c小鼠皮下接种1×10 6H22小鼠肝癌细胞，小鼠出现肿瘤后，瘤内注射2.5×10 8pfu AD5 CON或AD5 ApoA1，实时监测肿瘤大小。(C)每只小鼠肿瘤生长情况。 (D)小鼠的生存情况。**p<0.01。
图9为本发明的重组溶瘤腺病毒AD5 ApoA1在人源化小鼠模型中的抗肿瘤(LM3皮下瘤)的研究。(A)实验方案如图所示。NCG(NOD-Prkdc scid Il2rg null)小鼠皮下接种5×10 6LM3人肝癌细胞，小鼠出现肿瘤后经尾静脉注射2×10 6人外周血单个核细胞(PBMC)。之后瘤内注射2x10 8pfu AD5 CON或AD5 ApoA1。(B)检测肿瘤大小。数据代表三次独立性重复实验结果。*p<0.05。
图10ApoA1蛋白抑制乳腺癌侵袭相关蛋白的表达。(A)4T1-wt细胞和4T1-ApoA1细胞培养24h，提取mRNA进行RNAseq。图中表示相对表达量。相对表达量是以4T1-wt为1，4T1-ApoA1表达量比4T1-wt的值为4T1-ApoA1的相对表达量。(B)4T1-wt细胞和4T1-ApoA1细胞培养24h，QPCR检测Krt14的表达水平。Krt14，角蛋白14；Cdh3，钙粘连蛋白3；Smn1，Survival Of Motor Neuron 1,Telomeric；Mki67，细胞增殖标记分子ki-67。**p<0.01。
图11腺病毒上调肿瘤组织ApoA1受体分子Abca1的表达水平。Balb/c小鼠接种4T1肿瘤细胞，待肿瘤形成后，瘤内注射2.5x10 8pfu AD5 con或AD5 ApoA1病毒，48h后提取肿瘤组织RNA，进行RNAseq检测。图中表示相对表达量。相对表达量是以PBS组为1，其他组与PBS组的比值为该组的相对表达量。
图12为本发明的重组复制型溶瘤腺病毒AD5 ApoA1抑制stat1、stat3和NFkB信号通路的活化和TNF-alpha、IL-6和IL-1beta的分泌。(A)AD5 ApoA1或AD5 CON在MOI为5的感染复数下对人单核巨噬细胞THP-1或人肝癌细胞HCC-LM3分别感染24h。或再加入TNF-a刺激30min后，检测细胞内stat1、stat3和IkBa的磷酸化水平。 (B)QPCR检测HCC-LM3上清中TNF-alpha、IL-6和IL-1beta的表达水平。*p<0.05,**p<0.01,与AD5 CON组相比；#p<0.05，与PBS组相比。数据代表三次独立性重复实验结果。
图13为本发明的重组复制型溶瘤腺病毒AD5 ApoA1抑制IDO1的表达。AD5 ApoA1或AD5 CON在MOI为5的感染复数下对人肝癌细胞HCC-LM3和SMMC-7721分别感染24h。再加入IL-6刺激30min后，检测细胞内IDO1的表达量。数据代表三次独立性重复实验结果。
具体实施方式
下面结合具体实施例对本发明进行进一步的解释和说明，但应理解，所给出的实施例只作为举例说明，不以任何方式对本发明构成任何限制。
本发明所涉及的实验器材料和试剂如下：
(1)实验细胞系
人胚肾细胞株293T，人肝癌细胞株HCC-LM3、SMMC-7721，人肾透明细胞癌细胞株786-O,人肝癌细胞株HuH-7、HepG2，人膀胱癌细胞株T24，人肺癌细胞株H1299，小鼠肝癌细胞株H22，小鼠乳腺癌细胞株4T1，使用含10％胎牛血清，100U/I青霉素和1mg/ml链霉素的高糖DMEM培养基培养于37℃、5％CO 2的培养箱中。
(2)实验仪器
生物安全柜(
Figure PCTCN2020078360-appb-000001
advance，Class II Biological Safety Cabinet，The Baker Company)，二氧化碳培养箱(FORMA SERIES II WATER JACKET CO 2incubator，Thermo)，低温离心机(HERAEUS  MEGAFUGE 1.0R，Thermo)，垂直电泳槽(BIO-RAD)，电泳仪(BIO-RAD)，半干转-转膜仪(BIO-RAD)，免疫印迹曝光系统(Alpha Innotech)，PCR仪(PCR Thermal Cycler Dice，TaKaRa)，实时定量PCR仪及分析软件(ABI384，Sequence Detection Software，Version 1.3.1)，酶标仪(VERSA max microplate reader)，整套移液器(eppendorf和RAININ)，细胞计数仪(Countstar Automated cell counter，Inno-Alliance Biotech Inc.，Wilmington，USA)，流式细胞仪(FACSCalibur，Becton，Dickinson and Company，USA)，FlowJo software(Version 7.6.5，Tree Star Inc，Ashland，Oregon)，微孔板振荡器(QiLinBeiEr)，核酸纯度浓度检测仪(Biophotometer plus，eppendorf)，数显恒温水浴锅(国华电器)。
(3)主要实验试剂及耗材
引物均由金斯瑞公司合成。肿瘤细胞培养所需的DMEM高糖培养基，双抗，血清均购自Invitrogen(上海)公司。定量RT-PCR试剂Faststart Universal SYBR Green Master(Roche，04913914001)。Western Blot所需试剂耗材：蛋白酶抑制剂(Roche，11873580001)，细胞裂解液(碧云天：P0013)，PVDF膜(Roche，03010040001)，WB Immobilon ECL发光液(Millipore，WBKLS0500)，一抗稀释液(碧云天，P0023A)，HRP标记的二抗(Multisciences，GAR007 and GAM007，1:5000稀释)，其余所需试剂均为国产分析纯，购自南京大学化学化工学院。台盼蓝(碧云天，C0011)，Opti-MEM购自Invitrogen(上海)公司。Western Blot抗体:anti-His(金斯瑞，MB001，1:5000稀释)。
实施例1：AD5-ApoA1病毒质粒构建、病毒拯救与扩增
(1)AD5-ApoA1全长质粒构建：
将构建好的穿梭载体AD5-pShuttle-ApoA1用PmeI线性化后转入感受态pAdEasy-BJ5183中，使用含50ug/ml卡那霉素LB平板的进行筛选，挑取阳性克隆培养鉴定，鉴定正确的克隆质粒重新转化DH5a感受态进行二次筛选鉴定，鉴定正确后进行质粒大提获得AD5 ApoA1全长质粒。
(2)AD5-ApoA1病毒拯救：
AD5-ApoA1全长质粒使用PacI线性化，纯化后6孔板中1ug/well转染293T细胞，5％CO2、37℃培养，2天后将细胞消化后转入10cm平皿，2-3天换液，至80％细胞出现病变，使用10ml培养基将细胞吹下收集至15ml离心管，反复冻融2次，3000rpm/min离心15min，收集病毒上清-80℃保存做为毒种。
(3)病毒扩增：
取病毒种液50ul加入60％293T细胞10cm平皿中，5％CO2 37℃培养，细胞密度至90％以上，按照1传3比例传代，直至80％细胞出现病变，大约有10个平皿细胞，按上述方法收病毒，使用氯化铯密度梯度离心纯化病毒；使用TCID50方法进行滴度测定。
实施例2：AD5-ApoA1病毒的滴度测定
(1)293T细胞种于96孔板，每孔约1×103个细胞，待细胞贴壁后进行滴度测定。
(2)病毒梯度的稀释：准备EP管，每个EP管加入1170μl含胎牛血清的DMEM；往第一个EP管中加入130μl病毒溶液，混匀，标 记为10-1；从第一个EP管中吸取50μl于第二个EP管中，混匀，标记为10-2；依次类推，直至稀释到所需梯度为止。
(3)每孔加入100μl相应梯度的病毒稀释液，每个梯度重复10个孔，37℃培养过夜。
(4)5天后，将96孔板放于显微镜下观察GFP，记下每个梯度有GFP的孔数，用于病毒滴度的计算。
(5)病毒滴度TCID50的计算公式：
Log10(TCID 50)＝L+d(s-0.5)+log10(1/v)
L＝Log10 最高稀释度(如最高稀释度为10倍稀释，L＝1)
V＝最初每孔细胞培养液的体积(ml/well)
d＝Log10 稀释度(如为10倍稀释，d＝1)
s＝各个梯度GFP比率之和
实施例3：AD5-ApoA1病毒功能评价
(1)ApoA1的表达和分泌功能：
AD5 ApoA1病毒感染肿瘤细胞72小时后，收细胞和上清，使用dot Blot检测ApoA1的表达和分泌功能。
(2)病毒复制能力：
AD5 ApoA1和AD5 CON病毒相同MOI感染肿瘤细胞，在不同时间收细胞，反复冻融离心后得到等量病毒悬液，使用293T细胞进行病毒滴度测定；分析病毒复制能力变化。
(3)溶瘤功能：
分别使用AD5 ApoA1和AD5 CON病毒按照MOI 1到100病毒量感染肿瘤细胞，72小时后使用MTT检测细胞活性，评价AD5 ApoA1 的杀瘤作用。
实施例4：4T1-ApoA1稳转株构建
(1)慢病毒转染前18～24小时，将贴壁细胞以1×10 5(根据细胞大小而定，一般转染前细胞长到40-60％为宜)孔铺到24孔板中。使细胞在慢病毒转染时的数量为2×10 5/孔。
(2)加入polybrene6ug/ml，同时加入以CMV为启动子的ApoA1过表达慢病毒。继续培养48h。
(3)加入2ug/ml嘌呤霉素，筛选细胞。每天更换培养基，直到阴性对照孔细胞完全死亡。
实施例5：4T1克隆形成及侵袭实验
(1)细胞克隆获取
1)调整细胞密度至100个细胞/30微升。
2)10cm培养皿底部加入适量无菌PBS，盖子滴加30微升细胞悬液
3)培养2-3天，显微镜观察细胞成球状态。
4)待细胞克隆形成后，将细胞克隆接种于I型胶原包被的96孔板中，时时监测细胞克隆形态。
(2)Transwell实验
1)Matrix稀释30倍，每个小室加入40微升，凝固备用(前一天铺板)
2)细胞消化离心，换成无血清培养基或(2％血清)计数
3)每个小室加入5-10万细胞/200微升，下室加入700微升完全培养基。
4)24h左右固定染色，棉球擦去基质胶，显微镜拍照。
实施例6：体内研究AD5 ApoA1抗肿瘤效应与机制
选用6-8周龄Balb/c小鼠在右侧腋窝或皮下建立皮下瘤模型，4-6天后测量肿瘤大小至200mm 3，将小鼠随机分成3组，分别是：无处理组、对照AD5 CON病毒治疗组、AD5 ApoA1病毒治疗组；按照分组使用相应病毒瘤内注射，每只注射病毒量2.5×10 8pfu，跟踪测量肿瘤体积，体重，小鼠自然死亡后，记录小鼠生存期。
实施例7：细胞总蛋白的提取及浓度测定
以六孔板为例，去掉细胞培养上清，用PBS洗涤2遍，去掉PBS，每孔加入200μl的胰酶，消化吹打细胞，并将细胞收入至EP管中，1500rpm离心5min。
去掉上清，加入PBS重悬细胞，1500rpm离心5min。
去掉PBS，每孔根据细胞量加入相应的含蛋白酶抑制剂的细胞裂解液，涡旋30s，置于冰上10min，重复操作三次。4℃，12000g离心15min。收集上清于另一干净的EP管中。
蛋白浓度的测定：根据BCA蛋白浓度测定盒说明书进行检测。取2μl蛋白样品于96孔板中，加入18μl的PBS稀释样品，最后在加入200μl的测定工作液(工作液由试剂A:试剂B＝50:1)，放置于60℃的烘箱中，30min后，用酶标仪在562nm测定吸光度，根据标准曲线， 计算出蛋白样品的浓度。
每管加入1/4蛋白裂解液体积的5×loading buffer，混匀后，100℃金属浴5min，冷却后，-20℃保存备用。
实施例8：Western blot实验
配胶和电泳：按照不同要求配制不同浓度的SDS-PAGE分离胶和浓缩胶根据蛋白定量的计算结果，每个样品上样量调为30μg。电泳条件：浓缩胶80V 30min，分离胶120V，约80min，前提是将条带分开且不会跑出去。
转膜：准备滤纸和PVDF膜，先用甲醇浸泡PVDF膜，再和滤纸一同浸泡在转膜缓冲液中备用。从玻璃板中小心将胶取下，浸泡在转膜缓冲液中，按照负极-滤纸-PVDF膜-胶-滤纸-正极的三明治顺序放置，赶走气泡，根据所需条带大小不同，恒流110mA转膜60-70min。
封闭：转膜结束后，立即取出PVDF膜，放入5％脱脂奶粉中室温封闭1h。
一抗孵育：4℃孵育一抗过夜。
二抗孵育：用washing buffer洗涤条带，每次10min，共三次；再用相应的HPR标记的二抗室温孵育1h。
曝光：用washing buffer洗涤条带，每次10min，共三次；用化学发光液在WB曝光仪上曝光，并获取条带图像。
实施例9：台盼兰计数
以六孔板为例，去除细胞上清，用PBS洗涤2遍，去掉PBS，每孔加入200μl胰酶消化，轻轻吹打细胞并收集进入干净的EP管中， 1500rpm，离心5min。去掉上清，加入PBS重悬细胞，1500rpm，离心5min。去掉PBS，根据细胞数量加入一定量的PBS重悬细胞，从中取出10μl细胞重悬液，加入10μl 0.2％台盼蓝溶液混合，取混合液20μl于细胞计数板中，用细胞计数仪计数。
实施例10：实时定量PCR
实时定量PCR的10μl体系组成：2.6μl PCR water，上下游引物各0.2μl，2μl的模板和5μl的SYBR Green荧光染料。样品混合后，于ABI 384PCR仪上进行扩增。
结果和结论
图1的结果显示我们成功构建了复制型溶瘤腺病毒AD5 ApoA1。我们设计的ApoA1蛋白同时带有自身信号肽和组氨酸标签。实验证明我们设计的ApoA1的信号肽能够使ApoA1分泌到细胞外。
图2的结果显示，所构建的复制型溶瘤腺病毒AD5 ApoA1与对照病毒相比，在人肝癌细胞株HCC-LM3、SMMC-7721，人肾透明细胞癌细胞株786-O,人肝癌细胞株HuH-7、HepG2，人膀胱癌细胞株T24，人肺癌细胞株H1299中有更强的复制能力。且在Huh-7和T24中显示了更强的溶瘤能力。在体内实验中也发现注射相同剂量的病毒2天后，肿瘤内复制型AD5 ApoA1的病毒载量显著高于AD5 con。说明ApoA1的表达能够促进腺病毒的复制和溶瘤。
图3的结果显示高表达载脂蛋白ApoA1的乳腺癌细胞的划痕间隙比对照组划痕间隙大；表达ApoA1乳腺癌细胞球在基质胶中，侵袭迁移的范围和距离比对照组显著减少；小室侵袭实验发现，表达 ApoA1乳腺癌细胞比对照组透膜细胞数显著减少。说明ApoA1蛋白能够抑制乳腺癌细胞的迁移和侵袭。
图4的结果显示注射了AD5 ApoA1腺病毒的小鼠4T1皮下肿瘤体积较对照病毒和PBS处理组的肿瘤体积小。说明在小鼠模型中，复制型溶瘤腺病毒AD5 ApoA1显著抑制乳腺癌的生长。
图5的结果显示，注射了AD5 ApoA1腺病毒的小鼠4T1乳腺癌原位肿瘤体积较对照病毒和PBS处理组的肿瘤体积小，并且肿瘤腹腔侵袭和肺转移灶显著减少，且小鼠的生存率更优。说明复制型AD5 ApoA1显著抑制乳腺癌原位癌的侵袭迁移和远处转移，并显著延长乳腺癌小鼠的生存时间。
图6的结果显示，注射了AD5 ApoA1腺病毒的小鼠肺癌LLC皮下肿瘤体积较对照病毒和PBS处理组的肿瘤体积小，说明AD5 ApoA1腺病毒在小鼠体内显著抑制小鼠肺癌的生长。
图7的结果显示，注射了复制型AD5 ApoA1的小鼠结肠癌C26皮下肿瘤体积较对照处理组的肿瘤体积小，并且对于C26肿瘤引起的恶液质症状(体重急剧减轻)有明显的缓解作用。说明AD5 ApoA1腺病毒在小鼠体内显著抑制小鼠结肠癌C26皮下瘤的生长，且显著延缓恶液质的进展。
图8的结果显示，注射了AD5 ApoA1腺病毒的小鼠H22肝癌皮下肿瘤体积较对照病毒和PBS处理组的肿瘤体积小，且小鼠的生存率更优。说明AD5 ApoA1腺病毒在小鼠体内显著抑制肝癌的生长，并延长小鼠生存。
图9的结果显示，复制型AD5 ApoA1显著延长人源化肝癌小鼠的生存时间。
图10的结果显示ApoA1蛋白能够抑制乳腺癌细胞4T1的侵袭迁移相关分子尤其是角蛋白14(Krt14)的表达。
图11的结果显示复制型腺病毒能够上调ApoA1的特异性受体分子Abca1的表达，从而进一步增强ApoA1转运胆固醇的能力。
图12的结果显示复制型AD5 ApoA1能够显著抑制巨噬细胞和肿瘤细胞的促肿瘤炎症通路STAT3和NFkB的活化，并减少促肿瘤炎症因子IL-6、NFkB和IL-1beta的产生。
图13的结果显示AD5 ApoA1腺病毒在体外能够抑制肝癌细胞株HCC-LM3和SMMC-7721的吲哚胺2,3二氧化酶1(IDO1)的表达，抑制免疫耐受，增强抗肿瘤免疫。
由以上结果可知，本发明第一次提供了一种既可以抑制肿瘤生长又能阻断肿瘤侵袭转移的复制型溶瘤腺病毒AD5 ApoA1。该病毒不仅仅比Ad5 con病毒在肿瘤细胞内有更强的复制和溶瘤能力，还能够高表达载脂蛋白ApoA1，该蛋白能够分泌到细胞外，上调被感染肿瘤细胞高表达ApoA1的特异性受体ABCA1，能够大大增强AD5 ApoA1的转运胆固醇的作用，产生意料不到的协同作用，发挥多种抗肿瘤作用；抑制恶性肿瘤细胞侵袭迁移、阻断促肿瘤炎症通路，抑制介导肿瘤免疫逃逸的关键酶IDO-1，能够有效地恢复机体对肿瘤的免疫监视。不仅如此，本发明的重组复制型溶瘤腺病毒AD5-ApoA1具有预料不到的对抗恶液质进展的作用，能够有效地维持结肠癌小鼠的体重，并显著延长生存时间。总的来说，本发明的重组复制型溶瘤腺病毒AD5 ApoA1同时兼具多种抗肿瘤的作用：抑制肿瘤的侵袭转移、延缓恶性肿瘤的恶液质进程、上调感染肿瘤细胞高表达ApoA1的特异性受体ABCA1协同抗肿瘤、抑制IDO-1及促肿瘤炎症并恢复 抗肿瘤免疫监视。一个病毒，同时整合多种独特的在肿瘤治疗方面的作用与机制，相辅相成，具有预料不到的效果。
以上描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。

Claims (15)

  1. 一种调控脂代谢的复制型溶瘤腺病毒载体，包括或插入依次连接的下述外源基因：第一启动子、E1A早期激活复制原件、绝缘序列、第二启动子，目的基因序列、PA序列，其特征在于：
    所述的目的基因插入到溶瘤腺病毒E1B区；
    所述的目的基因序列为ApoA1基因或其简并序列；
    所述的目的基因序列前端包含信号肽识别序列。
  2. 根据权力要求1所述的复制型溶瘤腺病毒载体，其特征在于如下的(1)至(3)中的任一项或者多项组合：
    (1)所述的ApoA1目的基因核酸如SEQ ID NO：1所示；
    (2)所述的第一启动子为组成型启动子、特异型启动子或诱导型启动子之一；第二启动子为组成型启动子；
    (3)所述的腺病毒为C亚型。
  3. 根据权力要求1或2所述的复制型溶瘤腺病毒载体，其特征在于：组成型启动子包括CMV、SV40或EF1a启动子。
  4. 根据权力要求3所述的复制型溶瘤腺病毒载体，其特征在于：所述的组成型增强启动子为CMV，核酸序列如SEQ ID NO：2所示。
  5. 一种调控脂代谢的复制型溶瘤腺病毒，其含有权利要求1至4中任一项所述的复制型溶瘤腺病毒载体。
  6. 根据权力要求5所述的调控脂代谢的复制型溶瘤腺病毒，其具有在宿主细胞内复制的能力，其特征在于：该复制型溶瘤腺病毒也可以在宿主细胞内表达分泌可溶性ApoA1蛋白，达到抑制肿瘤的增殖、侵袭和转移效果。
  7. 根据权力要求6所述的调控脂代谢的复制型溶瘤腺病毒，其特征在于：所述的可溶性ApoA1蛋白的氨基酸序列如SEQ ID NO：3所示。
  8. 根据权力要求5至7所述的调控脂代谢的复制型溶瘤腺病毒，其特征在于：其由权利要求1至4中任一项所述的重组溶瘤腺病毒载体在293T细胞中重组得到。
  9. 权力要求1至4中任一项所述的复制型溶瘤腺病毒载体和权力要求5至7中任一项所述的复制型溶瘤腺病毒在制备预防和/或治疗肿瘤的药物应用。
  10. 根据权力要求9所述的应用，其特征在于：所述的治疗为单独治疗，辅助治疗或者联合治疗。
  11. 根据权力要求9或10所述的应用，其特征在于：所述的肿瘤为肝癌、乳腺癌、肺癌、结肠癌、胃癌、胰腺癌、宫颈癌、黑色素瘤、前列腺癌、卵巢癌、淋巴瘤、胆囊癌、食管癌、肾癌、鼻咽癌、喉癌、甲状腺肿瘤、纵隔肿瘤或神经胶质瘤。
  12. 根据权力要求9或10所述的应用，其特征在于：所述预防和/或治疗肿瘤的药物的应用包括在制备抗肿瘤侵袭转移药物的 应用。
  13. 根据权力要求9或10所述的应用，其特征在于：所述预防和/或治疗肿瘤的药物的应用包括在制备抑制肿瘤炎症通路及IDO-1并恢复抗肿瘤免疫监视药物的应用。
  14. 根据权力要求9或10所述的应用，其特征在于：所述预防和/或治疗肿瘤的药物的应用包括在制备延缓恶性肿瘤的恶液质进程药物的应用。
  15. 根据权力要求9或10所述的应用，其特征在于：所述预防和/或治疗肿瘤的药物的应用包括在制备上调所感染肿瘤细胞ApoA1特异性受体ABCA1表达药物的应用。
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