WO2022033468A1 - Vesicular stomatitis virus and therapeutic use thereof - Google Patents

Vesicular stomatitis virus and therapeutic use thereof Download PDF

Info

Publication number
WO2022033468A1
WO2022033468A1 PCT/CN2021/111760 CN2021111760W WO2022033468A1 WO 2022033468 A1 WO2022033468 A1 WO 2022033468A1 CN 2021111760 W CN2021111760 W CN 2021111760W WO 2022033468 A1 WO2022033468 A1 WO 2022033468A1
Authority
WO
WIPO (PCT)
Prior art keywords
vesicular stomatitis
stomatitis virus
tumor
cancer
cell surface
Prior art date
Application number
PCT/CN2021/111760
Other languages
French (fr)
Chinese (zh)
Inventor
吴可行
Original Assignee
上海行深生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海行深生物科技有限公司 filed Critical 上海行深生物科技有限公司
Priority to CN202180070834.8A priority Critical patent/CN116322728A/en
Publication of WO2022033468A1 publication Critical patent/WO2022033468A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to the field of oncolytic viruses, in particular to vesicular stomatitis virus, the use of vesicular stomatitis virus in preparing medicine, the use of tumor cell surface specific receptors in screening VSV virus for treating or preventing tumors, Methods for screening drugs, methods for predicting vesicular stomatitis virus oncolytic effects, and methods for screening subjects who can be used for vesicular stomatitis virus therapy.
  • oncolytic viruses In the past decade, the mechanism by which oncolytic viruses can kill tumors by inducing the body's anti-tumor immune response has gradually become clear. Since the German scientist Jean Rommelaere first called oncolytic virus therapy as tumor immunotherapy in 2011, oncolytic virus has been accepted by the public as an important branch of tumor immunotherapy. Compared with other tumor immunotherapies, oncolytic viruses have the advantages of high killing efficiency, good targeting, less side effects, multiple tumor killing pathways to avoid drug resistance, and low cost.
  • viruses are more likely to replicate and spread in cancer cells.
  • PLR protein kinase R
  • scientists have used the differences in many signaling pathways and metabolisms between cancer cells and normal cells to continuously improve the ability of oncolytic viruses by screening specific virus species and modifying virus genomes.
  • the targeting of tumors can reduce the harm to normal cells and improve safety.
  • the approved T-vec knocks out the ⁇ 34.5 gene of HSV-1 (herpes simplex virus type 1).
  • the ⁇ 34.5 gene can inhibit the clearance mechanism of the virus in normal cells.
  • JX594 (Pexa-Vec), currently in phase III clinical trials, has knocked out the TK (thymidine kinase) gene of vaccinia viruses, and the replication of the virus is related to the level of TK in cells, so JX594 knocked out TK is only It can replicate in cancer cells with high TK activity, but cannot replicate in normal cells (normal cells with low TK activity).
  • CG0070 adds the E2F-1 promoter before the adenovirus replication-competent gene E1A.
  • E2F-1 is regulated by retinoblastoma inhibitory protein (Rb), and Rb is deleted in bladder cancer, and the loss of Rb activates E2F-1
  • Rb retinoblastoma inhibitory protein
  • the transcriptional activity of the E1A gene allows the virus to replicate specifically in bladder cancer.
  • Reolysin is an unmodified wild-type reovirus whose proliferation depends on the activation of the Ras signaling pathway, so it can only specifically proliferate in Ras-activated cancer cells.
  • the current status of tumor therapy is that there is still a lack of a method that can not only improve the specificity of tumor killing (ie, high specificity relative to normal non-tumor cells), but also improve the broad spectrum of tumor therapy (ie, is suitable for multiple cancer treatment). Therefore, there is an urgent need in the art to develop a method for killing tumor cells that has both high tumor cell specificity and broad spectrum of tumor therapy.
  • VSV has been discovered for a long time, and VSV has a broad spectrum of cell invasiveness, the receptor for VSV has not been found for a long period of time, and some researchers even believe that its receptor is a lipid component on the cell membrane , such as phospholipid serine, phosphatidylinositol, ganglioside GM3, etc.
  • lipid component on the cell membrane such as phospholipid serine, phosphatidylinositol, ganglioside GM3, etc.
  • VSV-G is required for VSV binding to its putative receptor, endocytosis, and fusion with target cell membranes. After binding, VSV undergoes clathrin-mediated Endocytosis into cells (Reference: Techniques for dual staining of DNA and intracellular immunoglobulins in murine hybridoma cells: applications to cell-cycle analysis of hyperosmotic cultures. Sun et al., Cytotechnology. 2005 Jun; 48(1-3): 15-26.), which indicates that there must be a specific receptor for VSV on the cell membrane.
  • LDLR LDLR knockout cells were still able to infect VSV, suggesting that there are other receptors that mediate VSV endocytosis. Previous studies have shown that although VSV can infect most mammalian cells, VSV has a higher preference for tumor cells, suggesting that tumor cells may have highly expressed VSV receptors.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • the present invention proposes the use of the protein in the construction of vesicular stomatitis virus.
  • the protein is a tumor cell surface specific receptor, including at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.
  • the vesicular stomatitis (VSV virus) constructed by the specific receptors on the surface of tumor cells screened by the inventors of the present application has stronger specific binding ability to tumor cells, and has a more significant specific killing effect on tumor cells.
  • the above-mentioned use may further include at least one of the following additional technical features:
  • the ZDOCK score of the binding force between the G protein of the VSV virus and the tumor cell surface specific receptor is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100.
  • the ZDOCK score a parameter that characterizes the binding force between G proteins and cellular receptors, can be easily obtained. The inventors found that when the ZDOCK score is not less than 1800, the binding force between the virus carrying the G protein and the tumor cells carrying the corresponding receptor will be significantly improved.
  • the ZDOCK score can be determined by conventional software, for example, see Pierce BG, Houurai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657 .
  • the ZDOCK score of the G protein and the receptor LDLR is not less than 1600, preferably not less than 1650, more preferably not less than 1659, such as 1659.078.
  • the binding force of the tumor cell surface specific receptors screened by the inventors of the present application to G protein is much higher than the binding force of LDLR to G protein.
  • the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.
  • the VSV virus does not carry heterologous genes.
  • heterologous gene refers to a gene that has not been reported in wild-type vesicular stomatitis virus unless otherwise specified. Or in other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in the wild-type vesicular stomatitis virus.
  • the tumor cells include at least one selected from colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
  • the vesicular stomatitis virus comprises at least one selected from the Indiana strain serotype and the New Jersey strain serotype.
  • the present invention provides a vesicular stomatitis virus (VSV virus).
  • VSV virus vesicular stomatitis virus
  • the vesicular stomatitis virus expresses a G protein, and the G protein is suitable for binding to a tumor cell surface specific receptor, optionally, the tumor cell surface specific receptor includes a group selected from At least one of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.
  • the vesicular stomatitis virus (VSV virus) according to the embodiment of the present invention has stronger specific binding ability to tumor cells, and has a more significant specific killing effect on tumor cells.
  • the above-mentioned VSV virus may further include at least one of the following additional technical features:
  • the ZDOCK score of the binding force of the G glycoprotein of the VSV virus to the specific receptor on the surface of the tumor cell is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100.
  • the ZDOCK score a parameter that characterizes the binding force between G proteins and cellular receptors, can be easily obtained. The inventors found that when the ZDOCK score is not less than 1800, the binding force between the virus carrying the G protein and the tumor cells carrying the corresponding receptor will be significantly improved.
  • the ZDOCK score can be determined by conventional software, for example, see Pierce BG, Houurai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657 .
  • the ZDOCK score of the G protein and the receptor LDLR is not less than 1600, preferably not less than 1650, more preferably not less than 1659, such as 1659.078.
  • the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.
  • the VSV virus does not carry heterologous genes.
  • heterologous gene refers to a gene that has not been reported in wild-type vesicular stomatitis virus unless otherwise specified. Or in other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in the wild-type vesicular stomatitis virus.
  • the tumor cells include at least one selected from colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
  • the vesicular stomatitis virus comprises at least one selected from the Indiana strain serotype and the New Jersey strain serotype.
  • the present invention proposes the use of the aforementioned vesicular stomatitis virus in preparing a medicament for treating or preventing tumors.
  • the above-mentioned use may further include at least one of the following additional technical features:
  • the tumor cells carry tumor cell surface specific receptors, and the tumor cell surface specific receptors include at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.
  • the tumor cell surface specific receptor has an expression fold difference (log2FC) of not less than 2.0 in the tumor.
  • the use of the present tumor cell surface specific receptor in screening VSV virus for the treatment or prevention of tumors is selected from CHRNA5, SSTR5, KISS1R, HTR1D and One or more of CCR8.
  • the above-mentioned use may further include at least one of the following additional technical features:
  • the ZDOCK score of the binding force of the G glycoprotein of the VSV virus to the tumor cell surface specific receptor whose expression fold difference (log2FC) is greater than or equal to 2.0 in the tumor to be treated is not less than 1800, For example, not less than 1900, not less than 2000, preferably not less than 2100 is an indication that the virus to be screened is the target virus.
  • the VSV virus is a recombinant VSV virus; preferably, the VSV virus does not carry a heterologous gene.
  • the present invention proposes a method of screening subjects for vesicular stomatitis virus treatment.
  • the expression level of a cell surface receptor selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 is determined in the tumor of the subject.
  • the subjects screened according to the methods of the embodiments of the present invention are more suitable for receiving vesicular stomatitis virus treatment, and the curative effect will be more significant.
  • the above method may further include at least one of the following additional technical features:
  • the expression fold difference (log2FC) of the cell surface receptor in the tumor to be treated is not less than 2.0 is an indication that the subject is suitable for receiving vesicular stomatitis virus therapy.
  • the vesicular stomatitis virus is the aforementioned vesicular stomatitis virus.
  • the present invention provides a method for predicting the oncolytic effect of VSV virus.
  • the method includes: determining the binding capacity of the G glycoprotein of the VSV virus to be tested to a cell surface receptor of tumor cells, the cell surface receptor is selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 one or more of.
  • the above-mentioned method for predicting the oncolytic effect of VSV virus may further include at least one of the following additional technical features:
  • the ZDOCK score of the binding force between the G glycoprotein of the VSV virus and the cell surface receptor is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100 is the VSV virus to be tested Indication of good oncolytic effect.
  • the present invention provides a method for treating or preventing tumors. According to an embodiment of the present invention, comprising administering the vesicular stomatitis virus of the second aspect to a subject.
  • the methods according to the embodiments of the present invention can provide a subject with the ability to effectively prevent tumors or inhibit the growth of tumor cells.
  • the above-mentioned method for treating or preventing tumors may further include at least one of the following additional technical features:
  • the tumor comprises at least one selected from the group consisting of colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
  • Figure 1 shows a flowchart of the analysis of human membrane receptor genes based on a large sample of tumor tissue.
  • Figure 2 shows a jittered scatterplot of the proportion of patients with the corresponding receptor gene significantly up-regulated in each tumor.
  • Figure 3 shows the ZDOCK score results reflecting the binding strength of 16 candidate ligands to 5 tumor-specific receptors, respectively.
  • Figure 4 shows the ZDOCK score results of the binding strength of the screened ligands to 5 tumor-specific receptors.
  • Figure 5 shows the mRNA expression levels of CHRNA5, KISS1R, HTRID, CCR8 and SSTR5 in BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cell samples detected by qPCR.
  • Figure 6 shows the killing effect of viruses on BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells at different MOIs measured in cell killing experiments.
  • Figure 7 shows the killing effect of virus strains with different G proteins on NCL-H358 and NCL-H460 cells at different MOIs.
  • Figure 8 shows the effects of gene knockdown on CCR8, CHRNA5, KISS1R, HTR1D, and SSTR5 on the intracellular VSV virus replication in the in vitro knockdown experiment.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
  • the present invention provides a vesicular stomatitis virus.
  • the vesicular stomatitis virus expresses a G protein, and the G protein is suitable for binding to a tumor cell surface specific receptor, optionally, the tumor cell surface specific receptor includes a group selected from At least one of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.
  • the "specific receptors on the surface of tumor cells” mentioned in this application refer to receptors with significant up-regulation/down-regulation of differentially expressed receptors on the surface of tumor cells relative to receptors on the surface of normal cells, wherein the expression difference A fold (log2FC) greater than or equal to 2.0 was considered a significant up-regulation difference, and a p-value of t-test less than 0.01 was considered a statistically significant difference.
  • VSV virus vesicular stomatitis virus according to the embodiment of the present invention has stronger specific binding ability to tumor cells, and has a more significant specific killing effect on tumor cells.
  • VSV Vesicular stomatitis virus
  • VSV-NJ New Jersey
  • Indiana Indiana
  • Virus particles are bullet-shaped or cylindrical, with a size of 150-180 nm ⁇ 50-70 nm.
  • the virus has an envelope, and the envelope is evenly covered with fibers about 10nm long.
  • Inside the virus is a tightly coiled helix-symmetric nucleocapsid.
  • the virus is named after the classic vesicular lesions in the oral mucosa, tooth pads, tongue, lips, nostrils, hooves and nipples of sick animals.
  • Spread by insect vectors the disease is limited to its natural hosts such as horses, cattle and pigs. In humans, the infection is mild and asymptomatic.
  • the VSV genome is an unsegmented single-stranded negative-stranded RNA (ssRNA) virus with a length of approximately 11 KB. From the 3' end to the 5' end, there are five non-overlapping genes, N, NS, M, G, and L, which encode nuclear (N) protein, phosphate (P) protein, matrix (M) protein, sugar (G) protein, respectively. ) protein, and five different major proteins including RNA-dependent RNA polymerase (L) protein.
  • the 3' end of the N gene is a leader sequence (Leader), the 5' end is a trailing sequence (Trailor), and there is a spacer sequence between each gene.
  • the 3'-end leader RNA is the earliest viral transcript in infected cells.
  • N protein is required to initiate the synthesis of the genome and can effectively protect the viral RNA from digestion by various nucleases. N protein has high immunogenicity, stimulates the body to produce cellular immunity, and plays an important role in transcriptional replication. It may be necessary to maintain the extended form of genomic RNA and is related to replication regulation.
  • the P protein, VSV-NJ is 41% homologous to the VSV-IND strain, and its role is to form a polymerase complex with polymerase L, nucleoprotein N and genomic RNA to maintain the transcriptional activity of the virus.
  • the M protein plays a key role in viral pathogenesis and viral replication, is rich in basic amino acids, and contains a highly basic amino-terminal domain that inhibits transcription by binding to the nucleocapsid while assisting virus budding from the host , is the only polypeptide involved in the budding process.
  • G protein is the main surface antigen of the virus, which determines the virulence of the virus and is also the protective antigen of the virus. It stimulates the body to produce neutralizing antibodies.
  • the L gene encodes the RNA poly E protein, which may determine the transcriptional activity of RNA, and binds to the P protein to catalyze the replication of mRNA.
  • This protein is a core component of the polymerase complex and replicase complex and is involved in initiation, elongation, methylation, capping, poly(A) tail formation, and more.
  • there is extensive homology in the spacer sequences between each gene and these sequences share a common structure, namely 3'-AUAC(U)7NAUUGUCNN-UAG-5'.
  • the conserved sequence between these genes is a key signal to affect the activity of the polymerase or the cleavage activity of the enzyme, and during replication, these signals are masked and not functional.
  • the ZDOCK score of the binding force of the G glycoprotein of the VSV virus to the specific receptor on the surface of the tumor cell is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100.
  • the binding ability of the vesicular stomatitis virus described in the present application to specific receptors on the surface of tumor cells was evaluated by ZDOCK score function.
  • the generated docking results will be sorted according to the ZDOCK score. The higher the score, the stronger the combination and the higher the reliability of the result. There are also clustering results for these conformations.
  • the ZDOCK score is the shape complementation score calculated by the ZDOCK program. According to the parameter settings, the ZDOCK score will also include electrostatic and desolvation energy terms. The higher the ZDOCK score, the better.
  • the ZDOCK score a parameter that characterizes the binding force between G proteins and cellular receptors.
  • the ZDOCK score can be determined by conventional software, for example, see Pierce BG, Houurai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657 .
  • the ZDOCK score of the G protein and the receptor LDLR is not less than 1600, preferably not less than 1650, more preferably not less than 1659, or 1650-1660, for example 1659.078.
  • LDLR is a vesicular stomatitis virus entry receptor known in the art. The inventors used LDLR as a positive control and found that the binding capacity of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 screened in this application to the virus is much higher than that of LDLR .
  • the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.
  • the VSV virus does not carry heterologous genes.
  • the term "heterologous gene” described herein refers to a gene that has not been reported in wild-type vesicular stomatitis virus unless otherwise specified. Or in other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in the wild-type vesicular stomatitis virus. The inventors found that the killing effect of recombinant vesicular stomatitis virus without heterologous gene on tumor cells is significantly higher than that of recombinant vesicular stomatitis virus carrying heterologous gene.
  • the tumor cells include at least one selected from colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
  • the vesicular stomatitis virus comprises at least one selected from the Indiana strain serotype and the New Jersey strain serotype.
  • the present invention proposes the use of the aforementioned vesicular stomatitis virus in preparing a medicament for treating or preventing tumors.
  • the medicament prepared from the virus according to the embodiment of the present invention has a more significant effect in treating or preventing tumors.
  • the tumor cells carry tumor cell surface specific receptors, and the tumor cell surface specific receptors include at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.
  • the tumor cell surface specific receptor has an expression fold difference (log2FC) of not less than 2.0 in the tumor. The inventors found that the proportion of differential gene expression (log2FC) greater than 2 of 5 tumor-specific receptors CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 in 5 cancer types is much larger than that of LDLR.
  • the cancer includes at least one of lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, and leukemia.
  • the present invention proposes a pharmaceutical composition.
  • the pharmaceutical composition contains: the aforementioned recombinant vesicular stomatitis virus.
  • the pharmaceutical composition is in a form suitable for administration by inhalation or injection.
  • the pharmaceutical composition provided by the present invention comprises a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: the recombinant VSV virus specifically infecting tumor cells of the present invention.
  • the term “effective amount” or “effective dose” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent.
  • it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical compositions are preferably manufactured under sterile conditions.
  • additional tumor therapeutic agents may also be included in the pharmaceutical compositions of the present invention.
  • the effective amount of the active ingredient of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the administration way etc. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.
  • the pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or its combination.
  • the choice of carrier should match the mode of administration, as is well known to those of ordinary skill in the art.
  • a tumor cell surface specific receptor selected from the group consisting of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 in screening for VSV virus for the treatment or prevention of tumors one or more of.
  • the ZDOCK score of the binding ability of the G glycoprotein of the VSV virus to the tumor cell surface specific receptor whose expression fold difference (log2FC) is greater than or equal to 2.0 in the tumor to be treated is not less than 1800, For example, not less than 1900, not less than 2000, preferably not less than 2100 is an indication that the virus to be screened is the target virus.
  • the VSV virus screened for treating or preventing tumors according to the methods of the embodiments of the present invention has stronger specific targeting and affinity for tumors, and has a more significant killing effect.
  • the tumor to be treated is lung adenocarcinoma or lung squamous cell carcinoma, and the tumor cell surface specific receptor is selected from at least one of CHRNA5, SSTR5 and KISS1R.
  • the tumor to be treated is colon cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D, SSTR5 and KISS1R.
  • the tumor to be treated is liver cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D, SSTR5, CCR8 and KISS1R.
  • the tumor to be treated is gastric cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D and CCR8.
  • the tumor to be treated is pancreatic cancer, and the tumor cell surface specific receptor is selected from at least one of CHRNA5, SSTR5 and HTR1D.
  • CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 receptors on different tumor cells and normal cells are different.
  • the receptors that are specifically differentially expressed in different tumor cells are used to screen VSV viruses for the treatment or prevention of specific tumors more purposefully.
  • the VSV virus is a recombinant VSV virus; preferably, the VSV virus does not carry a heterologous gene. Furthermore, the killing effect of recombinant VSV virus on tumor was further improved.
  • the present invention proposes a method of screening subjects for vesicular stomatitis virus treatment.
  • the expression level of a cell surface receptor selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 is determined in the tumor of the subject.
  • the expression fold difference (log2FC) of the cell surface receptor in the tumor to be treated is not less than 2.0 is an indication that the subject is suitable for receiving vesicular stomatitis virus therapy.
  • the vesicular stomatitis virus is the aforementioned vesicular stomatitis virus.
  • the subjects screened according to the methods of the embodiments of the present invention are more suitable for receiving vesicular stomatitis virus treatment, and the curative effect will be more significant.
  • the present invention provides a method for predicting the oncolytic effect of VSV virus.
  • the method includes: determining the binding capacity of the G glycoprotein of the VSV virus to be tested to a cell surface receptor of tumor cells, the cell surface receptor is selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 one or more of.
  • the ZDOCK score of the binding force between the G glycoprotein of the VSV virus and the cell surface receptor is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100 is the VSV to be tested Indication of good viral oncolytic effect.
  • the method according to the embodiment of the present invention can predict the oncolytic effect of the VSV virus to be screened in advance, thereby providing scientific guidance for subsequent preclinical experiments.
  • Example 1 Analysis of human membrane receptor genes based on large tumor tissue samples
  • the present invention summarizes the receptor gene information expressed in human cells from existing research (reference: Synchronous birth is a dominant pattern in receptor-ligand evolution, Grandchamp and Monget, BMC Genomics. 2018 Aug 14; 19 ( 1):611.).
  • the inventors downloaded the gene expression matrix (normalized value), gene mutation information and related clinical data of cancer patients from UCSC Xena ( http://xena.ucsc.edu/ ).
  • Cancer types included in the data are: adrenal cortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and cervical endometrial adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, colon adenocarcinoma/Rectum adenocarcinoma, esophageal carcinoma , lymphoid neoplasms, diffuse large B-cell lymphoma, esophageal cancer, FFPE trial phase II, glioblastoma, glioma, head and neck squamous cell carcinoma, renal chromosome, pan-kidney cohort (KICH+KIRC+ KIRP), renal renal clear cell carcinoma, renal renal papillary cell carcinoma, acute myeloid leukemia, low-grade cerebral glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian se
  • the inventors first removed less than three sample tumor and normal tissue information from the downloaded data, and then performed differential expression analysis.
  • the inventors used limma software (version: 3.38.3) to perform differential expression analysis (reference: Limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies. Ritchie, ME, et al. Nucleic Acids Research, 2015, 43, e47 ).
  • the voom model of the limma R package was used in the analysis. Only when the gene satisfies the criterion
  • the fold difference (log2FC) and p-value of membrane receptor gene expression between groups were calculated using R language. Selecting
  • CHRNA5 neuropeptide type cholinergic receptor ⁇ 5
  • SSTR5 somatostatin receptor 5
  • KISS1R Kissin receptor
  • HTR1D serotonin receptor 1D
  • CCR8 CC chemokine receptor 8
  • LDLR is a vesicular stomatitis virus entry receptor known in the art
  • LDLR was used as a control in subsequent studies of the present invention.
  • the binding strength of five tumor-specific receptors and LDLR receptors to VSV virus was further analyzed and screened.
  • the inventors selected 16 kinds of vesicular stomatitis virus homologous ligands, and modeled and docked with different tumor-specific receptors obtained by screening in Example 1, respectively.
  • the generated docking results will be sorted according to the ZDOCK score. The higher the value, the stronger the binding and the higher the reliability of the result.
  • the clustering results of these conformations were comprehensively analyzed, and it was found that the ZDOCK score was the shape complementation score calculated by the ZDOCK program. According to the parameter settings, the ZDOCK score would also include electrostatic and desolvation energy terms. The higher the ZDOCK score, the better.
  • the inventors evaluated the strength of binding through the ZDOCK score function, and obtained a ligand with strong tumor-specific receptor binding ability (the results are shown in Figure 4), among which, the ligand with the best binding effect was DQ408670.
  • 1-lig-FL and X03633.1-lig-FL the capture number of the corresponding amino acid sequence is DQ408670.1, GENE ID: X03633.1.
  • the inventors combined the L, N, P, and M proteins derived from the Mudd summer subtype strain to capture the sequence numbers as GENE ID: DQ408670.1, GENE ID: X03633.1, GENE ID: KP872888.1 or GENE ID: The G protein of HQ593628.1 was used to construct recombinant vesicular stomatitis viruses REV DQ408670.1, REV X03633.1, REV KP872888.1 and REV HQ593628.1.
  • the packaging methods for the virus strains REV DQ408670.1, REV X03633.1, REV KP872888.1 and REV HQ593628.1 are as follows:
  • VSV In vitro recombination of VSV requires: a full-length plasmid (including G protein) containing the viral genome, and an auxiliary plasmid (N, P, L, M) of the backbone protein required for viral packaging, and the plasmid is transferred into BHK21 by the method of in vitro transfection In cells, the virus is assembled and matured in the cell and then buds and released outside the cell (Reference: Vesicular stomatitis virus-based vaccine protects hamsters again lethal challenge with Andes virus, Brown, KS, Safronetz, D., Marzi, A., Ebihara, H. & Feldmann, H. Journal of virology 85, 12781-12791, doi: 10.1128/JVI.00794-11 (2011)).
  • Virus amplification uses Vero cells, and a certain titer of virus is added to the cultured Vero cells.
  • the virus can infect the cells and complete self-replication in the cells.
  • the mature virus is released into the supernatant of the cell culture, and the cells are cultured.
  • the supernatant obtained can be concentrated to obtain a virus concentrate, which can be used for subsequent experiments after titer determination.
  • Example 3 different viruses constructed in Example 3 were used to verify the killing effect of different tumor cells.
  • the BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells in good condition were made into a cell suspension of 5 ⁇ 10 4 cells/mL and added to a 96-well plate at 100 ⁇ L/well, and the medium was filled with the edge to reduce evaporation, overnight. nourish. Dilute the known titer of virus with Opti-MEM to virus working solution of MOI: 0.01, MOI: 0.1 and MOI: 1, aspirate the culture solution in the 96-well plate, add 50ul virus dilution solution to each well, and each dilution solution Repeat 3 wells, and take Opti-MEM to repeat 3 wells as blank control.
  • the virus diluent was added for 2 hours and then the medium was changed, with 100 ⁇ L of 1% FBS medium per well. After 48/72h, add 10 ⁇ L CCK8 detection solution to each well, incubate at 37°C for 2h and read on OD450 microplate reader.
  • the experimental results are shown in Figure 7.
  • the CCK test results showed that the virus working solution of REV-DQ408670.1 and REV-X03633.1 had significantly better killing effect on NCL-H358 and NCL-H460 cells than REV-KP872888.1 and REV-HQ593628.1.
  • the expression levels of CCR8 and CHRNA5 were higher in NCL-H358, and the expression levels of CHRNA5 and HTR1D were higher in NCL-H460, as well as the binding heat map results of receptors and ligands in Figure 3, DQ408670.1ligant and X03633 .1G protein has strong binding ability to CCR8 and HTR1D receptors.
  • the comprehensive reaction shows that when the recombinant vesicular stomatitis virus has high binding force to the tumor cell receptor, the killing effect of the recombinant virus on the tumor cells highly expressing the receptor is more significant.
  • FIG. 6 shows the results of the CCK assay of REV-DQ408670.1.
  • REV-DQ408670.1 has positive effects on BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells at MOI: 0.01, MOI: 0.1 and MOI: 1. Significant killing effect.
  • the interfering RNA was designed according to the sequence of the target gene.
  • the shRNA was constructed on the pSGU6 vector, and lipo8000 was transfected into HEK-293T cells. After 48 hours, the cellular RNA was collected and reverse transcribed. The shRNA with significant knockdown effect was selected by q-PCR. . The selected shRNA was retransfected into HEK-293T cells, treated with REV-DQ408670.1 virus after 48 hours, and the cellular RNA was collected after 24 hours for reverse transcription. Effects of viral replication.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Provided is a vesicular stomatitis virus. The vesicular stomatitis virus expresses a G protein, and the G protein is suitable for binding to a specific receptor on the surface of tumor cells. Optionally, the specific receptor on the surface of tumor cells comprises at least one among CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8.

Description

水疱性口炎病毒及其治疗用途Vesicular stomatitis virus and its therapeutic use
优先权信息priority information
本申请请求2020年08月14日向中国国家知识产权局提交的、专利申请号为202010816788.9的专利申请的优先权和权益，并且通过参照将其全文并入此处。This application claims the priority and rights and interests of the patent application with the patent application number 202010816788.9 filed with the State Intellectual Property Office of China on August 14, 2020, which is hereby incorporated by reference in its entirety.
技术领域technical field
本发明涉及溶瘤病毒领域，具体涉及水疱性口炎病毒、水疱性口炎病毒在制备药物中的用途、肿瘤细胞表面特异性受体在筛选用于治疗或预防肿瘤的VSV病毒中的用途、筛选药物的方法、预测水疱性口炎病毒溶瘤效果的方法、筛选可用于水疱性口炎病毒治疗的受试者的方法。The present invention relates to the field of oncolytic viruses, in particular to vesicular stomatitis virus, the use of vesicular stomatitis virus in preparing medicine, the use of tumor cell surface specific receptors in screening VSV virus for treating or preventing tumors, Methods for screening drugs, methods for predicting vesicular stomatitis virus oncolytic effects, and methods for screening subjects who can be used for vesicular stomatitis virus therapy.
背景技术Background technique
近十几年，溶瘤病毒可以通过诱导机体的抗肿瘤免疫反应来杀伤肿瘤的机制逐渐明确。自2011年德国科学家Jean Rommelaere第一次将溶瘤病毒疗法称作肿瘤免疫治疗以来，溶瘤病毒目前已经被大众接受作为肿瘤免疫治疗的重要分支。而相较于其他肿瘤免疫疗法，溶瘤病毒具有杀伤效率高、靶向性好、副作用小、多种杀伤肿瘤途径避免耐药性和成本低廉等优势。In the past decade, the mechanism by which oncolytic viruses can kill tumors by inducing the body's anti-tumor immune response has gradually become clear. Since the German scientist Jean Rommelaere first called oncolytic virus therapy as tumor immunotherapy in 2011, oncolytic virus has been accepted by the public as an important branch of tumor immunotherapy. Compared with other tumor immunotherapies, oncolytic viruses have the advantages of high killing efficiency, good targeting, less side effects, multiple tumor killing pathways to avoid drug resistance, and low cost.
由于病毒基因组小，相对易于通过基因工程手段进行多种改造，且病毒的改造和包装是生物医学常规试验手段，技术相对成熟，价格也相对低廉。因此，溶瘤病毒更容易利用自身的特征和癌细胞与正常细胞的不同来做相应的改造，做到特异性靶向癌细胞。Due to the small genome of the virus, it is relatively easy to carry out various transformations by means of genetic engineering, and the transformation and packaging of viruses are routine biomedical testing methods. The technology is relatively mature and the price is relatively low. Therefore, it is easier for oncolytic viruses to use their own characteristics and the difference between cancer cells and normal cells to make corresponding transformations to specifically target cancer cells.
由于大部分癌细胞自身清除病毒的机制受损(如正常细胞清除病毒的关键因子蛋白激酶R(PKR)在癌细胞中缺失)，所以病毒更容易在癌细胞中复制扩散。另外，近几十年，随着研究的不断深入，科学工作者们利用癌细胞和正常细胞中很多信号通路和代谢等的不同，通过筛选特异病毒品种和改造病毒基因组，不断提高溶瘤病毒对肿瘤的靶向性，降低其对正常细胞的危害，提高安全性。例如：已获批的T-vec敲除了HSV-1(单纯疱疹病毒1型)的γ34.5基因，γ34.5基因可以抑制正常细胞对病毒的清除机制，γ34.5基因敲除后，病毒不能在正常细胞中复制；而癌细胞中这一机制缺失，γ34.5基因敲除后不影响病毒在癌细胞中的复制。目前处于III期临床的JX594(Pexa-Vec)敲除了牛痘病毒(vaccinia viruses)的TK(胸苷激酶，thymidine kinase)基因，而病毒的复制与细胞中TK水平有关，所以敲除了TK的JX594仅能在TK活性高的癌细胞中进行复制，不能在正常细胞中复制(正常细胞TK活性 低)。CG0070是在腺病毒主管复制的基因E1A前加了E2F-1启动子，E2F-1受视网膜母细胞瘤抑制蛋白(Rb)的调控，而Rb在膀胱癌中缺失，Rb的缺失激活E2F-1的转录活性，使得E1A基因表达，病毒可以特异性在膀胱癌中复制。Reolysin是未经改造的野生型呼肠孤病毒，其增值依赖Ras信号通路的激活，所以仅能在Ras激活的癌细胞中特异性增值。Because most cancer cells' own mechanisms for virus clearance are impaired (eg, protein kinase R (PKR), a key factor for virus clearance in normal cells is missing in cancer cells), viruses are more likely to replicate and spread in cancer cells. In addition, in recent decades, with the deepening of research, scientists have used the differences in many signaling pathways and metabolisms between cancer cells and normal cells to continuously improve the ability of oncolytic viruses by screening specific virus species and modifying virus genomes. The targeting of tumors can reduce the harm to normal cells and improve safety. For example: the approved T-vec knocks out the γ34.5 gene of HSV-1 (herpes simplex virus type 1). The γ34.5 gene can inhibit the clearance mechanism of the virus in normal cells. After the γ34.5 gene is knocked out, the virus It cannot replicate in normal cells; while this mechanism is missing in cancer cells, knockout of γ34.5 does not affect the replication of the virus in cancer cells. JX594 (Pexa-Vec), currently in phase III clinical trials, has knocked out the TK (thymidine kinase) gene of vaccinia viruses, and the replication of the virus is related to the level of TK in cells, so JX594 knocked out TK is only It can replicate in cancer cells with high TK activity, but cannot replicate in normal cells (normal cells with low TK activity). CG0070 adds the E2F-1 promoter before the adenovirus replication-competent gene E1A. E2F-1 is regulated by retinoblastoma inhibitory protein (Rb), and Rb is deleted in bladder cancer, and the loss of Rb activates E2F-1 The transcriptional activity of the E1A gene allows the virus to replicate specifically in bladder cancer. Reolysin is an unmodified wild-type reovirus whose proliferation depends on the activation of the Ras signaling pathway, so it can only specifically proliferate in Ras-activated cancer cells.
然而，目前的肿瘤治疗现状是，仍缺乏一种既能提高肿瘤杀伤特异性(即，相对于正常的非肿瘤细胞特异性高)、又能提高肿瘤治疗广谱性(即，同时适用于多种肿瘤治疗)的治疗手段。因此，本领域迫切需要开发一种同时具有较高的肿瘤细胞特异性和肿瘤治疗广谱性的杀伤肿瘤细胞的方法。However, the current status of tumor therapy is that there is still a lack of a method that can not only improve the specificity of tumor killing (ie, high specificity relative to normal non-tumor cells), but also improve the broad spectrum of tumor therapy (ie, is suitable for multiple cancer treatment). Therefore, there is an urgent need in the art to develop a method for killing tumor cells that has both high tumor cell specificity and broad spectrum of tumor therapy.
虽然VSV发现的时间比较久，并且VSV具有广谱的细胞侵染性，但是在很长的一段时间内都没有找到VSV的受体，甚至有研究者认为其受体为细胞膜上的脂质成分，如磷脂丝氨酸、磷脂酰肌醇、神经节苷脂GM3等(参考文献：Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH，Mastromarino et al.,J Gen Virol.1987 Sep；68(Pt 9):2359-69))。随着研究的深入，遗传学、生物化学和免疫化学结果表明，VSV-G是VSV与其假定受体结合、内吞以及与靶细胞膜融合所必需的，结合后，VSV经过网格蛋白介导的内吞作用进入细胞(参考文献：Techniques for dual staining of DNA and intracellular immunoglobulins in murine hybridoma cells:applications to cell-cycle analysis of hyperosmotic cultures.Sun et al.,Cytotechnology.2005 Jun；48(1-3):15-26.)，这表明一定存在细胞膜上VSV的特异性受体，直至2013年Finkelshtein等人才鉴定出LDLR作为VSV进入细胞的主要受体(参考文献：LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus.Finkelshtein et al.Proc Natl Acad Sci U S A.2013Apr 30；110(18):7306-11)。LDLR基因敲除的细胞依然可以感染VSV，表明有其他的受体存在可以介导VSV的内吞作用。之前的研究表明虽然VSV可以感染大部分的哺乳动物细胞，但是VSV对肿瘤细胞有更高的偏好性，表明肿瘤细胞可能存在高表达的VSV受体。Although VSV has been discovered for a long time, and VSV has a broad spectrum of cell invasiveness, the receptor for VSV has not been found for a long period of time, and some researchers even believe that its receptor is a lipid component on the cell membrane , such as phospholipid serine, phosphatidylinositol, ganglioside GM3, etc. (Reference: Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH, Mastromarino et al., J Gen Virol.1987 Sep ; 68(Pt 9):2359-69)). With further research, genetic, biochemical and immunochemical results have shown that VSV-G is required for VSV binding to its putative receptor, endocytosis, and fusion with target cell membranes. After binding, VSV undergoes clathrin-mediated Endocytosis into cells (Reference: Techniques for dual staining of DNA and intracellular immunoglobulins in murine hybridoma cells: applications to cell-cycle analysis of hyperosmotic cultures. Sun et al., Cytotechnology. 2005 Jun; 48(1-3): 15-26.), which indicates that there must be a specific receptor for VSV on the cell membrane. Until 2013, Finkelshtein and others identified LDLR as the main receptor for VSV to enter cells (Reference: LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Finkelshtein et al. Proc Natl Acad Sci U S A. 2013 Apr 30;110(18):7306-11). LDLR knockout cells were still able to infect VSV, suggesting that there are other receptors that mediate VSV endocytosis. Previous studies have shown that although VSV can infect most mammalian cells, VSV has a higher preference for tumor cells, suggesting that tumor cells may have highly expressed VSV receptors.
在使用VSV作为溶瘤病毒进行肿瘤治疗时，特异性受体的存在可以增强VSV的靶向性。那么如何找到这些特异性受体就成为了事情关键。随着测序技术的不断进步，以及生物信息学的发展，可以获取的癌症病人的公共测序数据可以帮助我们进行初级的筛查。The presence of specific receptors can enhance the targeting of VSV when using VSV as an oncolytic virus for tumor therapy. So how to find these specific receptors becomes the key. With the continuous advancement of sequencing technology and the development of bioinformatics, the availability of public sequencing data of cancer patients can help us conduct primary screening.
发明内容SUMMARY OF THE INVENTION
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。The present invention aims to solve one of the technical problems in the related art at least to a certain extent.
在本发明的第一方面，本发明提出了蛋白在构建水疱性口炎病毒中的用途。根据本发明的实施例，所述蛋白为肿瘤细胞表面特异性受体，包括选自CHRNA5、SSTR5、KISS1R、 HTR1D和CCR8的至少之一。针对本申请的发明人所筛选的肿瘤细胞表面特异性受体所构建的水疱性口炎(VSV病毒)与肿瘤细胞的特异性结合能力更强，对肿瘤细胞的特异性杀伤效果更加显著。In the first aspect of the present invention, the present invention proposes the use of the protein in the construction of vesicular stomatitis virus. According to an embodiment of the present invention, the protein is a tumor cell surface specific receptor, including at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8. The vesicular stomatitis (VSV virus) constructed by the specific receptors on the surface of tumor cells screened by the inventors of the present application has stronger specific binding ability to tumor cells, and has a more significant specific killing effect on tumor cells.
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
根据本发明的实施例，所述VSV病毒的G蛋白与所述肿瘤细胞表面特异性受体的结合力的ZDOCK score不小于1800，例如不小于1900，不小于2000，优选不小于2100。本领域技术人员能够理解，通过输入G蛋白和细胞受体的序列，可以容易地获得G蛋白和细胞受体之间的结合力表征参数ZDOCK score。发明人发现，当ZDOCK score不小于1800时，携带G蛋白的病毒与携带对应受体的肿瘤细胞之间的结合力将显著提高。根据本发明的实施例，ZDOCK score是可以常规软件确定的，例如参见Pierce BG,Hourai Y,Weng Z.(2011)Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library.PLoS One 6(9):e24657。According to an embodiment of the present invention, the ZDOCK score of the binding force between the G protein of the VSV virus and the tumor cell surface specific receptor is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100. Those skilled in the art can understand that by inputting the sequences of G proteins and cellular receptors, the ZDOCK score, a parameter that characterizes the binding force between G proteins and cellular receptors, can be easily obtained. The inventors found that when the ZDOCK score is not less than 1800, the binding force between the virus carrying the G protein and the tumor cells carrying the corresponding receptor will be significantly improved. According to an embodiment of the present invention, the ZDOCK score can be determined by conventional software, for example, see Pierce BG, Houurai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657 .
根据本发明的实施例，所述G蛋白与受体LDLR的ZDOCK score不小于1600，优选不低于1650，更优选不小于1659，例如1659.078。本申请的发明人所筛选的肿瘤细胞表面特异性受体与G蛋白的结合力远高于LDLR与G蛋白的结合力。According to an embodiment of the present invention, the ZDOCK score of the G protein and the receptor LDLR is not less than 1600, preferably not less than 1650, more preferably not less than 1659, such as 1659.078. The binding force of the tumor cell surface specific receptors screened by the inventors of the present application to G protein is much higher than the binding force of LDLR to G protein.
根据本发明的实施例，所述水疱性口炎病毒为重组水疱性口炎病毒。According to an embodiment of the present invention, the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.
根据本发明的具体实施例，所述VSV病毒不携带异源基因。根据本发明的实施例，在本文中所描述的术语“异源基因”如无特别说明，是指在野生型水疱性口炎病毒中未曾报道过的基因。或者换句话说，在重组水疱性口炎病毒中所编码的蛋白均为野生型水疱性口炎病毒中表达的。According to a specific embodiment of the present invention, the VSV virus does not carry heterologous genes. According to the embodiments of the present invention, the term "heterologous gene" described herein refers to a gene that has not been reported in wild-type vesicular stomatitis virus unless otherwise specified. Or in other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in the wild-type vesicular stomatitis virus.
根据本发明的实施例，所述肿瘤细胞包括选自肠癌、肺癌、胰腺癌、胃癌、肝癌、食管癌、乳腺癌、胆管癌、黑色素瘤细胞的至少之一。According to an embodiment of the present invention, the tumor cells include at least one selected from colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
根据本发明的实施例，所述水疱性口炎病毒包括选自印第安纳株血清型及新泽西株血清型的至少之一。According to an embodiment of the present invention, the vesicular stomatitis virus comprises at least one selected from the Indiana strain serotype and the New Jersey strain serotype.
在本发明的第二方面，本发明提出了一种水疱性口炎病毒(VSV病毒)。根据本发明的实施例，所述水疱性口炎病毒表达G蛋白，所述G蛋白适于与肿瘤细胞表面特异性受体结合，可选的，所述肿瘤细胞表面特异性受体包括选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的至少之一。根据本发明实施例的水疱性口炎病毒(VSV病毒)与肿瘤细胞的特异性结合能力更强，对肿瘤细胞的特异性杀伤效果更加显著。In a second aspect of the present invention, the present invention provides a vesicular stomatitis virus (VSV virus). According to an embodiment of the present invention, the vesicular stomatitis virus expresses a G protein, and the G protein is suitable for binding to a tumor cell surface specific receptor, optionally, the tumor cell surface specific receptor includes a group selected from At least one of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8. The vesicular stomatitis virus (VSV virus) according to the embodiment of the present invention has stronger specific binding ability to tumor cells, and has a more significant specific killing effect on tumor cells.
根据本发明的实施例，上述VSV病毒还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned VSV virus may further include at least one of the following additional technical features:
根据本发明的实施例，所述VSV病毒的G糖蛋白与所述肿瘤细胞表面特异性受体的结合力的ZDOCK score不小于1800，例如不小于1900，不小于2000，优选不小于2100。本 领域技术人员能够理解，通过输入G蛋白和细胞受体的序列，可以容易地获得G蛋白和细胞受体之间的结合力表征参数ZDOCK score。发明人发现，当ZDOCK score不小于1800时，携带G蛋白的病毒与携带对应受体的肿瘤细胞之间的结合力将显著提高。根据本发明的实施例，ZDOCK score是可以常规软件确定的，例如参见Pierce BG,Hourai Y,Weng Z.(2011)Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library.PLoS One 6(9):e24657。According to an embodiment of the present invention, the ZDOCK score of the binding force of the G glycoprotein of the VSV virus to the specific receptor on the surface of the tumor cell is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100. Those skilled in the art can understand that by inputting the sequences of G proteins and cellular receptors, the ZDOCK score, a parameter that characterizes the binding force between G proteins and cellular receptors, can be easily obtained. The inventors found that when the ZDOCK score is not less than 1800, the binding force between the virus carrying the G protein and the tumor cells carrying the corresponding receptor will be significantly improved. According to an embodiment of the present invention, the ZDOCK score can be determined by conventional software, for example, see Pierce BG, Houurai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657 .
根据本发明的实施例，所述G蛋白与受体LDLR的ZDOCK score不小于1600，优选不低于1650，更优选不小于1659，例如1659.078。According to an embodiment of the present invention, the ZDOCK score of the G protein and the receptor LDLR is not less than 1600, preferably not less than 1650, more preferably not less than 1659, such as 1659.078.
根据本发明的实施例，所述水疱性口炎病毒为重组水疱性口炎病毒。According to an embodiment of the present invention, the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.
根据本发明的具体实施例，所述VSV病毒不携带异源基因。根据本发明的实施例，在本文中所描述的术语“异源基因”如无特别说明，是指在野生型水疱性口炎病毒中未曾报道过的基因。或者换句话说，在重组水疱性口炎病毒中所编码的蛋白均为野生型水疱性口炎病毒中表达的。According to a specific embodiment of the present invention, the VSV virus does not carry heterologous genes. According to the embodiments of the present invention, the term "heterologous gene" described herein refers to a gene that has not been reported in wild-type vesicular stomatitis virus unless otherwise specified. Or in other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in the wild-type vesicular stomatitis virus.
根据本发明的实施例，所述肿瘤细胞包括选自肠癌、肺癌、胰腺癌、胃癌、肝癌、食管癌、乳腺癌、胆管癌、黑色素瘤细胞的至少之一。According to an embodiment of the present invention, the tumor cells include at least one selected from colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
根据本发明的实施例，所述水疱性口炎病毒包括选自印第安纳株血清型及新泽西株血清型的至少之一。According to an embodiment of the present invention, the vesicular stomatitis virus comprises at least one selected from the Indiana strain serotype and the New Jersey strain serotype.
在本发明的第三方面，本发明提出了前面所述的水疱性口炎病毒在制备药物中的用途，所述药物用于治疗或预防肿瘤。In the third aspect of the present invention, the present invention proposes the use of the aforementioned vesicular stomatitis virus in preparing a medicament for treating or preventing tumors.
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
根据本发明的实施例，所述肿瘤的细胞携带肿瘤细胞表面特异性受体，所述肿瘤细胞表面特异性受体包括选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的至少之一。According to an embodiment of the present invention, the tumor cells carry tumor cell surface specific receptors, and the tumor cell surface specific receptors include at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.
根据本发明的实施例，所述肿瘤细胞表面特异性受体在所述肿瘤中具有不小于2.0的表达差异倍数(log2FC)。According to an embodiment of the present invention, the tumor cell surface specific receptor has an expression fold difference (log2FC) of not less than 2.0 in the tumor.
在本发明的第四方面，本肿瘤细胞表面特异性受体在筛选用于治疗或预防肿瘤的VSV病毒中的用途，所述肿瘤细胞表面特异性受体选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的一种或多种。In the fourth aspect of the present invention, the use of the present tumor cell surface specific receptor in screening VSV virus for the treatment or prevention of tumors, the tumor cell surface specific receptor is selected from CHRNA5, SSTR5, KISS1R, HTR1D and One or more of CCR8.
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
根据本发明的实施例，所述VSV病毒的G糖蛋白与待治疗的肿瘤中表达差异倍数(log2FC)大于等于2.0的所述肿瘤细胞表面特异性受体的结合力的ZDOCK score不小于1800，例如不小于1900，不小于2000，优选不小于2100是待筛选病毒为目标病毒的指示。According to an embodiment of the present invention, the ZDOCK score of the binding force of the G glycoprotein of the VSV virus to the tumor cell surface specific receptor whose expression fold difference (log2FC) is greater than or equal to 2.0 in the tumor to be treated is not less than 1800, For example, not less than 1900, not less than 2000, preferably not less than 2100 is an indication that the virus to be screened is the target virus.
根据本发明的实施例，所述VSV病毒为重组VSV病毒；优选地，所述VSV病毒不携 带异源基因。According to an embodiment of the present invention, the VSV virus is a recombinant VSV virus; preferably, the VSV virus does not carry a heterologous gene.
在本发明的第五方面，本发明提出了一种筛选可用于水疱性口炎病毒治疗的受试者的方法。根据本发明的实施例，确定所述受试者肿瘤中细胞表面受体表达水平，所述细胞表面受体选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的一种或多种。根据本发明实施例的方法所筛选出受试者更加适于接受水疱性口炎病毒治疗，疗效会更加显著。In a fifth aspect of the present invention, the present invention proposes a method of screening subjects for vesicular stomatitis virus treatment. According to an embodiment of the present invention, the expression level of a cell surface receptor selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 is determined in the tumor of the subject. The subjects screened according to the methods of the embodiments of the present invention are more suitable for receiving vesicular stomatitis virus treatment, and the curative effect will be more significant.
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的实施例，所述细胞表面受体在所述待肿瘤中具有不小于2.0的表达差异倍数(log2FC)是所述受试者适于接受水疱性口炎病毒治疗的指示。According to an embodiment of the present invention, the expression fold difference (log2FC) of the cell surface receptor in the tumor to be treated is not less than 2.0 is an indication that the subject is suitable for receiving vesicular stomatitis virus therapy.
根据本发明的实施例，所述水疱性口炎病毒前面所述的水疱性口炎病毒。According to an embodiment of the present invention, the vesicular stomatitis virus is the aforementioned vesicular stomatitis virus.
在本发明的第六方面，本发明提出了一种预测VSV病毒溶瘤效果的方法。根据本发明的实施例，所述方法包括：确定待测VSV病毒的G糖蛋白与肿瘤细胞的细胞表面受体的结合力，所述细胞表面受体选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的一种或多种。In the sixth aspect of the present invention, the present invention provides a method for predicting the oncolytic effect of VSV virus. According to an embodiment of the present invention, the method includes: determining the binding capacity of the G glycoprotein of the VSV virus to be tested to a cell surface receptor of tumor cells, the cell surface receptor is selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 one or more of.
根据本发明的实施例，上述预测VSV病毒溶瘤效果的方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned method for predicting the oncolytic effect of VSV virus may further include at least one of the following additional technical features:
根据本发明的实施例，所述VSV病毒的G糖蛋白与所述细胞表面受体的结合力的ZDOCK score不小于1800，例如不小于1900，不小于2000，优选不小于2100是待测VSV病毒溶瘤效果佳的指示。According to an embodiment of the present invention, the ZDOCK score of the binding force between the G glycoprotein of the VSV virus and the cell surface receptor is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100 is the VSV virus to be tested Indication of good oncolytic effect.
在本发明的第七方面，本发明提出了一种治疗或预防肿瘤的方法。根据本发明的实施例，包括向受试者施用第二方面所述的水疱性口炎病毒。根据本发明实施例的方法可使得受试者具有有效预防肿瘤或抑制肿瘤细胞生长的能力。In the seventh aspect of the present invention, the present invention provides a method for treating or preventing tumors. According to an embodiment of the present invention, comprising administering the vesicular stomatitis virus of the second aspect to a subject. The methods according to the embodiments of the present invention can provide a subject with the ability to effectively prevent tumors or inhibit the growth of tumor cells.
根据本发明的实施例，上述治疗或预防肿瘤的方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned method for treating or preventing tumors may further include at least one of the following additional technical features:
根据本发明的实施例，所述肿瘤包括选自肠癌、肺癌、胰腺癌、胃癌、肝癌、食管癌、乳腺癌、胆管癌、黑色素瘤细胞的至少之一。According to an embodiment of the present invention, the tumor comprises at least one selected from the group consisting of colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the present invention will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the invention.
附图说明Description of drawings
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of embodiments taken in conjunction with the accompanying drawings, wherein:
图1显示了基于肿瘤组织大样本的人类膜受体基因的分析流程图。Figure 1 shows a flowchart of the analysis of human membrane receptor genes based on a large sample of tumor tissue.
图2显示了对应的受体基因在各肿瘤中显著上调的病人比例的抖动散点图。Figure 2 shows a jittered scatterplot of the proportion of patients with the corresponding receptor gene significantly up-regulated in each tumor.
图3显示了反映16个候选配体分别与5个肿瘤特异性受体结合强度的ZDOCK score结果。Figure 3 shows the ZDOCK score results reflecting the binding strength of 16 candidate ligands to 5 tumor-specific receptors, respectively.
图4显示了筛选出的配体与5个肿瘤特异性受体结合强度的ZDOCK score结果。Figure 4 shows the ZDOCK score results of the binding strength of the screened ligands to 5 tumor-specific receptors.
图5显示了qPCR检测得到的BXPC3、HCT-8、HepG2、Su8686、H358和PANC1细胞样品中的CHRNA5、KISS1R、HTRID、CCR8和SSTR5的mRNA的表达量。Figure 5 shows the mRNA expression levels of CHRNA5, KISS1R, HTRID, CCR8 and SSTR5 in BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cell samples detected by qPCR.
图6显示了细胞杀伤实验中所测得的病毒在不同的MOI情况下对BXPC3、HCT-8、HepG2、Su8686、H358和PANC1细胞的杀伤效果。Figure 6 shows the killing effect of viruses on BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells at different MOIs measured in cell killing experiments.
图7显示了具有不同G蛋白的病毒株在不同的MOI情况下对NCL-H358和NCL-H460细胞的杀伤效果。Figure 7 shows the killing effect of virus strains with different G proteins on NCL-H358 and NCL-H460 cells at different MOIs.
图8显示了体外敲减实验中，对CCR8、CHRNA5、KISS1R、HTR1D、SSTR5分别进行基因敲减之后，对细胞内VSV病毒复制的影响。Figure 8 shows the effects of gene knockdown on CCR8, CHRNA5, KISS1R, HTR1D, and SSTR5 on the intracellular VSV virus replication in the in vitro knockdown experiment.
发明详细描述Detailed description of the invention
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention and should not be construed as limiting the present invention.
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms "first" and "second" are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with "first", "second" may expressly or implicitly include at least one of that feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
水疱性口炎病毒vesicular stomatitis virus
在本发明的第一方面，本发明提出了一种水疱性口炎病毒。根据本发明的实施例，所述水疱性口炎病毒表达G蛋白，所述G蛋白适于与肿瘤细胞表面特异性受体结合，可选的，所述肿瘤细胞表面特异性受体包括选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的至少之一。需要说明的是，本申请所述的“肿瘤细胞表面特异性受体”是指肿瘤细胞表面受体相对于正常细胞表面受体具有显著性上调/下调的差异表达的受体，其中，表达差异倍数(log2FC)大于等于2.0被视为有显著性上调差异，t检验的p值小于0.01被判定为差异有统计学意义。根据本发明实施例的水疱性口炎病毒(VSV病毒)与肿瘤细胞的特异性结合能力更强，对肿瘤细胞的特异性杀伤效果更加显著。In the first aspect of the present invention, the present invention provides a vesicular stomatitis virus. According to an embodiment of the present invention, the vesicular stomatitis virus expresses a G protein, and the G protein is suitable for binding to a tumor cell surface specific receptor, optionally, the tumor cell surface specific receptor includes a group selected from At least one of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8. It should be noted that the "specific receptors on the surface of tumor cells" mentioned in this application refer to receptors with significant up-regulation/down-regulation of differentially expressed receptors on the surface of tumor cells relative to receptors on the surface of normal cells, wherein the expression difference A fold (log2FC) greater than or equal to 2.0 was considered a significant up-regulation difference, and a p-value of t-test less than 0.01 was considered a statistically significant difference. The vesicular stomatitis virus (VSV virus) according to the embodiment of the present invention has stronger specific binding ability to tumor cells, and has a more significant specific killing effect on tumor cells.
水疱性口炎病毒(Vesicular stomatitis Virus,VSV)属于弹状病毒科水疱病毒属的病毒，分为两个血清型:新泽西型(VSV-NJ)和印第安那型(VSV-IND)。病毒粒子为子弹状或圆柱状，大小为150～180nm×50～70nm。病毒有囊膜，囊膜上均匀密布有长约10nm的纤突。病毒内部为紧密盘旋的螺旋对称的核衣壳。该病毒根据患病动物的口腔粘膜， 牙垫，舌头，嘴唇，鼻孔，蹄和乳头中的经典水疱性病变命名。通过昆虫媒介传播，疾病仅限于其自然宿主，例如马，牛和猪。在人类中，感染是轻度且无症状的。Vesicular stomatitis virus (VSV) belongs to the Rhabdoviridae family of vesicular viruses and is divided into two serotypes: New Jersey (VSV-NJ) and Indiana (VSV-IND). Virus particles are bullet-shaped or cylindrical, with a size of 150-180 nm × 50-70 nm. The virus has an envelope, and the envelope is evenly covered with fibers about 10nm long. Inside the virus is a tightly coiled helix-symmetric nucleocapsid. The virus is named after the classic vesicular lesions in the oral mucosa, tooth pads, tongue, lips, nostrils, hooves and nipples of sick animals. Spread by insect vectors, the disease is limited to its natural hosts such as horses, cattle and pigs. In humans, the infection is mild and asymptomatic.
VSV基因组为不分节段的单股负链RNA(ssRNA)病毒，长度约为11KB。从3’端→5’端依次排列着N、NS、M、G、L五个不重叠的基因，分别编码核(N)蛋白、磷酸(P)蛋白、基质(M)蛋白、糖(G)蛋白、及RNA依赖的RNA聚合酶(L)蛋白等5种不同的主要蛋白。N基因的3’端是先导序列(Leader)，5’端是拖尾序列(Trailor)，各基因间有间隔序列。3’端先导RNA在感染细胞中是最早的病毒转录物，长度为47个核苷酸，不戴帽也不翻译，其功能尚未完全清楚，可能是抑制宿主RNA的合成。N蛋白是启动基因组合成所必需的，可有效的保护病毒RNA免受各种核酸酶的消化。N蛋白有高的免疫原性，刺激机体产生细胞免疫，且在转录复制中担任了重要的角色，它对维持基因组RNA呈伸展形式可能是必要的，与复制调节有关。P蛋白，VSV-NJ与VSV-IND病毒株的同源性为41％，其作用是与聚合酶L，核蛋白N组成聚合酶复合物以及基因组RNA共同维持病毒的转录活性。M蛋白在病毒致病机制和病毒复制方面起关键作用，富含碱性氨基酸，并含有高度碱性的氨基末端结构域，可通过与核衣壳结合而抑制转录，同时辅助病毒从宿主中出芽，是涉及出芽过程的惟一多肽。G蛋白是病毒的主要表面抗原，决定着病毒的毒力，也是病毒的保护性抗原。它可刺激机体产生中和抗体。L基因编码RNA poly E蛋白，它可能决定RNA的转录活性，与P蛋白结合以催化mRNA的复制。该蛋白是聚合酶复合物和复制酶复合物的核心成分，涉及起始、延伸、甲基化、戴帽、聚(A)尾形成等等。此外，在每个基因之间的间隔序列有广泛的同源性，这些序列有一个共同的结构，即3’-AUAC(U)7NAUUGUCNN-UAG-5’。这些基因之间的保守序列是一种关键信号，以影响多聚酶的活性或酶的切割活性，而在复制过程中，这些信号被掩盖，不起作用。The VSV genome is an unsegmented single-stranded negative-stranded RNA (ssRNA) virus with a length of approximately 11 KB. From the 3' end to the 5' end, there are five non-overlapping genes, N, NS, M, G, and L, which encode nuclear (N) protein, phosphate (P) protein, matrix (M) protein, sugar (G) protein, respectively. ) protein, and five different major proteins including RNA-dependent RNA polymerase (L) protein. The 3' end of the N gene is a leader sequence (Leader), the 5' end is a trailing sequence (Trailor), and there is a spacer sequence between each gene. The 3'-end leader RNA is the earliest viral transcript in infected cells. It is 47 nucleotides in length and is neither capped nor translated. Its function is not fully understood, but it may inhibit host RNA synthesis. The N protein is required to initiate the synthesis of the genome and can effectively protect the viral RNA from digestion by various nucleases. N protein has high immunogenicity, stimulates the body to produce cellular immunity, and plays an important role in transcriptional replication. It may be necessary to maintain the extended form of genomic RNA and is related to replication regulation. The P protein, VSV-NJ is 41% homologous to the VSV-IND strain, and its role is to form a polymerase complex with polymerase L, nucleoprotein N and genomic RNA to maintain the transcriptional activity of the virus. The M protein plays a key role in viral pathogenesis and viral replication, is rich in basic amino acids, and contains a highly basic amino-terminal domain that inhibits transcription by binding to the nucleocapsid while assisting virus budding from the host , is the only polypeptide involved in the budding process. G protein is the main surface antigen of the virus, which determines the virulence of the virus and is also the protective antigen of the virus. It stimulates the body to produce neutralizing antibodies. The L gene encodes the RNA poly E protein, which may determine the transcriptional activity of RNA, and binds to the P protein to catalyze the replication of mRNA. This protein is a core component of the polymerase complex and replicase complex and is involved in initiation, elongation, methylation, capping, poly(A) tail formation, and more. In addition, there is extensive homology in the spacer sequences between each gene, and these sequences share a common structure, namely 3'-AUAC(U)7NAUUGUCNN-UAG-5'. The conserved sequence between these genes is a key signal to affect the activity of the polymerase or the cleavage activity of the enzyme, and during replication, these signals are masked and not functional.
根据本发明的实施例，所述VSV病毒的G糖蛋白与所述肿瘤细胞表面特异性受体的结合力的ZDOCK score不小于1800，例如不小于1900，不小于2000，优选不小于2100。本申请所述的水疱性口炎病毒与肿瘤细胞表面特异性受体的结合力通过ZDOCK score函数来评价。生成的对接的结果会按照ZDOCK score分数进行排序，得分越高，则说明结合越强，结果可信度越高。同时还有这些构象的聚类结果。ZDOCK分数是ZDOCK程序计算的形状互补分数，根据参数设置，ZDOCK分数还将包括静电和去溶剂能项，ZDOCK分数越高越好。According to an embodiment of the present invention, the ZDOCK score of the binding force of the G glycoprotein of the VSV virus to the specific receptor on the surface of the tumor cell is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100. The binding ability of the vesicular stomatitis virus described in the present application to specific receptors on the surface of tumor cells was evaluated by ZDOCK score function. The generated docking results will be sorted according to the ZDOCK score. The higher the score, the stronger the combination and the higher the reliability of the result. There are also clustering results for these conformations. The ZDOCK score is the shape complementation score calculated by the ZDOCK program. According to the parameter settings, the ZDOCK score will also include electrostatic and desolvation energy terms. The higher the ZDOCK score, the better.
本领域技术人员能够理解，通过输入G蛋白和细胞受体的序列，可以容易地获得G蛋白和细胞受体之间的结合力表征参数ZDOCK score。发明人发现，当ZDOCK score不低于1800，例如不小于1900，不小于2000，优选不小于2100时，携带G蛋白的病 毒与携带对应受体的肿瘤细胞之间的结合力将显著提高。根据本发明的实施例，ZDOCK score是可以常规软件确定的，例如参见Pierce BG,Hourai Y,Weng Z.(2011)Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library.PLoS One 6(9):e24657。Those skilled in the art can understand that by inputting the sequences of G proteins and cellular receptors, the ZDOCK score, a parameter that characterizes the binding force between G proteins and cellular receptors, can be easily obtained. The inventors found that when the ZDOCK score is not less than 1800, such as not less than 1900, not less than 2000, preferably not less than 2100, the binding force between the virus carrying the G protein and the tumor cell carrying the corresponding receptor will be significantly improved. According to an embodiment of the present invention, the ZDOCK score can be determined by conventional software, for example, see Pierce BG, Houurai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657 .
根据本发明的实施例，所述G蛋白与受体LDLR的ZDOCK score不小于1600，优选不低于1650，更优选不小于1659，或为1650～1660，例如1659.078。LDLR是本领域已知的水疱性口炎病毒入胞受体，发明人将LDLR作为阳性对照，发现本申请所筛选出的CHRNA5、SSTR5、KISS1R、HTR1D和CCR8与病毒的结合力远高于LDLR。According to an embodiment of the present invention, the ZDOCK score of the G protein and the receptor LDLR is not less than 1600, preferably not less than 1650, more preferably not less than 1659, or 1650-1660, for example 1659.078. LDLR is a vesicular stomatitis virus entry receptor known in the art. The inventors used LDLR as a positive control and found that the binding capacity of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 screened in this application to the virus is much higher than that of LDLR .
根据本发明的实施例，所述水疱性口炎病毒为重组水疱性口炎病毒。According to an embodiment of the present invention, the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.
根据本发明的具体实施例，所述VSV病毒不携带异源基因。根据本发明的实施例，在本文中所描述的术语“异源基因”如无特别说明，是指在野生型水疱性口炎病毒中未曾报道过的基因。或者换句话说，在重组水疱性口炎病毒中所编码的蛋白均为野生型水疱性口炎病毒中表达的。发明人发现，不携带异源基因的重组水疱性口炎病毒对肿瘤细胞的杀伤效果显著高于携带异源基因的重组水疱性口炎病毒。According to a specific embodiment of the present invention, the VSV virus does not carry heterologous genes. According to the embodiments of the present invention, the term "heterologous gene" described herein refers to a gene that has not been reported in wild-type vesicular stomatitis virus unless otherwise specified. Or in other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in the wild-type vesicular stomatitis virus. The inventors found that the killing effect of recombinant vesicular stomatitis virus without heterologous gene on tumor cells is significantly higher than that of recombinant vesicular stomatitis virus carrying heterologous gene.
根据本发明的实施例，所述肿瘤细胞包括选自肠癌、肺癌、胰腺癌、胃癌、肝癌、食管癌、乳腺癌、胆管癌、黑色素瘤细胞的至少之一。According to an embodiment of the present invention, the tumor cells include at least one selected from colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
根据本发明的实施例，所述水疱性口炎病毒包括选自印第安纳株血清型及新泽西株血清型的至少之一。According to an embodiment of the present invention, the vesicular stomatitis virus comprises at least one selected from the Indiana strain serotype and the New Jersey strain serotype.
制药用途Pharmaceutical use
在本发明的第二方面，本发明提出了前面所述的水疱性口炎病毒在制备药物中的用途，所述药物用于治疗或预防肿瘤。根据本发明实施例的病毒所制备的药物，在治疗或预防肿瘤方面，具有更加显著的效果。In the second aspect of the present invention, the present invention proposes the use of the aforementioned vesicular stomatitis virus in preparing a medicament for treating or preventing tumors. The medicament prepared from the virus according to the embodiment of the present invention has a more significant effect in treating or preventing tumors.
根据本发明的实施例，所述肿瘤的细胞携带肿瘤细胞表面特异性受体，所述肿瘤细胞表面特异性受体包括选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的至少之一。根据本发明的实施例，所述肿瘤细胞表面特异性受体在所述肿瘤中具有不小于2.0的表达差异倍数(log2FC)。发明人发现，CHRNA5、SSTR5、KISS1R、HTR1D和CCR8这5种肿瘤特异性受体在5个癌种的差异基因表达在(log2FC)大于2的占比远大于LDLR。According to an embodiment of the present invention, the tumor cells carry tumor cell surface specific receptors, and the tumor cell surface specific receptors include at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8. According to an embodiment of the present invention, the tumor cell surface specific receptor has an expression fold difference (log2FC) of not less than 2.0 in the tumor. The inventors found that the proportion of differential gene expression (log2FC) greater than 2 of 5 tumor-specific receptors CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 in 5 cancer types is much larger than that of LDLR.
根据本发明的实施例，所述癌症包括肺癌，胃癌，肝癌，肠癌，食管癌，乳腺癌，宫颈癌，恶性淋巴瘤，鼻咽癌，白血病的至少之一。According to an embodiment of the present invention, the cancer includes at least one of lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, and leukemia.
进一步地，本发明提出了药物组合物。根据本发明的实施例，所述药物组合物含有：前面所述的重组水疱性口炎病毒。Further, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition contains: the aforementioned recombinant vesicular stomatitis virus.
根据本发明的实施例，所述药物组合物呈适于吸入或者注射方式给药的形式。According to an embodiment of the present invention, the pharmaceutical composition is in a form suitable for administration by inhalation or injection.
本发明提供的药物组合物，包括药学上可接受的载体和有效量的以下活性成分：本发明的特异性感染肿瘤细胞的重组VSV病毒。The pharmaceutical composition provided by the present invention comprises a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: the recombinant VSV virus specifically infecting tumor cells of the present invention.
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。As used herein, the term "effective amount" or "effective dose" refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. Usually the pharmaceutical preparation should match the mode of administration, and the dosage form of the pharmaceutical composition of the present invention is an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, and a sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions.
在一个实施方式中，本发明的药物组合物中还可以包括额外的肿瘤治疗剂。In one embodiment, additional tumor therapeutic agents may also be included in the pharmaceutical compositions of the present invention.
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the active ingredient of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the administration way etc. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.
本发明所述的药学上可接受的载体包括(但不限于)：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。The pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or its combination. The choice of carrier should match the mode of administration, as is well known to those of ordinary skill in the art.
筛选用于治疗或预防肿瘤的VSV病毒中的用途Use in screening VSV virus for the treatment or prevention of tumors
在本发明的第三方面，肿瘤细胞表面特异性受体在筛选用于治疗或预防肿瘤的VSV病毒中的用途，所述肿瘤细胞表面特异性受体选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的一种或多种。In a third aspect of the present invention, the use of a tumor cell surface specific receptor selected from the group consisting of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 in screening for VSV virus for the treatment or prevention of tumors one or more of.
根据本发明的实施例，所述VSV病毒的G糖蛋白与待治疗的肿瘤中表达差异倍数(log2FC)大于等于2.0的所述肿瘤细胞表面特异性受体的结合力的ZDOCK score不小于1800，例如不小于1900，不小于2000，优选不小于2100是待筛选病毒为目标病毒的指示。According to an embodiment of the present invention, the ZDOCK score of the binding ability of the G glycoprotein of the VSV virus to the tumor cell surface specific receptor whose expression fold difference (log2FC) is greater than or equal to 2.0 in the tumor to be treated is not less than 1800, For example, not less than 1900, not less than 2000, preferably not less than 2100 is an indication that the virus to be screened is the target virus.
根据本发明实施例的方法所筛选出的用于治疗或预防肿瘤的VSV病毒对肿瘤的特异性靶向和亲和力更强，杀伤效果更加显著。The VSV virus screened for treating or preventing tumors according to the methods of the embodiments of the present invention has stronger specific targeting and affinity for tumors, and has a more significant killing effect.
根据本发明的具体实施例，所述待治疗的肿瘤为肺腺癌或肺鳞癌，所述肿瘤细胞表面特异性受体选自CHRNA5、SSTR5和KISS1R的至少之一。根据本发明的具体实施例，所述待治疗的肿瘤为结肠癌，所述肿瘤细胞表面特异性受体选自HTR1D、SSTR5和KISS1R的至少之一。根据本发明的具体实施例，所述待治疗的肿瘤为肝癌，所述肿瘤细胞表面特异性受体选自HTR1D、SSTR5、CCR8和KISS1R的至少之一。根据本发明的具体实施例，所述待治疗的肿瘤为胃癌，所述肿瘤细胞表面特异性受体选自HTR1D和CCR8的至少之一。所述待治疗的肿瘤为胰腺癌，所述肿瘤细胞表面特异性受体选自CHRNA5、SSTR5和HTR1D的至少之一。发明人发现，CHRNA5、SSTR5、KISS1R、HTR1D和CCR8受体在不同肿瘤细胞与正常细胞上的表达差异存在不同，发明人根据差异的大小，确定了在不同肿瘤细胞中差异表达的不同受体，进而利用在不同肿瘤细胞中特异性差异表达的受体，更有目的地筛选用于治疗或预防特异性肿瘤的VSV病毒。According to a specific embodiment of the present invention, the tumor to be treated is lung adenocarcinoma or lung squamous cell carcinoma, and the tumor cell surface specific receptor is selected from at least one of CHRNA5, SSTR5 and KISS1R. According to a specific embodiment of the present invention, the tumor to be treated is colon cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D, SSTR5 and KISS1R. According to a specific embodiment of the present invention, the tumor to be treated is liver cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D, SSTR5, CCR8 and KISS1R. According to a specific embodiment of the present invention, the tumor to be treated is gastric cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D and CCR8. The tumor to be treated is pancreatic cancer, and the tumor cell surface specific receptor is selected from at least one of CHRNA5, SSTR5 and HTR1D. The inventors found that the expression differences of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 receptors on different tumor cells and normal cells are different. Furthermore, the receptors that are specifically differentially expressed in different tumor cells are used to screen VSV viruses for the treatment or prevention of specific tumors more purposefully.
根据本发明的实施例，所述VSV病毒为重组VSV病毒；优选地，所述VSV病毒不携带异源基因。进而重组VSV病毒对肿瘤的杀伤效果进一步提高。According to an embodiment of the present invention, the VSV virus is a recombinant VSV virus; preferably, the VSV virus does not carry a heterologous gene. Furthermore, the killing effect of recombinant VSV virus on tumor was further improved.
筛选可用于水疱性口炎病毒治疗的受试者的方法Methods of screening subjects for vesicular stomatitis virus therapy
在本发明的第四方面，本发明提出了一种筛选可用于水疱性口炎病毒治疗的受试者的方法。根据本发明的实施例，确定所述受试者肿瘤中细胞表面受体表达水平，所述细胞表面受体选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的一种或多种。根据本发明的实施例，所述细胞表面受体在所述待肿瘤中具有不小于2.0的表达差异倍数(log2FC)是所述受试者适于接受水疱性口炎病毒治疗的指示。根据本发明的实施例，所述水疱性口炎病毒前面所述的水疱性口炎病毒。根据本发明实施例的方法所筛选出受试者更加适于接受水疱性口炎病毒治疗，疗效会更加显著。In a fourth aspect of the present invention, the present invention proposes a method of screening subjects for vesicular stomatitis virus treatment. According to an embodiment of the present invention, the expression level of a cell surface receptor selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 is determined in the tumor of the subject. According to an embodiment of the present invention, the expression fold difference (log2FC) of the cell surface receptor in the tumor to be treated is not less than 2.0 is an indication that the subject is suitable for receiving vesicular stomatitis virus therapy. According to an embodiment of the present invention, the vesicular stomatitis virus is the aforementioned vesicular stomatitis virus. The subjects screened according to the methods of the embodiments of the present invention are more suitable for receiving vesicular stomatitis virus treatment, and the curative effect will be more significant.
预测VSV病毒溶瘤效果的方法Method for predicting the oncolytic effect of VSV virus
在本发明的第五方面，本发明提出了一种预测VSV病毒溶瘤效果的方法。根据本发明的实施例，所述方法包括：确定待测VSV病毒的G糖蛋白与肿瘤细胞的细胞表面受体的结合力，所述细胞表面受体选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的一种或多种。根据本发明的具体实施例，所述VSV病毒的G糖蛋白与所述细胞表面受体的结合力的ZDOCK score不小于1800，例如不小于1900，不小于2000，优选不小于2100是待测VSV病毒溶瘤效果佳的指示。根据本发明实施例的方法可提前预测待筛选VSV病毒溶瘤效果，进而为后续临床前实验提供科学指导。In the fifth aspect of the present invention, the present invention provides a method for predicting the oncolytic effect of VSV virus. According to an embodiment of the present invention, the method includes: determining the binding capacity of the G glycoprotein of the VSV virus to be tested to a cell surface receptor of tumor cells, the cell surface receptor is selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 one or more of. According to a specific embodiment of the present invention, the ZDOCK score of the binding force between the G glycoprotein of the VSV virus and the cell surface receptor is not less than 1800, for example, not less than 1900, not less than 2000, preferably not less than 2100 is the VSV to be tested Indication of good viral oncolytic effect. The method according to the embodiment of the present invention can predict the oncolytic effect of the VSV virus to be screened in advance, thereby providing scientific guidance for subsequent preclinical experiments.
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性 的，而不以任何方式限制本发明。The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative, and do not limit the present invention in any way.
实施例1 基于肿瘤组织大样本的人类膜受体基因的分析Example 1 Analysis of human membrane receptor genes based on large tumor tissue samples
以下将参考图1详细描述基于肿瘤组织大样本的人类膜受体基因的分析的方法。The method for the analysis of human membrane receptor genes based on a large sample of tumor tissue will be described in detail below with reference to FIG. 1 .
1.1人类膜受体基因及其表达数据预处理与分析1.1 Human membrane receptor genes and their expression data preprocessing and analysis
本发明从已有的研究中整理总结出在人类细胞中表达的受体基因信息(参考文献：Synchronous birth is a dominant pattern in receptor-ligand evolution，Grandchamp and Monget,BMC Genomics.2018 Aug 14；19(1):611.)。发明人从UCSC Xena( http://xena.ucsc.edu/)下载了癌症病人的的基因表达矩阵(归一化值)，基因突变信息以及相关临床资料。数据中包含的癌种由：肾上腺皮质癌、膀胱尿路上皮癌、乳腺浸润癌、宫颈鳞状细胞癌和宫颈内膜腺癌、胆管癌、结肠腺癌、结肠腺癌/Rectum腺癌食管癌、淋巴样肿瘤弥漫性大B细胞淋巴瘤、食道癌、FFPE试验第二阶段、胶质母细胞瘤、胶质瘤、头颈部鳞状细胞癌、肾染色体、泛肾队列(KICH+KIRC+KIRP)、肾脏肾透明细胞癌、肾脏肾乳头状细胞癌、急性髓性白血病、脑低级脑胶质瘤、肝肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮瘤、卵巢浆液性囊腺癌、胰腺腺癌、嗜铬细胞瘤和副神经节瘤、前列腺腺癌、直肠腺癌、肉瘤、皮肤黑色素瘤、胃腺癌、胃和食道癌、睾丸生殖细胞肿瘤、甲状腺癌、胸腺瘤、子宫体子宫内膜癌、子宫癌肉瘤、葡萄膜黑色素瘤。 The present invention summarizes the receptor gene information expressed in human cells from existing research (reference: Synchronous birth is a dominant pattern in receptor-ligand evolution, Grandchamp and Monget, BMC Genomics. 2018 Aug 14; 19 ( 1):611.). The inventors downloaded the gene expression matrix (normalized value), gene mutation information and related clinical data of cancer patients from UCSC Xena ( http://xena.ucsc.edu/ ). Cancer types included in the data are: adrenal cortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and cervical endometrial adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, colon adenocarcinoma/Rectum adenocarcinoma, esophageal carcinoma , lymphoid neoplasms, diffuse large B-cell lymphoma, esophageal cancer, FFPE trial phase II, glioblastoma, glioma, head and neck squamous cell carcinoma, renal chromosome, pan-kidney cohort (KICH+KIRC+ KIRP), renal renal clear cell carcinoma, renal renal papillary cell carcinoma, acute myeloid leukemia, low-grade cerebral glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous Cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, skin melanoma, gastric adenocarcinoma, gastric and esophageal cancer, testicular germ cell tumor, thyroid cancer, thymoma , uterine body endometrial cancer, uterine carcinosarcoma, uveal melanoma.
发明人首先从下载的数据中首先剔除少于三个的样本肿瘤及正常组织信息，然后进行差异表达分析。发明人使用limma软件(版本：3.38.3)来执行差异表达分析(参考文献：Limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies.Ritchie,M.E.,et al.Nucleic Acids Research,2015，43,e47)。分析中采用了limma R包的voom模型。只有当基因满足标准|log2FC|>1，P值<0.05才会被认定为是差异基因。The inventors first removed less than three sample tumor and normal tissue information from the downloaded data, and then performed differential expression analysis. The inventors used limma software (version: 3.38.3) to perform differential expression analysis (reference: Limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies. Ritchie, ME, et al. Nucleic Acids Research, 2015, 43, e47 ). The voom model of the limma R package was used in the analysis. Only when the gene satisfies the criterion |log2FC|>1, the P value <0.05 will be considered as a differential gene.
1.2数据分析1.2 Data Analysis
使用R语言计算各组间膜受体基因表达差异倍数(log2FC)和p值。选择|log2FC|大于等于2.0被视为有显著性上调/下调差异表达基因。t检验的p值小于0.01被判定为差异有统计学意义。使用ComplexHeatmap R包生成每个比较组对的log2FC矩阵的热图。The fold difference (log2FC) and p-value of membrane receptor gene expression between groups were calculated using R language. Selecting |log2FC| greater than or equal to 2.0 was considered to have significantly up-regulated/down-regulated differentially expressed genes. A p-value of t-test less than 0.01 was judged to be statistically significant. Heatmaps of log2FC matrices for each comparison group pair were generated using the ComplexHeatmap R package.
然后，我们根据一系列筛选条件，比如我们选择在肠癌，肺癌，胰腺癌，胃癌和肝癌癌种中选择大于等于70％的癌症样本中显著上调的基因，即|log2FC|≥2.0；本底表达量高等条件，选出10余个受体；另外，根据与备选配体的分子对接结果，最终选出5个受体。Then, we select genes that are significantly up-regulated in ≥70% of cancer samples in colorectal cancer, lung cancer, pancreatic cancer, gastric cancer and liver cancer according to a series of screening conditions, i.e. |log2FC|≥2.0; background Under the conditions of high expression level, more than 10 receptors were selected; in addition, according to the results of molecular docking with candidate ligands, 5 receptors were finally selected.
具体地，我们对每个基因在不同肿瘤样本中的log2FC值，使用ggplot2和ggbeeswarm软件绘制抖动散点图，用以展示该基因在各肿瘤中显著上调的病人比例。Specifically, we used ggplot2 and ggbeeswarm software to draw jitter scatter plots for the log2FC values of each gene in different tumor samples to show the proportion of patients whose genes were significantly up-regulated in each tumor.
在本实施例中，所用到的各癌种简称、英文名称，及其中文名称总结于下表1。In this example, the abbreviations, English names, and Chinese names of various cancer types used are summarized in Table 1 below.
表1：癌种简写和英文名称Table 1: Abbreviations and English names of cancer types
简写shorthand 英文名称English name 中文名称Chinese name
COADCOAD Colon adenocarcinomaColon adenocarcinoma 结肠癌colon cancer
LIHCLIHC Liver hepatocellular carcinomaLiver hepatocellular carcinoma 肝细胞肝癌hepatocellular carcinoma
LUADLUAD Lung adenocarcinomaLung adenocarcinoma 肺腺癌Lung adenocarcinoma
LUSCLUSC Lung squamous cell carcinomaLung squamous cell carcinoma 肺鳞癌lung squamous cell carcinoma
PAADPAAD Pancreatic adenocarcinomaPancreatic adenocarcinoma 胰腺癌Pancreatic cancer
STADSTAD Stomach adenocarcinomaStomach adenocarcinoma 胃癌stomach cancer
结果如图2所示。结果表明，CHRNA5、SSTR5、KISS1R、HTR1D和CCR8等10余种肿瘤特异性受体的差异基因表达在肿瘤病人中log2FC大于2的占比远大于LDLR。The results are shown in Figure 2. The results showed that the differential gene expression of more than 10 tumor-specific receptors such as CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 accounted for a much larger proportion of log2FC greater than 2 in tumor patients than LDLR.
1.3受体的结合力预测与筛选1.3 Binding prediction and screening of receptors
选择16条水疱性口炎病毒同源配体和大数据分析得到的10余种肿瘤特异性受体分别进行建模和对接，通过ZDOCK score函数来评价结合的强弱(结果如图3和4所示，其中(图3、4所示配体编号以及所对应的配体名称和氨基酸序列捕获号如表2所示)，发现CHRNA5、SSTR5、KISS1R、HTR1D和CCR8与配体结合力的ZDOCK score均大于1800，且这5种受体与配体的结合力远大于LDLR与配体的结合力，进而筛选出5个水疱性口炎病毒结合强的肿瘤特异性受体：CHRNA5(烟碱型胆碱受体α5)、SSTR5(生长抑素受体5)、KISS1R(吻素受体)、HTR1D(血清素受体1D)、CCR8(C-C趋化因子受体8)。16 vesicular stomatitis virus homologous ligands and more than 10 tumor-specific receptors obtained from big data analysis were selected for modeling and docking respectively, and the binding strength was evaluated by the ZDOCK score function (the results are shown in Figures 3 and 4). Among them (the ligand numbers shown in Figures 3 and 4 and the corresponding ligand names and amino acid sequence capture numbers are shown in Table 2), it was found that CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 had ZDOCKs with ligand binding ability The scores were all greater than 1800, and the binding force of these five receptors to ligands was much greater than that of LDLR and ligands. Then, five tumor-specific receptors with strong binding to vesicular stomatitis virus were screened: CHRNA5 (nicotine) type cholinergic receptor α5), SSTR5 (somatostatin receptor 5), KISS1R (kissin receptor), HTR1D (serotonin receptor 1D), CCR8 (CC chemokine receptor 8).
表2：Table 2:
Figure PCTCN2021111760-appb-000001
Figure PCTCN2021111760-appb-000001
Figure PCTCN2021111760-appb-000002
Figure PCTCN2021111760-appb-000002
鉴于LDLR是本领域已知的水疱性口炎病毒入胞受体，因此，在本发明的后续研究中，将LDLR作为对照。在后续的实验中，进一步分析筛选出5个肿瘤特异性受体以及LDLR受体与VSV病毒的结合强弱。Given that LDLR is a vesicular stomatitis virus entry receptor known in the art, LDLR was used as a control in subsequent studies of the present invention. In subsequent experiments, the binding strength of five tumor-specific receptors and LDLR receptors to VSV virus was further analyzed and screened.
实施例2 根据受体选择病毒配体Example 2 Selection of viral ligands according to receptors
发明人选择16种水疱性口炎病毒同源配体，与实施例1筛选获得的不同肿瘤特异性受体分别进行建模和对接，生成的对接的结果会按照ZDOCK score分数进行排序，得分越高，则说明结合越强，结果可信度越高。同时综合分析这些构象的聚类结果，发现，ZDOCK分数是ZDOCK程序计算的形状互补分数，根据参数设置，ZDOCK分数还将包括静电和去溶剂能项。ZDOCK分数越高越好。进而发明人通过ZDOCK score函数来评价结合的强弱，获得了筛选出肿瘤特异性受体结合能力强的配体(结果如图4所示)，其中，结合效果最优的配体为DQ408670.1-lig-FL和X03633.1-lig-FL，对应的氨基酸序列的捕获号为DQ408670.1，GENE ID:X03633.1。The inventors selected 16 kinds of vesicular stomatitis virus homologous ligands, and modeled and docked with different tumor-specific receptors obtained by screening in Example 1, respectively. The generated docking results will be sorted according to the ZDOCK score. The higher the value, the stronger the binding and the higher the reliability of the result. At the same time, the clustering results of these conformations were comprehensively analyzed, and it was found that the ZDOCK score was the shape complementation score calculated by the ZDOCK program. According to the parameter settings, the ZDOCK score would also include electrostatic and desolvation energy terms. The higher the ZDOCK score, the better. Furthermore, the inventors evaluated the strength of binding through the ZDOCK score function, and obtained a ligand with strong tumor-specific receptor binding ability (the results are shown in Figure 4), among which, the ligand with the best binding effect was DQ408670. 1-lig-FL and X03633.1-lig-FL, the capture number of the corresponding amino acid sequence is DQ408670.1, GENE ID: X03633.1.
实施例3 基于不同血清型蛋白的重组VSV病毒的构建及扩增Example 3 Construction and amplification of recombinant VSV viruses based on proteins of different serotypes
发明人将来源于Mudd summer亚型病毒株的L、N、P、M蛋白组合结合捕获序列号为GENE ID:DQ408670.1，GENE ID:X03633.1，GENE ID:KP872888.1或GENE ID:HQ593628.1的G蛋白构建重组水疱性口炎病毒REV DQ408670.1、REV X03633.1、REV KP872888.1和REV HQ593628.1。The inventors combined the L, N, P, and M proteins derived from the Mudd summer subtype strain to capture the sequence numbers as GENE ID: DQ408670.1, GENE ID: X03633.1, GENE ID: KP872888.1 or GENE ID: The G protein of HQ593628.1 was used to construct recombinant vesicular stomatitis viruses REV DQ408670.1, REV X03633.1, REV KP872888.1 and REV HQ593628.1.
病毒株REV DQ408670.1、REV X03633.1、REV KP872888.1和REV HQ593628.1的包装方法如下所述：The packaging methods for the virus strains REV DQ408670.1, REV X03633.1, REV KP872888.1 and REV HQ593628.1 are as follows:
体外重组VSV需要:包含病毒基因组的全长质粒(包含G蛋白)，以及病毒包装所需要的骨架蛋白的辅助质粒(N、P、L、M),通过体外转染的方法将质粒转入BHK21细胞中，病毒在细胞内组装成熟后出芽释放到细胞外(参考文献：Vesicular stomatitis virus-based vaccine protects hamsters against lethal challenge with Andes virus，Brown,K.S.,Safronetz,D.,Marzi,A.,Ebihara,H.&Feldmann,H.Journal of virology 85,12781-12791,doi:10.1128/JVI.00794-11(2011))。In vitro recombination of VSV requires: a full-length plasmid (including G protein) containing the viral genome, and an auxiliary plasmid (N, P, L, M) of the backbone protein required for viral packaging, and the plasmid is transferred into BHK21 by the method of in vitro transfection In cells, the virus is assembled and matured in the cell and then buds and released outside the cell (Reference: Vesicular stomatitis virus-based vaccine protects hamsters again lethal challenge with Andes virus, Brown, KS, Safronetz, D., Marzi, A., Ebihara, H. & Feldmann, H. Journal of virology 85, 12781-12791, doi: 10.1128/JVI.00794-11 (2011)).
病毒的扩增使用Vero细胞，将一定滴度的病毒加入到培养的Vero细胞中，病毒可以感 染细胞，并且在细胞中完成自我复制，成熟的病毒释放到细胞培养的上清中，将细胞培养的上清浓缩即可得到病毒浓缩液，滴度测定之后可用于后续实验。Virus amplification uses Vero cells, and a certain titer of virus is added to the cultured Vero cells. The virus can infect the cells and complete self-replication in the cells. The mature virus is released into the supernatant of the cell culture, and the cells are cultured. The supernatant obtained can be concentrated to obtain a virus concentrate, which can be used for subsequent experiments after titer determination.
实施例4 肿瘤细胞受体的检测以及细胞杀伤结果Example 4 Detection of tumor cell receptors and cell killing results
本实施例中利用实施例3构建的不同病毒对不同肿瘤细胞进行杀伤效果验证。In this example, different viruses constructed in Example 3 were used to verify the killing effect of different tumor cells.
4.1 q-PCR检测：4.1 q-PCR detection:
用Trizol法提取1×10 6的BXPC3、HCT-8、HepG2、Su8686、H358、NCL-H460(H460)和PANC1细胞样品，以500ng/μL RNA进行20μL体系的反转录，用SYBR GREEN法进行荧光定量PCR检测7个细胞样品中CHRNA5、KISS1R、HTRID、CCR8和SSTR5基因mRNA的表达。 1×10 6 samples of BXPC3, HCT-8, HepG2, Su8686, H358, NCL-H460 (H460) and PANC1 cells were extracted by Trizol method, and 500ng/μL RNA was used for reverse transcription in 20μL system, and SYBR GREEN method was used for reverse transcription. Fluorescence quantitative PCR was used to detect the mRNA expression of CHRNA5, KISS1R, HTRID, CCR8 and SSTR5 in 7 cell samples.
结果如图5所示。q-PCR检测结果显示，BXPC3、HCT-8、HepG2、Su8686、H358、NCL-H460和PANC1细胞样品CHRNA5受体基因mRNA表达水平较高，但不同细胞中，相对表达量高的受体存在差异，比如H460细胞中表达量最高的为CHRNA5、HTR1D受体，而其它细胞中CHRNA5和CCR8受体基因表达水平较高。The results are shown in Figure 5. The results of q-PCR showed that the mRNA expression level of CHRNA5 receptor gene was higher in BXPC3, HCT-8, HepG2, Su8686, H358, NCL-H460 and PANC1 cell samples, but there were differences in the receptors with high relative expression in different cells For example, CHRNA5 and HTR1D receptors are the most expressed in H460 cells, while the expression levels of CHRNA5 and CCR8 receptor genes are higher in other cells.
4.2细胞杀伤实验(CCK)：4.2 Cell Killing Assay (CCK):
将状态良好的BXPC3、HCT-8、HepG2、Su8686、H358和PANC1细胞制成5×10 4个/mL的细胞悬液按100μL/孔加入96孔板中，边缘补齐培养基减少蒸发，过夜培养。用Opti-MEM将已知滴度病毒稀释为MOI：0.01、MOI：0.1和MOI：1的病毒工作液，将96孔板中培养液吸弃，每孔加入50ul病毒稀释液，每个稀释液重复3复孔，另取Opti-MEM重复3孔作空白对照。病毒稀释液加入2h后换液，1％FBS培养基每孔100μL。48/72h后每孔加入10μLCCK8检测液，37℃孵育2h后OD450酶标仪读数。 The BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells in good condition were made into a cell suspension of 5×10 4 cells/mL and added to a 96-well plate at 100 μL/well, and the medium was filled with the edge to reduce evaporation, overnight. nourish. Dilute the known titer of virus with Opti-MEM to virus working solution of MOI: 0.01, MOI: 0.1 and MOI: 1, aspirate the culture solution in the 96-well plate, add 50ul virus dilution solution to each well, and each dilution solution Repeat 3 wells, and take Opti-MEM to repeat 3 wells as blank control. The virus diluent was added for 2 hours and then the medium was changed, with 100 μL of 1% FBS medium per well. After 48/72h, add 10 μL CCK8 detection solution to each well, incubate at 37°C for 2h and read on OD450 microplate reader.
实验结果如图7所示。CCK检测结果显示，REV-DQ408670.1、REV-X03633.1的病毒工作液对NCL-H358和NCL-H460细胞的杀伤效果显著优于REV-KP872888.1和REV-HQ593628.1。同时结合图5结果，NCL-H358中CCR8和CHRNA5表达量较高，NCL-H460中CHRNA5、HTR1D表达量较高，以及结合图3受体与配体的结合热图结果，DQ408670.1ligant和X03633.1G蛋白与CCR8、HTR1D受体的结合力强。综合反应了，当重组水疱性口炎病毒与肿瘤细胞受体具有高结合力时，重组病毒对高表达所述受体的肿瘤细胞的杀伤效果更加显著。The experimental results are shown in Figure 7. The CCK test results showed that the virus working solution of REV-DQ408670.1 and REV-X03633.1 had significantly better killing effect on NCL-H358 and NCL-H460 cells than REV-KP872888.1 and REV-HQ593628.1. At the same time, combined with the results in Figure 5, the expression levels of CCR8 and CHRNA5 were higher in NCL-H358, and the expression levels of CHRNA5 and HTR1D were higher in NCL-H460, as well as the binding heat map results of receptors and ligands in Figure 3, DQ408670.1ligant and X03633 .1G protein has strong binding ability to CCR8 and HTR1D receptors. The comprehensive reaction shows that when the recombinant vesicular stomatitis virus has high binding force to the tumor cell receptor, the killing effect of the recombinant virus on the tumor cells highly expressing the receptor is more significant.
图6显示了REV-DQ408670.1CCK检测结果，REV-DQ408670.1在MOI：0.01、MOI：0.1和MOI：1的病毒工作液均对BXPC3、HCT-8、HepG2、Su8686、H358和PANC1细胞具有显著的杀伤效果。Figure 6 shows the results of the CCK assay of REV-DQ408670.1. REV-DQ408670.1 has positive effects on BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells at MOI: 0.01, MOI: 0.1 and MOI: 1. Significant killing effect.
实施例5 体外敲减实验Example 5 In vitro knockdown experiment
根据目标基因的序列设计干扰RNA，shRNA构建于pSGU6载体上，lipo8000转染HEK-293T细胞，48h后收集细胞RNA，进行反转录，使用q-PCR的方法挑选出有显著敲减效果的shRNA。挑选出的shRNA重新转染HEK-293T细胞，48h后进行加REV-DQ408670.1病毒处理，24h后收集细胞RNA，进行反转录，使用q-PCR的方法验证对应基因敲减后对细胞内病毒复制的影响。The interfering RNA was designed according to the sequence of the target gene. The shRNA was constructed on the pSGU6 vector, and lipo8000 was transfected into HEK-293T cells. After 48 hours, the cellular RNA was collected and reverse transcribed. The shRNA with significant knockdown effect was selected by q-PCR. . The selected shRNA was retransfected into HEK-293T cells, treated with REV-DQ408670.1 virus after 48 hours, and the cellular RNA was collected after 24 hours for reverse transcription. Effects of viral replication.
实验结果如图8所示。从图中可以看出，CHRNA5、KISS1R、HTRID、CCR8和SSTR5分别进行基因敲减之后，能够显著影响细胞内VSV病毒的复制。The experimental results are shown in Figure 8. It can be seen from the figure that the gene knockdown of CHRNA5, KISS1R, HTRID, CCR8 and SSTR5 can significantly affect the replication of VSV virus in cells.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it should be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Embodiments are subject to variations, modifications, substitutions and variations.

Claims (26)

  1. 蛋白在构建水疱性口炎病毒中的用途，其特征在于，所述蛋白为肿瘤细胞表面特异性受体，包括选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的至少之一。The use of a protein in constructing vesicular stomatitis virus is characterized in that the protein is a specific receptor on the surface of tumor cells, including at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.
  2. 根据权利要求1所述的用途，其特征在于，所述水疱性口炎病毒的G蛋白与所述肿瘤细胞表面特异性受体的ZDOCK score不小于1800。The use according to claim 1, wherein the ZDOCK score of the G protein of the vesicular stomatitis virus and the tumor cell surface specific receptor is not less than 1800.
  3. 根据权利要求2所述的用途，其特征在于，所述G蛋白与受体LDLR的ZDOCK score不小于1600，优选不低于1650，更优选不小于1659，或为1650～1660。The use according to claim 2, wherein the ZDOCK score of the G protein and the receptor LDLR is not less than 1600, preferably not less than 1650, more preferably not less than 1659, or 1650-1660.
  4. 根据权利要求1所述的用途，其特征在于，所述水疱性口炎病毒为重组水疱性口炎病毒，任选的，所述水疱性口炎病毒不携带异源基因。The use according to claim 1, wherein the vesicular stomatitis virus is a recombinant vesicular stomatitis virus, and optionally, the vesicular stomatitis virus does not carry a heterologous gene.
  5. 根据权利要求1所述的用途，其特征在于，所述肿瘤细胞包括选自肠癌、肺癌、胰腺癌、胃癌、肝癌、食管癌、乳腺癌、胆管癌、黑色素瘤细胞的至少之一。The use according to claim 1, wherein the tumor cells comprise at least one selected from the group consisting of colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
  6. 根据权利要求1所述的用途，其特征在于，所述水疱性口炎病毒包括选自印第安纳株血清型及新泽西株血清型的至少之一。The use according to claim 1, wherein the vesicular stomatitis virus comprises at least one selected from the serotype of Indiana strain and the serotype of New Jersey strain.
  7. 一种水疱性口炎病毒，其特征在于，所述水疱性口炎病毒表达G蛋白，所述G蛋白适于与肿瘤细胞表面特异性受体结合，A vesicular stomatitis virus, characterized in that the vesicular stomatitis virus expresses a G protein, and the G protein is suitable for binding to specific receptors on the surface of tumor cells,
    可选的，所述肿瘤细胞表面特异性受体包括选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的至少之一。Optionally, the tumor cell surface specific receptor includes at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.
  8. 根据权利要求7所述的水疱性口炎病毒，其特征在于，所述G蛋白与所述肿瘤细胞表面特异性受体的ZDOCK score不小于1800。The vesicular stomatitis virus according to claim 7, wherein the ZDOCK score of the G protein and the tumor cell surface specific receptor is not less than 1800.
  9. 根据权利要求8所述的水疱性口炎病毒，其特征在于，所述G蛋白与受体LDLR的ZDOCK score不小于1600，优选不低于1650，更优选不小于1659，或为1650～1660。The vesicular stomatitis virus according to claim 8, wherein the ZDOCK score of the G protein and the receptor LDLR is not less than 1600, preferably not less than 1650, more preferably not less than 1659, or 1650-1660.
  10. 根据权利要求7所述的水疱性口炎病毒，其特征在于，所述水疱性口炎病毒为重组水疱性口炎病毒，The vesicular stomatitis virus according to claim 7, wherein the vesicular stomatitis virus is a recombinant vesicular stomatitis virus,
    任选的，所述水疱性口炎病毒不携带异源基因。Optionally, the vesicular stomatitis virus does not carry a heterologous gene.
  11. 根据权利要求7所述的水疱性口炎病毒，其特征在于，所述肿瘤细胞包括选自肠癌、肺癌、胰腺癌、胃癌、肝癌、食管癌、乳腺癌、胆管癌、黑色素瘤细胞的至少之一。The vesicular stomatitis virus according to claim 7, wherein the tumor cells comprise at least one selected from the group consisting of colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells one.
  12. 根据权利要求7所述的水疱性口炎病毒，其特征在于，所述水疱性口炎病毒包括选自印第安纳株血清型及新泽西株血清型的至少之一。The vesicular stomatitis virus according to claim 7, wherein the vesicular stomatitis virus comprises at least one selected from the serotype of Indiana strain and the serotype of New Jersey strain.
  13. 权利要求7～12任一项所述的水疱性口炎病毒在制备药物中的用途，所述药物用于治疗或预防肿瘤。Use of the vesicular stomatitis virus according to any one of claims 7 to 12 in the preparation of a medicament for treating or preventing tumors.
  14. 根据权利要求13所述的用途，其特征在于，所述肿瘤的细胞携带肿瘤细胞表面特 异性受体，所述肿瘤细胞表面特异性受体包括选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的至少之一。The use according to claim 13, wherein the tumor cells carry tumor cell surface specific receptors, and the tumor cell surface specific receptors include at least one selected from the group consisting of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 one.
  15. 根据权利要求14所述的用途，所述肿瘤细胞表面特异性受体在所述肿瘤中具有不小于2.0的表达差异倍数(log2FC)。The use of claim 14, wherein the tumor cell surface specific receptor has a fold difference in expression (log2FC) of not less than 2.0 in the tumor.
  16. 肿瘤细胞表面特异性受体在筛选用于治疗或预防肿瘤的VSV病毒中的用途，所述肿瘤细胞表面特异性受体选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的一种或多种。Use of tumor cell surface specific receptors selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 in screening for VSV virus for treating or preventing tumors.
  17. 一种筛选药物的方法，所述药物用于治疗或者预防肿瘤，所述方法包括：A method for screening a drug for treating or preventing tumors, the method comprising:
    (a)确定候选水疱性口炎病毒的G蛋白和待治疗肿瘤的肿瘤细胞表面特异性受体的结合力，所述肿瘤细胞表面特异性受体包括选自CHRNA5、SSTR5和KISS1R的至少之一；和(a) Determining the binding capacity of the G protein of the candidate vesicular stomatitis virus and the tumor cell surface specific receptor of the tumor to be treated, the tumor cell surface specific receptor including at least one selected from CHRNA5, SSTR5 and KISS1R ;and
    (b)基于步骤(a)中确定的所述结合力，确定所述候选水疱性口炎病毒是否适于治疗所述待治疗肿瘤。(b) determining whether the candidate vesicular stomatitis virus is suitable for treating the tumor to be treated based on the binding force determined in step (a).
  18. 根据权利要求17的方法，其特征在于，所述肿瘤细胞表面特异性受体在所述待治疗肿瘤中具有不小于2.0的表达差异倍数(log2FC)。The method according to claim 17, wherein the tumor cell surface specific receptor has an expression fold difference (log2FC) of not less than 2.0 in the tumor to be treated.
  19. 根据权利要求17所述的方法，其特征在于，所述结合力的ZDOCK score不低于1800是所述候选水疱性口炎病毒适于治疗所述待治疗肿瘤的指示。The method of claim 17, wherein a ZDOCK score of the binding force of not less than 1800 is an indication that the candidate vesicular stomatitis virus is suitable for treating the tumor to be treated.
  20. 根据权利要求17所述的方法，其特征在于，所述待治疗肿瘤为肺腺癌或肺鳞癌，所述肿瘤细胞表面特异性受体选自CHRNA5、SSTR5和KISS1R的至少之一；The method according to claim 17, wherein the tumor to be treated is lung adenocarcinoma or lung squamous cell carcinoma, and the tumor cell surface specific receptor is selected from at least one of CHRNA5, SSTR5 and KISS1R;
    所述待治疗的肿瘤为结肠癌，所述肿瘤细胞表面特异性受体选自HTR1D、SSTR5和KISS1R的的至少之一；The tumor to be treated is colon cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D, SSTR5 and KISS1R;
    所述待治疗的肿瘤为肝癌，所述肿瘤细胞表面特异性受体选自HTR1D、SSTR5、CCR8和KISS1R的至少之一；The tumor to be treated is liver cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D, SSTR5, CCR8 and KISS1R;
    所述待治疗的肿瘤为胃癌，所述肿瘤细胞表面特异性受体选自HTR1D和CCR8的至少之一；或者The tumor to be treated is gastric cancer, and the tumor cell surface specific receptor is selected from at least one of HTR1D and CCR8; or
    所述待治疗的肿瘤为胰腺癌，所述肿瘤细胞表面特异性受体选自CHRNA5、SSTR5和HTR1D的至少之一。The tumor to be treated is pancreatic cancer, and the tumor cell surface specific receptor is selected from at least one of CHRNA5, SSTR5 and HTR1D.
  21. 根据权利要求17所述的方法，其特征在于，所述水疱性口炎病毒为重组水疱性口炎病毒；The method of claim 17, wherein the vesicular stomatitis virus is a recombinant vesicular stomatitis virus;
    优选地，所述水疱性口炎病毒不携带异源基因。Preferably, the vesicular stomatitis virus does not carry a heterologous gene.
  22. 一种预测水疱性口炎病毒溶瘤效果的方法，其特征在于，包括：A method for predicting the oncolytic effect of vesicular stomatitis virus, comprising:
    确定待测水疱性口炎病毒的G蛋白和待治疗肿瘤的肿瘤细胞表面特异性受体的结合力，所述肿瘤细胞表面特异性受体包括选自CHRNA5、SSTR5和KISS1R的至少之一；和determining the binding capacity of the G protein of the vesicular stomatitis virus to be tested and a tumor cell surface specific receptor of the tumor to be treated, the tumor cell surface specific receptor comprising at least one selected from CHRNA5, SSTR5 and KISS1R; and
    基于所述结合力，确定所述待测水疱性口炎病毒的溶瘤效果。Based on the binding force, the oncolytic effect of the vesicular stomatitis virus to be tested is determined.
  23. 根据权利要求22所述的方法，其特征在于，所述结合力具有不低于1800的ZDOCK score是所述待测水疱性口炎病毒溶瘤效果佳的指示。The method according to claim 22, wherein the binding force has a ZDOCK score of not less than 1800, which is an indication that the oncolytic effect of the vesicular stomatitis virus to be tested is good.
  24. 一种筛选可用于水疱性口炎病毒治疗的受试者的方法，其特征在于，确定所述受试者肿瘤中细胞表面受体表达水平，所述细胞表面受体选自CHRNA5、SSTR5、KISS1R、HTR1D和CCR8的一种或多种，A method for screening subjects who can be used for vesicular stomatitis virus treatment, wherein the expression level of cell surface receptors in the tumor of the subject is determined, and the cell surface receptors are selected from CHRNA5, SSTR5, KISS1R , one or more of HTR1D and CCR8,
    可选的，所述细胞表面受体在所述待肿瘤中具有不小于2.0的表达差异倍数(log2FC)是所述受试者适于接受水疱性口炎病毒治疗的指示，Optionally, the expression fold (log2FC) of the cell surface receptor in the tumor to be treated is not less than 2.0 is an indication that the subject is suitable for receiving vesicular stomatitis virus therapy,
    可选的，所述水疱性口炎病毒为权利要求7～12任一项所述的水疱性口炎病毒。Optionally, the vesicular stomatitis virus is the vesicular stomatitis virus according to any one of claims 7 to 12.
  25. 一种治疗或预防肿瘤的方法，其特征在于，包括向受试者施用权利要求7～12任一项所述的水疱性口炎病毒。A method for treating or preventing tumors, comprising administering the vesicular stomatitis virus according to any one of claims 7 to 12 to a subject.
  26. 根据权利要求25所述的方法，其特征在于，所述肿瘤包括选自肠癌、肺癌、胰腺癌、胃癌、肝癌、食管癌、乳腺癌、胆管癌、黑色素瘤细胞的至少之一。The method of claim 25, wherein the tumor comprises at least one selected from the group consisting of colon cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.
PCT/CN2021/111760 2020-08-14 2021-08-10 Vesicular stomatitis virus and therapeutic use thereof WO2022033468A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202180070834.8A CN116322728A (en) 2020-08-14 2021-08-10 Vesicular stomatitis virus and therapeutic uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010816788.9 2020-08-14
CN202010816788 2020-08-14

Publications (1)

Publication Number Publication Date
WO2022033468A1 true WO2022033468A1 (en) 2022-02-17

Family

ID=80246924

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/111760 WO2022033468A1 (en) 2020-08-14 2021-08-10 Vesicular stomatitis virus and therapeutic use thereof

Country Status (2)

Country Link
CN (1) CN116322728A (en)
WO (1) WO2022033468A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1769433A (en) * 2004-11-04 2006-05-10 张庆勇 Recombinant vesicular stomatitis virus and its uses
WO2017198779A1 (en) * 2016-05-19 2017-11-23 Klinikum Rechts Der Isar Der Technischen Universität München Vsv/ndv hybrid viruses for oncolytic therapy of cancer
CN109937050A (en) * 2016-09-14 2019-06-25 鲁汶大学 The VSV-G and its vaccine of modification
WO2019133847A1 (en) * 2017-12-29 2019-07-04 Oncorus, Inc. Oncolytic viral delivery of therapeutic polypeptides
CN111494432A (en) * 2019-01-31 2020-08-07 惠君生物医药科技(杭州)有限公司 Pharmaceutical composition for treating tumor or cancer and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1769433A (en) * 2004-11-04 2006-05-10 张庆勇 Recombinant vesicular stomatitis virus and its uses
WO2017198779A1 (en) * 2016-05-19 2017-11-23 Klinikum Rechts Der Isar Der Technischen Universität München Vsv/ndv hybrid viruses for oncolytic therapy of cancer
CN109937050A (en) * 2016-09-14 2019-06-25 鲁汶大学 The VSV-G and its vaccine of modification
WO2019133847A1 (en) * 2017-12-29 2019-07-04 Oncorus, Inc. Oncolytic viral delivery of therapeutic polypeptides
CN111494432A (en) * 2019-01-31 2020-08-07 惠君生物医药科技(杭州)有限公司 Pharmaceutical composition for treating tumor or cancer and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A. AMMAYAPPAN, PENG K.-W., RUSSELL S. J.: "Characteristics of Oncolytic Vesicular Stomatitis Virus Displaying Tumor-Targeting Ligands", JOURNAL OF VIROLOGY, vol. 87, no. 24, 15 December 2013 (2013-12-15), US , pages 13543 - 13555, XP055122167, ISSN: 0022-538X, DOI: 10.1128/JVI.02240-13 *
GUO BINGNAN: "The Promotion of VSV Oncolytic Sensitivity by Suppressing Mx1 Gene Expression Through RNAi", SOOCHOW UNIVERSITY, vol. 36, no. 12, 31 December 2016 (2016-12-31), pages 1 - 72, XP055900160 *
WILLMON CANDICE L., SALOURA VASSILIKI, FRIDLENDER ZVI G., WONGTHIDA PHONPHIMON, DIAZ ROSA MARIA, THOMPSON JILL, KOTTKE TIMOTHY, FE: "Expression of IFN-β Enhances Both Efficacy and Safety of Oncolytic Vesicular Stomatitis Virus for Therapy of Mesothelioma", CANCER RESEARCH, vol. 69, no. 19, 1 October 2009 (2009-10-01), US , pages 7713 - 7720, XP055900164, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-09-1013 *

Also Published As

Publication number Publication date
CN116322728A (en) 2023-06-23

Similar Documents

Publication Publication Date Title
US11242527B1 (en) Combination vectors and methods for treating cancer
US20240141381A1 (en) Compositions and methods for treating phenylketonuria
AU2017207906B2 (en) Methods and compositions for the activation of gamma-delta T-cells
US11352646B2 (en) Vector system for expressing regulatory RNA
JP2023115125A (en) Vector having combination of promoter and enhancer for treating phenylketonuria
JP6250631B2 (en) Microvesicle and method for producing the same
US20240240202A1 (en) Compositions and therapeutic methods of microrna gene delivery
WO2021095838A1 (en) Nucleic acid lipid particle vaccine encapsulating hpv mrna
Shirvani et al. Non-coding RNA in SARS-CoV-2: progress toward therapeutic significance
CA3236236A1 (en) Compositions and systems for rna-programmable cell editing and methods of making and using same
JPWO2018193902A1 (en) Antiviral effect against hepatitis B virus by microRNA
Bongolo et al. Exosomes derived from microRNA‐27a‐3p overexpressing mesenchymal stem cells inhibit the progression of liver cancer through suppression of Golgi membrane protein 1
WO2022033469A1 (en) Recombinant oncolytic virus, and construction method therefor and use thereof
WO2022033468A1 (en) Vesicular stomatitis virus and therapeutic use thereof
WO2022033467A1 (en) Method for constructing oncolytic virus
US20240318147A1 (en) Recombinant oncolytic virus, and construction method therefor and use thereof
JP2022543610A (en) Genetically modified enterovirus vectors
WO2023020556A1 (en) Virus formulation, solution for formulating virus formulation, and use thereof
WO2023051607A1 (en) Virus culture method
WO2024082263A1 (en) System for controlling virus replication in response to hypoxic environment and use thereof
CN115350272A (en) Use of PBLD or agonist thereof as active ingredient against RNA virus
CN118660966A (en) Compositions and systems for RNA programmable cell editing and methods of making and using the same
WO2008136213A1 (en) Radiosensitivity-enhancing agent

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21855520

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21855520

Country of ref document: EP

Kind code of ref document: A1