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WO2022033468A1 - Virus de la stomatite vésiculaire et son utilisation thérapeutique - Google Patents

Virus de la stomatite vésiculaire et son utilisation thérapeutique Download PDF

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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
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Prior art keywords
vesicular stomatitis
stomatitis virus
tumor
cancer
cell surface
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Chinese (zh)
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吴可行
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Revoimmune Therapeutics Inc
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Revoimmune Therapeutics Inc
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Priority to CN202180070834.8A priority Critical patent/CN116322728A/zh
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    • 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.

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Abstract

L'invention concerne un virus de la stomatite vésiculaire. Le virus de la stomatite vésiculaire exprime une protéine G, et la protéine G est appropriée pour se lier à un récepteur spécifique sur la surface de cellules tumorales. Eventuellement, le récepteur spécifique sur la surface de cellules tumorales comprend au moins l'un parmi CHRNA5, SSTR5, KISS1R, HTR1D et CCR8.
PCT/CN2021/111760 2020-08-14 2021-08-10 Virus de la stomatite vésiculaire et son utilisation thérapeutique Ceased WO2022033468A1 (fr)

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