Vesicular stomatitis virus and therapeutic uses thereof

Abstract

A vesicular stomatitis virus is provided. The vesicular stomatitis virus expresses a G protein adapted to bind to a tumor cell surface-specific receptor, optionally including at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8.

Description

[0001] Priority information

[0002] This application claims priority and benefits to patent application No. 202010816788.9, filed with the China National Intellectual Property Administration on August 14, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of oncolytic viruses, specifically to vesicular stomatitis virus, the use of vesicular stomatitis virus in drug preparation, the use of tumor cell surface specific receptors in screening VSV viruses for the treatment or prevention of tumors, methods for screening drugs, methods for predicting the oncolytic effect of vesicular stomatitis virus, and methods for screening subjects who can be treated with vesicular stomatitis virus. Background Technology

[0004] Over the past decade or so, the mechanism by which oncolytic viruses kill tumors by inducing an anti-tumor immune response in the body has become increasingly clear. Since German scientist Jean Rommelaere first referred to oncolytic virus therapy as tumor immunotherapy in 2011, oncolytic viruses have been widely accepted as an important branch of tumor immunotherapy. Compared to other tumor immunotherapies, oncolytic viruses have advantages such as high killing efficiency, good targeting, fewer side effects, multiple tumor-killing pathways to avoid drug resistance, and low cost.

[0005] Because viral genomes are small, they are relatively easy to modify using genetic engineering techniques. Furthermore, viral modification and packaging are routine biomedical experimental methods, with relatively mature and inexpensive technologies. Therefore, oncolytic viruses can more easily be modified to specifically target cancer cells by leveraging their own characteristics and the differences between cancer cells and normal cells.

[0006] Because most cancer cells have impaired mechanisms for clearing viruses (e.g., the key factor for virus clearance in normal cells, protein kinase R (PKR), is absent in cancer cells), viruses can more easily replicate and spread within them. Furthermore, in recent decades, with ongoing research, scientists have leveraged the differences in signaling pathways and metabolism between cancer cells and normal cells. By screening specific viral strains and modifying viral genomes, they have continuously improved the targeting specificity of oncolytic viruses to tumors, reduced their harm to normal cells, and enhanced safety. For example, the approved T-vec gene knocks out the γ34.5 gene of HSV-1 (herpes simplex virus type 1). The γ34.5 gene can inhibit the virus clearance mechanism in normal cells; after knocking out the γ34.5 gene, the virus cannot replicate in normal cells. However, this mechanism is absent in cancer cells, and knocking out the γ34.5 gene does not affect viral replication in cancer cells. JX594 (Pexa-Vec), currently in Phase III clinical trials, knocks out the TK (thymidine kinase) gene of vaccinia viruses. Since viral replication is related to TK levels in cells, the TK-knockout JX594 can only replicate in cancer cells with high TK activity, not in normal cells (normal cells have low TK activity). CG0070 adds the E2F-1 promoter before the E1A gene, which is responsible for adenovirus replication. E2F-1 is regulated by retinoblastoma suppressor protein (Rb), which is absent in bladder cancer. The absence of Rb activates the transcriptional activity of E2F-1, leading to E1A gene expression, allowing the virus to specifically replicate in bladder cancer. Reolysin is an unmodified wild-type reovirus whose replication depends on the activation of the Ras signaling pathway, therefore it can only specifically replicate in Ras-activated cancer cells.

[0007] However, the current state of cancer treatment lacks a method that can simultaneously improve tumor-killing specificity (i.e., high specificity relative to normal non-tumor cells) and broaden the scope of cancer treatment (i.e., be applicable to multiple cancers). Therefore, there is an urgent need in this field to develop a method for killing tumor cells that possesses both high tumor cell specificity and broad-spectrum cancer treatment efficacy.

[0008] Although VSV was discovered a long time ago and has a broad spectrum of cell infectivity, its receptor was not found for a long time. Some researchers even thought that its receptor was a lipid component on the cell membrane, such as phosphatidylserine, 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)). As research progressed, genetic, biochemical, and immunochemical results showed that VSV-G is essential for VSV to bind to its putative receptor, undergo endocytosis, and fuse with the target cell membrane. After binding, VSV enters the cell via clathrin-mediated endocytosis (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.). This indicates the existence of a specific receptor for VSV on the cell membrane. It wasn't until 2013 that Finkelshtein et al. identified LDLR as the main receptor for VSV entry into cells (Reference: LDL receptor and its familymembers serve as the cellular receptors for vesicular stomatitisvirus. Finkelshtein et al. Proc Natl Acad Sci US A. 2013 Apr 30; 110(18):7306-11). Cells with the LDLR gene knocked out can still be infected with VSV, indicating the presence of other receptors that can mediate VSV endocytosis. Previous studies have shown that although VSV can infect most mammalian cells, it has a higher preference for tumor cells, suggesting that tumor cells may have highly expressed VSV receptors.

[0009] When using VSV as an oncolytic virus for tumor therapy, the presence of specific receptors can enhance VSV's targeting ability. Therefore, identifying these specific receptors becomes crucial. With the continuous advancement of sequencing technology and the development of bioinformatics, publicly available sequencing data from cancer patients can help us with initial screening. Summary of the Invention

[0010] The present invention aims to at least partially solve one of the technical problems in the related art.

[0011] In a first aspect, the present invention proposes the use of a protein in the construction of a vesicular stomatitis virus. According to embodiments of the 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 virus (VSV virus) constructed using the tumor cell surface-specific receptor screened by the inventors of this application exhibits stronger specific binding ability to tumor cells and a more significant specific killing effect on tumor cells.

[0012] According to embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0013] 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 specific receptor on the surface of the tumor cells is not less than 1800, for example, not less than 1900, not less than 2000, and preferably not less than 2100. Those skilled in the art will understand that the ZDOCK score, a characterization parameter of the binding force between the G protein and the cell receptor, can be easily obtained by inputting the sequences of the G protein and the cell receptor. The inventors have 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 is significantly increased. According to an embodiment of the present invention, the ZDOCK score can be determined using conventional software, for example, see Pierce BG, Houlai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657.

[0014] According to embodiments 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, for example 1659.078. The binding affinity of the tumor cell surface specific receptor screened by the inventors of this application to the G protein is much higher than that of LDLR to the G protein.

[0015] According to an embodiment of the present invention, the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.

[0016] According to a specific embodiment of the present invention, the VSV virus does not carry heterologous genes. According to embodiments of the present invention, unless otherwise specified, the term "heterologous gene" as used herein refers to a gene not previously reported in wild-type vesicular stomatitis virus. In other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in wild-type vesicular stomatitis virus.

[0017] According to embodiments of the present invention, the tumor cells include at least one selected from colorectal cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.

[0018] According to an embodiment of the present invention, the vesicular stomatitis virus includes at least one of the Indiana strain serotype and the New Jersey strain serotype.

[0019] In a second aspect, the present invention provides a vesicular stomatitis virus (VSV virus). According to embodiments of the invention, the vesicular stomatitis virus expresses a G protein adapted to bind to a specific receptor on the surface of tumor cells. Optionally, the specific receptor on the surface of tumor cells includes at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8. The vesicular stomatitis virus (VSV virus) according to embodiments of the present invention exhibits stronger specific binding ability to tumor cells and a more significant specific killing effect on tumor cells.

[0020] According to embodiments of the present invention, the above-mentioned VSV virus may further include at least one of the following additional technical features:

[0021] 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 specific receptor on the surface of the tumor cells is not less than 1800, for example, not less than 1900, not less than 2000, and preferably not less than 2100. Those skilled in the art will understand that the ZDOCK score, a characterization parameter of the binding force between the G protein and the cell receptor, can be easily obtained by inputting the sequences of the G protein and the cell receptor. The inventors have 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 is significantly increased. According to an embodiment of the present invention, the ZDOCK score can be determined using conventional software, for example, see Pierce BG, Houlai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657.

[0022] 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, for example 1659.078.

[0023] According to an embodiment of the present invention, the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.

[0024] According to a specific embodiment of the present invention, the VSV virus does not carry heterologous genes. According to embodiments of the present invention, unless otherwise specified, the term "heterologous gene" as used herein refers to a gene not previously reported in wild-type vesicular stomatitis virus. In other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in wild-type vesicular stomatitis virus.

[0025] According to embodiments of the present invention, the tumor cells include at least one selected from colorectal cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.

[0026] According to an embodiment of the present invention, the vesicular stomatitis virus includes at least one of the Indiana strain serotype and the New Jersey strain serotype.

[0027] In a third aspect, the present invention provides the use of the aforementioned vesicular stomatitis virus in the preparation of a medicament for the treatment or prevention of tumors.

[0028] According to embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0029] According to an embodiment of the present invention, the tumor cells carry tumor cell surface-specific receptors, which include at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.

[0030] According to an embodiment of the present invention, the tumor cell surface specific receptor has an expression differential fold of not less than 2.0 (log2FC) in the tumor.

[0031] In a fourth aspect of the invention, the use of the tumor cell surface specific receptor in screening VSV viruses for the treatment or prevention of tumors, said tumor cell surface specific receptor being selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.

[0032] According to embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0033] According to an embodiment of the present invention, the ZDOCK score of the binding affinity between the G glycoprotein of the VSV virus and the tumor cell surface specific receptor expressed at a differential fold change (log2FC) 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, and preferably not less than 2100, which is an indication that the virus to be screened is the target virus.

[0034] According to an embodiment of the present invention, the VSV virus is a recombinant VSV virus; preferably, the VSV virus does not carry heterologous genes.

[0035] In a fifth aspect, the present invention provides a method for screening subjects suitable for vesicular stomatitis virus (VSV) treatment. According to embodiments of the invention, the expression level of cell surface receptors in the tumors of the subjects is determined, wherein the cell surface receptors are selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8. Subjects screened by the method according to embodiments of the invention are more suitable for VSV treatment, and the therapeutic effect is more significant.

[0036] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0037] According to an embodiment of the present invention, a fold change in expression of the cell surface receptor of not less than 2.0 (log2FC) in the tumor is an indication that the subject is suitable for treatment with vesicular stomatitis virus.

[0038] According to an embodiment of the present invention, the vesicular stomatitis virus is the aforementioned vesicular stomatitis virus.

[0039] In a sixth aspect, 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 affinity of the G glycoprotein of the VSV virus to be tested to a cell surface receptor of tumor cells, wherein the cell surface receptor is selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8.

[0040] According to embodiments of the present invention, the method for predicting the oncolytic effect of VSV virus may further include at least one of the following additional technical features:

[0041] According to an embodiment of the present invention, a ZDOCK score of not less than 1800, for example not less than 1900, not less than 2000, and preferably not less than 2100, is an indicator of good oncolytic effect of the VSV virus to be tested.

[0042] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0043] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0044] Figure 1 A flowchart of the analysis of human membrane receptor genes based on a large sample of tumor tissue is shown.

[0045] Figure 2 A jittery scatter plot showing the proportion of patients whose corresponding receptor genes were significantly upregulated in each tumor.

[0046] Figure 3 The ZDOCK score results, which reflect the binding strength of the 16 candidate ligands to the 5 tumor-specific receptors, are shown.

[0047] Figure 4 The ZDOCK score results for the binding strength of the selected ligands to five tumor-specific receptors are shown.

[0048] Figure 5 The expression levels of CHRNA5, KISS1R, HTRID, CCR8, and SSTR5 in BXPC3, HCT-8, HepG2, Su8686, NCI-H358, and PANC1 cell samples obtained by qPCR are shown.

[0049] Figure 6The study showed the killing effects of the virus on BXPC3, HCT-8, HepG2, Su8686, NCI-H358 and PANC1 cells under different MOI conditions, as determined in the cell killing assay.

[0050] Figure 7 The study demonstrated the killing effects of viral strains with different G proteins on NCI-H358 and NCI-H460 cells under different MOI conditions.

[0051] Figure 8 The study demonstrated the effects of gene knockdown of CCR8, CHRNA5, KISS1R, HTR1D, and SSTR5 on intracellular VSV replication in an in vitro knockdown experiment.

[0052] Detailed description of the invention

[0053] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0054] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0055] vesicular stomatitis virus

[0056] In a first aspect, the present invention provides a vesicular stomatitis virus. According to embodiments of the present invention, the vesicular stomatitis virus expresses a G protein adapted to bind to a specific receptor on the surface of tumor cells. Optionally, the specific receptor on the surface of tumor cells includes at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8. It should be noted that the "specific receptor on the surface of tumor cells" described in this application refers to a receptor whose expression on the surface of tumor cells is significantly upregulated / downregulated compared to that on the surface of normal cells. A fold change in expression (log2FC) greater than or equal to 2.0 is considered a significant upregulation, and a p-value less than 0.01 in a t-test is considered statistically significant. The vesicular stomatitis virus (VSV virus) according to embodiments of the present invention exhibits stronger specific binding ability to tumor cells and a more significant specific killing effect on tumor cells.

[0057] Vesicular stomatitis virus (VSV) belongs to the genus *Vesicularvirus* of the family Rhabdoviridae. It has two serotypes: New Jersey (VSV-NJ) and Indiana (VSV-IND). The virus particles are bullet-shaped or cylindrical, measuring 150–180 nm × 50–70 nm. The virus has an envelope with uniformly distributed spikes approximately 10 nm long. Internally, the virus consists of a tightly coiled, helically symmetrical nucleocapsid. The virus is named after the classic vesicular lesions found in the oral mucosa, dental pads, tongue, lips, nostrils, hooves, and nipples of infected animals. It is transmitted via insect vectors, and the disease is limited to its natural hosts, such as horses, cattle, and pigs. In humans, infection is mild and asymptomatic.

[0058] The VSV genome is a non-segmented, single-stranded negative-sense RNA (ssRNA) virus, approximately 11 KB in length. Five non-overlapping genes—N, NS, M, G, and L—are arranged sequentially from the 3' to the 5' end, encoding five different major proteins: nuclear (N) protein, phosphoprotein (P) protein, matrix (M) protein, glycoprotein (G) protein, and RNA-dependent RNA polymerase (L) protein. The N gene has a leader sequence at the 3' end and a trailing sequence at the 5' end, with spacer sequences between the genes. The 3' leader RNA is the earliest viral transcript in infected cells, 47 nucleotides in length, uncapped and untranslated; its function is not fully understood, but it may inhibit host RNA synthesis. The N protein is essential for initiating genome synthesis and effectively protects viral RNA from digestion by various nucleases. The N protein has high immunogenicity, stimulating cellular immunity, and plays a crucial role in transcription and replication; it is likely necessary to maintain the extended form of the genomic RNA and is related to replication regulation. The P protein, sharing 41% homology with VSV-NJ and VSV-IND viral strains, functions as a polymerase complex with polymerase L and nucleoprotein N, working in conjunction with genomic RNA to maintain viral transcriptional activity. The M protein plays a crucial role in viral pathogenesis and replication, rich in basic amino acids and containing a highly basic N-terminal domain. It inhibits transcription by binding to the nucleocapsid and assists in viral budding from the host, being the only polypeptide involved in the budding process. The G protein is the main surface antigen of the virus, determining its virulence and serving as a protective antigen. It stimulates the body to produce neutralizing antibodies. The L gene encodes the RNA polyE protein, which may determine RNA transcriptional activity and binds to the P protein to catalyze mRNA replication. This protein is a core component of the polymerase and replicase complexes, involved in initiation, elongation, methylation, capping, and poly(A) tail formation, among other processes. Furthermore, there is extensive homology in the spacer sequences between each gene, sharing a common structure: 3'-AUAC(U)7NAUUGUCNN-UAG-5'. The conserved sequences among these genes are key signals that influence polymerase activity or enzyme cleavage activity, but these signals are masked and become ineffective during replication.

[0059] According to embodiments of the present invention, the ZDOCK score of the binding force between the G glycoprotein of the VSV virus and the specific receptor on the surface of the tumor cells is not less than 1800, for example, not less than 1900, not less than 2000, and preferably not less than 2100. The binding force between the vesicular stomatitis virus and the specific receptor on the surface of tumor cells described in this application is evaluated using the ZDOCK score function. The generated docking results are sorted according to the ZDOCK score; the higher the score, the stronger the binding and the higher the reliability of the result. Clustering results of these conformations are also provided. The ZDOCK score is the shape complementarity score calculated by the ZDOCK program. Depending on the parameter settings, the ZDOCK score will also include electrostatic and desolvation energy terms; a higher ZDOCK score is better.

[0060] Those skilled in the art will understand that the ZDOCK score, a characterization parameter of the binding force between the G protein and the cell receptor, can be easily obtained by inputting the sequences of the G protein and the cell receptor. The inventors have found that when the ZDOCK score is not less than 1800, for example not less than 1900, not less than 2000, and preferably not less than 2100, the binding force between a virus carrying the G protein and a tumor cell carrying the corresponding receptor will be significantly increased. According to embodiments of the present invention, the ZDOCK score can be determined using conventional software, for example, see Pierce BG, Houlai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library. PLoS One 6(9):e24657.

[0061] According to embodiments 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 known vesicular stomatitis virus entry receptor in the art. Using LDLR as a positive control, the inventors found that the binding affinity of CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8 selected in this application to the virus is much higher than that of LDLR.

[0062] According to an embodiment of the present invention, the vesicular stomatitis virus is a recombinant vesicular stomatitis virus.

[0063] According to a specific embodiment of the present invention, the VSV virus does not carry a heterologous gene. According to embodiments of the present invention, unless otherwise specified, the term "heterologous gene" as used herein refers to a gene not previously reported in wild-type vesicular stomatitis virus. In other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in wild-type vesicular stomatitis virus. The inventors have found that the recombinant vesicular stomatitis virus without a heterologous gene exhibits significantly higher cytotoxic effects against tumor cells than the recombinant vesicular stomatitis virus carrying a heterologous gene.

[0064] According to embodiments of the present invention, the tumor cells include at least one selected from colorectal cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, bile duct cancer, and melanoma cells.

[0065] According to an embodiment of the present invention, the vesicular stomatitis virus includes at least one of the Indiana strain serotype and the New Jersey strain serotype.

[0066] Pharmaceutical uses

[0067] In a second aspect, the present invention proposes the use of the aforementioned vesicular stomatitis virus in the preparation of a medicament for the treatment or prevention of tumors. The medicament prepared from the virus according to embodiments of the present invention exhibits a more significant effect in the treatment or prevention of tumors.

[0068] According to embodiments of the present invention, the tumor cells carry tumor cell surface-specific receptors, which include at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8. According to embodiments of the present invention, the tumor cell surface-specific receptors have an expression differential fold of not less than 2.0 (log2FC) in the tumor. The inventors have found that the proportion of differentially expressed (log2FC) values ​​of CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8 across five cancer types is significantly higher than that of LDLR.

[0069] According to embodiments of the present invention, the cancer includes at least one of lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal carcinoma, and leukemia.

[0070] Furthermore, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the aforementioned recombinant vesicular stomatitis virus.

[0071] According to embodiments of the present invention, the pharmaceutical composition is in a form suitable for administration by inhalation or injection.

[0072] The pharmaceutical composition provided by the present invention comprises a pharmaceutically acceptable carrier and an effective amount of the following active ingredient: the recombinant VSV virus of the present invention that specifically infects tumor cells.

[0073] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0074] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0075] The pharmaceutical compositions of the present invention contain 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, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. They are prepared, for example, using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0076] In one embodiment, the pharmaceutical composition of the present invention may further include additional tumor therapeutic agents.

[0077] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0078] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0079] Use of VSV viruses in screening for treatment or prevention of tumors

[0080] In a third aspect of the invention, there is the use of tumor cell surface-specific receptors in screening VSV viruses for the treatment or prevention of tumors, wherein the tumor cell surface-specific receptors are selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.

[0081] According to an embodiment of the present invention, the ZDOCK score of the binding affinity between the G glycoprotein of the VSV virus and the tumor cell surface specific receptor expressed at a differential fold change (log2FC) 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, and preferably not less than 2100, which is an indication that the virus to be screened is the target virus.

[0082] The VSV viruses screened by the method according to embodiments of the present invention for treating or preventing tumors have stronger specific targeting and affinity for tumors, and their killing effect is more significant.

[0083] 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 discovered that the expression of CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8 receptors differs between different tumor cells and normal cells. Based on the magnitude of the differences, the inventors identified the different receptors that are differentially expressed in different tumor cells. Furthermore, by utilizing the receptors that are specifically differentially expressed in different tumor cells, the inventors can more effectively screen for VSV viruses for the treatment or prevention of specific tumors.

[0084] According to an embodiment of the present invention, the VSV virus is a recombinant VSV virus; preferably, the VSV virus does not carry heterologous genes. Therefore, the tumor-killing effect of the recombinant VSV virus is further enhanced.

[0085] Methods for screening subjects who may be suitable for vesicular stomatitis virus treatment

[0086] In a fourth aspect, the present invention provides a method for screening subjects suitable for vesicular stomatitis virus (VSV) treatment. According to an embodiment of the invention, the expression level of cell surface receptors in the subject's tumor is determined, wherein the cell surface receptors are selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8. According to an embodiment of the invention, a fold change (log2FC) of not less than 2.0 in the expression of the cell surface receptors in the tumor is an indicator that the subject is suitable for VSV treatment. According to an embodiment of the invention, the VSV is the aforementioned VSV. Subjects screened by the method according to the embodiment of the invention are more suitable for VSV treatment, and the therapeutic effect is more significant.

[0087] Methods for predicting the oncolytic effect of VSV virus

[0088] In a fifth aspect, the present invention provides a method for predicting the oncolytic effect of VSV virus. According to an embodiment of the invention, the method includes: determining the binding affinity of the G glycoprotein of the VSV virus to be tested to a cell surface receptor of tumor cells, wherein the cell surface receptor is selected from one or more of CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8. According to a specific embodiment of the invention, a ZDOCK score of not less than 1800, for example not less than 1900, not less than 2000, and preferably not less than 2100, is an indicator of good oncolytic effect of the VSV virus to be tested. The method according to the embodiments of the invention can predict the oncolytic effect of the VSV virus to be screened in advance, thereby providing scientific guidance for subsequent preclinical experiments.

[0089] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0090] Example 1: Analysis of human membrane receptor genes based on a large sample of tumor tissue

[0091] The following will be referenced Figure 1 A detailed description of the method for analyzing human membrane receptor genes based on large samples of tumor tissue.

[0092] 1.1 Preprocessing and Analysis of Human Membrane Receptor Genes and Their Expression Data

[0093] This invention summarizes and organizes information on receptor genes 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 obtained information from UCSC Xena ( http: / / xena.ucsc.edu / The database downloaded gene expression matrices (normalized values), gene mutation information, and related clinical data from cancer patients. The cancer types included in the data are: adrenocortical carcinoma, urothelial carcinoma of the bladder, invasive breast cancer, cervical squamous cell carcinoma and cervical endometrial adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, colon adenocarcinoma / Rectum adenocarcinoma, esophageal cancer, lymphoid neoplasms, diffuse large B-cell lymphoma, esophageal cancer, FFPE trial phase II, glioblastoma, glioma, head and neck squamous cell carcinoma, kidney chromosomes, pan-renal cohort (KICH+KIRC+KIRP), and kidney... Clear cell carcinoma of the kidney, papillary cell carcinoma of the kidney, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma of the liver, lung adenocarcinoma, squamous cell carcinoma of the lung, mesothelioma, serous cystadenocarcinoma of the ovary, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, melanoma of the skin, gastric adenocarcinoma, gastric and esophageal cancer, testicular germ cell tumor, thyroid cancer, thymoma, endometrial cancer of the uterine body, uterine carcinosarcoma, uveal melanoma.

[0094] The inventors first removed tumor and normal tissue information from fewer than three samples from the downloaded data, and then performed differential expression analysis. The inventors used limma software (version 3.38.3) to perform the 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 from the limma R package was used in the analysis. Only genes meeting the criterion |log2FC|>1 and P-value <0.05 were considered differentially expressed genes.

[0095] 1.2 Data Analysis

[0096] The log2FC (fold change in expression) and p-value of the intercellular membrane receptor gene expression were calculated for each group using R. Genes with |log2FC| greater than or equal to 2.0 were considered to have significantly upregulated / downregulated expression. A p-value less than 0.01 in the t-test was considered statistically significant. A heatmap of the log2FC matrix for each comparison pair was generated using the ComplexHeatmap R package.

[0097] Then, based on a series of screening criteria, such as selecting genes that are significantly upregulated in 70% or more of cancer samples in colorectal cancer, lung cancer, pancreatic cancer, gastric cancer, and liver cancer (i.e., |log2FC|≥2.0) and high background expression levels, we selected more than 10 receptors. In addition, based on the molecular docking results with candidate ligands, we finally selected 5 receptors.

[0098] Specifically, we used ggplot2 and ggbeeswarm software to plot the log2FC values ​​of each gene in different tumor samples to show the proportion of patients in which the gene was significantly upregulated in each tumor.

[0099] In this embodiment, the abbreviations, English names, and Chinese names of the various cancer types used are summarized in Table 1 below.

[0100] Table 1: Abbreviations and English Names of Cancer Types

[0101]

[0102]

[0103] The results are as follows Figure 2 As shown in the figure. The results indicate that the proportion of differentially expressed genes of more than 10 tumor-specific receptors, such as CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8, with log2FC greater than 2 is much higher in cancer patients than in LDLR.

[0104] 1.3 Receptor Binding Ability Prediction and Screening

[0105] Sixteen homologous ligands of vesicular stomatitis virus and more than ten tumor-specific receptors obtained from big data analysis were selected for modeling and docking. The binding strength was evaluated using the ZDOCK score function. The results are as follows: Figure 3 and 4 As shown, where Figure 3 , 4The ligand numbers, corresponding ligand names, and amino acid sequence capture numbers shown in Table 2 are as follows. It was found that the ZDOCK scores of CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8 binding to the ligands were all greater than 1800, and the binding forces of these five receptors to the ligands were much greater than those of LDLR. Therefore, five tumor-specific receptors with strong binding to vesicular stomatitis virus were screened out: CHRNA5 (nicotinic cholinergic receptor α5), SSTR5 (somatostatin receptor 5), KISS1R (kissin receptor), HTR1D (serotonin receptor 1D), and CCR8 (CC chemokine receptor 8).

[0106] Table 2:

[0107]

[0108] Given that LDLR is a known vesicular stomatitis virus (VSV) entry receptor in the art, it was used as a control in subsequent studies of this invention. In subsequent experiments, five tumor-specific receptors were further analyzed and screened, along with the binding strength of the LDLR receptor to the VSV virus.

[0109] Example 2: Selection of viral ligands based on receptors

[0110] The inventors selected 16 homologous ligands of vesicular stomatitis virus and modeled and docked them with different tumor-specific receptors screened in Example 1. The docking results were sorted according to the ZDOCK score; a higher score indicates a stronger binding and higher reliability. Simultaneously, a comprehensive analysis of the clustering results of these conformations revealed that the ZDOCK score is a shape complementarity score calculated by the ZDOCK program. Depending on the parameter settings, the ZDOCK score also includes electrostatic and desolvation energy terms. A higher ZDOCK score is better. Furthermore, the inventors used the ZDOCK score function to evaluate the binding strength, obtaining ligands with strong binding ability to tumor-specific receptors (results are shown below). Figure 4 As shown in the figure, the ligands with the best binding effect are DQ408670.1-lig-FL and X03633.1-lig-FL, and the capture number of the corresponding amino acid sequence is DQ408670.1, GENEID:X03633.1.

[0111] Example 3: Construction and amplification of recombinant VSV virus based on different serotype proteins

[0112] The inventors combined the L, N, P, and M proteins derived from the Mudd Summer subtype virus strain with the G protein of the capture sequence number GENE ID:DQ408670.1, GENE ID:X03633.1, GENE ID:KP872888.1, or GENE ID:HQ593628.1 to construct recombinant vesicular stomatitis virus REV DQ408670.1, REV X03633.1, REV KP872888.1, and REVHQ593628.1.

[0113] The packaging methods for virus strains REV DQ408670.1, REV X03633.1, REV KP872888.1, and REV HQ593628.1 are as follows:

[0114] In vitro recombinant VSV requires: a full-length plasmid containing the viral genome (including G protein) and helper plasmids (N, P, L, M) containing the backbone proteins required for viral packaging. The plasmid is transferred into BHK21 cells via in vitro transfection. After the virus assembles and matures in the cells, it buds and is released outside the cells (Reference: Vesicular stomatitis virus-based vaccine protects hamsters against 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)).

[0115] Virus amplification is performed using Vero cells. A certain titer of virus is added to cultured Vero cells, where the virus can infect the cells and replicate itself. Mature virus is released into the cell culture supernatant. The cell culture supernatant is then concentrated to obtain a concentrated virus solution, which can be used for subsequent experiments after titer determination.

[0116] Example 4: Detection of tumor cell receptors and cell killing results

[0117] In this embodiment, the killing effect of different viruses constructed in Example 3 on different tumor cells was verified.

[0118] 4.1 q-PCR detection:

[0119] 1×10⁻⁶ was extracted using the Trizol method. 6BXPC3, HCT-8, HepG2, Su8686, NCI-H358, NCI-H460 (H460), and PANC1 cell samples were used for reverse transcription in a 20 μL system with 500 ng / μL RNA. The expression of CHRNA5, KISS1R, HTRID, CCR8, and SSTR5 gene mRNA in the seven cell samples was detected by real-time PCR using the SYBR Green method.

[0120] The results are as follows Figure 5 As shown in the figure. q-PCR results showed that the expression level of CHRNA5 receptor gene mRNA was high in BXPC3, HCT-8, HepG2, Su8686, H358, NCI-H460, and PANC1 cell samples. However, the relative expression levels of the receptors varied among different cell types. For example, the highest expression levels of CHRNA5 and HTR1D receptors were observed in H460 cells, while CHRNA5 and CCR8 receptor gene expression levels were higher in other cells.

[0121] 4.2 Cell Killing Assay (CCK):

[0122] Healthy BXPC3, HCT-8, HepG2, Su8686, NCI-H358, and PANC1 cells were divided into 5×10⁻⁶ cells. 4 Cell suspension of cells / mL was added at 100 μL / well to 96-well plates, with medium added to the edge to reduce evaporation, and incubated overnight. Virus working solutions with known titers were diluted with Opti-MEM to MOI: 0.01, MOI: 0.1, and MOI: 1. The culture medium in the 96-well plates was aspirated, and 50 μL of virus dilution was added to each well, with each dilution repeated in triplicate. Three additional wells were prepared with Opti-MEM as a blank control. The medium was changed 2 hours after adding the virus dilution, and 100 μL of 1% FBS medium was added to each well. After 48 / 72 hours, 10 μL of CCK8 assay solution was added to each well, and the plates were incubated at 37°C for 2 hours before reading the OD450 value using a microplate reader.

[0123] Experimental results are as follows Figure 7 As shown in the figure. CCK test results showed that the working solutions of REV-DQ408670.1 and REV-X03633.1 were significantly more effective than REV-KP872888.1 and REV-HQ593628.1 in killing NCI-H358 and NCI-H460 cells. Simultaneously, combined with... Figure 5 The results showed that CCR8 and CHRNA5 expression levels were high in NCI-H358, while CHRNA5 and HTR1D expression levels were high in NCI-H460, as well as binding... Figure 3The receptor-ligand binding heatmap results showed that the DQ408670.1 ligant and X03633.1 G proteins have strong binding affinity to CCR8 and HTR1D receptors. This collectively indicates that when the recombinant vesicular stomatitis virus exhibits high binding affinity to tumor cell receptors, the recombinant virus demonstrates a more significant killing effect on tumor cells highly expressing these receptors.

[0124] Figure 6 The results of REV-DQ408670.1 CCK assay show that REV-DQ408670.1 virus working solutions at MOI: 0.01, MOI: 0.1 and MOI: 1 all have significant killing effects on BXPC3, HCT-8, HepG2, Su8686, NCI-H358 and PANC1 cells.

[0125] Example 5 In vitro knockdown experiment

[0126] Interfering RNA (shRNA) was designed based on the target gene sequence. The shRNA was constructed in the pSGU6 vector and transfected into HEK-293T cells using lipo8000. After 48 hours, cellular RNA was collected and reverse transcribed. q-PCR was used to select shRNAs with significant knockdown effects. The selected shRNAs were then retransfected into HEK-293T cells. After 48 hours, the cells were treated with REV-DQ408670.1 virus. After 24 hours, cellular RNA was collected and reverse transcribed. q-PCR was used to verify the effect of corresponding gene knockdown on intracellular viral replication.

[0127] Experimental results are as follows Figure 8 As shown in the figure, knocking down CHRNA5, KISS1R, HTRID, CCR8, and SSTR5 significantly affected intracellular VSV replication.

[0128] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0129] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a protein in the screening of vesicular stomatitis virus, characterized in that, The proteins are tumor cell surface-specific receptors, including CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8, and the ZDOCK score of the G protein of the vesicular stomatitis virus and the tumor cell surface-specific receptor is not less than 1800.

2. The use according to claim 1, characterized in that, The vesicular stomatitis virus mentioned is a recombinant vesicular stomatitis virus.

3. The use according to claim 1, characterized in that, The vesicular stomatitis virus does not carry heterologous genes.

4. The use according to claim 1, characterized in that, The tumor cells include at least one of colorectal cancer, lung cancer, pancreatic cancer, and liver cancer.

5. The use according to claim 1, characterized in that, The vesicular stomatitis virus includes at least one of the Indiana strain serotype and the New Jersey strain serotype.

6. Use of tumor cell surface specific receptors in screening VSV viruses for the treatment or prevention of tumors, wherein the tumor cell surface specific receptors include CHRNA5, SSTR5, KISS1R, HTR1D and CCR8, and the tumor cells include at least one selected from colorectal cancer, lung cancer, pancreatic cancer and liver cancer.

7. A method for screening drugs for treating or preventing tumors, the method comprising: (a) Determining the binding affinity of the G protein of a candidate vesicular stomatitis virus to a tumor cell surface-specific receptor of the tumor to be treated, said tumor cell surface-specific receptor including those selected from CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8; and (b) Based on the binding affinity determined in step (a), determine whether the candidate vesicular stomatitis virus is suitable for treating the tumor to be treated. The ZDOCK score of the binding force being not less than 1800 is an indicator that the candidate vesicular stomatitis virus is suitable for treating the tumor to be treated. The tumor to be treated includes at least one of colorectal cancer, lung cancer, pancreatic cancer, and liver cancer.

8. The method according to claim 7, characterized in that, The tumor cell surface-specific receptor has an expression differential of not less than 2.0 log2FC in the tumor to be treated.

9. The method according to claim 7, characterized in that, 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; or 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.

10. The method according to claim 7, characterized in that, The vesicular stomatitis virus mentioned is a recombinant vesicular stomatitis virus.

11. The method according to claim 7, characterized in that, The vesicular stomatitis virus does not carry heterologous genes.

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