Viral formulations, solutions for formulating viral formulations, and uses thereof

Abstract

Provided are a virus preparation, a solution for preparing the virus preparation, and use thereof. The virus preparation contains: a virus; sucrose; magnesium chloride. The virus preparation contains a Tris-HCl buffer, and the pH is 7.2-7.6.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to viral preparations, solutions for preparing viral preparations, and their uses. Background Technology

[0002] 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.

[0003] Because viral genomes are small, they are relatively easy to modify using genetic engineering techniques. Furthermore, viral modification and packaging can be performed using conventional methods, which are relatively mature and inexpensive. Therefore, it is relatively easy to modify oncolytic viruses by utilizing their inherent characteristics and the differences between cancer cells and normal cells, in order to specifically target cancer cells.

[0004] 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 expression product of the γ34.5 gene can inhibit the virus clearance mechanism in normal cells; after γ34.5 gene knockout, the virus cannot replicate in normal cells. However, cancer cells lack this virus clearance mechanism; therefore, γ34.5 gene knockout 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 lower TK activity than cancer cells). CG0070 adds the E2F-1 promoter before the E1A gene, which is responsible for adenovirus replication. The E2F-1 promoter is regulated by retinoblastoma suppressor protein (Rb), which is absent in bladder cancer. Therefore, the absence of Rb activates the transcriptional activity of E2F-1, allowing the E1A gene to be expressed in bladder cancer cells, and the virus can specifically replicate in bladder cancer cells. Reolysin is an unmodified wild-type reovirus whose replication depends on the activation of the Ras signaling pathway, so it can only specifically replicate in Ras-activated cancer cells.

[0005] However, the current state of cancer treatment still lacks a treatment approach that can both enhance tumor-killing specificity (i.e., specifically kill tumor cells relative to normal non-tumor cells) and broaden the scope of cancer treatment (i.e., be applicable to multiple cancers simultaneously). There is an urgent need in this field to develop a recombinant oncolytic virus and its formulation that simultaneously possesses high tumor cell specificity and broad-spectrum cancer treatment efficacy. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, one object of this invention is to provide a recombinant oncolytic virus preparation that simultaneously possesses high tumor cell specificity and / or broad-spectrum tumor therapeutic efficacy.

[0007] In a first aspect, the present invention provides a viral preparation. According to an embodiment of the present invention, the viral preparation comprises: virus; sucrose; magnesium chloride. The viral preparation contains a Tris-HCl buffer solution and has a pH of 7.2–7.6. Therefore, the viral preparation exhibits minimal change in viral titer during storage and demonstrates excellent storage stability.

[0008] In addition, the viral preparations according to the above embodiments of the present invention may also have the following additional technical features:

[0009] According to an embodiment of the present invention, the virus is a recombinant oncolytic virus.

[0010] According to an embodiment of the present invention, the recombinant oncolytic virus is a vesicular stomatitis virus.

[0011] According to embodiments of the present invention, the recombinant oncolytic virus expresses a viral protein with high affinity for cell receptors, wherein the viral protein is selected from: (a) SEQ ID NO:1; (b) SEQ ID NO:2; or (c) an amino acid sequence having at least 80% homology with (a) or (b). Thus, the viral preparation exhibits high tumor cell specificity and / or broad-spectrum tumor therapeutic efficacy.

[0012] According to an embodiment of the present invention, the viral protein comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology with (a) or (b).

[0013] According to an embodiment of the present invention, the ZDOCK score of the binding force between the viral protein and the cell receptor is not less than 1800.

[0014] According to an embodiment of the present invention, the cell receptor includes at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8.

[0015] According to embodiments of the present invention, the recombinant oncolytic virus further expresses at least one of the following: nucleoproteins, phosphoproteins, matrix proteins, and RNA-dependent RNA polymerases.

[0016] According to an embodiment of the present invention, based on the total amount of the viral preparation, the viral preparation contains: 4.5 to 5.5% by weight of sucrose; and 1.5 to 2.5 mmol / L of magnesium chloride.

[0017] According to an embodiment of the present invention, the concentration of Tris in the Tris-HCl buffer is 50 mmol / L.

[0018] According to an embodiment of the present invention, the viral preparation, based on the total amount of the viral preparation, contains: 5% by weight sucrose; and 2 mmol / L magnesium chloride.

[0019] According to an embodiment of the present invention, the viral preparation is in a form suitable for administration by inhalation or injection.

[0020] In a second aspect, the present invention provides a solution for preparing viral preparations. According to an embodiment of the invention, the solution for preparing viral preparations contains: sucrose; magnesium chloride; Tris-HCl buffer, and the pH of said solution is 7.2 to 7.6. Therefore, the viral preparation prepared with this solution exhibits minimal change in viral titer during storage and demonstrates excellent storage stability.

[0021] In addition, the solution for preparing viral agents according to the above embodiments of the present invention may also have the following additional technical features:

[0022] According to an embodiment of the present invention, the solution for preparing the viral preparation contains 4.5 to 5.5% by weight of sucrose and 1.5 to 2.5 mmol / L of magnesium chloride.

[0023] According to an embodiment of the present invention, the solution for preparing the virus preparation contains 5% by weight of sucrose and 2 mmol / L of magnesium chloride.

[0024] According to an embodiment of the present invention, the concentration of Tris in the Tris-HCl buffer is 50 mmol / L.

[0025] In a third aspect, the present invention provides the use of the viral preparations of the above embodiments or the solutions for preparing the viral preparations of the above embodiments in the preparation of a medicament for the treatment or prevention of cancer or tumors.

[0026] According to embodiments of the present invention, the cancer or tumor includes at least one selected from lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal carcinoma, and leukemia.

[0027] In a fourth aspect, the present invention provides a method for preventing or treating cancer or tumors. According to an embodiment of the invention, the method includes: administering the aforementioned viral preparation or solution to a subject.

[0028] According to embodiments of the present invention, the cancer or tumor includes at least one selected from lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal carcinoma, and leukemia.

[0029] 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

[0030] 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:

[0031] Figure 1 A flowchart of the analysis of human membrane receptor genes based on a large sample of tumor tissue is shown.

[0032] Figure 2 A jittery scatter plot showing the proportion of patients whose corresponding receptor genes were significantly upregulated in each tumor.

[0033] Figure 3 The ZDOCK score results, reflecting the binding strength of candidate ligands to tumor-specific receptors, are shown.

[0034] Figure 4 The figure shows the experimental results of screening ligands based on the selected receptors.

[0035] Figure 5 The expression levels of CHRNA5, KISS1R, HTRID, CCR8, and SSTR5 in BXPC3, HCT-8, HepG2, Su8686, H358, NCL-H460, and PANC1 cell samples obtained by qPCR are shown.

[0036] Figure 6 The study demonstrated the cytotoxic effects of the virus on BXPC3, HCT-8, HepG2, Su8686, H358, and PANC1 cells under different MOI conditions, as measured in cell killing experiments.

[0037] Figure 7 The study demonstrated the killing effect of viral strains with different G proteins on NCL-H358 and NCL-H460 cells under different MOI conditions.

[0038] Figure 8 The study demonstrated the killing effect of the viral strain with inserted heterologous genes on NCL-H358 and NCL-H460 cells.

[0039] Figure 9 The figure shows the experimental results of the safety of the REV DQ408670.1 virus strain in normal cells.

[0040] Figure 10 The results of stability tests on the viral preparations of Example 7 and Comparative Examples 1-7 at 2-8°C are shown.

[0041] Figure 11 The results of stability tests on the viral preparations of Examples 7 and Comparative Examples 1-7 at 25±2°C are shown.

[0042] Figure 12 The results of stability tests on the viral preparations of Example 7 and Comparative Examples 1-7 at -60°C are shown.

[0043] Figure 13 The results of repeated freeze-thaw stability tests for Examples 7 and Comparative Examples 1-7 are shown. Detailed Implementation

[0044] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0045] In a first aspect, the present invention provides a viral preparation. According to an embodiment of the present invention, the viral preparation comprises: virus; sucrose; magnesium chloride. The viral preparation contains a Tris-HCl buffer solution and has a pH of 7.2 to 7.6.

[0046] The inventors discovered that the viral preparation with the composition described above exhibits excellent storage stability, showing minimal change in viral titer after prolonged storage at different temperatures or repeated freeze-thaw cycles. This may be because sucrose and magnesium chloride enhance viral protection, stabilize viral structure, and regulate osmotic pressure. Furthermore, compared to other common buffer solutions, Tris-HCl buffer has minimal interference with biochemical processes and offers a wider pH adjustment range. The virus in the preparation is relatively stable within the pH range of 7.2–7.6, and adjusting the pH of the buffer solution to 7.2–7.6 further improves viral stability.

[0047] Furthermore, according to embodiments of the present invention, based on the total amount of the viral preparation, the viral preparation contains: 4.5–5.5% by weight of sucrose; and 1.5–2.5 mmol / L of magnesium chloride. By controlling the sucrose and magnesium chloride content in the viral preparation within the above ranges, the storage stability of the viral preparation can be further improved.

[0048] Furthermore, according to embodiments of the present invention, based on the total amount of the viral preparation, the viral preparation contains: 5% by weight sucrose; and 2 mmol / L magnesium chloride. Therefore, the storage stability of the viral preparation is improved.

[0049] According to an embodiment of the present invention, the concentration of Tris in the above-mentioned Tris-HCl buffer is 50 mmol / L. Therefore, the storage stability of the viral preparation is improved.

[0050] In addition, the inventors discovered that if the sucrose content in the viral preparation is too high or too low, it may reduce the stability of the virus after repeated freeze-thaw cycles; if the magnesium chloride content in the viral preparation is too high or too low, it may reduce the stability of the virus at 2–8°C and ambient temperature (25°C); if the pH in the viral preparation is too high or too low, it may damage the viral structure and reduce viral activity.

[0051] According to embodiments of the present invention, the virus described above is a recombinant oncolytic virus. The recombinant oncolytic virus of the present invention exhibits good stability in viral preparations, with minimal change in viral titer after prolonged storage at different temperatures or repeated freeze-thaw cycles.

[0052] According to embodiments of the present invention, the recombinant oncolytic virus is a vesicular stomatitis virus. The vesicular stomatitis virus of the present invention exhibits good stability in viral preparations, with minimal changes in viral titer after prolonged storage at different temperatures or repeated freeze-thaw cycles.

[0053] According to embodiments of the present invention, the recombinant oncolytic virus expresses a viral protein with high affinity for cell receptors, wherein the viral protein is selected from: (a) SEQ ID NO:1; (b) SEQ ID NO:2; or (c) an amino acid sequence having at least 80% homology with (a) or (b). Therefore, the viral preparation exhibits high tumor cell specificity and / or broad-spectrum tumor therapeutic efficacy.

[0054] MKCFLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTGLQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISTDITFFSEDRELSSLGKEGTGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSGLHLSSKAQVFEHPHIQDAASQLPDDEILFFGDTGLSKNPIDFVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIYLYIKLKHTKKRQIYTDIEMNRLGR(SEQ ID NO:1)。

[0055] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQ SCGYATVTDAEAAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMAD KDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGP NGVLRTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:2)

[0056] According to embodiments of the present invention, the recombinant vesicular stomatitis virus expressing the above-mentioned viral protein has stronger specific targeting of tumor cells, a broader spectrum of tumor killing, and a more significant killing effect.

[0057] It should be noted that the "homology" mentioned in this application refers to the similarity of amino acid sequences, where differences in individual amino acids do not affect the protein's inherent function. "Homologous amino acid sequences" refer to amino acid sequences derived from the amino acid sequence of a polypeptide through the substitution, deletion, or addition of one or more amino acids. Specifically, the "percentage of sequence homology" mentioned in this application is calculated using the following formula:

[0058] 1 - Number of differentially expressed amino acids / Number of amino acids in the reference amino acid sequence × 100%,

[0059] The number of amino acids in the benchmark amino acid sequence refers to the number of amino acid sequences being compared. For example, the benchmark amino acid sequence in the statement "G protein has at least 80% sequence homology with either SEQ ID NO:1 or SEQ ID NO:2" is SEQ ID NO:1 or SEQ ID NO:2.

[0060] The aforementioned homologous amino acid sequences are biologically, chemically, or structurally similar and possess similar biological activities. Structural similarity refers to amino acids having side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity refers to amino acids having the same charge or being either hydrophilic or hydrophobic. For example, hydrophobic residues such as isoleucine, valine, leucine, or methionine can be substituted for each other. Alternatively, polar amino acids can be substituted for each other, such as arginine replacing lysine, glutamic acid replacing aspartic acid, glutamine replacing asparagine, serine replacing threonine, etc. Biological similarity refers to amino acid sequences with sequence homology having similar biological functions; for example, the recombinant vesicular stomatitis viruses according to embodiments of the present invention all possess broad-spectrum and specific high affinity and binding force to tumors.

[0061] 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.

[0062] 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 5' ends, encoding five different proteins: nuclear (N) protein, phosphoprotein (P) protein, matrix (M) protein, glycoprotein (G) protein, and RNA-dependent RNA polymerase (L) protein, respectively. 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 antigenicity and immunogenicity, stimulating the body to produce non-neutralizing antibodies for 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.

[0063] In the description of this invention, the terms "recombinant VSV virus", "recombinant vesicular stomatitis virus", and "recombinant virus of this invention" are used interchangeably and refer to the recombinant VSV virus that is capable of specifically infecting tumor cells as described above, wherein the recombinant VSV virus specifically infects tumor cells and specifically binds to tumor cell-specific receptors selected from the group consisting of CHRNA5, SSTR5, KISS1R, HTR1D and CCR8.

[0064] According to an embodiment of the present invention, the viral protein comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology with any one of SEQ ID NO:1 or SEQ ID NO:2.

[0065] According to embodiments of the present invention, the recombinant vesicular stomatitis virus does not carry a foreign gene. The inventors have found that the recombinant vesicular stomatitis virus without a foreign gene exhibits significantly higher cytotoxic effects against tumor cells than the recombinant vesicular stomatitis virus carrying a foreign gene. According to embodiments of the present invention, unless otherwise specified, the term "foreign 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.

[0066] According to an embodiment of the present invention, the ZDOCK score of the binding force between the viral protein and the cell receptor is not less than 1800. Those skilled in the art will understand that the ZDOCK score, a characterization parameter of the binding force between the viral protein and the cell receptor, can be easily obtained by inputting the sequences of the viral protein and the cell receptor. The inventors have found that when the ZDOCK score is 1800, for example, not less than 1900, not less than 2000, and preferably not less than 2100, the binding force between the virus carrying the viral protein and the tumor cell carrying the corresponding receptor will be significantly improved. According to an embodiment of the present invention, the ZDOCK score can be determined 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.

[0067] According to embodiments of the present invention, the viral protein includes at least one of the G protein selected from GenBank accession number X03633.1 and GenBank accession number DQ408670.1. The inventors of the present invention unexpectedly discovered that the G protein with GenBank accession number X03633.1 and the G protein with GenBank accession number DQ408670.1 have a significantly stronger binding affinity to receptors on tumor cells than other G proteins.

[0068] According to embodiments of the present invention, the recombinant vesicular stomatitis virus further expresses at least one of the following: nucleoproteins, phosphoproteins, matrix proteins, and RNA-dependent RNA polymerases. The inventors unexpectedly discovered that recombinant viruses constructed by combining these proteins with at least one of the G proteins from GenBank request number X03633.1 and GenBank request number DQ408670.1 possess stronger tumor-killing activity. The inventors believe that this may be because, for tumor cells, combinations of proteins from multiple different sources may elicit different immune responses than combinations of proteins from the same source, thereby further enhancing the killing effect on tumor cells.

[0069] Accordingly, the recombinant vesicular stomatitis virus carries: a nucleic acid molecule encoding the nucleoprotein; a nucleic acid molecule encoding the phosphoprotein; a nucleic acid molecule encoding the matrix protein; or a nucleic acid molecule encoding the RNA-dependent RNA polymerase.

[0070] Preferably, at least one of the nucleic acid molecule encoding the nucleoprotein, the nucleic acid molecule encoding the phosphoprotein, the nucleic acid molecule encoding the matrix protein, and the nucleic acid molecule encoding the RNA-dependent RNA polymerase is derived from the Mudd-Summer subtype of vesicular stomatitis virus. The inventors unexpectedly discovered that recombinant viruses constructed by combining these proteins with at least one of the G proteins from GenBank request number X03633.1 and GenBank request number DQ408670.1 exhibit stronger tumor-killing activity. The inventors believe that this may be because, for tumor cells, combinations of proteins from multiple different sources may trigger different immune responses than combinations of proteins from the same source, thereby further enhancing the killing effect on tumor cells.

[0071] According to an embodiment of the present invention, the viral preparation is in a form suitable for administration by inhalation or injection.

[0072] In a second aspect, the present invention provides a solution for preparing viral preparations. According to an embodiment of the invention, the solution for preparing viral preparations contains: sucrose; magnesium chloride; Tris-HCl buffer, and the pH of said solution is 7.2 to 7.6.

[0073] The inventors discovered that the viral preparation prepared with the solution described above exhibits excellent storage stability, with minimal changes in viral titer after prolonged storage at different temperatures or repeated freeze-thaw cycles. This may be because sucrose and magnesium chloride stabilize the viral structure and enhance viral protection; compared to other common buffer solutions, the Tris-HCl buffer offers a wider pH adjustment range, better stabilizing the viral preparation's pH; and the virus in the preparation is relatively stable within the pH range of 7.2–7.6, which can be further stabilized by adjusting the pH of the buffer solution to 7.2–7.6.

[0074] Furthermore, according to embodiments of the present invention, the solution used for preparing the viral preparation contains 4.5 to 5.5% by weight of sucrose and 1.5 to 2.5 mmol / L of magnesium chloride. By controlling the sucrose and magnesium chloride content in the solution within the above ranges, the storage stability of the viral preparation obtained from the solution can be further improved.

[0075] Furthermore, according to an embodiment of the present invention, the solution used for preparing the viral preparation contains 5% by weight of sucrose and 2 mmol / L of magnesium chloride. Therefore, the viral preparation prepared from this solution exhibits better storage stability.

[0076] According to an embodiment of the present invention, the concentration of Tris in the above-mentioned Tris-HCl buffer is 50 mmol / L. Therefore, the viral preparation prepared with this solution exhibits better storage stability.

[0077] Furthermore, the present invention proposes the use of the above-described pharmaceutical composition or the above-described recombinant vesicular stomatitis virus in the preparation of a medicament for the treatment or prevention of cancer or tumors.

[0078] According to embodiments of the present invention, the cancer or tumor includes at least one selected from lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal carcinoma, and leukemia.

[0079] Furthermore, the present invention provides a method for preventing or treating cancer or tumors. According to an embodiment of the present invention, the method includes: administering the above-mentioned viral preparation or the above-mentioned solution to a subject.

[0080] According to embodiments of the present invention, the cancer or tumor includes at least one selected from lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal carcinoma, and leukemia.

[0081] 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.

[0082] The embodiments of the recombinant vesicular stomatitis virus involved in this invention will be described below.

[0083] Example 1: Analysis of human membrane receptor genes based on a large sample of tumor tissue

[0084] 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.

[0085] 1.1 Preprocessing and Analysis of Human Membrane Receptor Genes and Their Expression Data

[0086] 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, BMC Genomics. Grandchamp and Monget, 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 cancer, 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.

[0087] The inventors first removed fewer than three samples of 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 the differential expression analysis (Reference: (Limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies. Nucleic Acids Research, 43, e47, Ritchie, ME, et al. (2015)). The Voom model of 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.

[0088] 1.2 Data Analysis

[0089] 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.

[0090] Then, the inventors selected more than 10 receptors based on a series of screening criteria, such as selecting genes that are significantly upregulated in more than 70% of cancer samples in colorectal cancer, lung cancer, pancreatic cancer, gastric cancer and liver cancer (i.e., genes with log2FC≥2.0) and high background expression levels.

[0091] Specifically, the inventors used ggplot2 and ggbeeswarm software to plot jittery scatter plots (e.g., log2FC values ​​of each gene in different tumor samples) for each gene. Figure 2 As shown in the figure, this is used to demonstrate the proportion of patients in various tumors where this gene is significantly upregulated.

[0092] In addition, the inventors performed molecular docking between the 13 selected receptors and the candidate ligands, and selected the 5 receptors with the best binding force as the final selection.

[0093] The results are as follows Figure 3 As shown (where, Figure 3 The ligand numbers and corresponding ligand names and amino acid sequence capture numbers shown in Table 1 are as follows. CHRNA5 (nicotinic cholinergic receptor α5), SSTR5 (somatostatin receptor 5), KISS1R (kissin receptor), HTR1D (serotonin receptor 1D), and CCR8 (CC chemokine receptor 8) are receptor proteins that are differentially expressed in tumor cells and normal cells.

[0094] Table 1. Ligand names and amino acid sequence capture numbers

[0095]

[0096]

[0097] Example 2: Selection of viral ligands based on receptors

[0098] The inventors selected 16 homologous ligands of vesicular stomatitis virus and modeled and docked them with the 5 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-F and X03633.1-lig-FL, and the capture number of the corresponding amino acid sequence is DQ408670.1, GENEID:X03633.1.

[0099] Example 3: Construction and amplification of recombinant vesicular stomatitis virus based on different serotype proteins

[0100] 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.

[0101] The packaging methods for virus strains REV DQ408670.1, REV X03633.1, REV KP872888.1, and REV HQ593628.1 are as follows:

[0102] In vitro recombinant VSV requires: a full-length plasmid containing the viral genome (including the 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. Journal of Virology 85, 12781-12791, doi:10.1128 / JVI.00794-11 (2011), Brown, KS, Safronetz, D., Marzi, A., Ebihara, H. & Feldmann, H.).

[0103] 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.

[0104] Example 4: Detection of tumor cell receptors and cell killing results

[0105] In this embodiment, the killing effect of different viruses constructed in Example 3 on different tumor cells was verified.

[0106] 3.1 q-PCR detection:

[0107] 1×10⁻⁶ was extracted using the Trizol method. 6 BXPC3, HCT-8, HepG2, Su8686, H358, NCL-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.

[0108] The results are as follows Figure 5As shown in the figure, qPCR results indicated that the expression levels of the CHRNA5 receptor gene mRNA were high in BXPC3, HCT-8, HepG2, Su8686, H358, NCL-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 cell types.

[0109] 3.2 Cell killing assay (CCK):

[0110] Healthy BXPC3, HCT-8, HepG2, Su8686, 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.

[0111] Figure 6 The results of CCK killing of REV DQ408670.1 on different cell types were shown. CCK detection results showed that REV DQ408670.1 virus working solutions with MOI: 0.01, MOI: 0.1 and MOI: 1 all had significant killing effects on BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells.

[0112] Experimental results are as follows Figure 7 As shown in the figure. CCK test results showed that REV DQ408670.1 and REV X03633.1, in their viral working solutions at MOI: 0.01, MOI: 0.1, and MOI: 1, had significantly better killing effects on NCL-H358 and NCL-H460 cells than REVKP872888.1 and REV HQ593628.1. Simultaneously, combined with... Figure 5 The results showed that CCR8 and CHRNA5 expression levels were high in NCL-H358, while CHRNA5 and HTR1D expression levels were high in NCL-H460. Figure 3The receptor-ligand binding heatmap results showed that the DQ408670.1 and X03633.1 G proteins had strong binding affinity to the CCR8 and HTR1D receptors. This suggests 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.

[0113] Example 5: Results of tumor cell killing by viral strains with different combinations of L, N, P, and M proteins based on selected G proteins.

[0114] Cell killing assay (CCK):

[0115] Using antisense genetics, the inventors constructed the REV DQ408670.1 virus strain, REV DQ408670.1-V1, and REV DQ408670.1-V2 virus strain. REV DQ408670.1-V1 is based on the REV DQ408670.1 virus strain with modifications to the L and M proteins, while REV DQ408670.1-V2 is based on the REV DQ408670.1 virus strain with modifications to the N and P proteins.

[0116] Healthy H358 and H460 cells were divided into 5×10⁶ cells. 4 Cell suspension of cells / mL was added at 100 μL / well to a 96-well plate, with medium added to the edge to reduce evaporation, and incubated overnight. Three known viral strains were diluted with Opti-MEM to an MOI of 0.01 as working virus solution. The culture medium in the 96-well plate was aspirated, and 50 μL of the virus dilution was added to each well, with each dilution repeated three times. Three additional wells were prepared as blank controls using Opti-MEM. The medium was changed 2 hours after adding the virus dilution, and 100 μL of 1% FBS medium was added to each well. After 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.

[0117] The experimental results are shown in Table 2. The results of the three virus strains REV DQ408670.1, REV DQ408670.1-V1 and REVDQ408670.1-V2 were similar, and all of them had significant killing effects on H358 and H460 cells in the virus working solution with an MOI of 0.01.

[0118] Table 2: Inhibition rate of virus against tumor cells at MOI: 0.01 (%)

[0119]

[0120]

[0121] Example 6: Results of tumor cell killing based on selected G protein and viral strain with inserted exogenous gene.

[0122] Cell killing assay (CCK):

[0123] Using antisense genetics, the inventors inserted the heterologous gene INFβ into the constructed REV DQ408670.1 virus strain to construct the virus strain FJ-INFβ.

[0124] Healthy H358 and H460 cells were divided into 5×10⁶ 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. Known titers of REV DQ408670.1 and FJ-INFβ virus strains were diluted with Opti-MEM to MOI: 0.01, MOI: 0.1, and MOI: 1, respectively. The culture medium in the 96-well plates was aspirated, and 50 μL of virus dilution was added to each well, with each dilution replicated 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 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 values ​​using an OD450 microplate reader.

[0125] The results are as follows Figure 8 As shown, the REV DQ408670.1 virus strain was significantly more effective at killing H358 and H460 cells than FJ-INFβ.

[0126] Example 6: Detection of the killing effect of REV DQ408670.1 virus strain on normal cells

[0127] Cell killing assay (CCK):

[0128] BEAS-2B normal lung cells in good condition were processed into 5×10 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 cultured overnight. Known titer REV DQ408670.1 virus was 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 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 values ​​using an OD450 microplate reader.

[0129] Experimental results are as follows Figure 9As shown in the figure. CCK test results showed that the REV DQ408670.1 virus strain had no significant killing effect on BEAS-2B cells in virus working solutions with MOI: 0.01, MOI: 0.1 and MOI: 1.

[0130] The embodiments of the viral preparations of the present invention are described below.

[0131] Example 7

[0132] The viral preparation formulation is as follows: 5% sucrose by weight, 2 mmol / L magnesium chloride, Tris-HCl buffer (containing 50 mmol / L Tris, and adjusted to pH 7.5 with HCl), and a titer of 9.8 g TCID. 50 / mL of recombinant vesicular stomatitis oncolytic virus.

[0133] Comparative Example 1

[0134] The viral preparation formulation is: 0.01 mol / L phosphate dissolved in physiological saline solution, pH 7.2, and a titer of 9.2 g TCID. 50 / mL of recombinant vesicular stomatitis oncolytic virus.

[0135] Comparative Example 2

[0136] The viral preparation formulation is basically the same as that in Example 7, except that the sucrose content is 3% by weight.

[0137] Comparative Example 3

[0138] The viral preparation formulation is basically the same as that in Example 7, except that the sucrose content is 7% by weight.

[0139] Comparative Example 4

[0140] The viral preparation formulation is basically the same as that in Example 7, except that the magnesium chloride content is 0.5 mol / L.

[0141] Comparative Example 5

[0142] The viral preparation formulation is basically the same as that in Example 7, except that the magnesium chloride content is 3.5 mol / L.

[0143] Comparative Example 6

[0144] The viral preparation formulation is basically the same as that in Example 7, except that the pH of the preparation is adjusted to 7.0 with HCl using Tris-HCl buffer.

[0145] Comparative Example 7

[0146] The viral preparation formulation is basically the same as that in Example 7, except that the pH of the preparation is adjusted to 8.0 with HCl using Tris-HCl buffer.

[0147] Experimental Example 1

[0148] The viral preparations of Examples 7 and Comparative Examples 1-7 were subjected to storage stability tests at 2-8°C. Viral titers were measured after 0, 3, 5, 7, 14, 21, 28, 35, and 42 days of storage. The test results for Examples 7 and Comparative Examples 1-7 are shown in Table 3 and... Figure 10 As shown.

[0149] Table 3. Virus titer detection results at 2–8℃ (lgTCID) 50 / mL)

[0150]

[0151] Experimental Example 2

[0152] The viral preparations from Examples 7 and Comparative Examples 1-7 were subjected to storage stability tests at 25±2℃. Viral titers were measured after 0, 3, 5, 7, 14, 21, 28, 35, and 42 days of storage. The test results for Examples 7 and Comparative Examples 1-7 are shown in Table 4. Figure 11 As shown.

[0153] Table 4. Virus titer detection results at 25±2℃ (lgTCID) 50 / mL)

[0154]

[0155]

[0156] Experimental Example 3

[0157] The viral preparations of Examples 7 and Comparative Examples 1-7 were subjected to storage stability tests at -60°C. Viral titers were measured after 0 days, 28 days, and 60 days of storage. The test results for Examples 7 and Comparative Examples 1-7 are shown in Table 5. Figure 12 As shown in the figure. The results show that the virus preparation of Example 7 has good stability under low temperature freezing conditions, and still has a high virus titer after 42 days of storage.

[0158] Table 5. Virus titer detection results at -60℃ (lgTCID) 50 / mL)

[0159]

[0160] Test Example 4

[0161] The viral preparations of Example 7 and Comparative Examples 1-7 were subjected to repeated freeze-thaw stability tests. Viral titers were measured after one, two, three, four, and five freeze-thaw cycles. The test results for Example 7 and Comparative Examples 1-7 are shown in Table 6. Figure 13 As shown.

[0162] Table 6. Virus titer detection results at different thaw cycles (lgTCID) 50 / mL)

[0163]

[0164] Results Discussion

[0165] For the viral preparations formulated in Example 7 and Comparative Examples 1-7, after the above test examples, the viral titer decreased by no more than 0.5 lg TCID compared to the initial labeled amount. 50 / mL, which is considered to be within the acceptable range for viral titer reduction.

[0166] The results of Experiments 1 and 2 show that, at temperatures of 2–8°C and 25±2°C, the viral titers in the viral preparations of Examples 7 and Comparative Examples 1–7 decreased with prolonged storage time. However, the viral preparation of Example 7 maintained its viral titer within the acceptable range for a longer period. The viral preparations of Comparative Examples 4 and 5 showed a greater decrease in viral titer due to excessively low or high magnesium chloride content.

[0167] The results of Experiment 3 show that the viral titers of the formulations in Examples 7 and Comparative Examples 1-3, 6 and 7 remained essentially unchanged after being placed at -60°C for 60 days, while the viral titers of the formulations in Comparative Examples 4 and 5 decreased significantly.

[0168] The results of Experiment 4 show that the viral titer of the virus preparation in Example 7 remained essentially unchanged with increasing thaw cycles, and after 5 thaw cycles, the viral titer was essentially the same as that of the unthawed virus. In Comparative Example 1, the viral titer of the virus preparation showed a decreasing trend with increasing thaw cycles, decreasing by approximately 90% compared to the unthawed virus titer after 5 thaw cycles. In Comparative Examples 2 and 3, the viral titers decreased significantly after repeated thaw cycles due to excessively low or high sucrose content in the formulations.

[0169] In addition, for the viral preparations formulated in Comparative Examples 6 and 7, the pH values ​​were too low or too high, which had a significant impact on the viral structure and activity. As a result, the viral titers of the viral preparations formulated in Comparative Examples 6 and 7 decreased significantly in Experiments 1 to 4.

[0170] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is 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.

[0171] 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 viral agent in the preparation of a drug for the treatment or prevention of tumors; The viral preparation consists of virus, sucrose, magnesium chloride, and Tris-HCl buffer solution; The virus is a recombinant oncolytic virus; The recombinant oncolytic virus expresses a viral protein with high affinity for cell receptors; The cell receptor includes at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8; The tumor cells of the tumor contain the cell receptor; The viral protein is selected from SEQ ID NO: 1, and the tumor is selected from lung cancer; or, the viral protein is selected from SEQ ID NO: 2, and the tumor is selected from adenocarcinoma, colon cancer, liver cancer, or lung cancer; The pH of the Tris-HCl buffer solution is 7.2~7.6; Based on the total amount of the viral agent, the sucrose content is 4.5-5.5% by weight, and the magnesium chloride content is 1.5-2.5 mmol / L; The recombinant oncolytic virus is a vesicular stomatitis virus.

2. The use according to claim 1, characterized in that, The ZDOCK score of the binding force between the viral protein and the cell receptor is not less than 1800.

3. The use according to claim 1, characterized in that, The recombinant oncolytic virus further expresses at least one of the following: nucleoproteins, phosphoproteins, matrix proteins, and RNA-dependent RNA polymerases.

4. The use according to claim 1, characterized in that, The concentration of Tris in the Tris-HCl buffer solution is 50 mmol / L.

5. The use according to claim 1, characterized in that, Based on the total amount of the viral preparation, the sucrose content is 5% by weight and the magnesium chloride content is 2 mmol / L.

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