WO2023020556A1 - 病毒制剂、用于配制病毒制剂的溶液及其用途 - Google Patents

病毒制剂、用于配制病毒制剂的溶液及其用途 Download PDF

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WO2023020556A1
WO2023020556A1 PCT/CN2022/113153 CN2022113153W WO2023020556A1 WO 2023020556 A1 WO2023020556 A1 WO 2023020556A1 CN 2022113153 W CN2022113153 W CN 2022113153W WO 2023020556 A1 WO2023020556 A1 WO 2023020556A1
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virus
cancer
virus preparation
solution
tris
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PCT/CN2022/113153
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French (fr)
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吴可行
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上海行深生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/766—Rhabdovirus, e.g. vesicular stomatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the invention relates to the field of biomedicine, in particular, the invention relates to virus preparations, solutions for preparing virus preparations and uses thereof.
  • oncolytic viruses In the past ten years, the mechanism by which oncolytic viruses can kill tumors by inducing the body's anti-tumor immune response has gradually become clear. Since German scientist Jean Rommelaere first called oncolytic virus therapy as tumor immunotherapy in 2011, oncolytic virus has been accepted by the public as an important branch of tumor immunotherapy. Compared with other tumor immunotherapies, oncolytic viruses have the advantages of high killing efficiency, good targeting, small side effects, multiple ways to kill tumors to avoid drug resistance, and low cost.
  • the approved T-vec has knocked out the ⁇ 34.5 gene of HSV-1 (herpes simplex virus type 1), the expression product of the ⁇ 34.5 gene can inhibit the virus clearance mechanism of normal cells, and the ⁇ 34.5 gene knockout After that, the virus will not be able to replicate in normal cells; while cancer cells will lose the virus clearance mechanism, therefore, the knockout of the ⁇ 34.5 gene will not affect the replication of the virus in cancer cells.
  • JX594 (Pexa-Vec), which is currently in phase III clinical trials, has knocked out the TK (thymidine kinase) gene of vaccinia viruses.
  • CG0070 adds the E2F-1 promoter before the gene E1A responsible for the replication of the adenovirus.
  • the E2F-1 promoter is regulated by the retinoblastoma suppressor protein (Rb), and Rb is missing in bladder cancer. Therefore, the Rb The deletion can activate the transcriptional activity of E2F-1, so that the E1A gene is expressed in bladder cancer cells, and the virus can also specifically replicate in bladder cancer cells.
  • Reolysin is an unmodified wild-type reovirus whose proliferation depends on the activation of Ras signaling pathway, so it can only proliferate specifically in Ras-activated cancer cells.
  • tumor-killing specificity that is, compared with normal non-tumor cells, specifically killing tumor cells
  • broad spectrum of tumor treatment that is, At the same time applicable to a variety of tumor treatment
  • an object of the present invention is to propose a recombinant oncolytic virus preparation with high tumor cell specificity and/or broad-spectrum tumor treatment.
  • the invention proposes a viral preparation.
  • the virus preparation contains: virus; sucrose; magnesium chloride.
  • the virus preparation contains Tris-HCl buffer, and the pH is 7.2-7.6. Therefore, the virus preparation has little change in virus titer during storage and has excellent storage stability.
  • virus preparations according to the above-mentioned embodiments of the present invention can also have the following additional technical features:
  • the virus is a recombinant oncolytic virus.
  • the recombinant oncolytic virus is vesicular stomatitis virus.
  • the recombinant oncolytic virus expresses a viral protein with high affinity for cell receptors, and 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 to (a) or (b). Therefore, the virus preparation has high tumor cell specificity and/or broad-spectrum tumor treatment.
  • the viral protein comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% homology to (a) or (b).
  • the ZDOCK score of the binding force between the viral protein and the cell receptor is not less than 1800.
  • the cell receptor includes at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D, and CCR8.
  • the recombinant oncolytic virus further expresses at least one selected from the group consisting of nucleoprotein, phosphoprotein, matrix protein and RNA-dependent RNA polymerase.
  • the virus preparation contains: 4.5-5.5% by weight of sucrose; and 1.5-2.5 mmol/L of magnesium chloride.
  • the concentration of Tris in the Tris-HCl buffer is 50mmol/L.
  • the virus preparation contains: 5% by weight of sucrose; and 2 mmol/L of magnesium chloride.
  • the virus preparation is in a form suitable for administration by inhalation or injection.
  • the invention proposes a solution for formulating a virus preparation.
  • the solution for preparing the virus preparation contains: sucrose; magnesium chloride; Tris-HCl buffer solution, and the pH of the solution is 7.2-7.6.
  • the virus preparation prepared from the solution has little change in virus titer during storage and has excellent storage stability.
  • solution for preparing virus preparations according to the above-mentioned embodiments of the present invention may also have the following additional technical features:
  • the solution for preparing the virus preparation contains 4.5-5.5% by weight of sucrose; and 1.5-2.5 mmol/L of magnesium chloride.
  • the solution for preparing the virus preparation contains 5% by weight of sucrose; and 2mmol/L of magnesium chloride.
  • the concentration of Tris in the Tris-HCl buffer is 50mmol/L.
  • the present invention proposes the use of the virus preparation of the above-mentioned embodiment or the solution for preparing the virus preparation of the above-mentioned embodiment in the preparation of medicine, and the medicine is used for treating or preventing cancer or tumor.
  • the cancer or tumor includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer and leukemia.
  • the present invention proposes a method of preventing or treating cancer or tumor.
  • the method includes: applying the above virus preparation or the above solution to the subject.
  • the cancer or tumor includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer and leukemia.
  • Figure 1 shows a flowchart of the analysis of human membrane receptor genes based on a large sample of tumor tissue.
  • Figure 2 shows the jittered scatterplot of the proportion of patients whose corresponding receptor genes were significantly upregulated in each tumor.
  • Figure 3 shows the ZDOCK score results reflecting the binding strength of candidate ligands to tumor-specific receptors.
  • Fig. 4 shows a diagram of the experimental results of screening ligands based on the screened receptors.
  • Figure 5 shows the mRNA expression levels of CHRNA5, KISS1R, HTRID, CCR8 and SSTR5 in BXPC3, HCT-8, HepG2, Su8686, H358, NCL-H460 and PANC1 cell samples detected by qPCR.
  • Figure 6 shows the killing effect of the virus on BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells under different MOI conditions measured in the cell killing experiment.
  • Figure 7 shows the killing effects of virus strains with different G proteins on NCL-H358 and NCL-H460 cells at different MOIs.
  • Figure 8 shows the killing effect of the virus strain inserted with the heterologous gene on NCL-H358 and NCL-H460 cells.
  • Figure 9 shows the experimental results of the REV DQ408670.1 virus strain being safe to normal cells.
  • Fig. 10 shows the stability test results of the virus preparations of Example 7 and Comparative Examples 1-7 at 2-8°C.
  • Figure 11 shows the results of the stability test at 25 ⁇ 2°C for the virus preparations of Example 7 and Comparative Examples 1-7.
  • Figure 12 shows the results of the stability test at -60°C of the virus preparations of Example 7 and Comparative Examples 1-7.
  • FIG. 13 shows the results of the repeated freezing stability test of Example 7 and Comparative Examples 1-7.
  • Embodiments of the present invention are described in detail below.
  • the embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
  • the invention proposes a viral preparation.
  • the virus preparation contains: virus; sucrose; magnesium chloride.
  • the virus preparation contains Tris-HCl buffer, and the pH is 7.2-7.6.
  • the reason may be that sucrose and magnesium chloride can increase the protection of viruses, stabilize the virus structure, and play a role in regulating osmotic pressure; in addition, compared with other common buffers, Tris-HCl buffer has little interference with biochemical processes, Adjust pH over a wider range.
  • the virus in the preparation is relatively stable in the range of pH 7.2-7.6, and the pH of the preparation buffer is adjusted to 7.2-7.6 to better stabilize the virus.
  • the virus preparation contains: 4.5-5.5% by weight of sucrose; and 1.5-2.5 mmol/L of magnesium chloride.
  • the virus preparation based on the total amount of the virus preparation, contains: 5% by weight of sucrose; and 2 mmol/L of magnesium chloride.
  • the storage stability of the viral preparation is better.
  • the concentration of Tris in the above-mentioned Tris-HCl buffer solution is 50 mmol/L.
  • the storage stability of the viral preparation is better.
  • the inventors have found that if the sucrose content in the virus preparation is too high or too low, the virus stability after repeated freezing and thawing may be reduced; Virus stability at ⁇ 8°C and ambient temperature (25°C); if the pH in the virus preparation is too high or too low, it may destroy the virus structure and reduce the virus activity.
  • the above virus is a recombinant oncolytic virus.
  • the recombinant oncolytic virus of the present invention has good stability in the virus preparation, and the virus titer in the preparation changes little after being stored at different temperatures for a long time or after repeated thawing.
  • the above-mentioned recombinant oncolytic virus is vesicular stomatitis virus.
  • the vesicular stomatitis virus of the present invention has good stability in the virus preparation, and the virus titer in the preparation changes little after being stored at different temperatures for a long time or after repeated thawing.
  • the above-mentioned recombinant oncolytic virus expresses a viral protein with high affinity to the cell receptor, and 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 to (a) or (b). Therefore, the virus preparation has high tumor cell specificity and/or broad-spectrum tumor treatment.
  • the recombinant vesicular stomatitis virus expressing the above-mentioned viral protein has a stronger specific targeting ability to tumor cells, a broader spectrum of tumor killing, and a more significant killing effect.
  • homology refers to the similarity of the amino acid sequence, and the difference of individual amino acids in the amino acid sequence does not affect the function of the protein itself.
  • homologous amino acid sequence refers to an amino acid sequence derived from a single or multiple amino acid substitutions, deletions, or additions in the amino acid sequence of a polypeptide. Specifically, “having a certain percentage of sequence homology” described in this application is calculated by the following formula:
  • the number of amino acids in the reference amino acid sequence refers to the number of amino acid sequences being compared, as described in "Protein G has at least 80% sequence homology with any of SEQ ID NO: 1 or SEQ ID NO: 2"
  • the reference amino acid sequence in is SEQ ID NO:1 or SEQ ID NO:2.
  • homologous amino acid sequences are biologically, chemically or structurally similar, and have similar biological activities.
  • Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine, or serine, or have side chains of similar size.
  • Chemical similarity means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example hydrophobic residues isoleucine, valine, leucine or methionine are substituted for each other. Or polar amino acids are substituted for each other, such as arginine for lysine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, and the like.
  • Biological similarity means that amino acid sequences with sequence homology are similar in biological function.
  • the recombinant vesicular stomatitis virus according to the embodiment of the present invention has high affinity and binding force with broad-spectrum and specificity for tumors. .
  • VSV Vesicular stomatitis virus
  • VSV-NJ New Jersey type
  • VSV-IND Indiana type
  • Virus particles are bullet-shaped or cylindrical, with a size of 150-180nm ⁇ 50-70nm.
  • the virus has a capsule, and the capsule is evenly and densely covered with fibrous processes with a length of about 10nm.
  • Inside the virus is a tightly coiled helical symmetrical nucleocapsid.
  • the virus is named for the classic vesicular lesions in the oral mucosa, tooth pads, tongue, lips, nostrils, hooves, and teats of affected animals.
  • Transmitted by an insect vector the disease is restricted to its natural hosts, such as horses, cattle, and pigs. In humans, infection is mild and asymptomatic.
  • the VSV genome is a non-segmented single-stranded negative-sense RNA (ssRNA) virus with a length of about 11 KB.
  • ssRNA single-stranded negative-sense RNA
  • N, NS, M, G, and L five non-overlapping genes, are arranged in sequence, encoding nuclear (N) protein, phosphate (P) protein, matrix (M) protein, sugar (G ) protein and RNA-dependent RNA polymerase (L) protein and other 5 different proteins.
  • the 3' end of the N gene is a leader sequence (Leader), the 5' end is a trailing sequence (Trailor), and there is a spacer sequence between each gene.
  • the 3' leader RNA is the earliest viral transcript in infected cells, with a length of 47 nucleotides. It is not capped or translated. Its function is not yet fully understood, and it may inhibit the synthesis of host RNA.
  • the N protein is necessary to initiate gene synthesis and effectively protects viral RNA from digestion by various nucleases. N protein has high antigenic immunogenicity, stimulates the body to produce non-neutralizing antibody cellular immunity, and plays an important role in transcription and replication. It may be necessary to maintain the extended form of genomic RNA and is related to replication regulation.
  • the homology of P protein, VSV-NJ and VSV-IND virus strain is 41%, and its role is to form a polymerase complex with polymerase L, nucleoprotein N and genomic RNA to maintain the transcriptional activity of the virus.
  • the M protein plays a key role in viral pathogenesis and viral replication, is rich in basic amino acids, and contains a highly basic amino-terminal domain, which can inhibit transcription by binding to the nucleocapsid and help virus budding from the host , is the only polypeptide involved in the budding process.
  • G protein is the main surface antigen of the virus, which determines the virulence of the virus and is also the protective antigen of the virus. It stimulates the body to produce neutralizing antibodies.
  • RNA poly E protein which may determine the transcriptional activity of RNA, and binds to P protein to catalyze the replication of mRNA.
  • This protein is a central component of the polymerase complex and replicase complex, involved in initiation, elongation, methylation, capping, poly(A) tail formation, and more.
  • the conserved sequence between these genes is a key signal to affect the activity of the polymerase or the cleavage activity of the enzyme, and during the replication process, these signals are masked and do not work.
  • the terms "recombinant VSV virus”, “recombinant vesicular stomatitis virus” and “recombinant virus of the present invention” are used interchangeably, and refer to the recombinant VSV virus capable of specifically infecting tumor cells as described above , the recombinant VSV virus specifically infects tumor cells, and the recombinant VSV virus specifically binds to specific receptors CHRNA5, SSTR5, KISS1R, HTR1D and CCR8 of tumor cells selected from the group below.
  • the viral protein comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% homology to any of SEQ ID NO:1 or SEQ ID NO:2 .
  • the recombinant vesicular stomatitis virus does not carry heterologous genes.
  • the term "heterologous gene" described herein refers to a gene that has not been reported in wild-type vesicular stomatitis virus unless otherwise specified. Or in other words, the proteins encoded in the recombinant vesicular stomatitis virus are all expressed in the wild-type vesicular stomatitis virus.
  • the ZDOCK score of the binding force between the viral protein and the cell receptor is not less than 1800.
  • the binding force characterization parameter ZDOCK score between the viral protein and the cellular receptor can be easily obtained.
  • ZDOCK score can be determined by conventional software, for example, see Pierce BG, Hourai Y, Weng Z. (2011) Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library.PLoS One 6(9):e24657 ..
  • the above-mentioned viral protein includes at least one selected from the G protein with GenBank accession number X03633.1 and the G protein with GenBank accession number DQ408670.1.
  • the inventors of the present invention unexpectedly found that the G protein with the GenBank accession number X03633.1 and the G protein with the GenBank accession number DQ408670.1 have a significantly stronger binding force to the receptor of tumor cells than other G proteins.
  • the recombinant vesicular stomatitis virus further expresses at least one selected from the group consisting of nucleoprotein, phosphoprotein, matrix protein and RNA-dependent RNA polymerase.
  • the inventors unexpectedly found that the recombinant virus constructed by combining these proteins with at least one of the G protein with the GenBank accession number X03633.1 and the G protein with the GenBank accession number DQ408670.1 has stronger tumor killing active. The inventors believe that it may be because for tumor cells, protein combinations from various sources may trigger different immune responses from protein combinations from the same source, thereby further enhancing the killing effect on tumor cells.
  • 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 encoding the RNA-dependent RNA polymerase nucleic acid molecules
  • said nucleic acid molecule encoding said nucleoprotein, said nucleic acid molecule encoding said phosphoprotein, said nucleic acid molecule encoding said matrix protein and said nucleic acid molecule encoding said RNA-dependent RNA polymerase At least one of the is derived from a Mudd summer subtype strain of vesicular stomatitis virus.
  • the inventors unexpectedly found that the recombinant virus constructed by combining these proteins with at least one of the G protein with the GenBank accession number X03633.1 and the G protein with the GenBank accession number DQ408670.1 has stronger tumor killing active. The inventors believe that it may be because for tumor cells, protein combinations from various sources may trigger different immune responses from protein combinations from the same source, thereby further enhancing the killing effect on tumor cells.
  • the virus preparation is in a form suitable for administration by inhalation or injection.
  • the invention proposes a solution for formulating a virus preparation.
  • the solution for preparing the virus preparation contains: sucrose; magnesium chloride; Tris-HCl buffer solution, and the pH of the solution is 7.2-7.6.
  • the virus preparation prepared from the above-mentioned solution has a small change in the virus titer in the preparation after being stored at different temperatures for a long time or after repeated thawing, showing excellent storage stability.
  • the reason may be that sucrose and magnesium chloride can stabilize the virus structure and enhance the protection of the virus; compared with other common buffers, the Tris-HCl buffer can adjust the pH range wider and better stabilize the pH of the virus preparation; the virus in the preparation
  • the pH range of 7.2-7.6 is relatively stable, and the virus can be better stabilized by adjusting the pH of the preparation buffer to 7.2-7.6.
  • the above-mentioned solution for preparing the virus preparation contains 4.5-5.5% by weight of sucrose; and 1.5-2.5 mmol/L of magnesium chloride.
  • the above-mentioned solution for preparing the virus preparation contains 5% by weight of sucrose; and 2 mmol/L of magnesium chloride.
  • sucrose sucrose
  • magnesium chloride magnesium chloride
  • the concentration of Tris in the above-mentioned Tris-HCl buffer solution is 50 mmol/L.
  • the storage stability of the virus preparation prepared from the solution is better.
  • the present invention proposes the use of the above-mentioned pharmaceutical composition or the above-mentioned recombinant vesicular stomatitis virus in the preparation of medicines for treating or preventing cancer or tumors.
  • the cancer or tumor includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer and leukemia.
  • the present invention proposes a method for preventing or treating cancer or tumor.
  • the method includes: applying the above virus preparation or the above solution to the subject.
  • the cancer or tumor includes at least one selected from lung cancer, gastric cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer and leukemia.
  • a method of analyzing human membrane receptor genes based on a large tumor tissue sample will be described in detail below with reference to FIG. 1 .
  • the present invention summarizes the receptor gene information expressed in human cells from the existing research (reference: (Synchronous birth is a dominant pattern in receptor-ligand evolution, BMC Genomics. Grandchamp and Monget, 2018 Aug14; 19( 1): 611.).
  • the inventor downloaded the gene expression matrix (normalized value), gene mutation information and relevant clinical data of cancer patients from UCSC Xena ( http://xena.ucsc.edu/ ).
  • cancer types are: adrenocortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, colon adenocarcinoma/Rectum adenocarcinoma esophageal carcinoma, lymphatic Tumor-like diffuse large B-cell lymphoma, esophageal cancer, FFPE trial phase II, glioblastoma, glioma, squamous cell carcinoma of the head and neck, renal chromosome, pan-renal cohort (KICH+KIRC+KIRP) , Renal clear cell carcinoma, Renal papillary cell carcinoma, Acute myeloid leukemia, Brain low-grade glioma, Hepatocellular carcinoma, Lung adenocarcinoma, Lung squamous cell carcinoma, Mesothelioma, Ovarian se
  • the inventor firstly removed less than three sample tumors and normal tissue information from the downloaded data, and then performed differential expression analysis.
  • the inventors used limma software (version: 3.38.3) to perform differential expression analysis (reference: (Limma Powers Differential Expression Analyzes for RNA-Sequencing and Microarray Studies. Nucleic Acids Research, 43, e47, Ritchie, M.E., et al. (2015)).
  • the voom model of the limma R package was used in the analysis. Only when the gene meets the standard
  • the inventors selected genes that were significantly up-regulated in more than or equal to 70% of cancer samples (ie, genes with log2FC ⁇ 2.0) in intestinal cancer, lung cancer, pancreatic cancer, gastric cancer, and liver cancer. More than 10 receptors were selected under the condition of high expression level.
  • the inventor used ggplot2 and ggbeeswarm software to draw a jitter scatter plot (as shown in Figure 2) for the log2FC value of each gene in different tumor samples, to show the proportion of patients whose gene was significantly upregulated in each tumor .
  • the inventors carried out molecular docking of the 13 screened receptors with candidate ligands, and selected the 5 receptors with the best binding force as the final selection.
  • the inventor selected 16 homologous ligands of vesicular stomatitis virus, and carried out modeling and docking with the 5 tumor-specific receptors screened in Example 1 respectively.
  • the generated docking results will be sorted according to the ZDOCK score score, and the score The higher the value, the stronger the binding and the higher the reliability of the result.
  • the clustering results of these conformations were comprehensively analyzed, and it was found that the ZDOCK score is the shape complementarity score calculated by the ZDOCK program. According to the parameter settings, the ZDOCK score will also include electrostatic and desolvation energy items. The higher the ZDOCK score, the better.
  • the inventors used the ZDOCK score function to evaluate the binding strength, and obtained ligands with strong binding ability to tumor-specific receptors (results shown in Figure 4).
  • the ligand with the best binding effect was DQ408670.
  • 1-lig-F and X03633.1-lig-FL the capture number of the corresponding amino acid sequence is DQ408670.1, GENE ID: X03633.1.
  • Example 3 Construction and amplification of genetically recombinant vesicular stomatitis virus based on different serotype proteins
  • the inventor combined L, N, P, and M proteins derived from the Mudd summer subtype virus strain to combine and capture the sequence numbers as GENE ID: DQ408670.1, GENE ID: X03633.1, GENE ID: KP872888.1 or GENE ID: The G protein of HQ593628.1 was used to construct recombinant vesicular stomatitis viruses REV DQ408670.1, REV X03633.1, REV KP872888.1 and REV HQ593628.1.
  • the packaging method for virus strains REV DQ408670.1, REV X03633.1, REV KP872888.1 and REV HQ593628.1 is as follows:
  • VSV In vitro recombination of VSV requires: a full-length plasmid (including G protein) containing the viral genome, and an auxiliary plasmid (N, P, L, M) of the backbone protein required for viral packaging, and the plasmid is transferred to BHK21 by in vitro transfection In the cell, the virus is assembled and matured in the cell and then buds out to the outside of the cell (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, K.S., Safronetz, D., Marzi, A., Ebihara, H. & Feldmann, H.).
  • Virus amplification uses Vero cells, and a certain titer of virus is added to the cultured Vero cells. The virus can infect the cells and complete self-replication in the cells. The mature virus is released into the cell culture supernatant, and the cells are cultured. The supernatant was concentrated to obtain a virus concentrate, which can be used in subsequent experiments after titer determination.
  • Example 3 different viruses constructed in Example 3 were used to verify the killing effect on different tumor cells.
  • Extract 1 ⁇ 10 6 BXPC3, HCT-8, HepG2, Su8686, H358, NCL-H460 (H460) and PANC1 cell samples by Trizol method perform reverse transcription of 20 ⁇ L system with 500ng/ ⁇ L RNA, and use SYBR GREEN method Real-time quantitative PCR was used to detect the mRNA expression of CHRNA5, KISS1R, HTRID, CCR8 and SSTR5 genes in 7 cell samples.
  • the medium was changed 2 hours after the virus dilution was added, and 100 ⁇ L of 1% FBS medium was added to each well. After 48/72 hours, add 10 ⁇ L of CCK8 detection solution to each well, incubate at 37°C for 2 hours, and then read the OD450 microplate reader.
  • FIG. 6 shows the CCK killing results of REV DQ408670.1 on different cells.
  • the CCK test results showed that the REV DQ408670.1 virus working solutions with MOI: 0.01, MOI: 0.1 and MOI: 1 were all effective against BXPC3, HCT-8, HepG2, Su8686, H358 and PANC1 cells have significant killing effect.
  • 1G protein has a strong binding force with CCR8 and HTR1D receptors. It has been comprehensively reflected that when the recombinant vesicular stomatitis virus has a high binding force to the tumor cell receptor, the killing effect of the recombinant virus on the tumor cell that highly expresses the receptor is more significant.
  • Example 5 Based on the selected G protein, the virus strains with different combinations of L, N, P, and M have the effect of killing tumor cells
  • CNK Cell Killing Assay
  • the inventor constructed REV DQ408670.1 virus strain, REV DQ408670.1-V1 and REV DQ408670.1-V2 virus strain, wherein, REV DQ408670.1-V1 is in REV DQ408670.1 virus strain
  • the L and M proteins were changed on the basis of the REV DQ408670.1-V2 strain, while the N and P proteins were changed on the basis of the REV DQ408670.1 strain.
  • the H358 and H460 cells in good condition were made into a cell suspension of 5 ⁇ 104 /mL and added to a 96-well plate at 100 ⁇ L/well, and the edge filling medium was reduced to reduce evaporation, and cultured overnight.
  • Example 6 Killing results of tumor cells based on selected G protein and virus strains inserted with exogenous genes
  • CNK Cell Killing Assay
  • the inventor inserted the heterologous gene INF ⁇ into the constructed REV DQ408670.1 virus strain to construct the virus strain FJ-INF ⁇ .
  • the H358 and H460 cells in good condition were made into a cell suspension of 5 ⁇ 104 /mL and added to a 96-well plate at 100 ⁇ L/well, and the edge filling medium was reduced to reduce evaporation, and cultured overnight.
  • the medium was changed 2 hours after the virus dilution was added, and 100 ⁇ L of 1% FBS medium was added to each well. After 72 hours, 10 ⁇ L of CCK8 detection solution was added to each well, and after incubation at 37°C for 2 hours, the OD450 microplate reader was read.
  • CNK Cell Killing Assay
  • the normal lung cells BEAS-2B in good condition were made into a cell suspension of 5 ⁇ 10 4 cells/mL and added to a 96-well plate at 100 ⁇ L/well, and the edge filling medium was reduced to reduce evaporation, and cultured overnight.
  • the medium was changed 2 hours after the virus dilution was added, and 100 ⁇ L of 1% FBS medium was added to each well. After 72 hours, 10 ⁇ L of CCK8 detection solution was added to each well, and after incubation at 37°C for 2 hours, the OD450 microplate reader was read.
  • Viral preparation prescription is: 5% by weight sucrose, 2mmol/L magnesium chloride, Tris-HCl buffer solution (containing 50mmol/L Tris, and adjust preparation pH to 7.5 with HCl), and titer is 9.8lgTCID 50 /mL recombinant vesicular oral cavity Inflammatory oncolytic virus.
  • the virus preparation prescription is: 0.01mol/L phosphate-washed normal saline solution, pH 7.2, and recombinant vesicular stomatitis oncolytic virus with a titer of 9.2lgTCID 50 /mL.
  • the prescription of virus preparation is basically the same as that of Example 7, the difference is that the sucrose content is 3% by weight.
  • the prescription of virus preparation is basically the same as that of Example 7, the difference is that the sucrose content is 7% by weight.
  • the virus preparation prescription is basically the same as that of Example 7, the difference being that the content of magnesium chloride is 0.5 mol/L.
  • the prescription of virus preparation is basically the same as that of Example 7, the difference is that the content of magnesium chloride is 3.5mol/L.
  • the prescription of the virus preparation is basically the same as in Example 7, the difference is that the Tris-HCl buffer uses HCl to adjust the pH of the preparation to 7.0.
  • the prescription of the virus preparation is basically the same as in Example 7, the difference is that the Tris-HCl buffer uses HCl to adjust the pH of the preparation to 8.0.
  • Example 7 and Comparative Examples 1 to 7 Take the virus preparations of Example 7 and Comparative Examples 1 to 7, and carry out the storage stability test at 2 to 8°C, and detect the virus drops after placing 0d, 3d, 5d, 7d, 14d, 21d, 28d, 35d, and 42d respectively. Degree, wherein, the test results of Example 7 and Comparative Examples 1 to 7 are shown in Table 3 and Figure 10.
  • Example 7 and Comparative Examples 1 to 7 Take the virus preparations of Example 7 and Comparative Examples 1 to 7, and carry out the storage stability test at 25 ⁇ 2°C, and detect the virus drops after placing 0d, 3d, 5d, 7d, 14d, 21d, 28d, 35d, and 42d respectively. Degree, wherein, the test results of Example 7 and Comparative Examples 1 to 7 are shown in Table 4 and Figure 11.
  • Example 7 and Comparative Examples 1-7 Take the virus preparations of Example 7 and Comparative Examples 1-7, carry out the storage stability test at -60°C, and detect the virus titer after placing them for 0d, 28d, and 60d respectively, wherein, Example 7 and Comparative Examples 1-7
  • the test results are shown in Table 5 and Figure 12. It can be seen from the results that under low-temperature freezing conditions, the virus preparation of Example 7 has better stability, and still has a higher virus titer after 42 days of storage.
  • Example 7 and Comparative Examples 1 to 7 were subjected to repeated thawing stability tests, and the virus titers were detected after thawing once, 2, 3, 4, and 5 times respectively, wherein, Example 7 and Comparative Example 1
  • the test results of ⁇ 7 are shown in Table 6 and Figure 13.
  • the virus titer decreased by no more than 0.5 lgTCID 50 /mL compared with the initial labeled amount, and the virus titer was considered to be within the acceptable range.
  • Example 7 From the results of Test Example 1 and Test Example 2, it can be seen that at a temperature of 2-8°C and 25 ⁇ 2°C, as the storage time prolongs, the virus in the virus preparations prescribed in Example 7 and Comparative Examples 1-7 Titers showed a downward trend. However, the virus preparation prescribed in Example 7 can keep the virus titer falling within the acceptable range for a longer period of time. For the virus preparations prescribed in Comparative Examples 4 and 5, the virus titer decreased greatly due to the low or high content of magnesium chloride.
  • Example 7 As can be seen from the results of Test Example 3, the virus preparations prescribed in Example 7 and Comparative Examples 1 to 3, 6 and 7 were placed at a temperature of -60°C for 60 days, and the virus titer did not change substantially, while the virus preparations prescribed in Comparative Examples 4 and 5 Prescription viral titers were significantly lower.
  • the virus titer of the prescription in Example 7 has basically no change with the increase of the number of times of repeated thawing, and the virus titer is basically the same as the unthawed virus titer after 5 times of repeated thawing. same.
  • the virus titer showed a downward trend as the number of times of repeated thawing increased, and after 5 times of repeated thawing, the virus titer decreased by about 90% compared with the unthawed virus titer.
  • the virus preparations prescribed in Comparative Examples 2 and 3 because the sucrose content in the prescriptions was too low or too high, the virus titers of the preparations dropped significantly after repeated thawing.

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Abstract

提供一种病毒制剂、用于配制病毒制剂的溶液及其用途。其中，病毒制剂含有：病毒；蔗糖；氯化镁。其中，所述病毒制剂含有Tris-HCl缓冲液，并且pH为7.2～7.6。

Description

病毒制剂、用于配制病毒制剂的溶液及其用途 技术领域
本发明涉及生物医药领域，具体的，本发明涉及病毒制剂、用于配制病毒制剂的溶液及其用途。
背景技术
近十几年，溶瘤病毒可以通过诱导机体的抗肿瘤免疫反应来杀伤肿瘤的机制逐渐明确。自2011年德国科学家Jean Rommelaere第一次将溶瘤病毒疗法称作肿瘤免疫治疗以来，溶瘤病毒目前已经被大众接受作为肿瘤免疫治疗的重要分支。相较于其他肿瘤免疫疗法，溶瘤病毒具有杀伤效率高、靶向性好、副作用小、多种杀伤肿瘤途径避免耐药性和成本低廉等优势。
由于病毒基因组小，比较容易通过基因工程手段进行多种改造，且可以通过常规手段进行病毒的改造和包装，这些技术比较成熟，成本也比较低。因此，容易通过利用溶瘤病毒自身的特征以及利用癌细胞和正常细胞的差异来对溶瘤病毒进行改造，以实现特异性靶向癌细胞。
由于大部分癌细胞自身清除病毒的机制受损(如正常细胞清除病毒的关键因子蛋白激酶R(PKR)在癌细胞中缺失)，所以病毒更容易在癌细胞中复制扩散。另外，近几十年，随着研究的不断深入，科学工作者们利用癌细胞和正常细胞中很多信号通路和代谢等的不同，通过筛选特定的病毒品种和改造病毒基因组，不断提高溶瘤病毒对肿瘤的靶向性，降低其对正常细胞的危害，提高安全性。例如：已获批的T-vec敲除了HSV-1(单纯疱疹病毒1型)的γ34.5基因，γ34.5基因的表达产物可以抑制正常细胞对病毒的清除机制，γ34.5基因敲除后，病毒将不能在正常细胞中复制；而癌细胞则缺失对病毒的清除机制，因此，γ34.5基因的敲除并不影响病毒在癌细胞中的复制。目前处于III期临床的JX594(Pexa-Vec)敲除了牛痘病毒(vaccinia viruses)的TK(胸苷激酶，thymidine kinase)基因，由于病毒的复制与细胞中TK水平有关，所以敲除了TK的JX594仅能在TK活性高的癌细胞中进行复制，不能在正常细胞中复制(正常细胞的TK活性比癌细胞的TK活性低)。CG0070是在腺病毒主管复制的基因E1A前加了E2F-1启动子，E2F-1启动子会受到视网膜母细胞瘤抑制蛋白(Rb)的调控，而Rb在膀胱癌中缺失，因此，Rb的缺失能够激活E2F-1的转录活性，使得E1A基因在膀胱癌细胞中表达，病毒也可以特异性地在膀胱癌细胞中复制。Reolysin是未经改造的野生型呼肠孤病毒，其增殖依赖Ras信号通路的激活，所以仅能在Ras激活的癌细胞中特异性增殖。
然而，目前的肿瘤治疗现状是，仍缺乏一种既能提高肿瘤杀伤特异性(即，相对于正常的非肿瘤细胞，特异性杀伤肿瘤细胞)，又能提高肿瘤治疗的广谱性(即，同时适用于多种肿瘤治疗)的治疗手段。本领域迫切需要开发一种同时具有较高的肿瘤细胞特异性和肿瘤治疗广谱性的重组溶瘤病毒及其制剂。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出一种同时具有较高的肿瘤细胞特异性和/或肿瘤治疗广谱性的重组溶瘤病毒制剂。
在本发明的第一方面，本发明提出了一种病毒制剂。根据本发明的实施例，该病毒制剂含有：病毒；蔗糖；氯化镁。其中，所述病毒制剂含有Tris-HCl缓冲液，并且pH为7.2～7.6。由此，该病毒制剂在贮 存中病毒滴度变化小，具有优秀的贮存稳定性。
另外，根据本发明上述实施例的病毒制剂还可以具有如下附加的技术特征：
根据本发明的实施例，所述病毒为重组溶瘤病毒。
根据本发明的实施例，所述重组溶瘤病毒为水疱性口炎病毒。
根据本发明的实施例，所述重组溶瘤病毒表达对细胞受体亲和力高的病毒蛋白，所述病毒蛋白选自：(a)SEQ ID NO:1；(b)SEQ ID NO:2；或(c)与(a)或(b)具有至少80％同源性的氨基酸序列。由此，该病毒制剂具有较高的肿瘤细胞特异性和/或肿瘤治疗广谱性。
根据本发明的实施例，所述病毒蛋白包含与(a)或(b)具有至少90％、至少95％、至少98％或至少99％同源性的氨基酸序列。
根据本发明的实施例，所述病毒蛋白与细胞受体的结合力的ZDOCK score不低于1800。
根据本发明的实施例，所述细胞受体包括选自CHRNA5、SSTR5、KISS1R、HTR1D、CCR8的至少之一。
根据本发明的实施例，所述重组溶瘤病毒进一步表达选自下列的至少之一：核蛋白、磷酸蛋白、基质蛋白以及RNA依赖的RNA聚合酶。
根据本发明的实施例，基于所述病毒制剂的总量，所述病毒制剂含有：4.5～5.5重量％的蔗糖；和1.5～2.5mmol/L的氯化镁。
根据本发明的实施例，所述Tris-HCl缓冲液中Tris的浓度为50mmol/L。
根据本发明的实施例，所述基于所述病毒制剂的总量，所述病毒制剂含有：5重量％的蔗糖；和2mmol/L的氯化镁。
根据本发明的实施例，所述病毒制剂呈适于吸入或者注射方式给药的形式。
在本发明的第二方面，本发明提出了一种用于配制病毒制剂的溶液。根据本发明的实施例，该用于配制病毒制剂的溶液含有：蔗糖；氯化镁；Tris-HCl缓冲液，并且所述溶液的pH为7.2～7.6。由此，该溶液配制得到的病毒制剂在贮存中病毒滴度变化小，具有优秀的贮存稳定性。
另外，根据本发明上述实施例的用于配制病毒制剂的溶液还可以具有如下附加的技术特征：
根据本发明的实施例，所述用于配制病毒制剂的溶液含有4.5～5.5重量％的蔗糖；和1.5～2.5mmol/L的氯化镁。
根据本发明的实施例，所述用于配制病毒制剂的溶液含有5重量％的蔗糖；和2mmol/L的氯化镁。
根据本发明的实施例，所述Tris-HCl缓冲液中Tris的浓度为50mmol/L。
在本发明的第三方面，本发明提出了上述实施例的病毒制剂或者上述实施例的用于配制病毒制剂的溶液在制备药物中的用途，所述药物用于治疗或者预防癌症或肿瘤。
根据本发明的实施例，所述癌症或者肿瘤包括选自肺癌、胃癌、肝癌、肠癌、食管癌、乳腺癌、宫颈癌、恶性淋巴瘤、鼻咽癌和白血病的至少之一。
在本发明的第四方面，本发明提出了一种预防或治疗癌症或肿瘤的方法。根据本发明的实施例，所述方法包括：向受试者施加上述病毒制剂或者上述溶液。
根据本发明的实施例，所述癌症或者肿瘤包括选自肺癌、胃癌、肝癌、肠癌、食管癌、乳腺癌、宫颈癌、恶性淋巴瘤、鼻咽癌和白血病的至少之一。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了基于肿瘤组织大样本的人类膜受体基因的分析流程图。
图2显示了对应的受体基因在各肿瘤中显著上调的病人比例的抖动散点图。
图3显示了反映候选配体分别与肿瘤特异性受体结合强度的ZDOCK score结果。
图4显示了依据筛选出的受体筛选配体的实验结果图。
图5显示了qPCR检测得到的BXPC3、HCT-8、HepG2、Su8686、H358、NCL-H460和PANC1细胞样品中的CHRNA5、KISS1R、HTRID、CCR8和SSTR5的mRNA的表达量。
图6显示了细胞杀伤实验中所测得的病毒在不同的MOI情况下对BXPC3、HCT-8、HepG2、Su8686、H358和PANC1细胞的杀伤效果。
图7显示了具有不同G蛋白的病毒株在不同的MOI情况下对NCL-H358和NCL-H460细胞的杀伤效果。
图8显示了插入了异源基因的病毒株对NCL-H358和NCL-H460细胞的杀伤效果。
图9显示了REV DQ408670.1病毒株对正常细胞安全的实验结果图。
图10显示了实施例7和对比例1～7的病毒制剂在2～8℃下的稳定性试验结果。
图11显示了实施例7和对比例1～7的病毒制剂在25±2℃下的稳定性试验结果。
图12显示了实施例7和对比例1～7的病毒制剂在-60℃下的稳定性试验结果。
图13显示了实施例7和对比例1～7的反复融冻稳定性试验结果。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本发明的第一方面，本发明提出了一种病毒制剂。根据本发明的实施例，该病毒制剂含有：病毒；蔗糖；氯化镁。其中，所述病毒制剂含有Tris-HCl缓冲液，并且pH为7.2～7.6。
发明人发现，具有如上所述组成的病毒制剂，在不同温度下贮存较长时间或经反复融冻后，制剂中的病毒滴度变化小，表现出了优秀的贮存稳定性。其原因可能在于，蔗糖和氯化镁能增加对病毒的保护，稳定病毒结构，同时起到调节渗透压的作用；另外，相对于其他常见缓冲液，Tris-HCl缓冲液对生物化学过程干扰很小，调节pH范围更广。制剂中病毒在pH7.2～7.6范围内较稳定，通过调节制剂缓冲液pH至7.2～7.6以更好地稳定病毒。
进一步地，根据本发明的实施例，基于病毒制剂的总量，病毒制剂含有：4.5～5.5重量％的蔗糖；和1.5～2.5mmol/L的氯化镁。通过控制病毒制剂中的蔗糖及氯化镁含量在上述范围，可以进一步提高病毒 制剂的贮存稳定性。
进一步地，根据本发明的实施例，基于病毒制剂的总量，病毒制剂含有：5重量％的蔗糖；和2mmol/L的氯化镁。由此，病毒制剂的贮存稳定性更佳。
根据本发明的实施例，上述Tris-HCl缓冲液中Tris的浓度为50mmol/L。由此，病毒制剂的贮存稳定性更佳。
另外，发明人发现，如果病毒制剂中的蔗糖含量过高或过低，则可能降低反复冻融后的病毒稳定性；如果病毒制剂中的氯化镁含量过高或过低，则可能降低病毒在2～8℃和环境温度(25℃)下的病毒稳定性；如果病毒制剂中的pH过高或过低，则可能破坏病毒结构，降低病毒活性。
根据本发明的实施例，上述病毒为重组溶瘤病毒。本发明的重组溶瘤病毒在病毒制剂中具有较好的稳定性，在不同温度下贮存较长时间或经反复融冻后，制剂中的病毒滴度变化小。
根据本发明的实施例，上述重组溶瘤病毒为水疱性口炎病毒。本发明的水疱性口炎病毒在病毒制剂中具有较好的稳定性，在不同温度下贮存较长时间或经反复融冻后，制剂中的病毒滴度变化小。
根据本发明的实施例，上述重组溶瘤病毒表达对细胞受体亲和力高的病毒蛋白，所述病毒蛋白选自：(a)SEQ ID NO:1；(b)SEQ ID NO:2；或(c)与(a)或(b)具有至少80％同源性的氨基酸序列。由此，该病毒制剂具有较高的肿瘤细胞特异性和/或肿瘤治疗广谱性。
Figure PCTCN2022113153-appb-000001
根据本发明的实施例，表达上述病毒蛋白的重组水疱性口炎病毒对肿瘤细胞的特异性靶向性更强，对肿瘤的杀伤更加广谱，杀伤效果更加显著。
需要说明的是，本申请所述的“同源性”是指氨基酸序列具有相似性，其氨基酸序列中个别氨基酸的差异不影响蛋白自有功能的发挥。“同源性氨基酸序列”是指一种多肽的氨基酸序列中，进行单一或多个氨基酸的取代、删除、添加而所衍生出的氨基酸序列。具体地，本申请所述的“具有某百分比序列同源 性”是通过如下公式计算获得的：
1-差异氨基酸的数量/基准氨基酸序列的氨基酸数×100％，
其中，基准氨基酸序列的氨基酸数量是指被比较的氨基酸序列的数量，如所述的“G蛋白与SEQ ID NO:1或SEQ ID NO:2中任一种具有至少80％序列同源性”中的基准氨基酸序列为SEQ ID NO:1或SEQ ID NO:2。
上述具有同源性的氨基酸序列在生物学上、化学上或者结构上具有相似性，并具有相似的生物学活性。结构上相似指的是氨基酸具有相似长度的侧链，如丙氨酸、甘氨酸或丝氨酸，或具有相似大小的侧链。化学相似性指的是氨基酸具有相同的荷电或者都是亲水或者疏水的。例如疏水残基异亮氨酸、缬氨酸、亮氨酸或者甲硫氨酸相互取代。或者极性氨基酸相互取代，例如用精氨酸取代赖氨酸、谷氨酸取代天冬氨酸、谷氨酰胺取代天冬酰胺、丝氨酸取代苏氨酸等等。生物学的相似性是指具有序列同源性的氨基酸序列在生物学功能上类似，如根据本发明实施例的重组水疱性口炎病毒均具有与肿瘤广谱和特异性的高亲和力和结合力。
水疱性口炎病毒(Vesicular stomatitis Virus，VSV)属于弹状病毒科水疱病毒属的病毒，分为两个血清型：新泽西型(VSV-NJ)和印第安那型(VSV-IND)。病毒粒子为子弹状或圆柱状，大小为150～180nm×50～70nm。病毒有囊膜，囊膜上均匀密布有长约10nm的纤突。病毒内部为紧密盘旋的螺旋对称的核衣壳。该病毒根据患病动物的口腔粘膜、牙垫、舌头、嘴唇、鼻孔、蹄和乳头中的经典水疱性病变命名。通过昆虫媒介传播，疾病仅限于其自然宿主，例如马、牛和猪。在人类中，感染是轻度且无症状的。
VSV基因组为不分节段的单股负链RNA(ssRNA)病毒，长度约为11KB。从3’端至5’端依次排列着N、NS、M、G、L五个不重叠的基因，分别编码核(N)蛋白、磷酸(P)蛋白、基质(M)蛋白、糖(G)蛋白及RNA依赖的RNA聚合酶(L)蛋白等5种不同的蛋白。N基因的3’端是先导序列(Leader)，5’端是拖尾序列(Trailor)，各基因间有间隔序列。3’端先导RNA在感染细胞中是最早的病毒转录物，长度为47个核苷酸，不戴帽也不翻译，其功能尚未完全清楚，可能是抑制宿主RNA的合成。N蛋白是启动基因组合成所必需的，可有效的保护病毒RNA免受各种核酸酶的消化。N蛋白有高的抗原性免疫原性，刺激机体产生非中和抗体细胞免疫，且在转录复制中担任了重要的角色，它对维持基因组RNA呈伸展形式可能是必要的，与复制调节有关。P蛋白，VSV-NJ与VSV-IND病毒株的同源性为41％，其作用是与聚合酶L，核蛋白N组成聚合酶复合物以及基因组RNA共同维持病毒的转录活性。M蛋白在病毒致病机制和病毒复制方面起关键作用，富含碱性氨基酸，并含有高度碱性的氨基末端结构域，可通过与核衣壳结合而抑制转录，同时辅助病毒从宿主中出芽，是涉及出芽过程的惟一多肽。G蛋白是病毒的主要表面抗原，决定着病毒的毒力，也是病毒的保护性抗原。它可刺激机体产生中和抗体。L基因编码RNA poly E蛋白，它可能决定RNA的转录活性，与P蛋白结合以催化mRNA的复制。该蛋白是聚合酶复合物和复制酶复合物的核心成分，涉及起始、延伸、甲基化、戴帽、聚(A)尾形成等等。此外，在每个基因之间的间隔序列有广泛的同源性，这些序列有一个共同的结构，即3’-AUAC(U)7NAUUGUCNN-UAG-5’。这些基因之间的保守序列是一种关键信号，以影响多聚酶的活性或酶的切割活性，而在复制过程中，这些信号被掩盖，不起作用。
在本发明的描述中，术语“重组VSV病毒”、“重组水疱性口炎病毒”、“本发明重组病毒”可互换使用，是指如上所述的能够特异性感染肿瘤细胞的重组VSV病毒，所述的重组VSV病毒特异性感染肿瘤细胞， 并且所述重组VSV病毒特异性结合于选自下组的肿瘤细胞的特异性受体CHRNA5、SSTR5、KISS1R、HTR1D和CCR8。
根据本发明的实施例，所述病毒蛋白包含与SEQ ID NO:1或SEQ ID NO:2中任一种具有至少90％、至少95％、至少98％或至少99％同源性的氨基酸序列。
根据本发明的实施例，所述重组水疱性口炎病毒不携带异源基因。发明人发现，不携带异源基因的重组水疱性口炎病毒对肿瘤细胞的杀伤效果显著高于携带外源基因的重组水疱性口炎病毒。根据本发明的实施例，在本文中所描述的术语“异源基因”如无特别说明，是指在野生型水疱性口炎病毒中未曾报道过的基因。或者换句话说，在重组水疱性口炎病毒中所编码的蛋白均为野生型水疱性口炎病毒中表达的。
根据本发明的实施例，所述病毒蛋白与细胞受体的结合力的ZDOCK score不低于1800。本领域技术人员能够理解，通过输入病毒蛋白和细胞受体的序列，可以容易地获得病毒蛋白和细胞受体之间的结合力表征参数ZDOCK score。发明人发现，当ZDOCK score 1800时，例如不小于1900，不小于2000，优选不小于2100，携带病毒蛋白的病毒与携带对应受体的肿瘤细胞之间的结合力将显著提高。根据本发明的实施例，ZDOCK score是可以常规软件确定的，例如参见Pierce BG,Hourai Y,Weng Z.(2011)Accelerating Protein Docking in ZDOCK Using an Advanced 3D Convolution Library.PLoS One 6(9):e24657.。
根据本发明的实施例，上述病毒蛋白包括选自GenBank索取号为X03633.1的G蛋白和GenBank索取号为DQ408670.1的G蛋白的至少之一。本发明的发明人意外地发现GenBank索取号为X03633.1的G蛋白和GenBank索取号为DQ408670.1的G蛋白与肿瘤细胞的受体具有显著强于其他G蛋白的结合力。
根据本发明的实施例，所述重组水疱性口炎病毒进一步表达选自下列的至少之一：核蛋白、磷酸蛋白、基质蛋白以及RNA依赖的RNA聚合酶。发明人意外地发现，通过将这些蛋白与GenBank索取号为X03633.1的G蛋白和GenBank索取号为DQ408670.1的G蛋白的至少之一进行组合构建得到的重组病毒，具有更强的肿瘤杀伤活性。发明人认为有可能是因为对于肿瘤细胞而言，多种不同来源的蛋白组合有可能会引发与相同来源的蛋白组合不同的免疫反应，从而进一步提高对肿瘤细胞的杀伤作用。
相应地，所述重组水疱性口炎病毒携带：编码所述核蛋白的核酸分子；编码所述磷酸蛋白的核酸分子；编码所述基质蛋白的核酸分子；或者编码所述RNA依赖的RNA聚合酶的核酸分子
优选地，所述编码所述核蛋白的核酸分子、所述编码所述磷酸蛋白的核酸分子、所述编码所述基质蛋白的核酸分子和所述编码所述RNA依赖的RNA聚合酶的核酸分子的至少之一衍生自水疱性口炎病毒Mudd summer亚型病毒株。发明人意外地发现，通过将这些蛋白与GenBank索取号为X03633.1的G蛋白和GenBank索取号为DQ408670.1的G蛋白的至少之一进行组合构建得到的重组病毒，具有更强的肿瘤杀伤活性。发明人认为有可能是因为对于肿瘤细胞而言，多种不同来源的蛋白组合有可能会引发与相同来源的蛋白组合不同的免疫反应，从而进一步提高对肿瘤细胞的杀伤作用。
根据本发明的实施例，所述病毒制剂呈适于吸入或者注射方式给药的形式。
在本发明的第二方面，本发明提出了一种用于配制病毒制剂的溶液。根据本发明的实施例，该用于配制病毒制剂的溶液含有：蔗糖；氯化镁；Tris-HCl缓冲液，并且所述溶液的pH为7.2～7.6。
发明人发现，具有如上所述组成的溶液配制得到的病毒制剂，在不同温度下贮存较长时间或经反复 融冻后，制剂中的病毒滴度变化小，表现出了优秀的贮存稳定性。其原因可能在于，蔗糖和氯化镁能稳定病毒结构，增强对病毒的保护；相对于其他常见缓冲液，Tris-HCl缓冲液调节pH范围更广，更好的稳定病毒制剂的pH；制剂中病毒在pH7.2～7.6范围内较稳定，通过调节制剂缓冲液pH至7.2～7.6以更好地稳定病毒。
进一步地，根据本发明的实施例，上述用于配制病毒制剂的溶液含有4.5～5.5重量％的蔗糖；和1.5～2.5mmol/L的氯化镁。通过控制溶液中的蔗糖及氯化镁含量在上述范围，可以进一步提高溶液配制得到的病毒制剂的贮存稳定性。
进一步地，根据本发明的实施例，上述用于配制病毒制剂的溶液含有5重量％的蔗糖；和2mmol/L的氯化镁。由此，该溶液配制得到的病毒制剂的贮存稳定性更佳。
根据本发明的实施例，上述Tris-HCl缓冲液中Tris的浓度为50mmol/L。由此，该溶液配制得到的病毒制剂的贮存稳定性更佳。
更进一步地，本发明提出了上述药物组合物或者上述重组水疱性口炎病毒在制备药物中的用途，所述药物用于治疗或者预防癌症或者肿瘤。
根据本发明的实施例，所述癌症或者肿瘤包括选自肺癌、胃癌、肝癌、肠癌、食管癌、乳腺癌、宫颈癌、恶性淋巴瘤、鼻咽癌和白血病的至少之一。
更进一步地，本发明提出了一种预防或治疗癌症或肿瘤的方法。根据本发明的实施例，所述方法包括：向受试者施加上述病毒制剂或者上述溶液。
根据本发明的实施例，所述癌症或者肿瘤包括选自肺癌、胃癌、肝癌、肠癌、食管癌、乳腺癌、宫颈癌、恶性淋巴瘤、鼻咽癌和白血病的至少之一。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。
下面首先对本发明中重组水疱性口炎病毒涉及的实施例进行描述。
实施例1 基于肿瘤组织大样本的人类膜受体基因的分析
以下将参考图1详细描述基于肿瘤组织大样本的人类膜受体基因的分析的方法。
1.1人类膜受体基因及其表达数据预处理与分析
本发明从已有的研究中整理总结出在人类细胞中表达的受体基因信息(参考文献：(Synchronous birth is a dominant pattern in receptor-ligand evolution，BMC Genomics.Grandchamp and Monget,2018 Aug14；19(1):611.)。发明人从UCSC Xena( http://xena.ucsc.edu/)下载了癌症病人的的基因表达矩阵(归一化值)，基因突变信息以及相关临床资料。数据中包含的癌种有：肾上腺皮质癌、膀胱尿路上皮癌、乳腺浸润癌、宫颈鳞状细胞癌和宫颈内膜腺癌、胆管癌、结肠腺癌、结肠腺癌/Rectum腺癌食管癌、淋巴样肿瘤弥漫性大B细胞淋巴瘤、食道癌、FFPE试验第二阶段、胶质母细胞瘤、胶质瘤、头颈部鳞状细胞癌、肾染色体、泛肾队列(KICH+KIRC+KIRP)、肾脏肾透明细胞癌、肾脏肾乳头状细胞癌、急性髓性白血病、脑低级脑胶质瘤、肝肝细胞癌、肺腺癌、肺鳞状细胞癌、间皮瘤、卵巢浆液性囊腺癌、胰腺腺癌、嗜铬细胞瘤和副神经节瘤、前列腺腺癌、直肠腺癌、肉瘤、皮肤黑色素瘤、胃腺癌、胃和食道癌、 睾丸生殖细胞肿瘤、甲状腺癌、胸腺瘤、子宫体子宫内膜癌、子宫癌肉瘤、葡萄膜黑色素瘤。
发明人首先从下载的数据中首先剔除少于三个的样本肿瘤及正常组织信息，然后进行差异表达分析。发明人使用limma软件(版本：3.38.3)来执行差异表达分析(参考文献：(Limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies.Nucleic Acids Research,43,e47，Ritchie,M.E.,et al.(2015))。分析中采用了limma R包的voom模型。只有当基因满足标准|log2FC|>1，P值<0.05才会被认定为是差异基因。
1.2数据分析
使用R语言计算各组间膜受体基因表达差异倍数(log2FC)和p值。选择|log2FC|大于等于2.0被视为有显著性上调/下调差异表达基因。t检验的p值小于0.01被判定为差异有统计学意义。使用ComplexHeatmap R包生成每个比较组对的log2FC矩阵的热图。
然后，发明人根据一系列筛选条件，比如选择在肠癌、肺癌、胰腺癌、胃癌和肝癌癌种中选择大于等于70％的癌症样本中显著上调的基因(即log2FC≥2.0的基因)、本底表达量高等条件，选出10余个受体。
具体地，发明人对每个基因在不同肿瘤样本中的log2FC值，使用ggplot2和ggbeeswarm软件绘制抖动散点图(如图2所示)，用以展示该基因在各肿瘤中显著上调的病人比例。
另外，发明人将筛选出来的13个受体与备选配体做分子对接，选择结合力最优的为最终选择的5个受体。
结果如图3所示(其中，图3所示配体编号以及所对应的配体名称和氨基酸序列捕获号如表1所示)，CHRNA5(烟碱型胆碱受体α5)、SSTR5(生长抑素受体5)、KISS1R(吻素受体)、HTR1D(血清素受体1D)、CCR8(C-C趋化因子受体8)为肿瘤细胞和正常细胞中差异表达的受体蛋白。
表1 配体名称及氨基酸序列捕获号
Figure PCTCN2022113153-appb-000002
Figure PCTCN2022113153-appb-000003
实施例2 根据受体选择病毒配体
发明人选择16种水疱性口炎病毒同源配体，与实施例1筛选获得的5种肿瘤特异性受体分别进行建模和对接，生成的对接的结果会按照ZDOCK score分数进行排序，得分越高，则说明结合越强，结果可信度越高。同时综合分析这些构象的聚类结果，发现，ZDOCK分数是ZDOCK程序计算的形状互补分数，根据参数设置，ZDOCK分数还将包括静电和去溶剂能项。ZDOCK分数越高越好。进而发明人通过ZDOCK score函数来评价结合的强弱，获得了筛选出肿瘤特异性受体结合能力强的配体(结果如图4所示)，其中，结合效果最优的配体为DQ408670.1-lig-F和X03633.1-lig-FL，对应的氨基酸序列的捕获号为DQ408670.1，GENE ID:X03633.1。
实施例3 基于不同血清型蛋白的基因重组水疱性口炎病毒的构建及扩增
发明人将来源于Mudd summer亚型病毒株的L、N、P、M蛋白组合结合捕获序列号为GENE ID:DQ408670.1、GENE ID:X03633.1、GENE ID:KP872888.1或GENE ID:HQ593628.1的G蛋白构建重组水疱性口炎病毒REV DQ408670.1、REV X03633.1、REV KP872888.1和REV HQ593628.1。
病毒株REV DQ408670.1、REV X03633.1、REV KP872888.1和REV HQ593628.1的包装方法如下所述：
体外重组VSV需要:包含病毒基因组的全长质粒(包含G蛋白)，以及病毒包装所需要的骨架蛋白的辅助质粒(N、P、L、M)，通过体外转染的方法将质粒转入BHK21细胞中，病毒在细胞内组装成熟后出芽释放到细胞外(参考文献：Vesicular stomatitis virus-based vaccine protects hamsters against lethal challenge with Andes virus.Journal of virology 85,12781-12791,doi:10.1128/JVI.00794-11(2011)，Brown,K.S.,Safronetz,D.,Marzi,A.,Ebihara,H.& Feldmann,H.)。
病毒的扩增使用Vero细胞，将一定滴度的病毒加入到培养的Vero细胞中，病毒可以感染细胞，并且在细胞中完成自我复制，成熟的病毒释放到细胞培养的上清中，将细胞培养的上清浓缩即可得到病毒浓缩液，滴度测定之后可用于后续实验。
实施例4 肿瘤细胞受体的检测以及细胞杀伤结果
本实施例中利用实施例3构建的不同病毒对不同肿瘤细胞进行杀伤效果验证。
3.1 q-PCR检测：
用Trizol法提取1×10 6的BXPC3、HCT-8、HepG2、Su8686、H358、NCL-H460(H460)和PANC1细胞样品，以500ng/μL RNA进行20μL体系的反转录，用SYBR GREEN法进行荧光定量PCR检测7个细胞样品中CHRNA5、KISS1R、HTRID、CCR8和SSTR5基因mRNA的表达。
结果如图5所示。qPCR检测结果显示，BXPC3、HCT-8、HepG2、Su8686、H358、NCL-H460和PANC1细胞样品CHRNA5受体基因mRNA表达水平较高，但不同细胞中，相对表达量高的受体存在差 异，比如H460细胞中表达量最高的为CHRNA5，HTR1D受体，而其它细胞中CHRNA5和CCR8受体基因表达水平较高。
3.2细胞杀伤实验(CCK)：
将状态良好的BXPC3、HCT-8、HepG2、Su8686、H358和PANC1细胞制成5×10 4个/mL的细胞悬液按100μL/孔加入96孔板中，边缘补齐培养基减少蒸发，过夜培养。用Opti-MEM将已知滴度病毒稀释为MOI：0.01、MOI：0.1和MOI：1的病毒工作液，将96孔板中培养液吸弃，每孔加入50μL病毒稀释液，每个稀释液重复3复孔，另取Opti-MEM重复3孔作空白对照。病毒稀释液加入2h后换液，1％FBS培养基每孔100μL。48/72h后每孔加入10μL CCK8检测液，37℃孵育2h后OD450酶标仪读数。
图6显示了REV DQ408670.1对不同细胞的CCK杀伤结果，CCK检测结果显示，MOI：0.01、MOI：0.1和MOI：1的REV DQ408670.1病毒工作液均对BXPC3、HCT-8、HepG2、Su8686、H358和PANC1细胞具有显著的杀伤效果。
实验结果如图7所示。CCK检测结果显示，REV DQ408670.1、REV X03633.1在MOI：0.01、MOI：0.1和MOI：1的病毒工作液对NCL-H358和NCL-H460细胞的杀伤效果显著优于REV KP872888.1和REV HQ593628.1。同时结合图5结果，NCL-H358中CCR8和CHRNA5表达量较高，NCL-H460中CHRNA5、HTR1D表达量较高，结合图3受体与配体的结合热图结果，DQ408670.1和X03633.1G蛋白与CCR8、HTR1D受体的结合力强。综合反应了，当重组水疱性口炎病毒与肿瘤细胞受体具有高结合力时，重组病毒对高表达所述受体的肿瘤细胞的杀伤效果更加显著。
实施例5 基于选定G蛋白，不同L、N、P、M组合的病毒株对肿瘤细胞杀伤结果
细胞杀伤实验(CCK)：
利用反义遗传学方法，发明人构建了REV DQ408670.1病毒株、REV DQ408670.1-V1和REV DQ408670.1-V2病毒株，其中，REV DQ408670.1-V1是在REV DQ408670.1病毒株的基础上变更了L、M蛋白，而REV DQ408670.1-V2是在REV DQ408670.1病毒株的基础上变更了N、P蛋白。
将状态良好的H358和H460细胞制成5×10 4个/mL的细胞悬液按100μL/孔加入96孔板中，边缘补齐培养基减少蒸发，过夜培养。用Opti-MEM将已知滴度3株病毒株稀释为MOI：0.01的病毒工作液，将96孔板中培养液吸弃，每孔加入50μL病毒稀释液，每个稀释液重复3复孔，另取Opti-MEM重复3孔作空白对照。病毒稀释液加入2h后换液，1％FBS培养基每孔100μL。72h后每孔加入10μL CCK8检测液，37℃孵育2h后OD450酶标仪读数。
实验结果如表2所示。3株病毒株REV DQ408670.1，REV DQ408670.1-V1和REV DQ408670.1-V2结果相似，在MOI：0.01的病毒工作液均对H358和H460细胞具有显著的杀伤效果。
表2：MOI:0.01时病毒对肿瘤细胞的抑制率％
  REV DQ408670.1 REV Q408670.1-V1 REV DQ408670.1-V2
H358 88.07 78.84 85.27
H460 83.33 81.33 79.36
实施例6 基于选定G蛋白以及插入外源基因的病毒株对肿瘤细胞杀伤结果
细胞杀伤实验(CCK)：
利用反义遗传学方法，发明人在构建的REV DQ408670.1病毒株中插入了异源基因INFβ，以构建病毒株FJ-INFβ。
将状态良好的H358和H460细胞制成5×10 4个/mL的细胞悬液按100μL/孔加入96孔板中，边缘补齐培养基减少蒸发，过夜培养。用Opti-MEM将已知滴度REV DQ408670.1病毒株和FJ-INFβ病毒株分别稀释为MOI：0.01、MOI：0.1和MOI：1的病毒工作液，将96孔板中培养液吸弃，每孔加入50μL病毒稀释液，每个稀释液重复3复孔，另取Opti-MEM重复3孔作空白对照。病毒稀释液加入2h后换液，1％FBS培养基每孔100μL。72h后每孔加入10μL CCK8检测液，37℃孵育2h后OD450酶标仪读数。
结果如图8所示，REV DQ408670.1病毒株对H358和H460细胞杀伤显著优于FJ-INFβ。
实施例6 REV DQ408670.1病毒株对正常细胞杀伤检测
细胞杀伤实验(CCK)：
将状态良好的肺正常细胞BEAS-2B制成5×10 4个/mL的细胞悬液按100μL/孔加入96孔板中，边缘补齐培养基减少蒸发，过夜培养。用Opti-MEM将已知滴度REV DQ408670.1病毒稀释为MOI：0.01、MOI：0.1和MOI：1的病毒工作液，将96孔板中培养液吸弃，每孔加入50μL病毒稀释液，每个稀释液重复3复孔，另取Opti-MEM重复3孔作空白对照。病毒稀释液加入2h后换液，1％FBS培养基每孔100μL。72h后每孔加入10μL CCK8检测液，37℃孵育2h后OD450酶标仪读数。
实验结果如图9所示。CCK检测结果显示，REV DQ408670.1病毒株在MOI：0.01、MOI：0.1和MOI：1的病毒工作液均对BEAS-2B细胞没有明显的杀伤效果。
下面进一步对本发明病毒制剂涉及的实施例进行描述。
实施例7
病毒制剂处方为：5重量％蔗糖、2mmol/L氯化镁、Tris-HCl缓冲液(含50mmol/L Tris，并用HCl调节制剂pH至7.5)，以及滴度为9.8lgTCID 50/mL的重组水疱性口炎溶瘤病毒。
对比例1
病毒制剂处方为：0.01mol/L磷酸盐冲生理盐水溶液，pH为7.2，以及滴度为9.2lgTCID 50/mL的重组水疱性口炎溶瘤病毒。
对比例2
病毒制剂处方与实施例7基本相同，区别在于，蔗糖含量为3重量％。
对比例3
病毒制剂处方与实施例7基本相同，区别在于，蔗糖含量为7重量％。
对比例4
病毒制剂处方与实施例7基本相同，区别在于，氯化镁含量为0.5mol/L。
对比例5
病毒制剂处方与实施例7基本相同，区别在于，氯化镁含量为3.5mol/L。
对比例6
病毒制剂处方与实施例7基本相同，区别在于，Tris-HCl缓冲液用HCl调节制剂pH至7.0。
对比例7
病毒制剂处方与实施例7基本相同，区别在于，Tris-HCl缓冲液用HCl调节制剂pH至8.0。
试验例1
取实施例7、对比例1～7的病毒制剂，在2～8℃下进行贮存稳定性试验，分别在放置0d、3d、5d、7d、14d、21d、28d、35d、42d后检测病毒滴度，其中，实施例7和对比例1～7的试验结果如表3和图10所示。
表3 2～8℃下病毒滴度检测结果(lgTCID 50/mL)
Figure PCTCN2022113153-appb-000004
试验例2
取实施例7、对比例1～7的病毒制剂，在25±2℃下进行贮存稳定性试验，分别在放置0d、3d、5d、7d、14d、21d、28d、35d、42d后检测病毒滴度，其中，实施例7和对比例1～7的试验结果如表4和图11所示。
表4 25±2℃下病毒滴度检测结果(lgTCID 50/mL)
Figure PCTCN2022113153-appb-000005
Figure PCTCN2022113153-appb-000006
试验例3
取实施例7、对比例1～7的病毒制剂，在-60℃下进行贮存稳定性试验，分别在放置0d、28d、60d后检测病毒滴度，其中，实施例7和对比例1～7的试验结果如表5和图12所示。由结果可以看出，在低温冷冻条件下，实施例7的病毒制剂具有较好的稳定性，保存42天后仍具有较高的病毒滴度。
表5 -60℃下病毒滴度检测结果(lgTCID 50/mL)
Figure PCTCN2022113153-appb-000007
试验例4
取实施例7、对比例1～7的病毒制剂进行反复融冻稳定性试验，分别在融冻1次、2、3、4、5后检测病毒滴度，其中，实施例7和对比例1～7的试验结果如表6和图13所示。
表6 不同融冻次数下病毒滴度检测结果(lgTCID 50/mL)
Figure PCTCN2022113153-appb-000008
结果讨论
对于实施例7和对比例1～7处方的病毒制剂，经以上试验例试验后，病毒滴度与初始标示量相比下降不超过0.5lgTCID 50/mL，认为病毒滴度下降在合格范围内。
从试验例1、试验例2的结果可以看出，在2～8℃、25±2℃的温度下，随着放置时间的延长，实施例7和对比例1～7处方的病毒制剂中病毒滴度呈下降趋势。但是，实施例7处方的病毒制剂可以更长时间保持病毒滴度下降在合格范围内。对比例4和5处方的病毒制剂，由于氯化镁含量过低或过高，病毒滴度下降较大。
从试验例3的结果可以看出，实施例7和对比例1～3、6和7处方的病毒制剂在-60℃的温度下放置60d，病毒滴度基本没有变化，而对比例4和5处方的病毒滴度明显降低。
从试验例4的结果可以看出，实施例7处方的病毒制剂随着反复融冻次数的增加，病毒滴度基本没有变化，反复融冻5次后病毒滴度与未融冻病毒滴度基本相同。对比例1处方的病毒制剂随着反复融冻次数的增加，病毒滴度呈下降趋势，反复融冻5次后病毒滴度与未融冻病毒滴度相比下降了90％左右。对比例2和3处方的病毒制剂，由于处方中蔗糖含量过低或过高，制剂经反复融冻后病毒滴度下降较大。
另外，对于对比例6和7处方的病毒制剂，由于处方中pH过低或过高，对病毒结构和活性的影响较大，对比例6和7处方的病毒制剂在试验例1～4中病毒滴度下降均较大。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (18)

  1. 一种病毒制剂，其特征在于，含有：
    病毒；
    蔗糖；
    氯化镁；
    其中，所述病毒制剂含有Tris-HCl缓冲液，并且pH为7.2～7.6。
  2. 根据权利要求1所述的病毒制剂，其特征在于，所述病毒为重组溶瘤病毒。
  3. 根据权利要求2所述的病毒制剂，其特征在于，所述重组溶瘤病毒为水疱性口炎病毒。
  4. 根据权利要求2或3所述的病毒制剂，其特征在于，所述重组溶瘤病毒表达对细胞受体亲和力高的病毒蛋白，所述病毒蛋白选自：
    (a)SEQ ID NO:1；
    (b)SEQ ID NO:2；或
    (c)与(a)或(b)具有至少80％同源性的氨基酸序列。
  5. 根据权利要求4所述的病毒制剂，其特征在于，所述病毒蛋白与细胞受体的结合力的ZDOCK score不低于1800。
  6. 根据权利要求5所述的病毒制剂，其特征在于，所述细胞受体包括选自CHRNA5、SSTR5、KISS1R、HTR1D、CCR8的至少之一。
  7. 根据权利要求2所述的病毒制剂，其特征在于，所述重组溶瘤病毒进一步表达选自下列的至少之一：核蛋白、磷酸蛋白、基质蛋白以及RNA依赖的RNA聚合酶。
  8. 根据权利要求1所述的病毒制剂，其特征在于，基于所述病毒制剂的总量，所述病毒制剂含有：
    4.5～5.5重量％的蔗糖；和
    1.5～2.5mmol/L的氯化镁。
  9. 根据权利要求1所述的病毒制剂，其特征在于，所述Tris-HCl缓冲液中Tris的浓度为50mmol/L。
  10. 根据权利要求1所述的病毒制剂，其特征在于，所述基于所述病毒制剂的总量，所述病毒制剂含有：
    5重量％的蔗糖；和
    2mmol/L的氯化镁。
  11. 一种用于配制病毒制剂的溶液，其特征在于，含有：
    蔗糖；
    氯化镁；
    Tris-HCl缓冲液，并且
    所述溶液的pH为7.2～7.6。
  12. 根据权利要求11所述的溶液，其特征在于，含有：
    4.5～5.5重量％的蔗糖；和
    1.5～2.5mmol/L的氯化镁。
  13. 根据权利要求12所述的溶液，其特征在于，含有：
    5重量％的蔗糖；和
    2mmol/L的氯化镁。
  14. 根据权利要求11所述的溶液，其特征在于，所述Tris-HCl缓冲液中Tris的浓度为50mmol/L。
  15. 权利要求1～10任一项所述的病毒制剂或者权利要求11～14任一项所述的溶液在制备药物中的用途，所述药物用于治疗或者预防癌症或肿瘤。
  16. 根据权利要求15所述的用途，其特征在于，所述癌症或者肿瘤包括选自肺癌、胃癌、肝癌、肠癌、食管癌、乳腺癌、宫颈癌、恶性淋巴瘤、鼻咽癌和白血病的至少之一。
  17. 一种预防或治疗癌症或肿瘤的方法，其特征在于，包括：向受试者施加权利要求1～10任一项所述的病毒制剂或者权利要求11～14任一项所述的溶液。
  18. 根据权利要求17所述的方法，其特征在于，所述癌症或者肿瘤包括选自肺癌、胃癌、肝癌、肠癌、食管癌、乳腺癌、宫颈癌、恶性淋巴瘤、鼻咽癌和白血病的至少之一。
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