CN115989321A - Virus preparation, solution for preparing virus preparation and use thereof - Google Patents
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- CN115989321A CN115989321A CN202280005157.6A CN202280005157A CN115989321A CN 115989321 A CN115989321 A CN 115989321A CN 202280005157 A CN202280005157 A CN 202280005157A CN 115989321 A CN115989321 A CN 115989321A
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Abstract
A viral formulation, a solution for formulating the viral formulation and uses thereof are provided. Wherein the virus preparation comprises: a virus; sucrose; magnesium chloride. Wherein the virus preparation contains Tris-HCl buffer solution, and the pH value is 7.2-7.6.
Description
The invention relates to the field of biomedicine, in particular to a virus preparation, a solution for preparing the virus preparation and application thereof.
In recent ten years, the mechanism of killing tumor by inducing the body's anti-tumor immune response is becoming clear. Since the first time that scientist Jean rommelae in germany called oncolytic virus therapy as tumor immunotherapy in 2011, oncolytic viruses have now been accepted by the general public as an important branch of tumor immunotherapy. Compared with other tumor immunotherapy methods, the oncolytic virus has the advantages of high killing efficiency, good targeting property, small side effect, multiple tumor killing ways, avoidance of drug resistance, low cost and the like.
Because the viral genome is small, various modifications can be easily carried out by genetic engineering means, and modification and packaging of the virus can be carried out by conventional means, the techniques are mature, and the cost is low. Thus, oncolytic viruses are readily engineered to specifically target cancer cells by exploiting the characteristics of the oncolytic virus itself and exploiting the differences between cancer cells and normal cells.
Since most cancer cells have an impaired mechanism for self-clearing of viruses (e.g., normal cells lack the key factor Protein Kinase R (PKR) for virus clearance in cancer cells), viruses are more likely to replicate and spread in cancer cells. In addition, in recent decades, with the continuous and intensive research, scientists use the difference of a plurality of signal pathways and metabolism in cancer cells and normal cells, and continuously improve the targeting of oncolytic virus to tumor, reduce the harm of oncolytic virus to normal cells and improve the safety by screening specific virus varieties and modifying virus genomes. For example: the approved T-vec has the gamma 34.5 gene of HSV-1 (herpes simplex virus 1) knocked out, the expression product of the gamma 34.5 gene can inhibit the virus clearance mechanism of normal cells, and after the gamma 34.5 gene is knocked out, the virus cannot replicate in the normal cells; cancer cells lack the mechanism for viral clearance, so that the knockout of the gamma 34.5 gene does not affect viral replication in cancer cells. JX594 (Pexa-Vec), which is currently in phase III clinical, knocks out the TK (thymidine kinase) gene of vaccinia virus (vaccinia viruses), and since replication of the virus is related to the TK level in cells, JX594 knocked out TK can only replicate in cancer cells with high TK activity and cannot replicate in normal cells (TK activity of normal cells is lower than that of cancer cells). CG0070 is that E2F-1 promoter is added before E1A of gene in charge of replication of adenovirus, E2F-1 promoter will be controlled by retinoblastoma arrestin (Rb), and Rb is deleted in bladder cancer, therefore, rb deletion can activate E2F-1 transcriptional activity, make E1A gene express in bladder cancer cell, the virus can replicate in bladder cancer cell specifically too. Reolysin is an unmodified wild-type reovirus, and proliferation thereof is dependent on activation of the Ras signaling pathway, so that it can only specifically proliferate in Ras-activated cancer cells.
However, in the current situation of tumor therapy, there is still no therapeutic means that can improve the specificity of tumor killing (i.e., specifically kill tumor cells relative to normal non-tumor cells) and can also improve the broad spectrum of tumor therapy (i.e., can be applied to multiple tumor therapies at the same time). The development of a recombinant oncolytic virus and a preparation thereof, which have high tumor cell specificity and broad spectrum of tumor treatment, is urgently needed in the field.
Disclosure of Invention
The present invention is directed to solving, at least in part, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a recombinant oncolytic virus preparation having high tumor cell specificity and/or broad spectrum of tumor therapy.
In a first aspect of the invention, a viral formulation is presented. According to an embodiment of the invention, the viral formulation comprises: a virus; sucrose; magnesium chloride. Wherein the virus preparation contains Tris-HCl buffer solution, and the pH value is 7.2-7.6. Therefore, the virus preparation has small change of virus titer in storage and excellent storage stability.
In addition, the virus preparation according to the above embodiment of the present invention may also have the following additional technical features:
according to an embodiment of the invention, the virus is a recombinant oncolytic virus.
According to an embodiment of the invention, the recombinant oncolytic virus is a vesicular stomatitis virus.
According to an embodiment of the invention, the recombinant oncolytic virus expresses a viral protein with high affinity for a cellular receptor selected from the group consisting of: (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 higher tumor cell specificity and/or broad spectrum of tumor treatment.
According to an embodiment of the invention, 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).
According to embodiments of the invention, the binding capacity of the viral proteins to cellular receptors is ZDOCK score of not less than 1800.
According to an embodiment of the invention, the cellular receptor comprises at least one selected from the group consisting of CHRNA5, SSTR5, KISS1R, HTR1D, CCR.
According to an embodiment of the invention, the recombinant oncolytic virus further expresses at least one selected from the group consisting of: nucleoprotein, phosphoprotein, matrix protein, and RNA-dependent RNA polymerase.
According to an embodiment of the invention, the virus preparation contains, based on the total amount of the virus preparation: 4.5 to 5.5 weight percent of sucrose; and 1.5-2.5 mmol/L magnesium chloride.
According to an embodiment of the present invention, the concentration of Tris in the Tris-HCl buffer is 50mmol/L.
According to an embodiment of the invention, the virus preparation comprises, based on the total amount of the virus preparation: 5% by weight of sucrose; and 2mmol/L magnesium chloride.
According to an embodiment of the invention, the viral formulation is in a form suitable for administration by inhalation or injection.
In a second aspect of the invention, the invention features a solution for formulating a viral formulation. According to an embodiment of the invention, the solution for formulating the virus preparation comprises: sucrose; magnesium chloride; tris-HCl buffer, and the pH of the solution is 7.2-7.6. Therefore, the virus preparation prepared from the solution has small change of virus titer in storage and has excellent storage stability.
In addition, the solution for preparing a virus preparation according to the above embodiment of the present invention may also have the following additional technical features:
according to an embodiment of the present invention, the solution for preparing a virus preparation contains 4.5 to 5.5 wt% of sucrose; and 1.5-2.5 mmol/L magnesium chloride.
According to an embodiment of the invention, the solution for formulating the virus preparation contains 5% by weight of sucrose; and 2mmol/L magnesium chloride.
According to an embodiment of the present invention, the concentration of Tris in the Tris-HCl buffer is 50mmol/L.
In a third aspect of the invention, the invention proposes the use of the viral formulation of the above example or the solution of the above example for formulating a viral formulation in the manufacture of a medicament for the treatment or prevention of cancer or a tumour.
According to an embodiment of the present invention, the cancer or tumor includes at least one selected from the group consisting of lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, and leukemia.
In a fourth aspect of the invention, the invention features a method of preventing or treating cancer or a tumor. According to an embodiment of the invention, the method comprises: applying the above virus preparation or the above solution to a subject.
According to an embodiment of the present invention, the cancer or tumor includes at least one selected from the group consisting of lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, and leukemia.
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.
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a flow chart of the analysis of human membrane receptor genes based on large samples of tumor tissue.
Figure 2 shows a jittered scatter plot of the proportion of patients whose corresponding receptor genes were significantly upregulated in each tumor.
Figure 3 shows the results of ZDOCK score reflecting the binding strength of candidate ligands to tumor specific receptors, respectively.
FIG. 4 is a graph showing the results of experiments for screening ligands according to the screened receptors.
FIG. 5 shows the expression levels of mRNA for CHRNA5, KISS1R, HTRID, CCR8 and SSTR5 in the BXPC3, HCT-8, hepG2, su8686, H358, NCL-H460 and PANC1 cell samples obtained by qPCR assay.
FIG. 6 shows the killing effect of the virus on BXPC3, HCT-8, hepG2, su8686, H358 and PANC1 cells at different MOIs as determined in a cell killing experiment.
FIG. 7 shows the killing effect of virus strains with different G proteins on NCL-H358 and NCL-H460 cells at different MOIs.
FIG. 8 shows the killing effect of a virus strain with an inserted heterologous gene on NCL-H358 and NCL-H460 cells.
FIG. 9 shows a graph of the results of an experiment in which the REV DQ408670.1 virus strain is safe for normal cells.
FIG. 10 shows the results of stability tests at 2 to 8 ℃ for the virus formulations of example 7 and comparative examples 1 to 7.
FIG. 11 shows the results of stability tests at 25. + -. 2 ℃ for the virus formulations of example 7 and comparative examples 1 to 7.
FIG. 12 shows the results of the stability test at-60 ℃ for the virus formulations of example 7 and comparative examples 1 to 7.
FIG. 13 shows the results of the repeated freeze stability test of example 7 and comparative examples 1 to 7.
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.
In a first aspect of the invention, a viral formulation is provided. According to an embodiment of the invention, the viral formulation comprises: a virus; sucrose; magnesium chloride. Wherein the virus preparation contains Tris-HCl buffer solution, and the pH value is 7.2-7.6.
The inventors have found that a virus preparation having the composition as described above shows little change in virus titer in the preparation and excellent storage stability when stored at different temperatures for a long period of time or after repeated thawing. The reason for this may be that sucrose and magnesium chloride can increase the protection of virus, stabilize virus structure, and at the same time, play a role in regulating osmotic pressure; in addition, compared with other common buffers, the Tris-HCl buffer has little interference on biochemical processes and has wider pH adjusting range. The virus in the preparation is stable in the range of pH7.2-7.6, and the virus can be better stabilized by adjusting the pH of the buffer solution of the preparation to 7.2-7.6.
Further, according to an embodiment of the present invention, the virus preparation contains, based on the total amount of the virus preparation: 4.5 to 5.5 weight percent of sucrose; and 1.5-2.5 mmol/L magnesium chloride. By controlling the contents of sucrose and magnesium chloride in the virus preparation to be within the above ranges, the storage stability of the virus preparation can be further improved.
Further, according to an embodiment of the present invention, the virus preparation contains, based on the total amount of the virus preparation: 5% by weight of sucrose; and 2mmol/L magnesium chloride. Thus, the storage stability of the virus preparation is better.
According to an embodiment of the present invention, the concentration of Tris in the above Tris-HCl buffer is 50mmol/L. Thus, the storage stability of the virus preparation is better.
In addition, the inventors have found that if the sucrose content in the virus preparation is too high or too low, the virus stability after repeated freeze-thawing may be reduced; if the magnesium chloride content in the virus preparation is too high or too low, the virus stability of the virus at 2-8 ℃ and ambient temperature (25 ℃) may be reduced; if the pH in the virus preparation is too high or too low, the virus structure may be destroyed, reducing the virus activity.
According to an embodiment of the invention, the virus is a recombinant oncolytic virus. The recombinant oncolytic virus has better stability in a virus preparation, and the virus titer in the preparation is small in change after the recombinant oncolytic virus is stored for a long time at different temperatures or repeatedly thawed.
According to an embodiment of the present invention, the 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 change in the preparation is small after storage for a long time at different temperatures or repeated thawing.
According to an embodiment of the present invention, the above recombinant oncolytic virus expresses a viral protein with high affinity for a cell receptor selected from the group consisting of: (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 higher tumor cell specificity and/or broad spectrum of tumor treatment.
According to the embodiment of the invention, the recombinant vesicular stomatitis virus expressing the viral protein has stronger specific targeting property on tumor cells, wider killing spectrum on tumors and more remarkable killing effect.
The term "homology" as used herein means that the amino acid sequences have similarity, and the difference between individual amino acids in the amino acid sequences does not affect the function of the protein itself. The "homologous amino acid sequence" refers to an amino acid sequence derived by substitution, deletion, or addition of a single or multiple amino acids in the amino acid sequence of a polypeptide. Specifically, the expression "having a certain percentage of sequence homology" as used herein is obtained by calculating the following formula:
1-the number of different amino acids per amino acid number of the reference amino acid sequence X100%,
wherein the number of amino acids of the reference amino acid sequence refers to the number of amino acid sequences to be compared, and the reference amino acid sequence in the case where the G protein has at least 80% sequence homology with any one of SEQ ID NO. 1 or SEQ ID NO. 2 is SEQ ID NO. 1 or SEQ ID NO. 2.
The above amino acid sequences having homology have similarity in biology, chemistry or structure and have similar biological activity. Structurally similar means that the amino acids have side chains of similar length, such as alanine, glycine or serine, or side chains of similar size. Chemical similarity refers to amino acids that are identically charged or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine. Or polar amino acids such as arginine for lysine, glutamic for aspartic acids, glutamine for asparagine, serine for threonine, and the like. Biological similarity means that amino acid sequences having sequence homology are biologically functionally similar, such as recombinant vesicular stomatitis viruses according to embodiments of the invention, all have high affinity and binding to tumors with broad spectrum and specificity.
Vesicular Stomatitis Virus (VSV) belongs to the genus Vesicular Virus of the rhabdoviridae family, and is divided into two serotypes: new Jersey type (VSV-NJ) and Indiana type (VSV-IND). The virus particle is bullet or cylinder, and the size is 150-180 nm x 50-70 nm. The virus has a cyst membrane, and fiber protrusions with the length of about 10nm are uniformly and densely distributed on the cyst membrane. Inside the virus is a tightly coiled, helically-symmetric nucleocapsid. The virus is named according to classical vesicular lesions in the oral mucosa, bite block, tongue, lips, nostrils, hooves, and papillae of the affected animal. Transmitted by insect vectors, the disease is limited to its natural host, such as horses, cattle and pigs. In humans, the infection is mild and asymptomatic.
The VSV genome is an unsegmented, single-stranded negative-strand RNA (ssRNA) virus, approximately 11KB in length. Five non-overlapping genes, N, NS, M, G, and L, are arranged in order from the 3 'end to the 5' end and encode 5 different proteins, such as a nuclear (N) protein, a phospho (P) protein, a matrix (M) protein, a carbohydrate (G) protein, and an RNA-dependent RNA polymerase (L) protein. The 3 'end of the N gene is a Leader sequence (Leader), the 5' end is a trailer sequence (trailer), and interval sequences are arranged among the genes. The 3' end lead RNA is the earliest virus transcript in infected cells, has the length of 47 nucleotides, is not capped and cannot be translated, has not completely understood function, and can inhibit the synthesis of host RNA. The N protein is necessary for starting gene combination, and can effectively protect the virus RNA from being digested by various nucleases. The N protein has high antigenicity and immunogenicity, stimulates the body to produce non-neutralizing antibody cell immunity, plays an important role in transcription and replication, and is possibly necessary for maintaining genome RNA in an extended form and is related to replication regulation. The homology of P protein, VSV-NJ and VSV-IND virus strain is 41%, and its function is to maintain the transcription activity of virus together with polymerase L, nucleoprotein N to form polymerase complex and genome RNA. The M protein plays a key role in the pathogenic mechanism of the virus and viral replication, is rich in basic amino acids, contains a highly basic amino terminal domain, inhibits transcription by binding to the nucleocapsid, and at the same time assists the budding of the virus from the host, and is the only polypeptide involved in the budding process. The G protein is the major surface antigen of the virus, which determines the virulence of the virus and is also the protective antigen of the virus. It can stimulate the body to produce neutralizing antibodies. The L gene encodes the RNA poly E protein, which may determine the transcriptional activity of the RNA, and binds to the P protein to catalyze the replication of the mRNA. The protein is the core component of the polymerase complex and replicase complex, involved in initiation, extension, methylation, capping, poly (a) tail formation, and the like. In addition, the spacer sequence between each gene has extensive homology, and these sequences have a common structure, i.e., 3'-AUAC (U) 7NAUUGUCNN-UAG-5'. The conserved sequence between these genes is a key signal to influence the activity of the polymerase or the cleavage activity of the enzyme, and during replication these signals are masked and not functional.
In the description of the present invention, the terms "recombinant VSV virus", "recombinant vesicular stomatitis virus", "recombinant virus of the invention" are used interchangeably and refer to a recombinant VSV virus as described above which is capable of specifically infecting tumor cells, which specifically infects tumor cells, and which specifically binds to the specific receptors CHRNA5, SSTR5, KISS1R, HTR D and CCR8 of tumor cells selected from the group.
According to an embodiment of the invention, 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.
According to an embodiment of the invention, the recombinant vesicular stomatitis virus does not carry a heterologous gene. The inventors found that the killing effect of the recombinant vesicular stomatitis virus not carrying a heterologous gene on tumor cells was significantly higher than that of the recombinant vesicular stomatitis virus carrying a foreign gene. According to an embodiment of the present invention, the term "heterologous gene" described herein refers to a gene that has not been reported in the 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 wild-type vesicular stomatitis virus.
According to embodiments of the invention, the binding capacity of the viral proteins to cellular receptors is ZDOCK score of not less than 1800. It will be appreciated by those skilled in the art that the binding capacity characterization parameter ZDOCK score between a viral protein and a cellular receptor can be readily obtained by importing the sequences of the viral protein and the cellular receptor. The inventors found that when ZDOCK score 1800 is, for example, not less than 1900, not less than 2000, preferably not less than 2100, the binding between a virus carrying a viral protein and a tumor cell carrying the corresponding receptor will be significantly improved. According to embodiments of the invention, the ZDCK score is determinable by conventional software, see, for example, pierce BG, hourai Y, weng Z. (2011) Accelering Protein Docking in ZDCK Using an Advanced 3D convention library of plos One 6 (9): e24657.
According to an embodiment of the present invention, the above viral proteins include at least one selected from the group consisting of a G protein with GenBank accession number X03633.1 and a G protein with GenBank accession number DQ 408670.1. The inventor of the invention unexpectedly finds that the G protein with GenBank accession number X03633.1 and the G protein with GenBank accession number DQ408670.1 have obviously stronger binding force with the receptor of tumor cells than other G proteins.
According to an embodiment of the invention, 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 inventor unexpectedly finds that the recombinant virus obtained by combining and constructing the proteins and at least one of the G protein with GenBank accession number X03633.1 and the G protein with GenBank accession number DQ408670.1 has stronger tumor killing activity. The inventors believe that it is possible that for tumor cells, a combination of proteins from different sources may elicit a different immune response than a combination of proteins from the same source, thereby further enhancing killing of the tumor cells.
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 said RNA-dependent RNA polymerase
Preferably, at least one of 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 is derived from a strain of the Mudd summer subtype of vesicular stomatitis virus. The inventor unexpectedly finds that the recombinant virus obtained by combining and constructing the proteins and at least one of the G protein with GenBank accession number X03633.1 and the G protein with GenBank accession number DQ408670.1 has stronger tumor killing activity. The inventors believe that it is possible that for tumor cells, a combination of proteins from different sources may elicit a different immune response than a combination of proteins from the same source, thereby further enhancing killing of the tumor cells.
According to an embodiment of the invention, the viral formulation is in a form suitable for administration by inhalation or injection.
In a second aspect of the invention, a solution for formulating a viral formulation is provided. According to an embodiment of the invention, the solution for formulating the virus preparation comprises: sucrose; magnesium chloride; tris-HCl buffer, and the pH of the solution is 7.2-7.6.
The inventors have found that a virus preparation prepared from a solution having the above-mentioned composition shows little change in virus titer in the preparation when stored at different temperatures for a long period of time or after repeated thawing, and exhibits excellent storage stability. The reason for this may be that sucrose and magnesium chloride stabilize the virus structure, enhancing the protection against viruses; compared with other common buffers, the Tris-HCl buffer has wider pH adjusting range and better stabilizes the pH of the virus preparation; the virus in the preparation is stable in the range of pH7.2-7.6, and the virus can be better stabilized by adjusting the pH of the buffer solution of the preparation to 7.2-7.6.
Further, according to an embodiment of the present invention, the above solution for preparing a virus preparation contains 4.5 to 5.5% by weight of sucrose; and 1.5-2.5 mmol/L magnesium chloride. By controlling the contents of sucrose and magnesium chloride in the solution within the above ranges, the storage stability of the virus preparation prepared from the solution can be further improved.
Further, according to an embodiment of the present invention, the above solution for preparing a virus preparation contains 5% by weight of sucrose; and 2mmol/L magnesium chloride. Thus, the storage stability of the virus preparation prepared from the solution is better.
According to an embodiment of the present invention, the concentration of Tris in the above Tris-HCl buffer is 50mmol/L. Thus, the virus preparation prepared from the solution has better storage stability.
Furthermore, the invention provides the application of the pharmaceutical composition or the recombinant vesicular stomatitis virus in preparing a medicament for treating or preventing cancer or tumor.
According to an embodiment of the present invention, the cancer or tumor includes at least one selected from the group consisting of lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, and leukemia.
Further, the present invention provides a method for preventing or treating cancer or tumor. According to an embodiment of the invention, the method comprises: applying the above virus preparation or the above solution to a subject.
According to an embodiment of the present invention, the cancer or tumor includes at least one selected from the group consisting of lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, and leukemia.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
The following first describes examples related to the recombinant vesicular stomatitis virus of the present invention.
Example 1 analysis of human Membrane receptor genes based on Large samples of tumor tissue
The method for analyzing human membrane receptor genes based on a large sample of tumor tissue will be described in detail below with reference to fig. 1.
1.1 human Membrane receptor Gene and Pre-processing and analysis of expression data thereof
The invention summarizes and summarizes receptor gene information expressed in human cells from the existing research (reference documents (synthetic birth is a dominant pattern in a receptor-ligand evolution, BMC genomics, grandchamp and Monget,2018 Aug14 (1): 611.). The inventor selects the gene sequence of UCSC Xena (A receptor gene in human cells)http://xena.ucsc.edu/) The gene expression matrix (normalized value) of cancer patients, gene mutation information and relevant clinical data are downloaded. The cancer species included in the data were: adrenocortical carcinoma, urothelial carcinoma of the bladder, breast infiltrating carcinoma, squamous cell carcinoma of the cervix and endometrial carcinoma of the cervix, cholangiocarcinoma, colon adenocarcinoma/Rectum adenocarcinoma esophageal carcinoma, diffuse large B-cell lymphoma, esophageal carcinoma, second stage of FFPE assay, glioblastoma, glioma, head and neck squamous cell carcinoma, renal chromosome, panrenal cohort (KICH + KIRC + KIRP), renal clear cell carcinoma, renal papillary cell carcinoma, acute myelogenous leukemia, brain lower brain glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma and paraganglioma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, skin melanoma, gastric adenocarcinoma, gastric and esophageal carcinoma, testicular germ cell tumor, thyroid carcinoma, thymoma, endometrial carcinoma of the uterusUterine carcinosarcoma, uveal melanoma.
The inventors first culled less than three sample tumor and normal tissue information from the downloaded data and then performed differential expression analysis. The inventors performed Differential Expression analysis using Limma software (version: 3.38.3) (references (Limma Powers Differential Expression analysis for RNA-Sequencing and Microarray students. Nucleic Acids Research,43, e47, ritchai, m.e., et al. (2015)). The vom model of the Limma R package was used in the analysis. Genes were considered to be Differential genes only if they met the criteria | log2FC | >1,P value < 0.05.
1.2 data analysis
The fold difference in membrane receptor gene expression (log 2 FC) and p-value between groups was calculated using the R language. Selection of | log2FC | greater than or equal to 2.0 is considered to have significant up/down-regulation of differentially expressed genes. A p-value of less than 0.01 for the t-test is judged to be statistically significant. A heatmap of the log2FC matrix for each comparison pair was generated using the ComplexHeatmap R packet.
Then, the inventors selected 10 receptors based on a series of screening conditions, for example, conditions such as selection of a gene whose up-regulation is significant in 70% or more of cancer samples among intestinal cancer, lung cancer, pancreatic cancer, gastric cancer and liver cancer (i.e., a gene whose log2FC is 2.0 or more), high background expression level, and the like.
Specifically, the inventors plotted a jittered scatter plot (as shown in fig. 2) using ggplot2 and ggbeeforward software for the log2FC value of each gene in different tumor samples to show the proportion of patients whose genes were significantly upregulated in each tumor.
In addition, the inventor carries out molecular docking on the 13 screened receptors and alternative ligands, and selects 5 receptors with optimal binding force as final selection.
The results are shown in fig. 3 (wherein the ligand numbers shown in fig. 3 and the corresponding ligand names and amino acid sequence capture numbers are shown in table 1), and CHRNA5 (nicotinic cholinergic receptor α 5), SSTR5 (somatostatin receptor 5), KISS1R (KISS receptor), HTR1D (serotonin receptor 1D), CCR8 (C-C chemokine receptor 8) are receptor proteins differentially expressed in tumor cells and normal cells.
TABLE 1 ligand name and amino acid sequence Capture number
Example 2 selection of viral ligands according to receptor
The inventor selects 16 vesicular stomatitis virus homologous ligands, and carries out modeling and docking with 5 tumor specific receptors obtained by screening in example 1 respectively, and the generated docking results are ranked according to the ZDOK score, and the higher the score is, the stronger the binding is, and the higher the reliability of the results is. By simultaneously analyzing the clustering results of these conformations, it was found that the ZDOCK score is the shape complementation score calculated by the ZDOCK program, and that the ZDOCK score will also include electrostatic and desolvation energy terms depending on the parameter settings. The higher the ZDOCK score, the better. The inventors further evaluated the binding strength by the ZDCK score function, and obtained ligands with strong binding capacity for screening tumor specific receptors (the result is shown in FIG. 4), wherein the ligands with the best binding effect are DQ408670.1-lig-F and X03633.1-lig-FL, the capture number of the corresponding amino acid sequence is DQ408670.1, and GENE ID: X03633.1.
Example 3 construction and amplification of genetically recombinant vesicular stomatitis Virus based on different serotype proteins
The inventors combined L, N, P, M protein from Mudd summer subtype strain with G protein with the capture sequence number of GENE ID: DQ408670.1, GENE ID: X03633.1, GENE ID: KP872888.1 or GENE ID: HQ593628.1 to construct recombinant vesicular stomatitis viruses REV DQ408670.1, REV X03633.1, REV KP872888.1 and REV HQ593628.1.
The packaging method for the strains REV DQ408670.1, REV X03633.1, REV KP872888.1 and REV HQ593628.1 is as follows:
recombinant VSV in vitro requires a full-length plasmid containing the viral genome (containing the G protein), and a helper plasmid of the backbone proteins required for viral packaging (N, P, L, M), which is transferred into BHK21 cells by in vitro transfection methods, and the virus is released extracellularly after maturation by intracellular assembly (ref: vesicular stock culture virus-based vaccine technique of culture obtained from fresh culture with animal virus. Journal of virology 85,12781-12791, doi 10.1128/JVI I.00794-11 (2011), brown, K.S., safrometz, D., marzi, A., ebihara, H. & Feldmann, H.).
Vero cells are used for amplification of the virus, the virus with a certain titer is added into the cultured Vero cells, the virus can infect the cells and complete self-replication in the cells, mature virus is released into cell culture supernatant, the cell culture supernatant is concentrated to obtain virus concentrated solution, and the virus concentrated solution can be used for subsequent experiments after the titer is determined.
Example 4 detection of tumor cell receptors and results of cell killing
In this example, different viruses constructed in example 3 were used to verify the killing effect of different tumor cells.
3.1 q-PCR detection:
extracting with Trizol method to obtain 1 × 10 extract 6 The BXPC3, HCT-8, hepG2, su8686, H358, NCL-H460 (H460) and PANC1 cell samples were subjected to reverse transcription in a 20. Mu.L system at 500 ng/. Mu.L of RNA, and subjected to quantitative fluorescence PCR by the SYBR GREEN method to detect the expression of mRNA of CHRNA5, KISS1R, HTRID, CCR8 and SSTR5 genes in 7 cell samples.
The results are shown in FIG. 5. The qPCR detection result shows that the CHRNA5 receptor gene mRNA expression level of BXPC3, HCT-8, hepG2, su8686, H358, NCL-H460 and PANC1 cell samples is high, but in different cells, the receptors with high relative expression level have difference, for example, the CHRNA5 and HTR1D receptor with the highest expression level in H460 cells, and CHRNA5 and CCR8 receptor genes with high expression level in other cells.
3.2 cell killing assay (CCK):
the BXPC3, HCT-8, hepG2, su8686, H358 and PANC1 cells in good state are addedMaking into 5 × 10 4 Cell suspension per mL was added at 100 μ L/well to 96-well plates, edge-filled with medium to reduce evaporation, and incubated overnight. Known titres of virus were diluted to MOI with Opti-MEM: 0.01, MOI:0.1 and MOI:1, removing culture medium from a 96-well plate by aspiration, adding 50 μ L of virus diluent into each well, repeating 3 wells for each diluent, and taking 3 repeated wells of Opti-MEM as blank control. The virus dilution was changed after 2 hours of addition, 1% of FBS medium per well, 100. Mu.L. After 48/72h, 10. Mu.L of CCK8 detection solution was added to each well, and after incubation at 37 ℃ for 2h, the OD450 microplate reader read.
Fig. 6 shows the results of CCK killing of different cells by REV DQ408670.1, and the results of CCK assay show MOI:0.01, MOI:0.1 and MOI:1, the REV DQ408670.1 virus working solution has obvious killing effect on BXPC3, HCT-8, hepG2, su8686, H358 and PANC1 cells.
The results of the experiment are shown in FIG. 7. The CCK detection result shows that the REV DQ408670.1 and the REV X03633.1 are in MOI:0.01, MOI:0.1 and MOI:1 has obviously better killing effect on NCL-H358 and NCL-H460 cells than REV KP872888.1 and REV HQ593628.1. And simultaneously, the results of combining with a graph 5 show that the expression levels of CCR8 and CHRNA5 in NCL-H358 are higher, the expression levels of CHRNA5 and HTR1D in NCL-H460 are higher, and the binding force of DQ408670.1 and X03633.1G proteins to CCR8 and HTR1D receptors is strong by combining the results of a heat map of the receptor and the ligand in a graph 3. Comprehensively reflects that when the recombinant vesicular stomatitis virus has high binding force with a tumor cell receptor, the killing effect of the recombinant virus on tumor cells highly expressing the receptor is more remarkable.
Example 5 results of tumor cell killing based on selected G proteins by different L, N, P, M combinations of viral strains
Cell killing assay (CCK):
the inventor constructs REV DQ408670.1 virus strain, REV DQ408670.1-V1 and REV DQ408670.1-V2 virus strain by using antisense genetics method, wherein REV DQ408670.1-V1 changes L, M protein on the basis of REV DQ408670.1 virus strain, and REV DQ408670.1-V2 changes N, P protein on the basis of REV DQ408670.1 virus strain.
Thinning the well-conditioned H358 and H460Cell preparation of 5 × 10 4 Cell suspension per mL was added at 100 μ L/well to 96-well plates, edge-filled with medium to reduce evaporation, and incubated overnight. The known titre of 3 strains were diluted to MOI with Opti-MEM: 0.01 of the virus working solution, the culture solution in the 96-well plate was aspirated away, 50. Mu.L of virus diluent was added to each well, 3 wells of each diluent were repeated, and 3 further wells of Opti-MEM were selected as blanks. Virus dilutions were changed after 2h addition, 1% fbs medium per well 100 μ L. After 72h, 10. Mu.L of CCK8 detection solution was added to each well, and after incubation at 37 ℃ for 2h, the OD450 microplate reader read.
The results of the experiment are shown in table 2. The results for the 3 strains REV DQ408670.1, REV DQ408670.1-V1 and REV DQ408670.1-V2 were similar, at MOI: the 0.01 virus working solution has a remarkable killing effect on H358 and H460 cells.
Table 2: MOI 0.01% inhibition rate of virus on tumor cells%
| 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 |
Example 6 results of killing tumor cells based on selected G proteins and foreign Gene-inserted Virus strains
Cell killing assay (CCK):
using an antisense genetics method, the inventors inserted a heterologous gene INF β into the constructed REV DQ408670.1 virus strain to construct the virus strain FJ-INF β.
Well conditioned H358 and H460 cells were made into 5X 10 cells 4 Cell suspension per mL was added at 100 μ L/well to 96-well plates, edge-filled with medium to reduce evaporation, and incubated overnight. Known titers of REV DQ408670.1 strain and FJ-INF β strain were diluted to MOI with Opti-MEM, respectively: 0.01, MOI:0.1 and MOI:1, removing culture medium from a 96-well plate by aspiration, adding 50 μ L of virus diluent into each well, repeating 3 wells for each diluent, and taking 3 repeated wells of Opti-MEM as blank control. Virus dilutions were changed after 2h addition, 1% fbs medium per well 100 μ L. After 72h, 10. Mu.L of CCK8 detection solution was added to each well, and after incubation at 37 ℃ for 2h, the OD450 microplate reader read.
The results are shown in figure 8, REV DQ408670.1 strain is significantly better at killing H358 and H460 cells than FJ-INF β.
Example 6 detection of normal cell killing by the REV DQ408670.1 Virus Strain
Cell killing assay (CCK):
making lung normal cell BEAS-2B into 5 × 10 4 Cell suspension at 100. Mu.L/well was added to 96-well plates, edge-filled with medium to reduce evaporation, and incubated overnight. Known titers of REV DQ408670.1 virus were diluted to MOI with Opti-MEM: 0.01, MOI:0.1 and MOI:1, and removing culture medium from the 96-well plate, adding 50. Mu.L of virus diluent into each well, repeating 3 wells for each diluent, and taking 3 repeated wells of Opti-MEM as blank control. Virus dilutions were changed after 2h addition, 1% fbs medium per well 100 μ L. After 72h, 10. Mu.L of CCK8 detection solution was added to each well, and after incubation at 37 ℃ for 2h, the OD450 microplate reader read.
The results of the experiment are shown in FIG. 9. The results of the CCK assay showed that REV DQ408670.1 strain was at MOI:0.01, MOI:0.1 and MOI: the virus working solution of 1 has no obvious killing effect on BEAS-2B cells.
Examples relating to the viral formulations of the present invention are further described below.
Example 7
The prescription of the virus preparation comprises: 5% by weight sucrose, 2mmol/L magnesium chloride, tris-HCl buffer (50 mmol/L Tris, pH adjusted to 7.5 with HCl), and a titer of 9.8lgTCID 50 a/mL recombinant vesicular stomatitis oncolytic virus.
Comparative example 1
The virus preparation prescription is as follows: 0.01mol/L phosphate saline solution, pH7.2, and titer 9.2lgTCID 50 mL of recombinant vesicular stomatitis oncolytic virus.
Comparative example 2
The virus formulation was formulated substantially the same as in example 7, except that the sucrose content was 3% by weight.
Comparative example 3
The virus formulation was formulated substantially the same as in example 7, except that the sucrose content was 7% by weight.
Comparative example 4
The virus preparation was formulated substantially as in example 7, except that the magnesium chloride content was 0.5mol/L.
Comparative example 5
The virus preparation was formulated substantially as in example 7, except that the magnesium chloride content was 3.5mol/L.
Comparative example 6
The virus formulation was formulated essentially as in example 7, except that Tris-HCl buffer was used to adjust the formulation pH to 7.0 with HCl.
Comparative example 7
The virus formulation was formulated essentially as in example 7, except that Tris-HCl buffer was used to adjust the formulation pH to 8.0 with HCl.
Test example 1
The virus preparations of example 7 and comparative examples 1 to 7 were subjected to storage stability test at 2 to 8 ℃ and tested for virus titer after being left for 0d, 3d, 5d, 7d, 14d, 21d, 28d, 35d and 42d, respectively, wherein the test results of example 7 and comparative examples 1 to 7 are shown in Table 3 and FIG. 10.
TABLE 3 Virus titer test results (lgTCID) at 2-8 deg.C 50 /mL)
Test example 2
The virus preparations of example 7 and comparative examples 1 to 7 were subjected to storage stability test at 25. + -. 2 ℃ and tested for virus titer after being left for 0d, 3d, 5d, 7d, 14d, 21d, 28d, 35d and 42d, respectively, wherein the test results of example 7 and comparative examples 1 to 7 are shown in Table 4 and FIG. 11.
TABLE 4 Virus titer test results (lgTCID) at 25. + -. 2 ℃ 50 /mL)
Test example 3
The virus preparations of example 7 and comparative examples 1 to 7 were subjected to a storage stability test at-60 ℃ to measure virus titers after standing for 0d, 28d and 60d, respectively, wherein the test results of example 7 and comparative examples 1 to 7 are shown in Table 5 and FIG. 12. As can be seen from the results, the virus preparation of example 7 has better stability under low temperature freezing condition, and still has higher virus titer after being stored for 42 days.
TABLE 5 results of virus titer detection (lgTCID) at 60 deg.C 50 /mL)
Test example 4
The virus preparations of example 7 and comparative examples 1 to 7 were subjected to repeated thawing stability tests, and virus titers were measured after 1, 2, 3, 4 and 5 thawing, respectively, wherein the test results of example 7 and comparative examples 1 to 7 are shown in table 6 and fig. 13.
TABLE 6 results of virus titer detection (lgTCID) at different thawing times 50 /mL)
Discussion of results
For the virus preparations prescribed in example 7 and comparative examples 1 to 7, the virus titer was reduced by not more than 0.5lgTCID from the initial labeled amount after the test of the above test examples 50 mL, the virus titer was considered to fall within the acceptable range.
As can be seen from the results of test examples 1 and 2, the virus titer tended to decrease in the virus preparations formulated in examples 7 and comparative examples 1 to 7 with the increase of the standing time at the temperatures of 2 to 8 ℃ and 25. + -. 2 ℃. However, the viral formulations prescribed in example 7 can maintain the viral titer reduction within the acceptable range for longer periods of time. The virus preparations formulated in comparative examples 4 and 5 showed a large reduction in virus titer due to the low or high magnesium chloride content.
As can be seen from the results of test example 3, the virus preparations formulated in example 7 and comparative examples 1 to 3, 6 and 7 were left at a temperature of-60 ℃ for 60 days with substantially no change in virus titer, while the virus titers formulated in comparative examples 4 and 5 were significantly reduced.
As can be seen from the results of test example 4, the virus titer of the virus preparation prescribed in example 7 was not substantially changed with the increase of the number of repeated thawing cycles, and the virus titer after 5 repeated thawing cycles was substantially the same as the virus titer without thawing. The virus titer of the virus preparation prescribed in comparative example 1 is decreased with the increase of the number of repeated thawing, and after 5 times of repeated thawing, the virus titer is decreased by about 90% compared with the virus titer without thawing. The virus preparations of the formulas of comparative examples 2 and 3 have a large reduction in virus titer after repeated thawing due to the excessively low or high sucrose content in the formulas.
In addition, for the virus preparations formulated in comparative examples 6 and 7, the influence on the virus structure and activity is large due to too low or too high pH in the formulation, and the virus preparations formulated in comparative examples 6 and 7 have large virus titer reductions in test examples 1 to 4.
In the description of the specification, reference to the description of "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
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, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (18)
- A viral formulation comprising:a virus;sucrose;magnesium chloride;wherein the virus preparation contains Tris-HCl buffer solution, and the pH value is 7.2-7.6.
- The viral formulation according to claim 1, wherein the virus is a recombinant oncolytic virus.
- The viral formulation according to claim 2, wherein the recombinant oncolytic virus is a vesicular stomatitis virus.
- The viral formulation according to claim 2 or 3, wherein the recombinant oncolytic virus expresses a viral protein with high affinity for a cellular receptor selected from the group consisting of:(a)SEQ ID NO:1；(b) 2, SEQ ID NO; or(c) An amino acid sequence having at least 80% homology to (a) or (b).
- The viral formulation according to claim 4, wherein the binding capacity of the viral protein to the cellular receptor has a ZDOCK score of not less than 1800.
- The viral preparation according to claim 5, wherein the cellular receptor comprises at least one selected from CHRNA5, SSTR5, KISS1R, HTR1D, CCR8.
- The viral formulation according to claim 2, wherein 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 viral formulation according to claim 1, wherein the viral formulation comprises, based on the total amount of the viral formulation:4.5 to 5.5 weight percent of sucrose; and1.5-2.5 mmol/L magnesium chloride.
- The virus preparation according to claim 1, wherein the concentration of Tris in the Tris-HCl buffer is 50mmol/L.
- The viral formulation according to claim 1, wherein the viral formulation comprises, based on the total amount of the viral formulation:5% by weight of sucrose; and2mmol/L magnesium chloride.
- A solution for formulating a viral formulation, comprising:sucrose;magnesium chloride;Tris-HCl buffer, andthe pH of the solution is 7.2-7.6.
- The solution of claim 11, comprising:4.5 to 5.5 weight percent of sucrose; and1.5-2.5 mmol/L magnesium chloride.
- The solution of claim 12, comprising:5% by weight of sucrose; and2mmol/L magnesium chloride.
- The solution of claim 11, wherein the Tris concentration in the Tris-HCl buffer is 50mmol/L.
- Use of a viral formulation according to any one of claims 1 to 10 or a solution according to any one of claims 11 to 14 for the preparation of a medicament for the treatment or prevention of cancer or a tumour.
- The use of claim 15, wherein the cancer or tumor comprises at least one selected from the group consisting of lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, and leukemia.
- A method of preventing or treating cancer or a tumor, comprising: administering to a subject a viral formulation according to any one of claims 1 to 10 or a solution according to any one of claims 11 to 14.
- The method of claim 17, wherein the cancer or tumor comprises at least one selected from the group consisting of lung cancer, stomach cancer, liver cancer, intestinal cancer, esophageal cancer, breast cancer, cervical cancer, malignant lymphoma, nasopharyngeal cancer, and leukemia.
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