An oncolytic virus and its uses

Abstract

This application relates to the technical field of biomedicine, specifically disclosing an oncolytic virus and its uses. The oncolytic virus comprises an M protein and a G protein; the M protein, compared to the amino acid sequence shown in SEQ ID NO 1, contains amino acid substitutions at positions 51, 221, and 226; the G protein, compared to the amino acid sequence shown in SEQ ID NO 2, contains at least one amino acid substitution; the application also includes an expression vector for the oncolytic virus, virus-producing cells for producing the oncolytic virus, a pharmaceutical composition comprising the oncolytic virus; and methods for preparing and using the aforementioned oncolytic virus, oncolytic virus expression vector, virus-producing cells, and / or pharmaceutical composition. The oncolytic virus provided in this application exhibits good safety and cure rate.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, more specifically, it relates to an oncolytic virus and use thereof. BACKGROUND

[0002] Oncolytic virus is a kind of tumor-killing virus with replication ability, which has been widely accepted as an important branch of tumor immunotherapy. Oncolytic virus can specifically target tumor cells, for example, by taking advantage of the inactivation or defect of tumor suppressor genes in tumor cells, thereby selectively infecting tumor cells. After the oncolytic virus infects the tumor cells, it will replicate in the tumor cells and eventually destroy the tumor cells, thereby killing the tumor cells. At the same time, the oncolytic virus can also provide the necessary immune stimulation signal to enhance the host's anti-cancer response, thereby attracting more immune cells to continue to kill the residual tumor cells.

[0003] Although oncolytic virus has good application prospects in tumor immunotherapy, wild-type oncolytic virus often causes problems such as nervous system inflammation in the body, and there is a high risk of pathogenicity in the process of using wild-type virus to infect tumor cells. Therefore, in order to further promote the clinical application of oncolytic virus, it is necessary to modify the wild-type oncolytic virus to obtain attenuated oncolytic virus. The attenuated oncolytic virus is used for clinical application, thereby reducing the pathogenic risk of oncolytic virus and improving the safety of oncolytic virus.

[0004] However, in the process of modifying the oncolytic virus, if only random genetic modification is made to the wild-type oncolytic virus, although the toxicity can be reduced, the modified oncolytic virus may have poor cure rate, or even the modified oncolytic virus cannot be packaged, which is not conducive to promoting the clinical application of oncolytic virus. Therefore, it is of important scientific research value and application significance in the field of tumor immunotherapy to provide a modified oncolytic virus with good safety performance and high cure rate. SUMMARY

[0005] The present application provides an oncolytic virus and use thereof, the oncolytic virus provided by the present application has good safety and cure rate.

[0006] In a first aspect, the present application provides an oncolytic virus, which adopts the following technical solution:

[0007] In the present application, the oncolytic virus comprises an M protein and a G protein; the M protein comprises amino acid substitutions at positions 51, 221 and 226 compared with the amino acid sequence shown in SEQ ID NO 1; and the G protein comprises at least one amino acid substitution compared with the amino acid sequence shown in SEQ ID NO 2.

[0008] In certain embodiments, the amino acid substitution of the M protein comprises a methionine at position 51 mutated to an arginine (M51R); and / or, a valine at position 221 mutated to a phenylalanine (V221F); and / or, a serine at position 226 mutated to an arginine (S226R).

[0009] In certain embodiments, the amino acid substitution of the M protein comprises a methionine at position 51 mutated to an arginine (M51R).

[0010] In certain embodiments, the amino acid substitution of the M protein comprises a valine at position 221 mutated to a phenylalanine (V221F).

[0011] In certain embodiments, the amino acid substitution of the M protein comprises a serine at position 226 mutated to an arginine (S226R).

[0012] In certain embodiments, the M protein has the amino acid substitutions of M51R and V221F.

[0013] In certain embodiments, the M protein has the amino acid substitutions of M51R and S226R.

[0014] In certain embodiments, the M protein has the amino acid substitutions of M51R, V221F and S226R.

[0015] In certain embodiments, the M protein has the amino acid substitutions of V221F and S226R.

[0016] In certain embodiments, the M protein comprises an amino acid sequence as set forth in SEQ ID NO 5.

[0017] In certain embodiments, the M protein comprises an amino acid sequence as set forth in SEQ ID NO 3.

[0018] In certain embodiments, the M protein comprises an amino acid sequence as set forth in SEQ ID NO 4.

[0019] In certain embodiments, the G protein comprises an amino acid substitution at one or more of the following positions: position 438, position 453, position 471 and position 487, as compared to the amino acid sequence set forth in SEQ ID NO 2.

[0020] In some embodiments, the G protein, compared to the amino acid sequence shown in SEQ ID NO 2, includes amino acid substitutions at one or more of the following sites: position 53, position 141, position 172, position 217, position 232, position 331, position 371, position 436, position 438, position 453, position 471, and position 487.

[0021] In some embodiments, the amino acid substitutions of the G protein include a valine mutation at position 53 to isoleucine (V53I); and / or alanine mutation at position 141 to valine (A141V); and / or alanine mutation at position 172 to tyrosine (A172Y); and / or leucine mutation at position 217 to glutamic acid (L217E); and / or alanine mutation at position 232 to glycine (A232G); and / or valine mutation at position 331 to alanine. (V331A); and / or, valine at position 371 is mutated to glutamic acid (V371E); and / or, glycine at position 436 is mutated to aspartic acid (G436D); and / or, threonine at position 438 is mutated to serine (T438S); and / or, proline at position 453 is mutated to leucine (P453L); and / or, threonine at position 471 is mutated to isoleucine (T471I); and / or, threonine at position 487 is mutated to histidine (T487H).

[0022] In some embodiments, the amino acid substitution of the G protein includes a mutation of valine at position 53 to isoleucine (V53I).

[0023] In some embodiments, the amino acid substitution of the G protein includes a mutation of alanine at position 141 to valine (A141V);

[0024] In some embodiments, the amino acid substitution of the G protein includes a mutation of alanine at position 172 to tyrosine (A172Y).

[0025] In some embodiments, the amino acid substitution of the G protein includes a mutation of leucine at position 217 to glutamic acid (L217E).

[0026] In some embodiments, the amino acid substitution of the G protein includes a mutation of alanine at position 232 to glycine (A232G);

[0027] In some embodiments, the amino acid substitution of the G protein includes a mutation of valine at position 331 to alanine (V331A);

[0028] In some embodiments, the amino acid substitution of the G protein includes a mutation of valine at position 371 to glutamic acid (V371E).

[0029] In some embodiments, the amino acid substitution of the G protein includes a mutation of glycine at position 436 to aspartic acid (G436D).

[0030] In some embodiments, the amino acid substitution of the G protein includes a mutation of threonine at position 438 to serine (T438S).

[0031] In some embodiments, the amino acid substitution of the G protein includes a mutation of proline at position 453 to leucine (P453L).

[0032] In some embodiments, the amino acid substitution of the G protein includes a mutation of threonine at position 471 to isoleucine (T471I);

[0033] In some embodiments, the amino acid substitution of the G protein includes a mutation of the tyrosine residue at position 487 to histidine (Y487H).

[0034] In some embodiments, the amino acid substitutions of the G protein include T438S, P453L, T471I, and T487H. In some embodiments, the amino acid substitutions of the G protein include V53I and A141V.

[0035] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, and A172Y.

[0036] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, and L217E.

[0037] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, and A232G.

[0038] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, A232G, and V331A.

[0039] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, A232G, V331A, and V371E.

[0040] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, A232G, V331A, V371E, and G436D.

[0041] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, and T438S.

[0042] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, and P453L.

[0043] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, and T471I.

[0044] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H.

[0045] In some embodiments, the amino acid substitutions of the G protein include A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H.

[0046] In some embodiments, the amino acid substitutions of the G protein include A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H.

[0047] In some embodiments, the amino acid substitutions of the G protein include L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H.

[0048] In some embodiments, the amino acid substitutions of the G protein include A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H.

[0049] In some embodiments, the amino acid substitutions of the G protein include V331A, V371E, G436D, T438S, P453L, T471I, and T487H.

[0050] In some embodiments, the amino acid substitutions of the G protein include V371E, G436D, T438S, P453L, T471I, and T487H.

[0051] In some embodiments, the amino acid substitutions of the G protein include G436D, T438S, P453L, T471I, and T487H.

[0052] In some embodiments, the amino acid substitutions of the G protein include T438S, P453L, T471I, and T487H.

[0053] In some embodiments, the amino acid substitutions of the G protein include P453L, T471I, and T487H.

[0054] In some embodiments, the amino acid substitutions of the G protein include T471I and T487H.

[0055] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H.

[0056] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 17.

[0057] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 6.

[0058] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 7.

[0059] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 8.

[0060] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 9.

[0061] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 10.

[0062] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 11.

[0063] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 12.

[0064] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 13.

[0065] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 14.

[0066] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 15.

[0067] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 16.

[0068] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 18.

[0069] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 19.

[0070] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 20.

[0071] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 21.

[0072] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 22.

[0073] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 23.

[0074] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 24.

[0075] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 25.

[0076] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 26.

[0077] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 27.

[0078] In some embodiments, the G protein comprises an amino acid sequence as shown in SEQ ID NO 28.

[0079] In some embodiments, the oncolytic virus is obtained by site-directed mutation based on a rod-shaped virus.

[0080] In some embodiments, the oncolytic virus is obtained by site-directed mutation of Vesicular Stomatitis Virus (VSV).

[0081] In some embodiments, the oncolytic virus is obtained by site-directed mutation based on the Indiana MuddSummer subtype of VSV virus.

[0082] In some embodiments, the oncolytic virus contains or expresses an exogenous target protein.

[0083] In some embodiments, the oncolytic virus comprises a nucleic acid molecule containing a nucleic acid sequence encoding the M protein with amino acid substitutions and a nucleic acid sequence encoding the G protein with amino acid substitutions.

[0084] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the exogenous target protein.

[0085] In some embodiments, the nucleic acid sequence encoding the exogenous target protein in the nucleic acid molecule is located between the nucleic acid sequence encoding the M protein with amino acid substitutions and the nucleic acid sequence encoding the G protein with amino acid substitutions.

[0086] In some embodiments, the nucleic acid molecule encodes the N protein (nucleoprotein N), L protein (large polymerase protein L), and P protein (phosphoprotein P) of the oncolytic virus.

[0087] Secondly, this application provides an oncolytic virus expression vector, which adopts the following technical solution:

[0088] An oncolytic virus expression vector, said oncolytic virus expression vector being capable of generating any of the oncolytic viruses described in this application.

[0089] Thirdly, this application provides a virus production cell, employing the following technical solution:

[0090] A virus-producing cell capable of producing any of the oncolytic viruses described in this application.

[0091] Fourthly, this application provides a pharmaceutical composition, which adopts the following technical solution:

[0092] A pharmaceutical composition comprising any of the oncolytic viruses described in this application, and optionally a pharmaceutically acceptable carrier.

[0093] Fifthly, this application provides methods for preparing the above-mentioned oncolytic virus, oncolytic virus expression vector, virus production cells and / or pharmaceutical composition.

[0094] Sixthly, this application provides the use of the above-mentioned oncolytic virus, oncolytic virus expression vector, virus production cell and / or pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and / or symptoms.

[0095] In some embodiments, the oncolytic virus, oncolytic virus expression vector, virus-producing cells, and / or pharmaceutical composition are used in a method for slowly and continuously killing abnormally proliferating cells.

[0096] In some embodiments, the disease and / or symptom includes: abnormally proliferating cells selected from tumor cells or related cells of tumor tissue; preferably, the tumor cells are cancer cells; more preferably, the cancer cells are metastatic cancer cells.

[0097] In some implementations, the tumor includes solid tumors and / or hematomas.

[0098] In summary, this application has the following beneficial effects:

[0099] The oncolytic viruses provided in this application all exhibit good infectivity against LLC cells, MC38 cells, and HeLa cells, and good in vitro killing ability against LLC cells, MC38 cells, and 4T1 cells. They are also difficult to eliminate from LLC cells, MC38 cells, and HeLa cells. Furthermore, the oncolytic viruses provided in this application all exhibit poor infectivity, in vitro killing ability, and ease of elimination against MEF cells. Therefore, the oncolytic viruses provided in this application can be effectively used for the infection and killing of tumor and cancer cells, and are difficult to eliminate from tumor and cancer cells, further improving the cure rate of oncolytic viruses against tumor and cancer cells. Simultaneously, the oncolytic viruses provided do not damage normal cells and are more easily eliminated from normal cells, further ensuring the safety of normal cells. Attached Figure Description

[0100] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0101] Figure 1 Results of the detection of the infectivity of the oncolytic virus prepared for this application and the wild-type oncolytic virus on LLC cells.

[0102] Figure 2 Results of the detection of the infectivity of the oncolytic virus prepared in this application and the wild-type oncolytic virus against MC38 cells.

[0103] Figure 3 The results of the detection of the infectivity of the oncolytic virus prepared in this application and the wild-type oncolytic virus against HeLa cells.

[0104] Figure 4 Results of the detection of the infectivity of the oncolytic virus prepared in this application and the wild-type oncolytic virus to MEF cells.

[0105] Figure 5 Results of in vitro killing ability of the oncolytic virus prepared in this application and the wild-type oncolytic virus against LLC cells.

[0106] Figure 6 The results show the in vitro killing ability of the oncolytic virus prepared in this application and the wild-type oncolytic virus against MC38 cells.

[0107] Figure 7 The results show the in vitro killing ability of the oncolytic virus prepared in this application and the wild-type oncolytic virus against 4T1 cells.

[0108] Figure 8 Results of in vitro killing ability of the oncolytic virus prepared in this application and the wild-type oncolytic virus against MEF cells.

[0109] Figure 9 Results of the detection of the ease of clearance of the oncolytic virus prepared in this application and the wild-type oncolytic virus in LLC cells.

[0110] Figure 10 Results of the detection of the oncolytic virus prepared in this application and the ease of clearance of the wild-type oncolytic virus in MC38 cells.

[0111] Figure 11 Results of the detection of the ease of clearance of the oncolytic virus prepared in this application and the wild-type oncolytic virus in HeLa cells.

[0112] Figure 12 Results of the detection of the ease of clearance of the oncolytic virus prepared in this application and the wild-type oncolytic virus in MEF cells.

[0113] In the above figures, the horizontal axis 0 represents wild-type oncolytic virus; the horizontal axes 1-26 represent the oncolytic viruses prepared in preparation examples 1-26, respectively.

[0114] The vertical axis is Log 10 TCID50 represents the TCID50 value calculated using the Karber method, Log 10 A higher TCID50 value indicates a better ability of the oncolytic virus to infect the cell; Log 10The smaller the TCID50 value, the weaker the ability of the oncolytic virus to infect the cell;

[0115] The vertical axis OD 570 OD represents the OD value of a cell. 570 The higher the OD value, the weaker the oncolytic virus's ability to kill the cell; 570 The smaller the value, the better the oncolytic virus's ability to kill the cell.

[0116] The vertical axis represents the IFN-β level, indicating the expression of the IFN-β gene. A higher IFN-β level indicates that the oncolytic virus is more easily cleared from the cell; a lower IFN-β level indicates that the oncolytic virus is less easily cleared from the cell.

[0117] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Detailed Implementation

[0118] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0119] Terminology Definition

[0120] In this application, the term "oncolytic virus" generally refers to a virus capable of replicating in and killing tumor cells. Oncolytic viruses include, but are not limited to: VSV virus, poxvirus, herpes simplex virus, measles virus, Semleeki Forest virus, poliovirus, reovirus, Seneca Valley virus, echovirus, Coxsackie virus, Newcastle disease virus, and Malaba virus. In some embodiments, the oncolytic virus is modified to improve selectivity for tumor cells. In some embodiments, the oncolytic virus is modified to reduce its immunogenicity. In some embodiments, the oncolytic virus described in this application is a VSV virus. In some embodiments, the VSV virus is a mutant of the Indiana MuddSummer subtype of VSV virus. In some embodiments, site-directed gene mutations can be performed on the M and G proteins of the VSV virus.

[0121] In some embodiments, the oncolytic virus described in this application may be a genetically modified oncolytic virus, such as one or more modified genes to enhance its tumor selectivity and / or preferentially replicate in dividing cells. The genetic modification may involve modifying genes involved in DNA replication, nucleic acid metabolism, host orientation, surface attachment, virulence, lysis, and diffusion processes, or it may involve integrating exogenous genes. The exogenous genes may include exogenous immune regulatory genes, exogenous selection genes, exogenous reporter genes, etc. The modified oncolytic virus may also be an amino acid-modified oncolytic virus, such as through the insertion, deletion, or substitution of one or more amino acids.

[0122] In this application, the term "M protein" generally refers to the VSV viral matrix protein. The M protein is an important virulence factor of VSV and is also a known VSV protein that can interfere with the innate immune response in mice. The term "M protein" also includes its homologs, orthologs, variants, functionally active fragments, etc. In this application, the wild-type VSV virus Indiana MuddSummer subtype M protein may contain the amino acid sequence shown in SEQ ID NO 1. In this application, the oncolytic virus M protein may contain the amino acid sequences shown in SEQ ID NO 3-5.

[0123] In this application, the term "G protein" generally refers to the glycoprotein of water VSV virus, also known as the envelope protein. The term "G protein" also includes its homologs, orthologs, variants, functionally active fragments, etc. In this application, the G protein of the wild-type VSV virus Indiana MuddSummer subtype may contain the amino acid sequence shown in SEQ ID NO 2. In this application, the glycoprotein G of the oncolytic virus may contain the amino acid sequences shown in SEQ ID NO 6-28.

[0124] In this application, the term "L protein" generally refers to the VSV viral RNA polymerase protein. The L gene of VSV virus encodes the RNApoly E protein. The term "L protein" also includes its homologs, orthologs, variants, functionally active fragments, etc.

[0125] In this application, the term "N protein" generally refers to the nucleocapsid protein of the VSV virus. The term "N protein" also includes its homologs, orthologs, variants, functionally active fragments, etc.

[0126] In this application, protein mutation sites are typically described as "amino acid + amino acid position + (mutated amino acid)". In this application, the mutation may include, but is not limited to, the addition, substitution, deletion, and / or removal of amino acids. For example, the term "M51R" typically refers to a mutation at position 51, where methionine M is replaced by arginine R.

[0127] In this application, the term "amino acid substitution" generally refers to replacing an amino acid residue present in the parental sequence with another amino acid residue. The amino acid in the parental sequence can be substituted, for example, via chemical peptide synthesis or by recombination methods known in the art. Therefore, "substitution at position xx" generally means replacing the amino acid present at position xx with an alternative amino acid residue. In this application, the amino acid substitution may include amino acid mutations.

[0128] In this application, the term "mutation" generally refers to an alteration of the nucleotide or amino acid sequence of a wild-type molecule. Amino acid changes can include substitution, deletion, omission, insertion, addition, truncation, or protein processing or cleavage.

[0129] In this application, the term "nucleic acid molecule" generally refers to a nucleotide of any length. In this application, the term "nucleic acid molecule" may encode a protein contained in the oncolytic virus. In this application, the nucleic acid molecule may contain DNA and / or RNA. In some cases, the RNA may contain single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA), and the single-stranded RNA may contain sense RNA, anti-sense RNA, or ambiguous RNA.

[0130] In this application, the term "expression vector" generally refers to a nucleic acid vector. Under appropriate conditions, it is typically capable of expressing a target gene and / or a target protein. In some embodiments of this application, the expression vector comprises a nucleic acid molecule for expressing one or more components of a virus (e.g., an oncolytic virus). For example, the expression vector may include at least one viral genomic element and may be packaged into a virus or packaged as a viral particle.

[0131] In this application, the term "virus-producing cell" generally refers to a cell, cell line, or cell culture that may contain or already contains the nucleic acid molecules or expression vectors described in this application, or is capable of expressing the oncolytic virus described in this application. The cell may include progeny of a single host cell. The cell can be obtained by in vitro transfection using the expression vectors described in this application.

[0132] In this application, the term "pharmaceutical composition" generally refers to a formulation present in a form that allows for the effective biological activity of the active ingredient and does not contain any additional ingredients that would have unacceptable toxicity to the subject to whom the formulation is to be administered. In some embodiments, these formulations may comprise the active component of a drug and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical product comprises a drug product administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. The pharmaceutical product may be administered via various routes, such as intravenous, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration.

[0133] In this application, the term "prevention" generally refers to preventing the occurrence, onset, recurrence, and / or spread of a disease or one or more symptoms thereof by taking certain measures in advance. In this application, the term "treatment" generally refers to eliminating or improving a disease, or one or more symptoms associated with a disease. In some embodiments, treatment generally refers to administering one or more drugs to a patient suffering from the disease so that the disease is eliminated or alleviated. In some embodiments, "treatment" may be the administration of the drug combination and / or pharmaceutical product after the onset of symptoms of a specific disease, in the presence or absence of other drugs. For example, using the drug combination and / or pharmaceutical product described in this application to prevent the occurrence, development, recurrence, and / or metastasis of a tumor.

[0134] In this application, the term "tumor" generally refers to any new pathological tissue growth. Tumors may be benign or malignant. In this application, the tumor may be a solid tumor and / or a hematoma. When used for research purposes, these tissues can be isolated from readily available resources using methods well known to those skilled in the art. Invention Details

[0136] This application provides an oncolytic virus based on wild-type VSV virus, specifically the Indiana strain or the Indiana MuddSummer subtype of VSV virus, obtained by mutating sites on the amino acid sequences of its M protein and / or G protein. The amino acid sequence of its M protein is shown in SEQ ID NO 1; the amino acid sequence of its G protein is shown in SEQ ID NO 2. In this application, the M protein can be modified, and the G protein can also be modified.

[0137] This application modifies the VSV virus as follows to obtain an oncolytic virus:

[0138] The oncolytic virus comprises an M protein and a G protein; the M protein contains amino acid substitutions at positions 51, 221, and 226 compared to the amino acid sequence shown in SEQ ID NO 1; the G protein contains at least one amino acid substitution compared to the amino acid sequence shown in SEQ ID NO 2.

[0139] The amino acid substitutions of the M protein include a mutation at position 51 of methionine to arginine (M51R); and / or, a mutation at position 221 of valine to phenylalanine (V221F); and / or, a mutation at position 226 of serine to arginine (S226R); for example, the M protein contains the amino acid sequence shown in SEQ ID NO 5. In this application, the M protein may also contain amino acid substitutions at other positions.

[0140] In this application, the amino acid substitutions of the G protein may include a mutation at position 53 of valine to isoleucine (V53I); and / or a mutation at position 141 of alanine to valine (A141V); and / or a mutation at position 172 of alanine to tyrosine (A172Y); and / or a mutation at position 217 of leucine to glutamic acid (L217E); and / or a mutation at position 232 of alanine to glycine (A232G); and / or a mutation at position 331 of valine to alanine (V331A). ; and / or, valine at position 371 is mutated to glutamic acid (V371E); and / or, glycine at position 436 is mutated to aspartic acid (G436D); and / or, threonine at position 438 is mutated to serine (T438S); and / or, proline at position 453 is mutated to leucine (P453L); and / or, threonine at position 471 is mutated to isoleucine (T471I); and / or, threonine at position 487 is mutated to histidine (T487H); the G protein may contain the amino acid sequence shown in SEQ ID NO 17.

[0141] In this application, the G protein may contain amino acid mutations at positions 53 and 141.

[0142] In this application, the G protein may contain amino acid mutations at positions 53, 141, and 172.

[0143] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, and 217.

[0144] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, and 232.

[0145] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, and 331.

[0146] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, and 371.

[0147] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, and 436.

[0148] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, and 438.

[0149] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, and 453.

[0150] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, 453, and 471.

[0151] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0152] In this application, the G protein may contain amino acid mutations at positions 141, 172, 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0153] In this application, the G protein may contain amino acid mutations at positions 172, 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0154] In this application, the G protein may contain amino acid mutations at positions 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0155] In this application, the G protein may contain amino acid mutations at positions 232, 331, 371, 436, 438, 453, 471, and 487.

[0156] In this application, the G protein may contain amino acid mutations at positions 331, 371, 436, 438, 453, 471, and 487.

[0157] In this application, the G protein may contain amino acid mutations at positions 371, 436, 438, 453, 471, and 487.

[0158] In this application, the G protein may contain amino acid mutations at positions 436, 438, 453, 471, and 487.

[0159] In this application, the G protein may contain amino acid mutations at positions 436, 453, 471, and 487.

[0160] In this application, the G protein may contain amino acid mutations at positions 453, 471, and 487.

[0161] In this application, the G protein may contain amino acid mutations at positions 471 and 487.

[0162] In this application, the G protein may contain a mutation at amino acid position 487.

[0163] In this application, the G protein may also contain amino acid substitutions at other positions.

[0164] In some embodiments, the G protein contains at least one or more amino acid substitutions in a conserved region. For example, the conserved region may contain amino acids 437-461 of the G protein. In some embodiments, the G protein contains at least one or more amino acid substitutions in a truncated region of the cytoplasmic domain. For example, the truncated region of the cytoplasmic domain may contain amino acids 483-511 of the G protein.

[0165] In this application, the G protein may contain at least amino acid substitutions at positions 438, 453, 471, and 487.

[0166] The oncolytic virus may further comprise a nucleic acid molecule and a foreign target protein. The nucleic acid molecule may comprise a nucleic acid sequence encoding the M protein with amino acid substitutions and a nucleic acid sequence encoding the G protein with amino acid substitutions; the nucleic acid molecule may also comprise a nucleic acid sequence encoding the foreign target protein. Further, the nucleic acid sequence encoding the foreign target protein is located between the nucleic acid sequence encoding the M protein with amino acid substitutions and the nucleic acid sequence encoding the G protein with amino acid substitutions. Still further, the nucleic acid molecule encodes the N protein (nucleoprotein N), L protein (large polymerase protein L), and P protein (phosphoprotein P) of the oncolytic virus.

[0167] In this application, the oncolytic virus described herein can be obtained through a viral packaging process and a viral rescue process. Specifically, the process may include infecting BSR-T7 cells with a poxvirus vTF7-3 expressing T7 RNA polymerase, followed by lipofectamine transfection using expression plasmids and backbone plasmids that respectively clone the VSVN, VSV P, and VSV L genes to obtain the target oncolytic virus.

[0168] This application also provides an oncolytic virus expression vector, a virus production cell, and a pharmaceutical composition.

[0169] The oncolytic virus expression vector may contain nucleic acid sequences encoding the M and G proteins of the oncolytic virus; the oncolytic virus expression vector may also contain nucleic acid sequences encoding the N, P, and L proteins of the oncolytic virus.

[0170] The virus-producing cells are capable of producing the aforementioned oncolytic virus; the virus-producing cells may include BSR-T7 cells.

[0171] The pharmaceutical composition comprises the oncolytic virus described above, and optionally a pharmaceutically acceptable carrier.

[0172] In some embodiments, the pharmaceutical composition may include suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the pharmaceutical composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of this application include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0173] In some embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration and are generally safe and non-toxic.

[0174] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously. This continuous (or uninterrupted) administration may be achieved using a small pump system worn by the patient to measure the amount of therapeutic agent flowing into the patient, as described in WO2015 / 036583.

[0175] Furthermore, this application also provides a method for preparing the aforementioned oncolytic virus, which may include methods for preparing oncolytic virus expression vectors, virus production cells, and / or pharmaceutical compositions. Any method suitable for producing oncolytic viruses can be used to generate the oncolytic virus of this application. For example, cells can be transfected with a poxvirus expressing T7 RNA polymerase, and then transfected with plasmids expressing the oncolytic virus N, L, and P proteins, as well as a backbone plasmid, to obtain the oncolytic virus of this application through a virus rescue process.

[0176] This application also provides the use of the above-mentioned oncolytic viruses, oncolytic virus expression vectors, virus production cells and / or pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of diseases and / or symptoms.

[0177] The oncolytic viruses provided in this application all exhibit good infectivity against LLC cells, MC38 cells, and HeLa cells. In particular, the oncolytic viruses numbered JBS104-JBS126, based on the oncolytic virus numbered JBS103, have undergone site-directed mutations in the amino acids of their G protein, further enhancing their infectivity against LLC, MC38, and HeLa cells. Meanwhile, the oncolytic viruses prepared above show poor infectivity against normal MEF cells, indicating that the oncolytic viruses prepared in this application can be effectively used to infect tumor and cancer cells without damaging normal cells, demonstrating broad application prospects.

[0178] The oncolytic viruses provided in this application all exhibit good in vitro killing ability against LLC cells, MC38 cells, and 4T1 cells. In particular, the oncolytic viruses numbered JBS104-JBS126, based on the oncolytic virus numbered JBS103, have undergone site-directed mutations in the amino acids of their G protein, further enhancing their in vitro killing ability against LLC, MC38, and 4T1 cells. Simultaneously, the oncolytic viruses prepared above have almost no effect on MEF cells, indicating that the oncolytic viruses prepared in this application can be effectively used to damage and kill abnormal cells such as tumors and cancer cells without harming normal cells.

[0179] Oncolytic viruses designated JBS101 and JBS102 are not easily cleared from LLC, MC38, and HeLa cells. In contrast, oncolytic virus designated JBS103 is more easily cleared from these cells. The oncolytic viruses designated JBS104-JBS126 in this application, based on the oncolytic virus designated JBS103, further involve site-directed mutations in the amino acids of the G protein, making them even more difficult to clear from LLC, MC38, and HeLa cells. This further ensures that the oncolytic viruses can better exert their infective and killing abilities in these cells. Simultaneously, the oncolytic viruses provided in this application are more easily cleared from MEF cells, further ensuring the safety of MEF cells and thus improving the safety of the oncolytic viruses.

[0180] Without being limited by any theory, the embodiments described below are merely for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0181] Preparation Example

[0182] Preparation Examples 1-3

[0183] Preparation Examples 1-3 each provide an oncolytic virus, differing mainly in the site-directed mutation of the amino acid in the M protein of the wild-type oncolytic virus. The specific construction methods for the oncolytic viruses corresponding to each preparation example are as follows:

[0184] (1) Constructing plasmids

[0185] Using the pRV-core plasmid (BioVector NTCC plasmid vector bacterial cell gene depository) as a template, the mutation sites shown in Table 1 were introduced using PCR technology. The pRV-core plasmid was then subjected to PCR with primers carrying each mutation site; the PCR products were then subjected to 1% agarose gel electrophoresis; and the gel was then extracted using a gel extraction kit to obtain plasmids with different mutation sites of the M protein, thus obtaining the constructed plasmid pRV-core Mut.

[0186] Table 1. Mutation status of oncolytic virus M protein in preparation examples 1-3.

[0187]

[0188] (2) Virus rescue

[0189] Using a calcium phosphate transfection kit (Thermo Fisher Scientific), the constructed plasmid pRV-core Mut was transfected into BSR-T7 cells (purchased from ATCC, the American Type Culture Collection Center, also known as the American Type Culture Collection Center) via cell transfection technology.

[0190] The four plasmids, pRV-core Mut, pP, pN, and pL, were mixed in a mass ratio of 10:5:4:1, with a total plasmid volume of 5 μg. The plasmids were diluted with 200 μl of opti-MEM medium (Thermo Fisher Scientific) and 7.5 μl of transfection reagent Plus Reagent (Life Technologies) was added to obtain a premixed transfection plasmid solution. The premixed solution contained pP (plasmid carrying the rod-shaped virus phosphoprotein gene), pN (plasmid carrying the rod-shaped virus nucleoprotein gene), and pL (plasmid carrying the rod-shaped virus polymerase protein gene). The parent vectors for the pN, pP, and pL plasmids were all pCAGGS (purchased from ATCC).

[0191] Dilute 10 μl of Lipofectamine LTX (Thermo Fisher Scientific) with 200 μl of opti-MEM medium to obtain an LTX mixture;

[0192] Plasmid transfection was performed according to the instructions for Lipofectamine LTX. After 6 hours, BSR-T7 cells were washed twice with PBS and then seeded in DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum for 3 days.

[0193] The cell supernatant obtained from culturing BSR-T7 cells was transferred to Vero cells (Thermo Fisher Scientific), and the Vero cells were cultured at 37°C for 3 days. The green fluorescence in the cells was observed under a fluorescence microscope to determine the virus rescue status. The rescued mutant rod-shaped virus library was further passaged in Vero cells, and monoclonal virus strains were selected in the established plaque screening system.

[0194] (3) M protein gene sequencing. Viral genomic RNA was extracted using a Trizol kit, and reverse transcription was performed using random primers. The cDNA transcribed from the reverse transcription was then subjected to PCR using primers designed for the M protein gene sequence.

[0195] The primer sequences are:

[0196] 5'-AAAAAAGTAACAGATATCAC-3';

[0197] 5'-ACATTTTTCCAGTTTCCTTTTTGG-3'.

[0198] The product was recovered after 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequencing results are shown in Table 1.

[0199] Preparation Example 4-26

[0200] Preparation Examples 4-26 each provide an oncolytic virus, differing mainly in the site-directed amino acid mutation sites on the M and G proteins of the wild-type oncolytic virus. The mutation site in the M protein is the same as the corresponding mutation site in Preparation Example 3. The construction methods for the oncolytic viruses in each preparation example are the same as those described in Preparation Examples 1-3, with the following differences:

[0201] The mutation sites shown in Table 2 were introduced into the plasmid constructed in step (1) using PCR technology;

[0202] Step (3) involves sequencing the G protein gene. Viral genomic RNA was extracted using a Trizol kit, and reverse transcription was performed using random primers. The cDNA transcribed from the reverse transcription was then subjected to PCR using primers designed for the G protein gene sequence.

[0203] The primer sequences are:

[0204] 5'-CCATGGCCTGTTGCTCCACCAGCTT-3';

[0205] 5'-AAGCTTCAGCAGTGGCTCACAGCAG-3'.

[0206] The product was recovered after 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequencing results are shown in Table 2.

[0207] Table 2. Mutation status of oncolytic virus G protein in preparation examples 4-26

[0208]

[0209]

[0210] Preparation Example 27

[0211] This preparation example provides a packaging process for the oncolytic virus prepared using Examples 1-26 above, specifically including the following steps:

[0212] 1) BSR-T7 cells (purchased from ATCC) were infected with poxvirus vTF7-3 expressing T7 RNA polymerase (BioVector NTCC plasmid vector bacterial strain cell gene deposit center).

[0213] The specific steps are as follows: Plate BSR-T7 cells in a 6-well plate, controlling the cell count per well to be 3 × 10⁶. 5 After 14-16 hours of plating, poxvirus vTF7-3 expressing T7 RNA polymerase was added to infect BSR-T7 cells with poxvirus vTF7-3. Six hours after infection, BSR-T7 cells were washed once with DPBS buffer (Thermo Fisher Scientific) and transfected.

[0214] 2) Transfection process

[0215] The specific steps include: mixing the four plasmids (pRV-coreMut, pP, pN, and pL) in a mass ratio of 10:5:4:1, with a total plasmid volume of 5 μg; diluting the plasmids with 200 μl of opti-MEM medium (Thermo Fisher Scientific) and adding 7.5 μl of transfection reagent Plus Reagent (Life Technologies) to obtain a premixed transfection plasmid solution; wherein pP (plasmid carrying the rod-shaped virus phosphoprotein gene), pN (plasmid carrying the rod-shaped virus nucleoprotein gene), and pL (plasmid carrying the rod-shaped virus polymerase protein gene); the parent vectors corresponding to the three plasmids pN, pP, and pL are all pCAGGS (purchased from ATCC);

[0216] Dilute 10 μl of Lipofectamine LTX (Thermo Fisher Scientific) with 200 μl of opti-MEM medium to obtain an LTX mixture;

[0217] Mix 200 μl of LTX mixture with 200 μl of transfection plasmid premix and incubate at room temperature for 15 min to obtain LTX-DNA mixture;

[0218] Replace the DPBS buffer in the 6-well plate from step 1) with Opti-MEM medium. Add the LTX-DNA mixture dropwise to the 6-well plate containing BSR-T7 cells. Gently shake the 6-well plate to distribute the LTX-DNA mixture evenly within the 6-well plate. After 6-8 hours of transfection, remove the transfection reagent and add 3 ml of fresh complete culture medium (Thermo Fisher Scientific). After 72 hours, harvest the cell supernatant from the BSR-T7 cells and filter it using a 0.22 μm filter to obtain the corresponding oncolytic virus droplets for each of Examples 1-26.

[0219] Example

[0220] Example 1

[0221] This embodiment utilizes the oncolytic viruses prepared in Examples 1-26 and wild-type oncolytic viruses to detect the infectivity of different cells. The detection method is the TCID50 assay, in which 200 pfu of each of the oncolytic viruses prepared in Examples 1-26 and wild-type oncolytic viruses are added to the culture medium of different cells, and the half-maximal infectious dose (TCID50) produced by each oncolytic virus is detected. The cells tested include: LLC cells (mouse non-small cell lung cancer cells), MC38 cells (mouse colon cancer cells), HeLa cells (human cervical cancer cells), and MEF cells (human fibroblasts). The specific detection method is as follows:

[0222] (1) Add 3 mL of Vero (LLC / MC38 / HeLa / MEF) cell suspension to each of the 6-well culture plates to achieve a cell count of 4 × 10⁻⁶ cells / wells. 5 Cells / well, 6 wells for each type of Vero cell, 2 wells for MEF cells as a control; the 6-well culture plate was incubated at 37℃ and 5% CO2 for 16 h;

[0223] (2) Add 200 pfu of the oncolytic virus prepared in the preparation example to each well of a 6-well culture plate, and harvest 100 μl of supernatant from MEF cells and each type of Vero cell after 24 h; add the harvested supernatant to the wells of a 96-well culture plate to make the cell mass of each type of cell reach 1 × 10⁻⁶. 4 The number of cells / ml was increased, and the 96-well culture plate was incubated at 37°C and 5% CO2 for 16 hours.

[0224] (3) Dilute the supernatant obtained in step (2) 10-fold in a 1.5ml EP tube. -1 ~10 -11 There are 11 titers in total; the diluted supernatant is inoculated into a 96-well plate, with 8 wells in a row for each dilution, and 100 μl inoculated into each well;

[0225] (4) After 48 hours, observe the fluorescence of cells in each well. If fluorescence is present, the well is considered infected. Calculate TCID50 according to the Karber method.

[0226] Test results as follows Figures 1-4 As shown, the horizontal axis 0 represents wild-type oncolytic virus; the horizontal axes 1-26 represent the oncolytic viruses prepared in preparation examples 1-26, respectively; the vertical axis Log 10 TCID50 represents the TCID50 value calculated using the Karber method, Log 10 A higher TCID50 value indicates a better ability of the oncolytic virus to infect the cell; Log 10 The smaller the TCID50 value, the weaker the ability of the oncolytic virus to infect the cell.

[0227] Figure 1 Results of the detection of the infectivity of the oncolytic virus prepared for this application and the wild-type oncolytic virus on LLC cells.

[0228] Figure 2 Results of the detection of the infectivity of the oncolytic virus prepared in this application and the wild-type oncolytic virus against MC38 cells.

[0229] Figure 3 The results of the detection of the infectivity of the oncolytic virus prepared in this application and the wild-type oncolytic virus against HeLa cells.

[0230] Figure 4 Results of the detection of the infectivity of the oncolytic virus prepared in this application and the wild-type oncolytic virus to MEF cells.

[0231] As shown in the accompanying figures, the oncolytic viruses prepared in Examples 1-26 of this application all exhibit good infectivity against LLC cells, MC38 cells, and HeLa cells. In particular, Examples 4-26, based on Example 3, further enhanced the infectivity of the oncolytic virus against LLC cells, MC38 cells, and HeLa cells by site-directed mutation of the amino acids on the G protein. Meanwhile, the oncolytic viruses prepared in the above examples all showed poor infectivity against MEF cells, indicating that the oncolytic viruses prepared in this application can be effectively used to infect tumor and cancer cells without damaging normal cells, demonstrating broad application prospects.

[0232] Example 2

[0233] In this embodiment, oncolytic viruses prepared in Examples 1-26 and wild-type oncolytic viruses were used to conduct in vitro cell killing assays on different cells. The detection method was the MTT assay, in which 200 pfu of each of the oncolytic viruses prepared in Examples 1-26 and wild-type oncolytic viruses were added to the culture medium of different cells, and cell viability was detected by the MTT assay after 24 hours. The cells tested included LLC cells, MC38 cells, MEF cells, and 4T1 cells (mouse mammary cells). The specific detection method was as follows:

[0234] (1) Add 100 μl of Vero (LLC / MC38 / 4T1 / MEF) cell suspension to each well of a 96-well culture plate to achieve a cell count of 1 × 10⁻⁶. 4 The 96-well culture plate was incubated at 37°C and 5% CO2 for 16 hours.

[0235] (2) The oncolytic virus prepared in the preparation example was diluted to MOI (multiple of infection) of 0.001, 0.01, 0.1 and 1.0 respectively. The oncolytic virus of each dilution gradient was inoculated into the 96-well culture plate of step (1), with 100 μl inoculated into 4 wells for each dilution gradient. The 96-well culture plate was placed in an environment of 37°C and 5% CO2 for 40 h.

[0236] (3) Take out the cell supernatant from the 96-well culture plate in step (2), and add fresh culture medium and MTT solution to the 96-well culture plate at a volume of 20 μL / well. Place the 96-well culture plate in an environment of 37°C and 5% CO2 for 4 h.

[0237] (4) Centrifuge the 96-well culture plate at room temperature for 5 min at a speed of 2500 rpm / min, and gently aspirate the supernatant with a 1 mL disposable sterile syringe; then add DMSO to each well of the 96-well culture plate at a volume of 100 μL / well, and incubate at 37°C for 10 min. Using a multi-functional microplate reader, shake for 2 min, and measure the OD value of each well on the 96-well culture plate at a wavelength of 570 nm or 490 nm.

[0238] Test results as follows Figures 5-8 As shown, the horizontal axis 0 represents wild-type oncolytic virus; the horizontal axes 1-26 represent the oncolytic viruses prepared in preparation examples 1-26, respectively; and the vertical axis OD... 570 OD represents the OD value of a cell. 570 The higher the OD value, the weaker the oncolytic virus's ability to kill the cell; 570 The smaller the value, the better the oncolytic virus's ability to kill the cell.

[0239] Figure 5 Results of in vitro killing ability of the oncolytic virus prepared in this application and the wild-type oncolytic virus against LLC cells.

[0240] Figure 6 The results show the in vitro killing ability of the oncolytic virus prepared in this application and the wild-type oncolytic virus against MC38 cells.

[0241] Figure 7 The results show the in vitro killing ability of the oncolytic virus prepared in this application and the wild-type oncolytic virus against 4T1 cells.

[0242] Figure 8 Results of in vitro killing ability of the oncolytic virus prepared in this application and the wild-type oncolytic virus against MEF cells.

[0243] As shown in the accompanying figures, the oncolytic viruses prepared in Examples 1-26 of this application all exhibit good in vitro killing ability against LLC cells, MC38 cells, and 4T1 cells. In particular, Examples 4-26, based on Example 3, further enhanced the in vitro killing ability of the oncolytic virus against LLC cells, MC38 cells, and 4T1 cells through site-directed mutation of the amino acids on the G protein. Simultaneously, the oncolytic viruses prepared above have almost no effect on MEF cells, indicating that the oncolytic viruses prepared in this application can be effectively used to damage and kill abnormal cells such as tumors and cancer cells without harming normal cells.

[0244] While wild-type oncolytic viruses have demonstrated good in vitro killing ability against LLC, MC38, and 4T1 cells, they also significantly damage and kill MEF cells, limiting their clinical application. Therefore, this application's modification of the wild-type oncolytic virus ensures both its safety against normal cells and its ability to kill tumor and cancer cells, demonstrating broad clinical application prospects.

[0245] Example 3

[0246] This embodiment examines the clearance efficiency of oncolytic viruses prepared in Examples 1-26 and wild-type oncolytic viruses in different cells. The detection index is the expression level of the IFN-β gene. IFN-β is the gene for a soluble glycoprotein produced by cells with broad antiviral, antitumor, and immunomodulatory effects. The expression level of the IFN-β gene can determine the cell's ability to clear oncolytic viruses: high IFN-β expression indicates that the oncolytic virus is easily cleared from the cell; low IFN-β expression indicates that the oncolytic virus is not easily cleared from the cell. The cells tested include LLC cells, MC38 cells, HeLa cells, and MEF cells. The specific detection method is as follows:

[0247] (1) Add 100 μl of Vero (LLC / MC38 / HeLa / MEF) cell suspension and MEF cell suspension to 96-well culture plates respectively, so that the cell mass reaches 1×10⁻⁶. 4 The 96-well culture plate was incubated at 37°C and 5% CO2 for 16 hours.

[0248] (2) The oncolytic virus prepared in the preparation example was diluted to MOI (multiple of infection) of 0.001, 0.01, 0.1 and 1.0 respectively. The oncolytic virus of each dilution gradient was inoculated into the 96-well culture plate of step (1), with 100 μl inoculated into 4 wells for each dilution gradient. The 96-well culture plate was placed in an environment of 37°C and 5% CO2 for 40 h.

[0249] (3) The cells obtained in step (2) were lysed, and total RNA was extracted from each cell using TRIzol (Invitrogen). The RNA was then reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with DNA Eraser (Takara) and stained with LightCycler 480 SYBR Green I Master (Roche) dye. The Ct values ​​of each gene were detected using a LightCycler 480 quantitative PCR instrument. The relative expression level of the target gene IFN-β was calculated using the ΔΔCt method.

[0250] Test results as follows Figures 9-12 As shown, the horizontal axis 0 represents wild-type oncolytic virus; the horizontal axes 1-26 represent the oncolytic viruses prepared in preparation examples 1-26, respectively; the vertical axis IFN-β level represents the expression of the IFN-β gene. The higher the IFN-β level, the easier it is to clear the oncolytic virus in the cell; the lower the IFN-β level, the less likely it is to clear the oncolytic virus in the cell.

[0251] Figure 9Results of the detection of the ease of clearance of the oncolytic virus prepared in this application and the wild-type oncolytic virus in LLC cells.

[0252] Figure 10 Results of the detection of the oncolytic virus prepared in this application and the ease of clearance of the wild-type oncolytic virus in MC38 cells.

[0253] Figure 11 Results of the detection of the ease of clearance of the oncolytic virus prepared in this application and the wild-type oncolytic virus in HeLa cells.

[0254] Figure 12 Results of the detection of the ease of clearance of the oncolytic virus prepared in this application and the wild-type oncolytic virus in MEF cells.

[0255] As shown in the accompanying figures, Preparation Examples 1-2 of this application were not easily cleared from LLC cells, MC38 cells, and HeLa cells. Compared to Preparation Examples 1 and 2, Preparation Example 3 was more easily cleared from LLC cells, MC38 cells, and HeLa cells. Preparation Examples 4-26 of this application, based on Preparation Example 3, further performed site-directed mutations on the amino acids of the G protein of the oncolytic virus, thereby making the oncolytic virus even more difficult to clear from LLC cells, MC38 cells, and HeLa cells, further ensuring that the oncolytic virus can better exert its infective and killing capabilities in LLC cells, MC38 cells, and HeLa cells; at the same time, it made the oncolytic virus easier to clear from MEF cells, further ensuring the safety of MEF cells, thereby improving the safety of the oncolytic virus.

[0256] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents. sequence list <110> Shanghai Rongrui Pharmaceutical Technology Co., Ltd. <120> An oncolytic virus and its uses <160> 28 <170> SIPOSequenceListing 1.0 <210> 1 <211> 229 <212> PRT <213> Artificial Sequence <400> 1 MSSLLLILGLLGLGLLSLLLGIAPPPTGGATAMGTAPSAPIALSTPGVAGMATHAPAGLA TGLPPPTVLMTVASAAPPATTSAVAAAVSHTAHMTIGMAGLAPPTLILAPLGSSALLATP AVLAAGGGPGTHAHCGGAATLPHAMGLTPPMLAVPGHPAAPPAIGLTLGTIGLTMTITAA GSLGAAPMITAHPASSLPSAPAGLALMPGLIVGLLASGATVLASVSHPL <210> 2 <211> 511 <212> PRT <213> Artificial Sequence <400> 2 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 3 <211> 229 <212> PRT <213> Artificial Sequence <400> 3 MSSLLLILGLLGLGLLSLLLGIAPPPTGGATAMGTAPSAPIALSTPGVAGRATHAPAGLA TGLPPPTVLMTVASAAPPATTSAVAAAVSHTAHMTIGMAGLAPPTLILAPLGSSALLATP AVLAAGGGPGTHAHCGGAATLPHAMGLTPPMLAVPGHPAAPPAIGLTLGTIGLTMTITAA GSLGAAPMITAHPASSLPSAPAGLALMPGLIVGLLASGATVLASVSHPL <210> 4 <211> 229 <212> PRT <213> Artificial Sequence <400> 4 MSSLLLILGLLGLGLLSLLLGIAPPPTGGATAMGTAPSAPIALSTPGVAGRATHAPAGLA TGLPPPTVLMTVASAAPPATTSAVAAAVSHTAHMTIGMAGLAPPTLILAPLGSSALLATP AVLAAGGGPGTHAHCGGAATLPHAMGLTPPMLAVPGHPAAPPAIGLTLGTIGLTMTITAA GSLGAAPMITAHPASSLPSAPAGLALMPGLIVGLLASGATFLASVSHPL <210> 5 <211> 229 <212> PRT <213> Artificial Sequence <400> 5 MSSLLLILGLLGLGLLSLLLGIAPPPTGGATAMGTAPSAPIALSTPGVAGRATHAPAGLA TGLPPPTVLMTVASAAPPATTSAVAAAVSHTAHMTIGMAGLAPPTLILAPLGSSALLATP AVLAAGGGPGTHAHCGGAATLPHAMGLTPPMLAVPGHPAAPPAIGLTLGTIGLTMTITAA GSLGAAPMITAHPASSLPSAPAGLALMPGLIVGLLASGATFLASVRHPL <210> 6 <211> 511 <212> PRT <213> Artificial Sequence <400> 6 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 7 <211> 511 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 7 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 8 <211> 511 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 8 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 9 <211> 511 <212> PRT <213> Artificial Sequence <400> 9 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 10 <211> 511 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 10 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 11 <211> 511 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 11 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 12 <211> 511 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 12 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 13 <211> 511 <231> PRT <213> Artificial Sequence <400> 13 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 14 <211> 511 <212> PRT <213> Artificial Sequence <400> 14 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 15 <211> 511 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 15 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 16 <211> 511 <212> PRT <213> Artificial Sequence <400> 16 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGITLCILLLHTLLAGITTAIGMAALGL <210> 17 <211> 511 <212> PRT <213> Artificial Sequence <400> 17 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALIGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 18 <211> 511 <212> PRT <213> Artificial Sequence <400> 18 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAVIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 19 <211> 511 <212> PRT <213> Artificial Sequence <400> 19 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAYICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 20 <211> 511 <212> PRT <213> Artificial Sequence <400> 20 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLEGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 21 <211> 511 <212> PRT <213> Artificial Sequence <400> 21 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGGLACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 22 <211> 511 <212> PRT <213> Artificial Sequence <400> 22 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPAPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 23 <211> 511 <212> PRT <213> Artificial Sequence <400> 23 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAELTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 24 <211> 511 <212> PRT <213> Artificial Sequence <400> 24 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPADGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 25 <211> 511 <212> PRT <213> Artificial Sequence <400> 25 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGSLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 26 <211> 511 <212> PRT <213> Artificial Sequence <400> 26 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIGPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGLVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 27 <211> 511 <212> PRT <213> Artificial Sequence <400> 27 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPIIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL <210> 28 <211> 511 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 28 MLCLLTLAPLPIGVACLPTIVPPHAGLGATLAVPSATHTCPSSALATHAALVGTALGVL MPLSHLAIGAAGTMCHASLTVTTCAPATTGPLTITHSIASPTPSVGGCLGSIGGTLGGTT LAPGPPGSCGTATVTAAGAAIVGVTPHHVLVAGTTGGTVASGPIAGLCSAAICPTVHAS TTTHSATLVLGLCASALISMAITPPSGAGGLSSLGLLGTGPASATPATGTGALACLMGTC LHTGVALPSGVTPGMAALALPAAAAPPGCPGGSSISAPSGTSVAVSLIGAVGAILATSLC GGTTSLIAAGLPISPVALSTLAPLAPGTGPVPTIIAGTLLTPGTATIAVAIAAPILSAMV GMISGTTTGAVLTAATAPTGAVGIPAGVLATSSGTLPPLTMIGHGMLASALHLSSLAGV PGHPHIGAAASGLPAGGTLPPGATGLSLAPIGPVGGTPSSTLSSIASPPPTIGLIIGLPL VLAVGIHLCILLLHTLLAGITTAIGMAALGL

Claims

1. An oncolytic virus, characterized in that, the oncolytic virus is a VSV virus; the oncolytic virus comprises an M protein and a G protein; the M protein has a mutation of methionine to arginine at position 51 (M51R), a mutation of valine to phenylalanine at position 221 (V221F), and a mutation of serine to arginine at position 226 (S226R) compared to the amino acid sequence set forth in SEQ ID NO: 1; the G protein has amino acid substitutions selected from any one of the following group: 1) the G protein has amino acid substitutions V53I; 2) the G protein has amino acid substitutions V53I and A141V; 3) the G protein has amino acid substitutions V53I, A141V and A172Y; 4) the G protein has amino acid substitutions V53I, A141V, A172Y and L217E; 5) the G protein has amino acid substitutions V53I, A141V, A172Y, L217E and A232G; 6) the G protein has amino acid substitutions V53I, A141V, A172Y, L217E, A232G and V331A; 7) the G protein has amino acid substitutions V53I, A141V, A172Y, L217E, A232G, V331A and V371E; 8) the G protein has amino acid substitutions V53I, A141V, A172Y, L217E, A232G, V331A, V371E and G436D; 9) the G protein has amino acid substitutions V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D and T438S; 10) the G protein has amino acid substitutions V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S and P453L; 11) the G protein has amino acid substitutions V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L and T471I; 12) the G protein has amino acid substitutions V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I and T487H; 13) the G protein has amino acid substitutions A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I and T487H; 14) the G protein has amino acid substitutions A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I and T487H; 15) the G protein has amino acid substitutions L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H; 16) the G protein has amino acid substitutions A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H; 17) the G protein has amino acid substitutions V331A, V371E, G436D, T438S, P453L, T471I, and T487H; 18) the G protein has amino acid substitutions V371E, G436D, T438S, P453L, T471I, and T487H; 19) the G protein has amino acid substitutions G436D, T438S, P453L, T471I, and T487H; 20) the G protein has amino acid substitutions T438S, P453L, T471I, and T487H; 21) the G protein has amino acid substitutions P453L, T471I, and T487H; 22) the G protein has amino acid substitutions T471I and T487H; 23) the G protein has amino acid substitution T487H.

2. The oncolytic virus of claim 1, wherein: the M protein is the amino acid sequence set forth in SEQ ID NO 5.

3. The oncolytic virus of claim 1, wherein: the G protein has amino acid substitutions V53I, A141V, A172Y, L217E, A232G, V331A, V371E, G436D, T438S, P453L, T471I, and T487H.

4. The oncolytic virus of claim 3, wherein: the G protein is the amino acid sequence set forth in SEQ ID NO 17.

5. The oncolytic virus of claim 1, wherein: the oncolytic virus comprises a Rhabdovirus.

6. The oncolytic virus of claim 1, wherein: the oncolytic virus comprises a Vesicular Stomatitis Virus (VSV).

7. The oncolytic virus of claim 6, wherein: the oncolytic virus comprises a VSV virus of the Indiana Mudd Summer subtype.

8. The oncolytic virus of claim 1, wherein: the oncolytic virus comprises or expresses an exogenous protein of interest.

9. The oncolytic virus of claim 1, wherein: the oncolytic virus comprises a nucleic acid molecule comprising a nucleic acid sequence encoding the M protein having amino acid substitutions and a nucleic acid sequence encoding the G protein having amino acid substitutions.

10. The oncolytic virus of claim 9, wherein: the nucleic acid molecule comprises a nucleic acid sequence encoding the exogenous protein of interest.

11. The oncolytic virus of claim 10, wherein: the nucleic acid sequence encoding the exogenous protein of interest is located between the nucleic acid sequence encoding the L protein and the nucleic acid sequence encoding the G protein having amino acid substitutions in the nucleic acid molecule.

12. The oncolytic virus of claim 11, wherein: the nucleic acid molecule encodes the N protein, the L protein, and the P protein of the oncolytic virus.

13. An oncolytic virus expression vector characterized in that, the viral production cell is capable of producing the oncolytic virus of any one of claims 1-12.

14. A viral production cell, characterized in that: the pharmaceutical composition comprises the oncolytic virus of any one of claims 1-12, and optionally a pharmaceutically acceptable carrier.

15. A pharmaceutical composition, characterized by: ​ 16. Use of the oncolytic virus of any one of claims 1-12, the oncolytic virus expression vector of claim 13, the virus production cell of claim 14, and / or the pharmaceutical composition of claim 15 in the manufacture of a medicament for the prevention and / or treatment of lung cancer, colon cancer, breast cancer, cervical cancer.

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