Oncolytic viruses and their use

Abstract

Provided is an oncolytic virus, the oncolytic virus comprising an M protein and a G protein, the M protein comprising amino acid substitutions at positions 51, 221 and 226 compared to the amino acid sequence shown in SEQ ID NO 1, and the G protein comprising at least one amino acid substitution compared to the amino acid sequence shown in SEQ ID NO 2. Further provided are expression vectors for the oncolytic virus, virus-producing cells that produce the oncolytic virus, pharmaceutical compositions comprising the oncolytic virus, and methods for producing and using the oncolytic virus, oncolytic virus expression vectors, virus-producing cells and / or pharmaceutical compositions.

Description

[Technical Field]

[0001] This application relates to the technology of biopharmaceuticals, and more specifically to oncolytic viruses and their use. [Background technology]

[0002] Oncolytic viruses are a type of tumor-killing virus with replication capabilities and are now widely accepted as an important branch of tumor immunotherapy. Oncolytic viruses can specifically target and infect tumor cells, for example by inactivating or deleting tumor suppressor genes within tumor cells, thereby selectively infecting them. Once infected, oncolytic viruses replicate rapidly within the tumor cells, ultimately destroying them and killing them. At the same time, oncolytic viruses can also provide further immune-stimulating signals necessary to enhance the host's own anti-cancer response, thereby attracting more immune cells to continue killing any remaining tumor cells.

[0003] While oncolytic viruses (TCRs) have relatively good potential for application in tumor immunotherapy, wild-type TCRs often cause problems such as inflammation of the nervous system, and there is a significant pathogenicity risk in the process of infecting tumor cells using wild-type viruses. Therefore, in order to further advance the clinical application of TCRs, it is necessary to modify wild-type TCRs to obtain attenuated TCRs. By using attenuated TCRs in clinical applications, the pathogenicity risk of TCRs can be reduced, and the safety of TCRs can be improved.

[0004] However, in the modification process of oncolytic viruses, simply performing random gene modifications on wild-type oncolytic viruses may reduce their toxicity, but the modified oncolytic viruses may not have a good cure rate. Consequently, the modified oncolytic viruses cannot be packaged, which is detrimental to promoting the clinical application of oncolytic viruses. Therefore, providing modified oncolytic viruses that have good safety performance and high cure rates simultaneously has significant scientific value and application implications in the field of tumor immunotherapy. [Overview of the project] [Problems that the invention aims to solve]

[0005] This application provides oncolytic viruses and their use, and the oncolytic viruses provided herein have good safety and cure rates. [Means for solving the problem]

[0006] In the first embodiment, the present application provides an oncolytic virus and employs the following technical approach: In this application, the oncolytic virus comprises an M protein and a G protein, wherein the M protein includes amino acid substitutions at positions 51, 221, and 226 compared to the amino acid sequence shown in SEQ ID NO 1, and the G protein includes at least one amino acid substitution compared to the amino acid sequence shown in SEQ ID NO 2.

[0007] In some embodiments, the amino acid substitutions of the M protein include a mutation from methionine to arginine at position 51 (M51R), and / or a mutation from valine to phenylalanine at position 221 (V221F), and / or a mutation from serine to arginine at position 226 (S226R).

[0008] In some embodiments, the amino acid substitution of the M protein includes a mutation from methionine to arginine at position 51 (M51R).

[0009] In some embodiments, the amino acid substitution of the M protein includes a mutation from valine to phenylalanine at position 221 (V221F).

[0010] In some embodiments, the amino acid substitution of the M protein includes a mutation from serine to arginine at position 226 (S226R).

[0011] In some embodiments, the M protein has the amino acid substitutions M51R and V221F.

[0012] In some embodiments, the M protein has the amino acid substitutions M51R and S226R.

[0013] In some embodiments, the M protein has the amino acid substitutions M51R, V221F, and S226R.

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

[0015] In some embodiments, the M protein includes the amino acid sequence shown in SEQ ID NO 5.

[0016] In some embodiments, the M protein includes the amino acid sequence shown in SEQ ID NO 3.

[0017] In some embodiments, the M protein includes the amino acid sequence shown in SEQ ID NO 4.

[0018] In some embodiments, the G protein includes amino acid substitutions at one or more positions, specifically at positions 438, 453, 471, and 487, compared to the amino acid sequence shown in SEQ ID NO 2.

[0019] In some embodiments, the G protein comprises amino acid substitutions at one or more sites at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, 453, 471, and 487 as compared to the amino acid sequence shown in SEQ ID NO 2.

[0020] In some embodiments, the amino acid substitutions of the G protein include a mutation from valine to isoleucine at position 53 (V53I), and / or a mutation from alanine to valine at position 141 (A141V), and / or a mutation from aspartic acid to tyrosine at position 172 (D172Y), and / or a mutation from lysine to glutamic acid at position 217 (K217E), and / or a mutation from aspartic acid to glycine at position 232 (D232G), and / or a mutation from valine to alanine at position 331 (V331A), and / or a mutation from valine to glutamic acid at position 371 (V371E), and / or a mutation from glycine to aspartic acid at position 436 (G436D), and / or a mutation from threonine to serine at position 438 (T438S), and / or a mutation from phenylalanine to leucine at position 453 (F453L), and / or a mutation from threonine to isoleucine at position 471 (T471I), and / or a mutation from tyrosine to histidine at position 487 (Y487H).

[0021] In some embodiments, the amino acid substitution of the G protein includes a mutation from valine to isoleucine at position 53 (V�3I).

[0022] In some embodiments, the amino acid substitution of the G protein includes a mutation from alanine to valine at position 141 (A141V).

[0023] In some embodiments, the amino acid substitution of the G protein includes a mutation from aspartic acid to tyrosine at position 172 (D172Y).

[0024] In some embodiments, the amino acid substitution of the G protein includes a mutation from lysine to glutamic acid at position 217 (K217E).

[0025] In some embodiments, the amino acid substitution of the G protein includes a mutation from aspartic acid to glycine at position 232 (D232G).

[0026] In some embodiments, the amino acid substitution of the G protein includes a mutation from valine to alanine at position 331 (V331A).

[0027] In some embodiments, the amino acid substitution of the G protein includes a mutation from valine to glutamic acid at position 371 (V371E).

[0028] In some embodiments, the amino acid substitution of the G protein includes a mutation from glycine to aspartic acid at position 436 (G436D).

[0029] In some embodiments, the amino acid substitution of the G protein includes a mutation from threonine to serine at position 438 (T438S).

[0030] In some embodiments, the amino acid substitution of the G protein includes a mutation from phenylalanine to leucine at position 453 (F453L).

[0031] In some embodiments, the amino acid substitution of the G protein includes a mutation from threonine to isoleucine at position 471 (T471I).

[0032] In some embodiments, the amino acid substitution of the G protein includes a mutation from tyrosine to histidine at position 487 (Y487H).

[0033] In some embodiments, the amino acid substitutions of the G protein include T438S, F453L, T471I, and Y487H.

[0034] 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 D172Y.

[0036] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, and K217E.

[0037] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, and D232G.

[0038] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, and V331A.

[0039] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, and V371E.

[0040] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, and G436D.

[0041] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, and T438S.

[0042] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, and F453L.

[0043] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, and T471I.

[0044] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H.

[0045] In some embodiments, the amino acid substitutions of the G protein include A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H.

[0046] In some embodiments, the amino acid substitutions of the G protein include D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H.

[0047] In some embodiments, the amino acid substitutions of the G protein include K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H.

[0048] In some embodiments, the amino acid substitutions of the G protein include D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H.

[0049] In some embodiments, the amino acid substitutions of the G protein include V331A, V371E, G436D, T438S, F453L, T471I, and Y487H.

[0050] In some embodiments, the amino acid substitutions of the G protein include V371E, G436D, T438S, F453L, T471I, and Y487H.

[0051] In some embodiments, the amino acid substitutions of the G protein include G436D, T438S, F453L, T471I, and Y487H.

[0052] In some embodiments, the amino acid substitutions of the G protein include T438S, F453L, T471I, and Y487H.

[0053] In some embodiments, the amino acid substitutions of the G protein include F453L, T471I, and Y487H.

[0054] In some embodiments, the amino acid substitutions of the G protein include T471I and Y487H.

[0055] In some embodiments, the amino acid substitutions of the G protein include V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H.

[0056] In some embodiments, the amino acid substitutions of the G protein are V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H.

[0057] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 17.

[0058] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 6.

[0059] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 7.

[0060] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 8.

[0061] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 9.

[0062] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 10.

[0063] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 11.

[0064] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 12.

[0065] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 13.

[0066] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 14.

[0067] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 15.

[0068] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 16.

[0069] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 18.

[0070] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 19.

[0071] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 20.

[0072] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 21.

[0073] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 22.

[0074] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 23.

[0075] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 24.

[0076] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 25.

[0077] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 26.

[0078] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 27.

[0079] In some embodiments, the G protein includes the amino acid sequence shown in SEQ ID NO 28.

[0080] In some embodiments, the oncolytic virus is obtained after site-directed mutation based on a Corynebacter virus.

[0081] In some embodiments, the oncolytic virus is obtained by performing site-directed mutations on the Vesicular Stomatitis Virus (VSV) virus.

[0082] In some embodiments, the oncolytic virus is obtained after site-directed mutation based on the VSV virus Indiana MuddSummer subtype.

[0083] In some embodiments, the oncolytic virus contains or expresses an exogenous target protein.

[0084] In some embodiments, the oncolytic virus comprises a nucleic acid molecule including 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.

[0085] In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the foreign target protein.

[0086] In some embodiments, the nucleic acid sequence encoding the foreign target protein in the nucleic acid molecule is located between the nucleic acid sequence encoding the M protein having amino acid substitutions and the nucleic acid sequence encoding the G protein having amino acid substitutions.

[0087] In some embodiments, the nucleic acid molecule encodes the N protein (nucleoprotein N), L protein (macropolymerase protein L), and P protein (phosphate protein P) of the oncolytic virus.

[0088] In a second embodiment, the present application provides an oncolytic virus expression vector and employs the following technical approach: An oncolytic virus expression vector, wherein the oncolytic virus expression vector can produce any of the oncolytic viruses described in this application.

[0089] In a third aspect, the present application provides a virus-producing cell and employs the following technical solution: A virus-producing cell, the virus-producing cell is capable of producing any of the oncolytic viruses described in this application.

[0090] In a fourth aspect, the present application provides a pharmaceutical composition employing the following technical solution: A pharmaceutical composition comprising any oncolytic virus described in this application and optionally a pharmaceutically acceptable carrier.

[0091] In a fifth embodiment, the present application provides a method for producing the above-mentioned oncolytic virus, oncolytic virus expression vector, virus-producing cells, and / or pharmaceutical composition.

[0092] In a sixth aspect, the present application provides the use of the above-mentioned oncolytic virus, oncolytic virus expression vector, virus-producing cells and / or pharmaceutical composition in the manufacture of pharmaceuticals for the prevention and / or treatment of diseases and / or conditions.

[0093] In some embodiments, the oncolytic virus, oncolytic virus expression vector, virus-producing cells, and / or pharmaceutical composition are used in a method that slowly and continuously kills abnormally proliferating cells.

[0094] In some embodiments, the disease and / or condition comprises abnormally proliferating cells selected from tumor cells or cells associated with tumor tissue, preferably the tumor cells being cancer cells, and more preferably the cancer cells being metastatic cancer cells.

[0095] In some embodiments, the tumor includes solid tumors and / or hematological malignancies. [Effects of the Invention]

[0096] In summary, this application has the following beneficial effects. The oncolytic viruses provided in this application all possess good invasion and infection capabilities against LLC cells, MC38 cells, and HeLa cells, and good in vitro killing capabilities against LLC cells, MC38 cells, and 4T1 cells, and are not easily removed within LLC cells, MC38 cells, and HeLa cells. Furthermore, the oncolytic viruses provided in this application all possess low invasion and infection capabilities, in vitro killing capabilities, and ease of removal against MEF cells. Therefore, the oncolytic viruses provided in this application can be suitably used for invasion, infection, and killing of tumor and cancer cells, and are difficult to remove within tumor and cancer cells, further improving the cure rate of oncolytic viruses against tumor and cancer cells. At the same time, the oncolytic viruses provided above do not damage normal cells, and are more easily removed when present within normal cells, further ensuring the safety of normal cells. [Brief explanation of the drawing]

[0097] The specific features of the invention described herein are set forth in the attached claims. The features and advantages of the invention described herein can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the attached drawings is as follows: [Figure 1] This shows the detection results of the ability of oncolytic viruses and wild-type oncolytic viruses manufactured according to this invention to invade and infect LLC cells. [Figure 2] This shows the detection results of the ability of oncolytic viruses and wild-type oncolytic viruses manufactured according to this invention to invade and infect MC38 cells. [Figure 3] This shows the detection results of the ability of oncolytic viruses and wild-type oncolytic viruses manufactured according to this invention to invade and infect HeLa cells. [Figure 4] This shows the detection results of the ability of oncolytic viruses and wild-type oncolytic viruses manufactured according to this invention to invade and infect MEF cells. [Figure 5]This shows the results of detecting the in vitro killing ability of oncolytic viruses and wild-type oncolytic viruses produced according to this invention against LLC cells. [Figure 6] This shows the results of detecting the in vitro killing ability of oncolytic viruses and wild-type oncolytic viruses produced according to this invention against MC38 cells. [Figure 7] This shows the results of detecting the in vitro killing ability of oncolytic viruses and wild-type oncolytic viruses produced according to this invention against 4T1 cells. [Figure 8] This shows the results of detecting the in vitro killing ability of oncolytic viruses and wild-type oncolytic viruses produced according to this invention against MEF cells. [Figure 9] This shows the detection results for the difficulty of removing oncolytic viruses and wild-type oncolytic viruses produced according to this invention within LLC cells. [Figure 10] This shows the detection results for the difficulty of removing oncolytic viruses and wild-type oncolytic viruses produced according to this invention in MC38 cells. [Figure 11] This shows the detection results for the difficulty of removing oncolytic viruses and wild-type oncolytic viruses produced according to this invention in HeLa cells. [Figure 12] This shows the detection results for the difficulty of removing oncolytic viruses and wild-type oncolytic viruses produced according to this invention in MEF cells.

[0098] In the above diagram, the horizontal axis 0 represents wild-type oncolytic viruses, and the horizontal axes 1 to 26 represent the oncolytic viruses produced in production examples 1 to 26, respectively. Vertical axis: Log 10 TCID50 represents the TCID50 value calculated using the Karber method, Log 10 A higher TCID50 value indicates a higher ability of the oncolytic virus to invade and infect the cells in question. 10 A lower TCID50 value indicates a lower ability of the oncolytic virus to invade and infect the cells in question. Vertical axis OD 570This represents the OD value of the cell, OD 570 A higher value indicates that the oncolytic virus has a lower ability to kill the cells in question, and OD 570 A smaller value indicates a higher killing capacity of the oncolytic virus against the cells in question.

[0099] The vertical axis, IFN-β level, represents the expression status of the IFN-β gene. A higher IFN-β level indicates that the oncolytic virus is easily removed within the cell, while a lower IFN-β level indicates that the oncolytic virus is not easily removed within the cell.

[0100] Those skilled in the art will readily recognize other aspects and advantages of the Application from the following detailed description. Only exemplary embodiments of the Application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of the Application allows those skilled in the art to modify the specific embodiments disclosed without departing from the spirit and scope of the invention. Accordingly, the drawings and description of the Application are merely illustrative and not limiting. [Modes for carrying out the invention]

[0101] The embodiments of the present invention will be described below with reference to specific examples, but those skilled in the art will readily understand other advantages and effects of the present invention from the contents disclosed herein. Term definition

[0102] In this application, the term "oncolytic virus" typically refers to a virus capable of replicating and killing tumor cells within tumor cells. Oncolytic viruses include, but are not limited to, VSV virus, poxvirus, herpes simplex virus, measles virus, Semryki forest virus, poliovirus, reovirus, Seneca Valley virus, echogenic enterovirus, coxsackievirus, Newcastle disease virus, and Maraba virus. In some embodiments, the oncolytic virus is modified to improve its selectivity for tumor cells. In some embodiments, the oncolytic virus is modified to reduce its immunogenicity. In some embodiments, the oncolytic virus described herein is the VSV virus. In some embodiments, the VSV virus is a variant of the VSV virus Indiana MuddSummer subtype strain. In some embodiments, site-directed gene mutations can be made in the M and G proteins of the VSV virus.

[0103] In some embodiments, the oncolytic virus described herein may be a genetically modified oncolytic virus, for example, modified by modification of one or more genes, thereby improving its tumor selectivity and / or preferentially replicating within dividing cells. The genetically modified gene may be a modification of a gene involved in DNA replication, nucleic acid metabolism, host targeting, surface adhesion, toxicity, cleavage, and diffusion processes, or it may be a modification of incorporating a foreign gene. The foreign gene may include a foreign immunomodulatory gene, a foreign screening gene, a foreign reporter gene, and the like. The modified oncolytic virus may be an oncolytic virus modified at the amino acid level, such as by insertion, deletion, or substitution of one or more amino acids.

[0104] In this application, the term "M protein" usually refers to the VSV virus matrix protein. The M protein is an important toxic factor of the VSV virus and is also a protein known to interfere with the mouse innate immune response within the VSV virus. The term "M protein" also includes its homologs, orthologs, variants, and functionally active fragments. In this application, the wild-type VSV virus Indiana MuddSummer subtype M protein may include the amino acid sequence shown in SEQ ID NO 1. In this application, the M protein of the oncolytic virus may include the amino acid sequences shown in SEQ ID NO 3-5.

[0105] In this application, the term "G protein" usually refers to the glycoprotein of the water VSV virus, also known as the envelope protein. The term "G protein" also includes its homologs, orthologs, variants, and functionally active fragments. In this application, the wild-type VSV virus Indiana MuddSummer subtype G protein may include the amino acid sequence shown in SEQ ID NO 2. In this application, the glycoprotein G of the oncolytic virus may include the amino acid sequences shown in SEQ ID NO 6-28.

[0106] In this application, the term "L protein" usually refers to the VSV virus RNA polymerase protein. The L gene of the VSV virus encodes the RNA poly E protein. The term "L protein" also includes its homologs, orthologs, variants, and functionally active fragments.

[0107] In this application, the term "N protein" usually refers to the nucleocapsid protein of the VSV virus. The term "N protein" also includes its homologs, orthologs, variants, and functionally active fragments.

[0108] In this application, the site of a protein mutation is usually expressed as "amino acid + amino acid position + (mutated amino acid)". In this application, such mutation may include, but is not limited to, an increase, substitution, deletion, and / or deletion of an amino acid. For example, the term "M51R" usually refers to a mutation from methionine M to arginine R at position 51.

[0109] In this application, the term "amino acid substitution" usually refers to the substitution of one amino acid residue present in the parent sequence with another amino acid residue. The amino acids in the parent sequence may be substituted, for example, by synthesis via chemical peptides or by recombination methods known in the art. Therefore, "substitution at position xx" usually refers to the substitution of an amino acid located at position xx with a substitute amino acid residue. In this application, such amino acid substitution may include amino acid mutations.

[0110] In this application, the term "mutation" generally refers to altering the nucleotide or amino acid sequence of a wild-type molecule. Amino acid alterations may include amino acid substitution, deletion, insertion, addition, truncation, or protein processing or cleavage.

[0111] In this application, the term "nucleic acid molecule" usually refers to a nucleotide of any length. In this application, the term "nucleic acid molecule" may also encode a protein contained in the oncolytic virus. In this application, the nucleic acid molecule may include DNA and / or RNA. In some cases, the RNA may include single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA), and the single-stranded RNA may include sense RNA, antisense RNA, or ambisense RNA.

[0112] In this application, the term “expression vector” usually refers to a nucleic acid vector. Under appropriate conditions, it can usually express a target gene and / or target protein. In some embodiments of this application, the expression vector comprises nucleic acid molecules for expressing one or more components of a virus (e.g., an oncolytic virus). For example, the expression vector may contain at least one viral genome element and may be packaged in a virus or packaged as a viral particle.

[0113] In this application, the term "virus-producing cell" usually refers to a cell, cell line, or cell culture that may contain, or contains, the nucleic acid molecule or expression vector described in this application, or that is capable of expressing the oncolytic virus described in this application. The cell may include offspring of a single host cell. The cell can be obtained by transfecting it in vitro with the expression vector described in this application.

[0114] In this application, the term “pharmaceutical composition” usually refers to a formulation that exists in a form that enables the biological activity of an active ingredient and does not include additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. In some embodiments, these formulations may include pharmaceutically acceptable ingredients and pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical product includes a pharmaceutical product for parenteral, transdermal, intracavitary, intra-arterial, intrathecal and / or intranasal administration, or direct injection into tissue. The pharmaceutical product may be administered in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0115] In this application, the term “prevention” usually refers to preventing the onset, attack, recurrence, and / or spread of a disease or one or more of its symptoms by taking some measures in advance. In this application, the term “treatment” usually refers to eliminating or improving one or more of a disease or one or more of its symptoms. In some embodiments, treatment usually refers to administering one or more drugs to a patient suffering from such a disease so that the disease is eliminated or goes into remission. In some embodiments, “treatment” may also refer to administering the drug combination and / or pharmaceutical product in the presence or absence of other drugs after an attack of symptoms of a particular disease. For example, the drug combination and / or pharmaceutical product described in this application may be used to prevent the onset, progression, recurrence, and / or metastasis of a tumor.

[0116] In this application, the term “tumor” generally refers to any novel pathological tissue growth. Tumors may be benign or malignant. In this application, said tumors may be solid tumors and / or hematological malignancies. When used in research, these tissues can be isolated from readily available resources by methods well known to those skilled in the art.

[0117] This application provides an oncolytic virus, which is obtained by mutating a site in the amino acid sequence of its M protein and / or G protein, based on the wild-type VSV virus, specifically the VSV virus Indiana strain and the VSV virus Indiana MuddSummer subtype strain. The amino acid sequence of the M protein is shown in SEQ ID NO 1, and the amino acid sequence of the G protein is shown in SEQ ID NO 2. In this application, the M protein and the G protein may be modified.

[0118] In order to obtain an oncolytic virus, the present invention modifies the VSV virus as follows.

[0119] The oncolytic virus comprises an M protein and a G protein, wherein the M protein includes amino acid substitutions at positions 51, 221, and 226 compared to the amino acid sequence shown in SEQ ID NO 1, and the G protein includes at least one amino acid substitution compared to the amino acid sequence shown in SEQ ID NO 2.

[0120] The amino acid substitutions of the M protein include a mutation from methionine to arginine at position 51 (M51R), and / or a mutation from valine to phenylalanine at position 221 (V221F), and / or a mutation from serine to arginine at position 226 (S226R). For example, the M protein includes the amino acid sequence shown in SEQ ID NO 5. In this application, the M protein may further include amino acid substitutions at other positions.

[0121] In this application, the amino acid substitutions of the G protein are a mutation from valine to isoleucine at position 53 (V53I), and / or a mutation from alanine to valine at position 141 (A141V), and / or a mutation from aspartic acid to tyrosine at position 172 (D172Y), and / or a mutation from lysine to glutamic acid at position 217 (K217E), and / or a mutation from aspartic acid to glycine at position 232 (D232G), and / or a mutation from valine to alanine at position 331 (V331A), and / Alternatively, the G protein may include a mutation from valine to glutamic acid at position 371 (V371E), and / or a mutation from glycine to aspartic acid at position 436 (G436D), and / or a mutation from threonine to serine at position 438 (T438S), and / or a mutation from phenylalanine to leucine at position 453 (F453L), and / or a mutation from threonine to isoleucine at position 471 (T471I), and / or a mutation from tyrosine to histidine at position 487 (Y487H), and the G protein may also include the amino acid sequence shown in SEQ ID NO 17.

[0122] In this application, the G protein may contain amino acid mutations at positions 53 and 141.

[0123] In this application, the G protein may contain amino acid mutations at positions 53, 141, and 172.

[0124] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, and 217.

[0125] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, and 232.

[0126] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, and 331.

[0127] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331 and 371.

[0128] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371 and 436.

[0129] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, and 438.

[0130] In this application, the G protein may contain amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, and 453.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] In this application, the G protein may contain amino acid mutations at positions 172, 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0135] In this application, the G protein may contain amino acid mutations at positions 217, 232, 331, 371, 436, 438, 453, 471, and 487.

[0136] In this application, the G protein may contain amino acid mutations at positions 232, 331, 371, 436, 438, 453, 471, and 487.

[0137] In this application, the G protein may contain amino acid mutations at positions 331, 371, 436, 438, 453, 471, and 487.

[0138] In this application, the G protein may contain amino acid mutations at positions 371, 436, 438, 453, 471, and 487.

[0139] In this application, the G protein may contain amino acid mutations at positions 436, 438, 453, 471, and 487.

[0140] In this application, the G protein may contain amino acid mutations at positions 436, 453, 471, and 487.

[0141] In this application, the G protein may contain amino acid mutations at positions 453, 471, and 487.

[0142] In this application, the G protein may contain amino acid mutations at positions 471 and 487.

[0143] In this application, the G protein may contain an amino acid mutation at position 487.

[0144] In this application, the G protein may further include amino acid substitutions at other positions.

[0145] In some embodiments, the G protein includes one or more amino acid substitutions in at least a conserved region. For example, the conserved region includes amino acids at positions 437 to 461 of the G protein. In some embodiments, the G protein includes one or more amino acid substitutions in at least a truncated region of the cytoplasm. For example, the truncated region of the cytoplasm may include amino acids at positions 483 to 511 of the G protein.

[0146] In this application, the G protein may contain at least amino acid substitutions at positions 438, 453, 471 and 487.

[0147] 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 having amino acid substitutions and a nucleic acid sequence encoding the G protein having amino acid substitutions, and the nucleic acid molecule may further comprise a nucleic acid sequence encoding the foreign target protein. Furthermore, the nucleic acid sequence encoding the foreign target protein in the nucleic acid molecule is located between the nucleic acid sequence encoding the M protein having amino acid substitutions and the nucleic acid sequence encoding the G protein having amino acid substitutions. Furthermore, the nucleic acid molecule encodes the N protein (nucleoprotein N), L protein (macropolymerase protein L), and P protein (phosphate protein P) of the oncolytic virus.

[0148] In this application, the oncolytic virus described herein can be obtained by a virus packaging process and a virus rescue process. The specific process may include inoculating BSR-T7 cells with poxvirus vTF7-3 expressing T7 RNA polymerase, performing lipofectamine transfection using expression plasmids and backbone plasmids containing cloned VSV N, VSV P, and VSV L genes, respectively, to obtain the target oncolytic virus.

[0149] This application further provides oncolytic virus expression vectors, virus-producing cells, and pharmaceutical compositions.

[0150] The oncolytic virus expression vector may include nucleic acid sequences encoding the M protein and G protein of the oncolytic virus, and the oncolytic virus expression vector may further include nucleic acid sequences encoding the N protein, P protein, and L protein of the oncolytic virus.

[0151] The virus-producing cells can produce the above-mentioned oncolytic viruses, and the virus-producing cells may also include BSR-T7 cells.

[0152] The pharmaceutical composition comprises the above-mentioned oncolytic virus and optionally a pharmaceutically acceptable carrier.

[0153] In some embodiments, the pharmaceutical composition may contain one or more suitable formulations of pharmaceutically effective adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the pharmaceutical composition are preferably nontoxic to receptors at the doses and concentrations used. The pharmaceutical compositions of this application include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0154] In some embodiments, the pharmaceutically acceptable carrier may contain any and all solvents, dispersions, coatings, isotonic agents, and absorption retarders suitable for pharmaceutical administration, and is generally safe and non-toxic.

[0155] In some embodiments, the pharmaceutical composition may be administered parenterally, transdermally, intracavitarially, intraarterially, intrathecally, and / or intranasally, or by direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the pharmaceutical composition may be administered without interruption. Such uninterrupted (or continuous) administration may be achieved by a small pump system attached to the patient, as described in WO2015 / 036583, which can measure the amount of therapeutic drug flowing into the patient's body.

[0156] Furthermore, the present invention provides a method for producing the above-mentioned oncolytic virus, which may include a method for producing an oncolytic virus expression vector, virus-producing cells, and / or a pharmaceutical composition. Any method suitable for the production of an oncolytic virus can be used to produce the oncolytic virus of the present invention. For example, the oncolytic virus of the present invention can be obtained by transfecting cells with a poxvirus expressing T7 RNA polymerase, adding plasmids expressing the oncolytic virus N protein, L protein, and P protein, and a backbone plasmid, and then transfecting the cells through a virus rescue process.

[0157] This application further provides the use of the above-mentioned oncolytic viruses, oncolytic virus expression vectors, virus-producing cells and / or pharmaceutical compositions in the manufacture of pharmaceuticals for the prevention and / or treatment of diseases and / or medical conditions.

[0158] The oncolytic viruses provided in this application all possess good ability to invade and infect LLC cells, MC38 cells, and HeLa cells. In particular, the oncolytic viruses numbered JBS104-JBS126 are based on the oncolytic virus numbered JBS103, and undergo site-directed mutations in the amino acids on the G protein of the oncolytic virus, thereby further improving the ability of the oncolytic viruses to invade and infect LLC cells, MC38 cells, and HeLa cells. At the same time, since the ability of the oncolytic viruses produced above to invade and infect normal cells (MEF cells) is low, it is clear that the oncolytic viruses produced in this application can be suitably used for the invasion and infection of tumor and cancer cells without damaging normal cells, and have great potential for a wide range of applications.

[0159] 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 are based on the oncolytic virus numbered JBS103, and undergo site-directed mutations in the amino acids on the G protein of the oncolytic virus, thereby further improving the in vitro killing ability of the oncolytic viruses against LLC cells, MC38 cells, and 4T1 cells. At the same time, since the oncolytic viruses produced above have almost no effect on MEF cells, it can be seen that the oncolytic viruses produced in this application can be suitably used to damage and kill abnormal cells such as tumors and cancer cells without damaging normal cells.

[0160] The oncolytic viruses numbered JBS101 and JBS102 cannot be easily removed from LLC cells, MC38 cells, and HeLa cells. In contrast, the oncolytic virus numbered JBS103 is more easily removed from LLC cells, MC38 cells, and HeLa cells. On the other hand, the oncolytic viruses numbered JBS104-JBS126 of this application are based on the oncolytic virus numbered JBS103, but with site-directed mutations in the amino acids on the G protein of the oncolytic virus, thereby making the oncolytic virus more difficult to remove from LLC cells, MC38 cells, and HeLa cells, and ensuring that the oncolytic virus can better exert its ability to invade, infect, and kill within LLC cells, MC38 cells, and HeLa cells. At the same time, the oncolytic viruses provided in this application are more easily removed from MEF cells, further ensuring the safety of MEF cells, and thereby improving the safety of the oncolytic virus.

[0161] To avoid being limited by any theory, the following examples are used solely to illustrate the individual technical solutions of the present invention and not to limit the scope of the present invention.

[0162] Manufacturing example Manufacturing Examples 1-3 Production Examples 1-3 each provide oncolytic viruses, the differences of which lie primarily in the site-directed amino acid mutations on the M protein of the wild-type oncolytic virus. The specific construction methods for the oncolytic viruses corresponding to each production example are as follows.

[0163] (1) Plasmid construction Using the pRV-core plasmid (BioVector NTCC Plasmid Vector Bacterial Species Cell Gene Depositary Center) as a template, mutation sites as shown in Table 1 were introduced using PCR technology. PCR was performed on the pRV-core plasmid with each primer carrying a different mutation site, and the PCR products were subjected to 1% agarose gel electrophoresis. Further gel cleavage and recovery were performed using a gel recovery kit to obtain plasmids with different mutation sites in the M protein, thereby obtaining the constructed plasmid pRV-core Mut.

[0164] Table 1: Mutation status of the M protein of oncolytic viruses in production examples 1-3. JPEG0007857683000001.jpg35152

[0165] (2) Virus Rescue Using a calcium phosphate transfection kit (Thermo Fisher Scientific), the constructed plasmid pRV-core Mut was transfected into BSR-T7 cells using cell transfection technology (purchased from ATCC, also known as the American Type Culture Collection). The four plasmids were mixed according to the mass ratio of pRV-core Mut, pP, pN, and pL of 10:5:4:1, resulting in 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 transfection plasmid sprimmix. Here, pP (plasmid carrying the Corynebacterial viral phosphate protein gene), pN (plasmid carrying the Corynebacterial viral nucleoprotein gene), and pL (plasmid carrying the Corynebacterial viral polymerase protein gene). The parent vectors corresponding to the three plasmids pN, pP, and pL are all pCAGGS (purchased from ATCC). Dilute 10 μl of Lipofectamine LTX (Thermo Fisher Scientific) in 200 μl of opti-MEM medium to obtain an LTX mixture. Transfection with the plasmid was performed according to the instructions for Lipofectamine LTX, and after 6 hours, the BSR-T7 cells were washed twice with PBS, and then inoculated into 10% fetal bovine serum DMEM medium (Thermo Fisher Scientific) and cultured for 3 days. The supernatant obtained from culturing BSR-T7 cells was transferred to Vero cells (Thermo Fisher Scientific), and the Vero cells were cultured for 3 days under environmental conditions of 37°C. The status of virus rescue was determined by observing the green fluorescence inside the cells with a fluorescence microscope. Furthermore, the rescued mutant Corynebacter virus library was passaged using Vero cells, and monoclonal virus strains were selected using an established plaque screening system.

[0166] (3) Sequencing of the M protein gene. Viral genomic RNA was extracted using a Trizol kit, reverse transcription was performed using random primers, and PCR was performed on the reverse-transcribed cDNA using primers designed for the M protein gene sequence. The primer sequence is as follows: 5'-AAAAAAGTAACAGATATCAC-3', 5'-ACATTTTTCCAGTTTCCTTTTTGG-3'.

[0167] 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.

[0168] Manufacturing Examples 4-26 Production Examples 4-26 each provide oncolytic viruses, the differences being primarily in the site-directed mutations of amino acids on the M and G proteins of the wild-type oncolytic virus, where the mutation site of the M protein is the same as the corresponding mutation site in Production Example 3. The construction method for the oncolytic viruses corresponding to each production example is the same as the construction method described in Production Examples 1-3, except for the following points. In step (1), the plasmid was constructed using PCR technology to introduce the mutation sites shown in Table 2. Step (3) was the sequencing of the G protein gene. Viral genomic RNA was extracted using a Trizol kit, reverse transcription was performed using random primers, and PCR was performed on the reverse-transcribed cDNA using primers designed for the G protein gene sequence. The primer sequence is as follows: 5'-CCATGGCCTGTTGCTCCACCAGCTT-3', 5'-AAGCTTTCAGCAGTGGCTCACAGCAG-3'.

[0169] 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.

[0170] Table 2: Mutation status of G protein of oncolytic virus in production examples 4-26. JPEG0007857683000002.jpg246154JPEG0007857683000003.jpg23154

[0171] Manufacturing example 27 This manufacturing example provides a packaging process for oncolytic viruses produced using Examples 1 to 26 described above, and specifically includes the following steps.

[0172] 1) BSR-T7 cells (purchased from ATCC) were inoculated with poxvirus vTF7-3 expressing T7 RNA polymerase (BioVector NTCC plasmid vector bacterial species cell gene depository center). The specific steps are as follows: Plate BSR-T7 cells into a 6-well plate, with 3 × 10⁶ cells per well. 5 The cells were controlled to reach a certain number, and after 14-16 hours of plating, poxvirus vTF7-3 expressing T7 RNA polymerase was added to infect the BSR-T7 cells with poxvirus vTF7-3. After 6 hours of infection, the BSR-T7 cells were rinsed once with DPBS buffer (Thermo Fisher Scientific) and then transfected.

[0173] 2) Transfection process Specifically, the following steps are involved: Mixing the four plasmids according to the mass ratio of pRV-core Mut, pP, pN, and pL of 10:5:4:1, resulting in 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) is performed to obtain a transfection plasmid premix. Here, pP (plasmid carrying the Corynebacterial viral phosphate protein gene), pN (plasmid carrying the Corynebacterial viral nucleoprotein gene), and pL (plasmid carrying the Corynebacterial viral polymerase protein gene) are used; the parent vectors corresponding to the three plasmids pN, pP, and pL are all pCAGGS (purchased from ATCC). Dilute 10 μl of Lipofectamine LTX (Thermo Fisher Scientific) in 200 μl of opti-MEM medium to obtain an LTX mixture. Mix 200 μl of LTX mixture with 200 μl of transfection plasmid premix, incubate at room temperature for 15 minutes to obtain LTX-DNA mixture. Replace the DPBS buffer solution in the 6-well plate in step 1) with Opti-MEM medium, drop the LTX-DNA mixture into the 6-well plate for culturing BSR-T7 cells, gently shake the 6-well plate to evenly distribute the LTX-DNA mixture in the 6-well plate. After 6 - 8 h of transfection, aspirate and remove the transfection reagent, add 3 ml of fresh complete medium (Thermo Fisher Scientific), collect the cell supernatant of BSR-T7 cells after 72 h, filter it through a 0.22 μm filter to obtain the oncolytic virus titers corresponding to each of Production Examples 1 - 26.

[0174] Example Example 1 In this example, the invasion and infection abilities of different cells were detected using the oncolytic viruses produced in Production Examples 1 - 26 and the wild-type oncolytic virus respectively. The detection method was the TCID50 detection method, that is, 200 pfu of each of the oncolytic viruses produced in Production Examples 1 - 26 and the wild-type oncolytic virus were added to the culture solutions of different cells respectively, and the median tissue culture infective dose (TCID50) by each oncolytic virus was detected. The detected cells included 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 was as follows: (1) Add 3 mL of Vero (LLC / MC38 / Hela / MEF) cell suspension to each 6-well culture plate so that the cell number reaches 4×10 5 cells / well. There are a total of 6 wells for various Vero cells and 2 wells for MEF cells as a control. Culture the 6-well culture plate under environmental conditions of 37 °C and 5% CO2 for 16 h. (2) Add 200 pfu of the oncolytic virus produced in the production example to each well of the 6-well culture plate. After 24 h, collect 100 μl of the supernatant of MEF cells and various Vero cells, add the collected samples to the wells of a 96-well culture plate respectively, so that the cell number of each type of cell reaches 1×10 4Adjust the concentration to cells / ml and culture in a 96-well culture plate at 37°C under 5% CO2 environmental conditions for 16 hours. (3) Dilute the supernatant collected in step (2) 10 times in a 1.5 ml EP tube. -1 ~10 -11 The total titer was 11, and the diluted supernatant was inoculated into a 96-well culture plate, with one row of 8 wells in each dilution gradient, and 100 μl was inoculated per well. (4) After 48 hours, the fluorescence status of the cells in each well was observed. If fluorescence was present, the well was recorded as infected, and the TCID50 was calculated according to the Karber method.

[0175] The detection results are shown in Figures 1 to 4, where the horizontal axis 0 represents wild-type oncolytic viruses, the horizontal axis 1 to 26 represents oncolytic viruses produced in production examples 1 to 26, respectively, and the vertical axis is Log. 10 TCID50 represents the TCID50 value calculated using the Karber method, Log 10 A higher TCID50 value indicates a higher ability of the oncolytic virus to invade and infect the cells in question. 10 A lower TCID50 value indicates a lower ability of the oncolytic virus to invade and infect the cells in question.

[0176] Figure 1 shows the detection results of the ability of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention to invade and infect LLC cells.

[0177] Figure 2 shows the detection results of the ability of the oncolytic virus and wild-type oncolytic virus produced according to the present invention to invade and infect MC38 cells.

[0178] Figure 3 shows the detection results of the ability of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention to invade and infect HeLa cells.

[0179] Figure 4 shows the detection results of the ability of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention to invade and infect MEF cells.

[0180] From the drawings above, it can be seen that the oncolytic viruses produced in Production Examples 1 to 26 of this application all exhibit good ability to invade and infect LLC cells, MC38 cells, and HeLa cells. In particular, Production Examples 4 to 26 involve site-directed mutations in the amino acids on the G protein of the oncolytic virus, based on Production Example 3, thereby further improving the ability of the oncolytic virus to invade and infect LLC cells, MC38 cells, and HeLa cells. At the same time, since all of the oncolytic viruses produced above exhibit low ability to invade and infect MEF cells, it can be seen that the oncolytic viruses produced by this application can be suitably used for the invasion and infection of tumor and cancer cells without damaging normal cells, and have the potential for a wide range of applications.

[0181] Example 2 In this example, in vitro toxicity tests were performed on different cell types using oncolytic viruses produced in Production Examples 1-26 and wild-type oncolytic viruses, respectively. The detection method was the MTT detection method; specifically, 200 pfu each of Production Examples 1-26 and wild-type oncolytic viruses were added to the culture media of different cells, and cell activity was detected after 24 hours using the MTT detection method. The detected cells included LLC cells, MC38 cells, MEF cells, and 4T1 cells (mouse mammary cells). The specific detection method is as follows: (1) Add 100 μl of Vero (LLC / MC38 / 4T1 / MEF) cell suspension to each 96-well culture plate, and count the number of cells to 1 × 10 4 Prepare the cultures so that the number of cells per well is 1, and incubate the 96-well culture plates for 16 hours under environmental conditions of 37°C and 5% CO2. (2) The oncolytic viruses produced in the production example were diluted to have MOI (Moritomoles of Infection) values ​​of 0.001, 0.01, 0.1, and 1.0, respectively. The oncolytic viruses from each dilution gradient were inoculated into the 96-well culture plate from step (1), with 4 wells in each dilution gradient, and 100 μl in each well. The 96-well culture plate was then cultured for 40 hours under environmental conditions of 37°C and 5% CO2. (3) Remove the cell supernatant from the 96-well culture plate in step (2), and add fresh medium and MTT solution to the 96-well culture plate, with an addition amount of 20 μL / well. Incubate the 96-well culture plate for 4 hours under environmental conditions of 37°C and 5% CO2. (4) The 96-well culture plate was centrifuged at room temperature for 5 minutes, the rotation speed was set to 2500 rpm / min, and the supernatant was gently aspirated and removed with a 1 mL disposable sterile syringe. DMSO was then added to each well of the 96-well culture plate at a volume of 100 uL / well, and the plate was left to stand for 10 minutes under ambient conditions of 37°C. The plate was shaken for 2 minutes using a multi-function microplate reader, and the OD value of each well on the 96-well culture plate was measured at a wavelength of 570 nm or 490 nm.

[0182] The detection results are shown in Figures 5 to 8, where the horizontal axis 0 represents wild-type oncolytic viruses, the horizontal axis 1 to 26 represents oncolytic viruses produced in production examples 1 to 26, respectively, and the vertical axis OD 570 This represents the OD value of the cell, OD 570 A higher value indicates that the oncolytic virus has a lower ability to kill the cells in question, and OD 570 A smaller value indicates a higher killing capacity of the oncolytic virus against the cells in question.

[0183] Figure 5 shows the results of detecting the in vitro killing ability of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention against LLC cells.

[0184] Figure 6 shows the results of detecting the in vitro killing ability of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention against MC38 cells.

[0185] Figure 7 shows the results of detecting the in vitro killing ability of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention against 4T1 cells.

[0186] Figure 8 shows the results of detecting the in vitro killing ability of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention against MEF cells.

[0187] From the drawings above, it can be seen that the oncolytic viruses produced in Production Examples 1 to 26 of this application all exhibit good in vitro killing ability against LLC cells, MC38 cells, and 4T1 cells. In particular, Production Examples 4 to 26 involve site-directed mutations in the amino acids on the G protein of the oncolytic virus, based on Production Example 3, thereby improving the in vitro killing ability of the oncolytic virus against LLC cells, MC38 cells, and 4T1 cells. At the same time, since the oncolytic viruses produced above have almost no effect on MEF cells, it can be seen that the oncolytic viruses produced according to this application can be suitably used to damage and kill abnormal cells such as tumors and cancer cells without damaging normal cells.

[0188] Wild-type oncolytic viruses exhibit good in vitro killing ability against LLC cells, MC38 cells, and 4T1 cells. However, while they damage and kill these cells, they also significantly damage and kill MEF cells, limiting the clinical application of wild-type oncolytic viruses. Therefore, the modification of wild-type oncolytic viruses described in this application ensures the safety of the oncolytic virus against normal cells while simultaneously ensuring its killing ability against tumors and cancer cells, thus offering potential for broad clinical application.

[0189] Example 3 This example detected the difficulty of intracellular removal of oncolytic viruses and wild-type oncolytic viruses produced in Production Examples 1-26. The detection indicator was the expression level of the IFN-β gene. The IFN-β gene is a soluble glycoprotein gene produced by cells that has broad-spectrum antiviral, antitumor, and immunomodulatory effects. The cell's ability to remove oncolytic viruses can be determined by the expression level of the IFN-β gene. High IFN-β expression indicates that oncolytic viruses are easily removed within the cell, while low IFN-β expression indicates that oncolytic viruses are not easily removed within the cell. The detected cells included LLC cells, MC38 cells, HeLa cells, and MEF cells. The specific detection method is as follows: (1) Add 100 μl of Vero(LLC / MC38 / Hela / MEF) cells and 100 μl of MEF cell suspension to each of the 96-well culture plates, and reduce the cell count to 1 × 10⁶ 4 Prepare the cultures so that the number of cells per well is 1, and incubate the 96-well culture plates for 16 hours under environmental conditions of 37°C and 5% CO2. (2) The oncolytic viruses produced in the production example were diluted to have MOI (Moritomoles of Infection) values ​​of 0.001, 0.01, 0.1, and 1.0, respectively. The oncolytic viruses from each dilution gradient were inoculated into the 96-well culture plate from step (1), with 4 wells in each dilution gradient, and 100 μl in each well. The 96-well culture plate was then cultured for 40 hours under environmental conditions of 37°C and 5% CO2. (3) The cells from each group obtained by culturing in step (2) were disrupted, and total RNA was extracted from each cell using TRIzol (Invitrogen). This was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with DNA Eraser (Takara) reverse transcription kit, and stained with LightCycler 480SYBR 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.

[0190] The detection results are shown in Figures 9 to 12. Here, the horizontal axis 0 represents wild-type oncolytic viruses, and the horizontal axis 1 to 26 represents oncolytic viruses produced in production examples 1 to 26, respectively. The vertical axis IFN-β level represents the expression status of the IFN-β gene. A higher IFN-β level indicates that the oncolytic virus is easily removed within the cell, while a lower IFN-β level indicates that the oncolytic virus is not easily removed within the cell.

[0191] Figure 9 shows the detection results for the difficulty of removal of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention within LLC cells.

[0192] Figure 10 shows the detection results for the difficulty of removal of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention in MC38 cells.

[0193] Figure 11 shows the detection results for the difficulty of removal of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention in HeLa cells.

[0194] Figure 12 shows the detection results for the difficulty of removal of oncolytic viruses and wild-type oncolytic viruses produced according to the present invention in MEF cells.

[0195] From the above drawings, it can be seen that Production Examples 1 and 2 of this application cannot be easily removed in LLC cells, MC38 cells, and HeLa cells. Production Example 3 is more easily removed in LLC cells, MC38 cells, and HeLa cells than Production Examples 1 and 2. Production Examples 4 to 26 of this application are based on Production Example 3 and further involve site-directed mutations in the amino acids on the G protein of the oncolytic virus, thereby making it more difficult to remove the oncolytic virus in LLC cells, MC38 cells, and HeLa cells, and further ensuring that the oncolytic virus can better exert its ability to invade, infect, and kill in LLC cells, MC38 cells, and HeLa cells, while at the same time making it easier to remove the oncolytic virus in MEF cells, further ensuring the safety of MEF cells, and thereby improving the safety of the oncolytic virus.

[0196] The detailed description provided herein is for illustrative purposes only and does not limit the scope of the attached claims. Various variations of the embodiments described herein are now obvious to those skilled in the art and remain within the scope of the attached claims and corresponding methods.

Claims

1. An oncolytic virus comprising an M protein and a G protein, wherein the M protein includes amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO 1, the M protein including the amino acid substitutions includes the amino acid sequence shown in SEQ ID NO 5, and the G protein includes at least one amino acid substitution compared to the amino acid sequence shown in SEQ ID NO 2, and the amino acid substitutions of the G protein are 1) The G protein whose amino acid substitution is V53I, 2) The G protein in which the amino acid substitutions are V53I and A141V, 3) The G protein having amino acid substitutions V53I, A141V and D172Y, 4) The G protein having amino acid substitutions V53I, A141V, D172Y and K217E, 5) The G protein having amino acid substitutions V53I, A141V, D172Y, K217E and D232G, 6) The G protein whose amino acid substitutions are V53I, A141V, D172Y, K217E, D232G and V331A, 7) The G protein whose amino acid substitutions are V53I, A141V, D172Y, K217E, D232G, V331A and V371E, 8) The G protein whose amino acid substitutions are V53I, A141V, D172Y, K217E, D232G, V331A, V371E and G436D, 9) The G protein whose amino acid substitutions are V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D and T438S, 10) The G protein whose amino acid substitutions are V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S and F453L, 11) The G protein whose amino acid substitutions are V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L and T471I, 12) The G protein whose amino acid substitutions are V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H, 13) The G protein whose amino acid substitutions are A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H, 14) The G protein whose amino acid substitutions are D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H, 15) The G protein whose amino acid substitutions are K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H, 16) The G protein whose amino acid substitutions are D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H, 17) The G protein whose amino acid substitutions are V331A, V371E, G436D, T438S, F453L, T471I and Y487H, 18) The G protein whose amino acid substitutions are V371E, G436D, T438S, F453L, T471I and Y487H, 19) The G protein whose amino acid substitutions are G436D, T438S, F453L, T471I and Y487H, 20) The G protein whose amino acid substitutions are T438S, F453L, T471I and Y487H, 21) The G protein having amino acid substitutions F453L, T471I and Y487H, 22) The G protein whose amino acid substitutions are T471I and Y487H, 23) Any one selected from the group consisting of G proteins in which the amino acid substitution is Y487H, A tumor-lytic virus characterized by the following:

2. The amino acid substitutions of the G protein are V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I, and Y487H. The oncolytic virus according to feature 1.

3. The aforementioned G protein contains the amino acid sequence shown in SEQ ID NO 17, The oncolytic virus according to feature 2.

4. The aforementioned oncolytic viruses include Corynebacterium viruses. The oncolytic virus according to feature 1.

5. The aforementioned oncolytic viruses include vesicular stomatitis virus. The oncolytic virus according to feature 1.

6. The aforementioned oncolytic virus includes the VSV virus Indiana MuddSummer subtype. The oncolytic virus according to feature 1.

7. The aforementioned oncolytic virus contains or expresses an exogenous target protein. The oncolytic virus according to feature 1.

8. The oncolytic virus comprises a nucleic acid molecule including 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. The oncolytic virus according to feature 1.

9. The nucleic acid molecule includes a nucleic acid sequence encoding an external target protein. The oncolytic virus according to feature 8.

10. The nucleic acid sequence encoding the foreign target protein in the nucleic acid molecule is located between the nucleic acid sequence encoding the L protein having amino acid substitutions and the nucleic acid sequence encoding the G protein having amino acid substitutions. The oncolytic virus according to feature 9.

11. The nucleic acid molecule encodes the N protein, L protein, and P protein of the oncolytic virus. The oncolytic virus according to claim 10.

12. A nucleic acid molecule for expressing the oncolytic virus described in claim 1, which can express the oncolytic virus described in claim 1. A tumor-lytic virus expression vector characterized by the following features.

13. A nucleic acid molecule for expressing the oncolytic virus described in claim 1, capable of producing the oncolytic virus described in claim 1. A virus-producing cell characterized by the following features.

14. The oncolytic virus described in claim 1, and a pharmaceutically acceptable carrier, A pharmaceutical composition characterized by the following features.

15. Use of the oncolytic virus according to any one of claims 1 to 3, the oncolytic virus expression vector according to claim 12, the virus-producing cell according to claim 13, or the pharmaceutical composition according to claim 14 in the manufacture of a pharmaceutical for the prevention and / or treatment of a disease and / or medical condition.

16. The oncolytic virus, the oncolytic virus expression vector, the virus-producing cells, and / or the pharmaceutical composition are used in a method for continuously killing abnormally proliferating cells. The use described in feature 15.

17. The abnormally proliferating cells are selected from tumor cells or related cells of tumor tissue. The use described in feature 16.

18. The tumor cells are cancer cells or metastatic cancer cells. The use described in feature 17.

19. Tumors include solid tumors and / or hematological malignancies. The use described in claim 17.

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