M1 virus variant and its use

Abstract

To provide an M1 virus, and further provide a series of uses of the virus.SOLUTION: The uses include, but are not limited to, viral vectors, anti-tumor agents, and pharmaceutical compositions. The virus can effectively inhibit the growth of various tumor cells, and at the same time, has tumor targeting properties and is non-toxic to normal cells. The virus can be administrated by means of intravenous injection, having operational convenience.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This invention belongs to the field of biopharmaceuticals and, more specifically, relates to M1 virus variants and their use. [Background technology]

[0002] Accumulated genetic and epigenetic changes in normal cells promote the transformation of normal cells into malignant tumor cells. This complex pathological process determines the diversity of mechanisms involved in the development, maintenance, and metastasis of various tumors (1-3). Traditional treatments for tumors include surgical resection, chemotherapy, and radiotherapy. However, surgical resection has the problem of recurrence, and radiotherapy and chemotherapy have the problem of serious side effects (4). In recent years, targeted therapies and tumor immunotherapies, including IL-2 regulation, adoptive cell therapy, and modulation of immune checkpoints such as PD-1, have achieved some efficacy in clinical treatment. However, targeted therapies are prone to drug resistance, and immunotherapies have low response rates and can cause serious immune-related adverse events (5,6). Therefore, there is a need for research and development of new anti-cancer treatments that are low in toxicity, effective, and have low drug resistance.

[0003] Oncolytic viruses not only kill tumor cells but also alert the host's immune system to the presence of cancer. Viral therapy is a method based on the characteristic of oncolytic viruses that they tend to attack cancerous tissue more readily than healthy tissue. In 2005, the China Food and Drug Administration approved the sale of the first oncolytic virus therapy drug (brand name: Oncorine). This is a genetically modified virus that preferentially attacks tumor cells and is already being used to treat head and neck cancer. In 2015, the U.S. Food and Drug Administration (FDA) approved T-VEC for the treatment of melanoma. This uses a genetically modified herpesvirus and was approved in Australia and the European Union the following year. In addition, several oncolytic viruses are being studied. In recent decades, viral therapy has made great progress, but it still faces many challenges.

[0004] First, there are problems with the therapeutic effect. The antitumor effect or antitumor spectrum of oncolytic viruses is limited. Many oncolytic viruses cannot adequately inhibit or kill tumor cells and need to be used in combination with other chemotherapeutic agents or immune checkpoint inhibitors, and are sometimes used as adjunct therapy to radiotherapy. For example, the M1 virus described in Chinese Patent Application No. 201410425510.3, when used as an antitumor drug, is most effective against colorectal cancer, liver cancer, bladder cancer, and breast cancer, less effective against pancreatic cancer, nasopharyngeal cancer, prostate cancer, and melanoma, even less effective against glioma, cervical cancer, and lung cancer, and least effective against gastric cancer.

[0005] Furthermore, there are safety concerns. Some viruses are dangerous to humans, and these dangerous viruses need to be modified and attenuated before being used in viral therapy. However, even after modification and attenuation, oncolytic viruses can become "escape viruses," that is, viruses that change again after release or bind to pathogens already present in the patient's body and rapidly infect healthy tissues.

[0006] Furthermore, there is the problem of virus delivery, that is, how to deliver the virus to the lesion. Existing oncolytic viruses (e.g., T-VEC, approved by the U.S. Food and Drug Administration (FDA) for the treatment of melanoma) are mostly injected into tumor tissue. However, many solid tumors and micrometastases cannot be injected directly, or non-solid tumors such as hematological malignancies are distributed throughout the body and do not have fixed injection sites. Treating these types of tumors with existing viral therapies is difficult.

[0007] Therefore, the development of oncolytic viruses remains a major challenge.

[0008] Alphaviruses belong to the Togaviridae family and are a type of enveloped, single-stranded positive-sense RNA virus. Chikungunya virus, a member of the alphavirus genus, is a pathogenic virus that can infect humans and is highly virulent, reported to cause fever, rash, arthritis, and even fatal encephalitis after infection (7,8). Another virus in the alphavirus genus, Venezuelan equine encephalitis virus, has been reported to be used as a vector to transduce dendritic cells and treat tumors (9). However, this encephalitis virus has also caused fever, seizures, miscarriage, and even death in humans (10).

[0009] The Alphavirus M1 virus belongs to the genus Alphavirus and was isolated from the Culex pipiens mosquito on Hainan Island, China in 1964. The method for isolating the virus has been published in the literature (11). The M1 virus has a high homology of 97.8% with getavirus and belongs to the geta-like virus group (12). In 2008, the whole genome of the M1 strain was sequenced (13). The M1 virus has been reported to have oncolytic effects in patent CN201410425510.3, but there is room for improvement in the antitumor spectrum and antitumor strength of the existing wild-type M1 virus. [Overview of the project]

[0010] In some embodiments, an M1 virus is provided, wherein the amino acid residue at position 358 of the NS3 protein of the M1 virus is not M, and / or the amino acid residue at position 4 of the envelope protein E2 is not E or K.

[0011] In some embodiments, the amino acid residue at position 358 of the NS3 protein (nonstructural protein 3) of the M1 virus is G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, E, K, R, or H, and / or the amino acid residue at position 4 of the envelope protein E2 (envelope protein 2) of the M1 virus is M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R, or H.

[0012] In some embodiments, the NS3 protein contained in the M1 virus has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 31, and / or the E2 protein contained in the M1 virus has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 31. In some embodiments, the M1 virus is provided. The amino acid residue corresponding to position 358 of the NS3 protein encoded by that nucleic acid sequence is not M, and / or the amino acid residue corresponding to position 4 of the E2 protein is not E or K.

[0013] In some embodiments, the nucleic acid sequence of the M1 virus has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9% or 100% sequence identity with the M1 sequence represented by SEQ ID NO: 5, SEQ ID NO: 15, GenBank accession number EU015061.1, GenBank accession number EF011023.1, or CCTCC V201423.

[0014] In some embodiments, preferably, the amino acid residue at the position corresponding to the 358th position of the NS3 protein is G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, E, K, R or H, and / or the amino acid residue at the position corresponding to the 4th position of the E2 protein is M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R or H.

[0015] In some embodiments, the M1 virus has mutations relative to the wild-type M1 virus or pseudo-wild-type M1 virus.

[0016] In some embodiments, the M1 virus has mutations relative to the M1 virus whose sequence is shown in SEQ ID NO: 5 or the M1 virus shown in SEQ ID NO: 15.

[0017] In some embodiments, the mutations are M358G, M358A, M358L, M358I, M358V, M358P, M358S, M358Q, M358T, M358C, M358N, M358F, M358Y, M358W, M358D, M358E, M358K, M358R or M358H in the NS3 protein, and / or E These are K4M, K4G, K4A, K4L, K4I, K4V, K4P, K4S, K4Q, K4T, K4C, K4N, K4F, K4Y, K4W, K4D, K4R, K4H, E4M, E4G, E4A, E4L, E4I, E4V, E4P, E4S, E4Q, E4T, E4C, E4N, E4F, E4Y, E4W, E4D, E4R, or E4H in the two proteins.

[0018] In some embodiments, the M1 virus is characterized in which amino acid residue M at position 358 of the NS3 protein of the M1 virus having the sequence shown in Sequence ID No. 5 is mutated to G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, E, K, R, or H, and / or amino acid residue K at position 4 of the E2 protein is mutated to M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R, or H.

[0019] In some embodiments, the M1 virus is characterized in which amino acid residue M at position 358 of the NS3 protein of the M1 virus shown in SEQ ID NO: 15 is mutated to L, and / or amino acid residue E at position 4 of the E2 protein is mutated to D.

[0020] In some embodiments, nucleic acid sequences containing any of the M1 viruses described above are provided.

[0021] In some embodiments, an amino acid sequence corresponding to the NS3 protein of the M1 virus is provided, where the amino acid residue at position 358 is not M.

[0022] In some embodiments, the amino acid residue at position 358 is G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, E, K, R, or H.

[0023] In some embodiments, the amino acid sequence corresponding to the NS3 protein of the M1 virus has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 18.

[0024] In some embodiments, an amino acid sequence corresponding to the E2 protein of the M1 virus is further provided, in which the amino acid residue at the position corresponding to the fourth position is neither E nor K.

[0025] In some embodiments, the amino acid residue at the position corresponding to the fourth position is M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R, or H.

[0026] In some embodiments, the amino acid sequence corresponding to the E2 protein of the M1 virus has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 31.

[0027] In some embodiments, nucleic acid sequences encoding amino acid sequences corresponding to the NS3 protein of the M1 virus are provided.

[0028] In some embodiments, nucleic acid sequences encoding amino acid sequences corresponding to the E2 protein of the M1 virus are provided.

[0029] In some embodiments, a vector is provided. The vector comprises nucleic acid encoding the E2 protein and / or NS3 protein of the M1 virus. The amino acid residue corresponding to position 358 of the NS3 protein is not M, and the amino acid residue corresponding to position 4 of the E2 protein is not E or K.

[0030] In some embodiments, the amino acid residue at position 358 of the NS3 protein is G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, E, K, R, or H.

[0031] In some embodiments, the amino acid residue at the position corresponding to the 4th position of the E2 protein is M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R, or H.

[0032] In some embodiments, the NS3 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 18.

[0033] In some embodiments, the E2 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 31.

[0034] In some embodiments, the vector further comprises coding sequences for the M1 virus's NS1 protein, NS2 protein, NS4 protein, C protein, E3 protein, 6K protein, and / or E1 protein.

[0035] In some embodiments, the amino acid sequence of the NS1 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 16.

[0036] In some embodiments, the amino acid sequence of the NS2 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 17.

[0037] In some embodiments, the amino acid sequence of the NS4 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 19.

[0038] In some embodiments, the amino acid sequence of the C protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 20.

[0039] In some embodiments, the amino acid sequence of the E3 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 30.

[0040] In some embodiments, the amino acid sequence of the 6K protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 32.

[0041] In some embodiments, the amino acid sequence of the E1 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 33.

[0042] In some embodiments, the vector further comprises an exogenous gene for the M1 virus.

[0043] In some embodiments, the exogenous gene expresses an antitumor-related molecule.

[0044] In some embodiments, the vector is selected from viruses.

[0045] In some embodiments, the vector is selected from retroviruses, Newcastle disease virus, rabies virus, varicella stomatitis virus, maraba virus, alphavirus, Newcastle disease virus, reovirus, adenovirus, adeno-associated virus, herpes simplex virus, cowpox virus, or measles virus.

[0046] In some embodiments, the vector is selected from a plasmid.

[0047] In some embodiments, a vector containing the nucleic acid sequence is provided.

[0048] In some embodiments, the vector is selected from a plasmid.

[0049] In some embodiments, a viral vector is provided. The virus is one of the M1 viruses described above.

[0050] In some embodiments, an exogenous gene is inserted into the vector.

[0051] In some embodiments, the exogenous gene expresses an antitumor-related molecule.

[0052] In some embodiments, the use of the M1 virus, the nucleic acid sequence, the amino acid sequence of the NS3 protein of the M1 virus, the amino acid sequence of the E2 protein of the M1 virus, the nucleic acid sequence of the NS3 protein or the E2 protein, the vector, or the viral vector in the manufacture of an antitumor drug is further provided.

[0053] In some embodiments, an antitumor agent comprising the M1 virus, the nucleic acid sequence, the amino acid sequence of the NS3 protein of the M1 virus, the amino acid sequence of the E2 protein of the M1 virus, the nucleic acid sequence, the vector, or the viral vector is further provided.

[0054] In some embodiments, compositions are further provided that include an effective amount of the M1 virus, the nucleic acid sequence, the amino acid sequence of the NS3 protein of the M1 virus, the amino acid sequence of the E2 protein of the M1 virus, the vector or the viral vector, and a pharmaceutically acceptable vector.

[0055] In some embodiments, the composition further comprises an immune checkpoint inhibitor.

[0056] In some embodiments, the composition further comprises a chemotherapeutic agent.

[0057] Of course, in some embodiments, the M1 virus may not contain immune checkpoint inhibitors and / or chemotherapeutic agents. In one embodiment of the present invention, the M1 virus alone has a very high tumor-suppressing effect even when not used in combination with any other anticancer agent (chemotherapeutic agent, immune checkpoint inhibitor, or other conventional substance or tool having an antitumor effect).

[0058] In some embodiments, the composition comprises at least 10 1 Contains individual virus particles or PFUs.

[0059] In some embodiments, the composition contains 10 1 to 10 30 virus particles or PFUs.

[0060] In some embodiments, the composition contains 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , 10 20 , 10 21 , 10 22 virus particles or PFUs. <​​​​​​​​​​​​​​​​​​​​​​​In some embodiments, the solid tumors include liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, stomach cancer, adrenocortical cancer, accessory cortical cancer, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, and carcinoid tumor. Tumors, cardiac tumors, cholangiocarcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germ cell tumors, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumors, extragonadal germ cell tumors, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia, mycosis fungoides It is one or more of the following selected conditions: nasal cavity / paranasal sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papilloma, paraganglioma, paranasal sinus / nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal / laryngeal cancer, pituitary tumor, pleuroblastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, César syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, hemangioma, vulvar cancer, and solitary myeloma.

[0067] In some embodiments, the hematological malignancy is one or more selected from B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia.

[0068] In some embodiments, the tumor is one or more selected from liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, and gastric cancer.

[0069] In some embodiments, the obtained M1 virus can effectively treat a variety of tumors. These tumors include liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, and gastric cancer. In vitro experiments have demonstrated that the M1 virus of the present invention can induce tumor cell death. In vivo experiments have demonstrated that the M1 virus of the present invention can significantly inhibit the growth of liver cancer and colorectal cancer in the body. Whether in vitro or in vivo, the efficacy of the M1 virus of the present invention is almost always higher than that of the wild-type virus.

[0070] In some embodiments, the resulting M1 virus exhibits good selectivity and safety. As is evident from cell experiments, the M1 virus of the present invention does not exhibit cytotoxicity to normal cells and can selectively kill tumor cells. This indicates that the M1 virus of the present invention has tumor selectivity. As is evident from animal experiments, injection of the M1 virus of the present invention into the tail vein did not affect the body weight or mental state of nude mice, and no viral distribution was observed in normal organs, thus demonstrating the safety of the M1 virus of the present invention.

[0071] In some embodiments, tumor growth can be significantly suppressed by administering the obtained M1 virus intratumor or intravenously. Intratumor injection of commercially available oncolytic viruses requires specialized training of physicians and nurses, has low patient acceptance, and is unsuitable for deep organ tumors and micrometastases. In contrast, the M1 virus of the present invention can be administered intravenously. As a result, the M1 virus of the present invention is more convenient and feasible in clinical application and has a wider range of applications.

[0072] In this specification, unless the context requires otherwise, terms such as “inclusion” and “containment” mean including the aforementioned step, element, or combination of step and element, but not excluding other steps, elements, or combinations of step and element; i.e., they are open limitations.

[0073] For example, in some embodiments, "included" in the mutations "included" in the M1 virus means that, in addition to the mutations (e.g., the mutation at the 358th amino acid residue of the non-structural protein NS3 and / or the 4th amino acid residue of the structural protein E2), other mutations (in particular silent mutations), such as mutations that do not affect the function of the virus, or mutations that do not affect the basic function and enhance a certain ability of the virus, reduce toxicity, or improve stability without interfering with the basic function of mutant M1.

[0074] In some embodiments, the M1 virus is a bisite mutation, namely M358L in the NS3 protein and one of K4N, K4D, E4N, or E4D in the E2 protein. This means that the mutant contains only these mutations and no other mutations.

[0075] In some embodiments, the wild-type M1 virus corresponding to the M1 virus is the virus shown in deposit number CCTCC V201423 (see Chinese Patent No. 104814984A for details).

[0076] In some embodiments, the wild-type M1 virus corresponding to the M1 virus has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and at least 99.8% identity with the viral genome sequence shown in deposit number CCTCC V201423 (see Chinese Patent No. 104814984A for details).

[0077] In some embodiments, the wild-type M1 virus corresponding to the M1 virus is as shown in gene bank accession number EU015061.1 or EF011023.1 (as per the filing date / priority date information).

[0078] In some embodiments, the wild-type M1 virus corresponding to the M1 virus is considered to have at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and at least 99.8% identity with the viral genome sequence published by Gene Bank accession numbers EU015061.1, EF011023.1 (according to the information on the filing date / priority date), or by Zhai YG (13), and is considered to be a virus that is likely to originate from the same strain as CCTCC V201423.

[0079] In some embodiments, the protein is an isolated polypeptide.

[0080] In some embodiments, the nucleic acid is a separated polynucleic acid.

[0081] In some embodiments, the M1 virus is an isolated virus. [Brief explanation of the drawing]

[0082] [Figure 1] The nucleic acid sequencing of the mutation sites of four types of mutant viruses (rM1-NS3M, rM1-E2M, rM1-N3E2M, and rM1-c6v1) is shown. Figure 1A shows the nucleic acid sequencing near the amino acid residue at position 358 of the NS3 protein. The wild-type nucleic acid sequence at this site is CCA, with the corresponding amino acid being M, while the mutant nucleic acid sequence is CCC, with the corresponding amino acid being L. Figure 1B shows the nucleic acid sequencing near the amino acid residue at position 4 of the E2 protein. The wild-type nucleic acid sequence at this site is AAA or GAA, with the corresponding amino acid being K or E, while the mutant nucleic acid sequence is AAC or GAC, with the corresponding amino acid being N or D. [Figure 2] This shows the enhanced tumor lytic effect of site-directed mutant viruses (rM1-NS3M, rM1-E2M, rM1-N3E2M) in the HCT 116 cell line. Figure 2A shows the cell morphology observed 48 hours after infection of HCT 116 cells at 1 MOI. Figure 2B shows the results of comparing cell viability 72 hours after infection with rM1-WT, rM1-NS3M, rM1-E2M, and rM1-N3E2M viruses at 0.001-10 MOI using the MTT method. [Figure 3] This report presents safety and efficacy studies of the rM1-N3E2M virus in animal models. Nude mice were inoculated with HCT116 subcutaneously to form tumors, and the virus was intravenously injected 14 days after tumor inoculation. Tumor volume and nude mouse body weight were repeatedly measured, and statistical analysis using ANOVA showed that *p<0.05, which is statistically significant. Figure 3A shows the changes in tumor growth in the nude mouse subcutaneous tumor model. Figure 3B shows the changes in nude mouse body weight in the nude mouse subcutaneous tumor model. [Figure 4] Infection with the rM1-N3E2M virus upregulated the expression of the cell apoptosis indicator Cl-casp3 and downregulated the expression of the cell proliferation indicator Ki67 in tumor tissue. In nude mouse subcutaneous tumor models, tumor tissue was dissected after 3 days of treatment with rM1-WT and rM1-N3E2M, and the expression of Cl-casp3 and Ki67 was measured by immunohistochemical staining. [Figure 5] This indicates that no toxic lesions caused by the rM1-N3E2M virus were observed in normal organ tissues. [Figure 6] This paper presents experimental results on the safety and efficacy of the M1-c6v1 virus in animal models. C57 BL / 6 mice were inoculated with B16-F10 subcutaneously, and the virus was intravenously injected 11 days after tumor inoculation. Tumor volume and body weight of nude mice were repeatedly measured, and statistical analysis by ANOVA showed *p<0.05, which is statistically significant. Figure 6A shows the changes in tumor growth in the mouse subcutaneous tumor model. Figure 6B shows the changes in body weight of nude mice in the mouse subcutaneous tumor model. [Figure 7-1]Figures 7A-7Z are sequencing diagrams of the mutation sites in site-specific variants of the M1 virus. Figure 7A is rM1-WT(E2-4K)(AAA). Figure 7B is the variant (E2-4L,AAA→CTG). Figure 7C is the variant (E2-4I,AAA→ATT). Figure 7D is the variant (E2-4V,AAA→GTG). Figure 7E is the variant (E2-4S,AAA→AGC). Figure 7F is the variant (E2-4C,AAA→TGC). Figure 7G is the variant (E2-4L,AAA→CTG). Figure 7H is the variant (E2-4D,AAA→GAT). [Figure 7-2] Figure 7I shows rM1-WT (NS3-358M, ATG). Figure 7J shows the mutant strain (NS3-358G, ATG → GGC). Figure 7K shows the mutant strain (NS3-358A, ATG → GCG). Figure 7L shows the mutant strain (NS3-358L, ATG → CTG). Figure 7M shows the mutant strain (NS3-358I, ATG → ATT). Figure 7N shows the mutant strain (NS3-358V, ATG → GTG). Figure 7O shows the mutant strain (NS3-358P, ATG → CCG). Figure 7P shows the mutant strain (NS3-358S, ATG → AGC). [Figure 7-3] Figure 7Q shows the mutant strain (NS3-358Q, ATG→CAG). Figure 7R shows the mutant strain (NS3-358T, ATG→ACC). Figure 7S shows the mutant strain (NS3-358C, ATG→TGC). Figure 7T shows the mutant strain (NS3-358N, ATG→AAC). Figure 7U shows the mutant strain (NS3-358F, ATG→TTT). Figure 7V shows the mutant strain (NS3-358Y, ATG→TAT). Figure 7W shows the mutant strain (NS3-358D, ATG→GAT). Figure 7X shows the mutant strain (NS3-358K, ATG→AAA). [Figure 7-4] Figure 7Y shows the mutant strain (NS3-358R, ATG→CGT). Figure 7Z shows the mutant strain (NS3-358H, ATG→CAT). [Figure 8-1]Figures 8A-8Z show the killing curves for each site-specific mutation (single-site mutation) strain of the M1 virus against HCT116. Figure 8A is the killing curve for rM-WT(E2-4K). Figure 8B is the killing curve for mutant strain E2-4L. Figure 8C is the killing curve for mutant strain E2-4I. Figure 8D is the killing curve for mutant strain E2-4V. [Figure 8-2] Figure 8E shows the lethality curve for mutant strain E2-4S. Figure 8F shows the lethality curve for mutant strain E2-4C. Figure 8G shows the lethality curve for mutant strain E2-4M. Figure 8H shows the lethality curve for mutant strain E2-4D. [Figure 8-3] Figure 8I shows the lethality curve for the rM-WT strain (NS3-358M). Figure 8J shows the lethality curve for the mutant strain NS3-358G. Figure 8K shows the lethality curve for the mutant strain NS3-358A. Figure 8L shows the lethality curve for the mutant strain NS3-358L. [Figure 8-4] Figure 8M shows the lethality curve for mutant strain NS3-358I. Figure 8N shows the lethality curve for mutant strain NS3-358V. Figure 8O shows the lethality curve for mutant strain NS3-358P. Figure 8P shows the lethality curve for mutant strain NS3-358S. [Figure 8-5] Figure 8Q shows the killing curve for mutant strain NS3-358Q. Figure 8R shows the killing curve for mutant strain NS3-358T. Figure 8S shows the killing curve for mutant strain NS3-358C. Figure 8T shows the killing curve for mutant strain NS3-358N. [Figure 8-6] Figure 8U shows the killing curve for mutant strain NS3-358F. Figure 8V shows the killing curve for mutant strain NS3-358Y. Figure 8W shows the killing curve for mutant strain NS3-358D. Figure 8X shows the killing curve for mutant strain NS3-358K. [Figure 8-7] Figure 8Y shows the killing curve for mutant strain NS3-358R. Figure 8Z shows the killing curve for mutant strain NS3-358H. [Modes for carrying out the invention]

[0083] The technical means of the present invention will be further described below with reference to specific embodiments. The following specific embodiments do not limit the scope of protection of the present invention. Non-essential modifications and adjustments made by those skilled in the art based on the concept of the present invention are also included in the scope of protection of the present invention.

[0084] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as those generally understood by those skilled in the art.

[0085] Unless otherwise specified, the mutations of the present invention include, but are not limited to, spontaneous mutations, forced mutations, or selective mutations, and include gene modifications, sequence additions or deletions, or partial substitutions.

[0086] The aforementioned "mutation" includes, but is not limited to, artificial mutations (mutaogenesis or genetic engineering) in specific sites of the wild-type virus strain. In this art, wild-type and mutation are relative concepts, and it is known that wild-type viruses themselves have differences in nucleic acid or amino acid residue sequences.

[0087] As used herein, the term "and / or" means to include any and all possible combinations of one or more related items. When used in a list of two or more items, the term "and / or" means that any one of the items shown may be used alone, or two or more of the items shown may be used in combination. For example, when terms such as composition, combination, and structure are used to include (or contain) components A, B, C and / or D, the composition includes A alone, B alone, C alone, D alone, a combination of A and B, a combination of A and C, a combination of A and D, a combination of B and C, a combination of B and D, a combination of C and D, a combination of A, B and C, a combination of A, B and D, a combination of A, C and D, or a combination of A, B and C and D.

[0088] In this specification, for example, "M358G" of the NS3 protein refers to a mutation in the NS3 protein of the M1 virus, specifically the M358G mutation occurring at the 358th amino acid residue, changing from M to G. For example, "M358A" refers to a mutation in the NS3 protein of the M1 virus, specifically the M358A mutation occurring at the 358th amino acid residue, changing from M to A. The same applies to other cases.

[0089] In this specification, for example, K4M in the E2 protein refers to a K4M mutation occurring at the fourth amino acid residue of the E2 protein of the M1 virus, where K is mutated to M. For example, K4A in the E2 protein refers to a K4A mutation occurring at the fourth amino acid residue of the E2 protein of the M1 virus, where K is mutated to A. The same applies to other cases.

[0090] [Table 1] JPEG0007894402000002.jpg68170

[0091] NS3 protein: nonstructural protein 3.

[0092] E2 protein: Envelope protein 2.

[0093] In this specification, the numbers of each sequence are shown in Table A below. [Table 2] JPEG0007894402000004.jpg199170

[0094] The wild-type M1 virus refers to a virus strain obtained from nature, and its corresponding genome is the wild-type genome. The wild-type genome may have diversity in certain regions. For example, the amino acid residue at position 4 of the E2 protein of known wild-type M1 viruses is either E or K.

[0095] A pseudowild-type M1 virus refers to an M1 virus that does not perfectly match the sequence of a known wild-type M1 virus, but whose biological characteristics do not differ significantly from those of a known wild-type M1 virus.

[0096] In some examples, the wild-type M1 virus is used, for example, the M1 virus with deposit number CCTCC V201423 and its monoclonal virus M1-c6 virus, or, for example, EU015061.1, EF011023.1.

[0097] For example, in some embodiments, mutations may occur in one or more of the following sites based on the wild type: the second and / or 786th amino acid residue in the unstructured protein NS2, the 30th and / or 393rd amino acid residue in the unstructured protein NS3, the 381st amino acid residue in the unstructured protein NS3, the 154th amino acid residue in the structural protein C, and the 246th amino acid residue in the structural protein E2.

[0098] rM1-WT is an M1 virus whose genome sequence is shown in SEQ ID NO: 5. The sequences of its NS1 protein are shown in SEQ ID NO: 6, its NS2 protein in SEQ ID NO: 7, its NS3 protein in SEQ ID NO: 8, its NS4 protein in SEQ ID NO: 9, its C protein in SEQ ID NO: 10, its E3 protein in SEQ ID NO: 11, its E2 protein in SEQ ID NO: 12, its 6K protein in SEQ ID NO: 13, and its E1 protein in SEQ ID NO: 14. The amino acid residue at position 358 of the NS3 protein is M, and the amino acid residue at position 4 of the E2 protein is K.

[0099] rM1-NS3M refers to the recombinant M1 virus. This virus is derived from rM1-WT and has undergone an M358L mutation (i.e., a mutation from M to L) at the 358th amino acid residue of the NS3 protein.

[0100] rM1-E2M refers to the recombinant M1 virus. This virus is derived from rM1-WT and has undergone a K4N mutation (i.e., a K to N mutation) in the amino acid residue at position 4 of the E2 protein.

[0101] rM1-N3E2M refers to the recombinant M1 virus. This virus is derived from rM1-WT, with an M358L mutation (i.e., a mutation from M to L) occurring at the 358th amino acid residue of the NS3 protein, and a K4N mutation (i.e., a mutation from K to N) occurring at the 4th amino acid residue of the E2 protein.

[0102] M1-c6 is an M1 virus whose genome sequence is shown in SEQ ID NO: 15. The sequences of its NS1 protein are shown in SEQ ID NO: 16, its NS2 protein in SEQ ID NO: 17, its NS3 protein in SEQ ID NO: 18, its NS4 protein in SEQ ID NO: 19, its C protein in SEQ ID NO: 20, its E3 protein in SEQ ID NO: 30, its E2 protein in SEQ ID NO: 31, its 6K protein in SEQ ID NO: 32, and its E1 protein in SEQ ID NO: 33. The amino acid residue at position 358 of the NS3 protein is M, and the amino acid residue at position 4 of the E2 protein is E.

[0103] M1-c6v1 is a recombinant M1-c6 virus. This virus is derived from M1-c6 by developing an M358L mutation (i.e., a mutation from M to L) at the 358th amino acid residue of the NS3 protein, and an E4D mutation (i.e., a mutation from E to D) at the 4th amino acid residue of the E2 protein.

[0104] The amino acid residue at position 358 of the NS3 protein is located at position 358 in the known wild-type M1 virus, with the sequence of three amino acid residues upstream of it (N-terminus) being YET, and the sequence of three amino acid residues downstream of it (C-terminus) being EVV. The number "358" is not an absolute restriction, and whether a specific amino acid residue (e.g., a mutated amino acid residue) is located at the said position should be determined depending on the functional domain or motif in which it is located. For example, in the sequences of other pseudowild-type M1 viruses, the said position is not necessarily at position 358 in the sequence order of the NS3 protein. In this case, "358" should not be considered an absolute restriction, and whether a specific amino acid residue belongs to the amino acid residue at position 358 of the NS3 protein as described herein should be determined depending on the functional domain or motif in which it is located.

[0105] Regarding the amino acid residue at position 4 of the E2 protein, in known wild-type M1 viruses, the said site is located at position 4 of the E2 protein, with the sequence of three amino acid residues upstream of it at the N-terminus being SVT, and the sequence of three amino acid residues downstream of it at the C-terminus being HFN. The number "4" is not an absolute restriction, and it should be determined whether a particular amino acid residue (e.g., a mutated amino acid residue) is located at the said site depending on the functional domain or motif in which it is located. For example, in the sequences of other pseudowild-type M1 viruses, the said site is not necessarily at position 4 in the sequence order of the E2 protein's primary structure. In this case, "4" should not be considered an absolute restriction, and it should be determined whether the particular amino acid residue belongs to the amino acid residue at position 4 of the NS3 protein described herein, depending on the functional domain or motif in which it is located.

[0106] A pharmaceutically acceptable vector is a molecular entity or composition that does not cause allergic or similar adverse reactions when administered to humans, and includes any of the following: solvents, dispersion media, mediators, coats, diluents, antimicrobial and antifungal agents, isotonic and absorption retarders, buffers, vector solutions, suspensions, colloids, etc. The use of these pharmacoactive substances in media and reagents is known in the art. Unless the active ingredient is incompatible with any of the common media or reagents, they are expected to be used in therapeutic compositions.

[0107] Immune checkpoint inhibitors are molecules that reduce, inhibit, interfere with, or modulate one or more checkpoint proteins, either whole or partially. Checkpoint proteins modulate the activation or function of T cells. Several types of checkpoint proteins are known, such as CTLA-4, its ligands CD80 and CD86, and PD1, its ligands PDL1 and PDL2 (Pardoll, Nature Reviews Cancer 12:252-264, 2012). These proteins are responsible for inhibiting the co-stimulation or interaction of T cell responses. Immune checkpoint proteins regulate and maintain self-resistance and the duration and breadth of physiological immune responses. Immune checkpoint inhibitors contain or are derived from antibodies.

[0108] Chemotherapy agents are compounds that can be used to treat cancer.

[0109] Treatment refers to alleviating symptoms, temporarily or permanently eliminating the cause of symptoms, or preventing or delaying a particular disease or condition.

[0110] The effective dose refers to an amount sufficient to provide the desired therapeutic effect, including the alphavirus or proteasome inhibitor used in the present invention. The required precise dose varies depending on the subject and depends on the species being treated, the subject's age, general medical condition, the severity of the condition being treated, the specific drug administered, and the mode of administration. However, for certain situations, those skilled in the art can adjust the dose of the pharmaceutical composition of the present invention according to the severity of symptoms, the frequency of recurrence, and the physiological response to the treatment plan.

[0111] Example 1: Construction and identification of M1 site-directed mutant virus strains material 1. Cloning: Competent TOP10, plasmid extraction kit, DNA product recovery kit; Gibson AssemblyMaster Mix; Phanta Max Super-Fidelity DNA Polymerase. 2. Restriction enzymes SpeI, SwaI, XhoI, ApaI, XbaI 3. Site-specific mutation primers are shown in Table 2. [Table 3] 4. Wild-type M1 virus full-length genome plasmid: pBR-M1-WT is a plasmid vector containing the entire M1 genome, the sequence of which is shown in Sequence ID No. 5. Based on the sequence shown in Sequence ID No. 5, the entire gene is synthesized, and the M1 sequence is cloned between the ClaI and EcoRI enzyme cleavage sites of the pBR322 vector (with added polyclonal sites) using recombinant DNA technology to form the pBR-M1-WT vector. The pBR-M1-c6 vector is constructed using a similar method. M1-c6 is the M1 virus, the sequence of which is shown in Sequence ID No. 15, with the amino acid residue at position 358 of its NS3 protein being M and the amino acid residue at position 4 of its E2 protein being E. 5. Mutant virus full-length genome plasmids: Mutations are introduced into the pBR-M1-WT vector using site-directed mutagenesis technology to form pBR-M1-NS3M (i.e., containing the M358L mutation at the 358th amino acid residue of the NS3 protein), pBR-M1-E2M (i.e., containing the K4N mutation at the 4th amino acid residue of the E2 protein), and pBR-M1-N3E2M (i.e., containing the M358L mutation at the 358th amino acid residue of the NS3 protein and the K4N mutation at the 4th amino acid residue of the E2 protein). Mutations are also introduced into pBR-M1-c6 to form pBR-M1-c6v1 (i.e., containing the M358L mutation at the 358th amino acid residue of the NS3 protein and the E4D mutation at the 4th amino acid residue of the E2 protein). 6. DH5α competent cells, high-purity plasmid extraction kit; JM110 competent cells; Lipofectamine RNAiMAX Transfection Reagent. 7. RNA extraction: TRIzol; chloroform, isopropanol, anhydrous ethanol. 8. Reverse transcription: Random hexamer, dNTP, MMLV, RNaseOUT. 9. PCR: Q5 high-fidelity enzyme, PCR primers (Table 3). Primers are synthesized by Invitrogen. 10. Agarose gel DNA recovery kit. 11. DNA electrophoresis: Agarose, SYBR green, DNA marker.

[0112] [Table 4]

[0113] method 1. Construction of a plasmid vector containing the full-length viral gene. pBR-M1-WT and pBR-M1-c6 strains were added to 5 mL of LB medium and shaken overnight at 37°C. Plasmids were extracted using a plasmid extraction kit, and the plasmid DNA concentration was measured using Nanodrop. The PCR amplification system is shown in Table 4. [Table 5]

[0114] Reaction conditions: Pre-denaturation was performed at 98°C for 3 minutes, followed by 35 cycles of 98°C for 15 seconds, 58°C for 15 seconds, and 72°C for 45 seconds. The mixture was then expanded at 72°C for 5 minutes, after which the temperature was cooled to 4°C. The presence or absence of product and the accuracy of its size were detected by agarose gel electrophoresis.

[0115] Enzymatic cleavage of vector plasmids: Enzymatic cleavage systems for NS3 and E2 mutations were constructed (Table 5). [Table 6]

[0116] The reaction was carried out at 37°C for 1 hour, the cleaved plasmid was collected using the kit, and its concentration was measured.

[0117] The vector and PCR fragments were assembled using the Gibson AssemblyMaster Mix kit. The reaction system consisted of PCR fragment 1 + PCR fragment 2 + plasmid + 2 × Mix + H2O = 1 + 1 + 10 + 7 (μL), and the reaction conditions were 50°C for 1 hour.

[0118] Transformed clones: 10 μL of the conjugation product was taken, and 100 μL of DH5a competent cells were transformed and screened using an ampicillin-resistant plate. Clones were selected and sequenced. The bisite-mutated viruses were those that had undergone further mutations at the NS3 site in the E2-mutated vector plasmid.

[0119] 2. Production of site-directed mutant viruses The pBR-M1-NS3M, pBR-M1-E2M, pBR-M1-N3E2M, and pBR-M1-c6v1 strains were inoculated into 5 mL of LB medium and shaken overnight at 37°C. Plasmids were extracted using a kit and their concentrations were measured. JM110 was transformed (due to the low transformation efficiency of JM110 competent strains, more plasmid is needed to transform 100 μL of competent strains. After adding 300 μL of LB solution and allowing to recover for 1 hour, the entire mixture was plated) and screened using ampicillin-resistant plates. Monoclones were selected and added to 500 μL of LB / ampicillin medium, shaken at 37°C for 12 hours, further expanded culture, and shaken at 37°C for 14-16 hours. Once the bacterial suspension concentration was appropriate, the strains were preserved with a final glycerin concentration of 15-30%. Plasmids were extracted using a plasmid extraction kit capable of removing endotoxins and their concentrations were measured. Plasmids were linearized with XbaI endonuclease (Table 6). [Table 7]

[0120] The mixture was divided into two test tubes and reacted at 37°C for 2 hours. Proteinase K digestion: Dilution was performed 10-fold with Proteinase K (20 mg / ml), 2.5 μL was added to each tube, followed by 5 μL of 10% SDS, and incubated at 50°C for 30 minutes. Linear plasmid DNA was recovered after enzymatic digestion using the kit, and its concentration was measured. In vitro transfer: The reaction was carried out at 37°C for 2 hours. The product should not be frozen and stored afterward, but used immediately in the next step. The transfer system is shown in Table 7. [Table 8]

[0121] DNA template removal: RQ1 RNase-Free DNase (1U / μL) was added to a 1U / μg DNA template and reacted at 37°C for 15 minutes. RNA transfection: 3 x 10⁶ Vero cells 1 day prior 5Cells were inoculated into a 6-well plate at 1 cell / well and cultured in 1.5 ml of complete medium. 125 μL of Opti-MEM and 3.75 μL of LipomRNA, and 125 μL of Opti-MEM and 2.5 μg of RNA were uniformly mixed, and both were placed in a single test tube and mixed uniformly. The mixture was allowed to stand at room temperature for 5 minutes. The RNA and transfection reagent complex was placed in a cell culture dish, and the presence of cellular lesion morphology was observed for the next 2-4 days. The supernatant was collected, and the virus was further amplified in Vero cells.

[0122] 3. RNA extraction using the TRIzol method TRIzol was added, and the cells were thoroughly lysed by pipetting. Once precipitate formed, the mixture was centrifuged at 12000g at 4°C for 10 minutes, and the supernatant was collected. 200 μL of chloroform was added, and the mixture was mixed homogeneously by vigorous shaking. The mixture was then allowed to stand at room temperature for 3 minutes. Centrifuged at 12000g at 4°C for 15 minutes, and approximately 500 μL of the upper aqueous phase was aspirated and transferred to a new EP tube. 500 μL of isopropanol was added, the mixture was gently inverted to mix homogeneously, and the mixture was allowed to stand at room temperature for 10 minutes. Centrifuged at 12000g at 4°C for 10 minutes, and the supernatant was discarded. The precipitate was washed with 500 μL of pre-cooled 75% ethanol, and the mixture was centrifuged at 12000g at 4°C for 5 minutes. The supernatant was discarded. After drying, the RNA precipitate was dissolved with an appropriate amount of DEPC water. The RNA concentration was measured using Nanodrop.

[0123] 4. Reverse transcription Reverse transcription was performed using MMLV reverse transcriptase. [Table 9]

[0124] Each reaction component was added according to Table 8, and after initial thermal denaturation at 65°C for 5 minutes, the mixture was immediately placed on ice and allowed to stand for 2-3 minutes. 4 μL of 5×buffer reaction solution, 2 μL of RNaseOUT reagent, 1 μL of DTT, and 1 μL of reverse transcriptase MMLV were added, and the reaction was carried out at 25°C for 10 min, 37°C for 50 min, and 70°C for 15 min.

[0125] 5. PCR The cDNA after reverse transcription was diluted 2-5 times, and the DNA was amplified by PCR using the Q5 high-fidelity enzyme (the M1 genome was amplified by dividing it into 10 fragments). [Table 10]

[0126] Reaction conditions: Initial thermal denaturation was performed at 98°C for 30 seconds, followed by 35 cycles of 98°C for 10 seconds, 58°C for 30 seconds, and 72°C for 1 minute. The reaction was then extended at 72°C for 2 minutes, after which the temperature was cooled to 4°C.

[0127] Detection of PCR products by 1% agarose gel electrophoresis 0.5 g of agarose was weighed, added to 50 mL of water, heated to completely dissolve the agarose, cooled to approximately 50°C, and SYBR green dye was added at a ratio of 1 / 10,000. The mixture was then placed in a gel plate, a comb was inserted, and after complete coagulation, it was placed in an electrophoresis tank. 5 μL of sample was added to each well, and electrophoresis was performed at a voltage of 100 V for approximately 40 mins. DNA products were observed and photographed using a Tianneng Imager.

[0128] [Table 11]

[0129] 6. The PCR product was mailed to Thermo Scientific for sequencing.

[0130] result As shown in Figure 1, and as can be seen from the comparison of gene sequences, all site-directed mutant viruses were successfully constructed. These are the following four types of mutant viruses. (1) rM1-NS3M: This variant is a modified version of the M1 virus whose genome sequence is shown in Sequence ID No. 5, in which the amino acid residue at position 358 of the NS3 protein is mutated, changing from methionine (M) to leucine (L). (2) rM1-E2M: The genome sequence of the M1 virus shown in Sequence ID No. 5 has a mutation in the amino acid residue at position 4 of the E2 protein, changing from lysine (K) to asparagine (N). (3) rM1-N3E2M: The genome sequence of the M1 virus shown in Sequence ID No. 5 has a mutation in the amino acid residue at position 358 of the NS3 protein, changing from methionine (M) to leucine (L), and a mutation in the amino acid residue at position 4 of the E2 protein, changing from lysine (K) to asparagine (N). (4) rM1-c6v1: The M1 virus (i.e., M1-c6) whose genome sequence is shown in Sequence ID No. 15 has a mutation in the amino acid residue at position 358 of the NS3 protein, changing from methionine (M) to leucine (L), and a mutation in the amino acid residue at position 4 of the E2 protein, changing from glutamic acid (E) to aspartic acid (D).

[0131] conclusion Four mutant viruses, rM1-NS3M, rM1-E2M, rM1-N3E2M, and rM1-c6v1, were successfully constructed through site-directed mutation. Of these, mutant viruses rM1-NS3M, rM1-E2M, and rM1-N3E2M were obtained by site-directed mutation from the M1 virus whose sequence is shown in SEQ ID NO: 5, while rM1-c6v1 was obtained by site-directed mutation from the M1 virus M1-c6 whose sequence is shown in SEQ ID NO: 15.

[0132] Example 2: Antitumor effects of four types of mutant M1 viruses against various types of tumor cells 1. Experimental materials and equipment 1.1. Main chemicals, reagents and preparations Main chemicals and reagents • M1 viruses: rM1-WT, rM1-NS3M, rM1-E2M, rM1-N3E2M, M1-c6, M1-c6v1 ·Cell Counting Kit-8 (CCK-8): Donren Chemical Technology (Shanghai) Co., Ltd. (Model number: CK-04) • Potassium chloride: Merck Chemical Technology (Shanghai) Co., Ltd. (Batch number: K46837809603) • Potassium dihydrogen phosphate: Merck Chemical Technology (Shanghai) Co., Ltd. (Batch number: AM1217539805) • Sodium bicarbonate: Merck Chemical Technology (Shanghai) Co., Ltd. (Batch number: K49804023804) • Disodium hydrogen phosphate dodecahydrate: Chengdu Huayi Medicinal Supplement Manufacturing Co., Ltd. (Batch number: 20170402) Anhydrous calcium chloride: Hebei Huachen Pharmaceutical Co., Ltd. (Batch number: 171009) Magnesium chloride hexahydrate: Chengdu Huayi Medicinal Supplement Manufacturing Co., Ltd. (Batch number: 20170807) • Mannitol: French company Rocket (Batch number: E939X) • Trehalose: Pfanstiehl, USA (Batch number: 36358A) • Human albumin: Shenzhen Weiguang Bio-Products Co., Ltd. (Batch number: 20171144B) 1.2. Main Equipment

[0133] [Table 12]

[0134] 2. Source of experimental cells The cells were purchased from the ATCC or the Cell Library of the Committee for the Preservation of Typical Cultures of the Chinese Academy of Sciences.

[0135] 3. Experimental Method ·Cell culture According to the instructions for each cell type, appropriate culture conditions were used, and cell proliferation subculturing was performed at the subculturing ratios specified in the instructions.

[0136] • Experimental grouping and treatment The inhibitory effect of M1-c6v1 on the proliferation of each cell type was detected. The specific groupings and treatments are shown in Table 12.

[0137] [Table 13]

[0138] After subculturing the cells in the logarithmic growth phase, the cells were digested and inoculated into 48-well plates at the corresponding density. Experiments were planned according to the group classifications described above, and the experiment was repeated three times for each group. No cells were inoculated into the blank control group. Samples were added 24 hours after the cells had adhered to the container wall, and the cells were cultured for a further 72 hours before measuring cell viability.

[0139] • Measurement of cell viability using the CCK-8 method The original culture medium was removed from the 48-well plate, and 200 μL / well of chromogenic solution (100% complete medium + 10% CCK-8 solution) was slowly added along the wall. After incubation at 37°C for 0.5–3 hours, the absorbance value of each well was measured at a wavelength of 450 nm using an absorbance microplate reader.

[0140] • Calculation of the median inhibitory concentration (IC50) of the M1 virus for each cell. The formula "Inhibition of Cell Growth (IR) = (Average OD)" is calculated from the absorbance values ​​measured for each well. 陰性 -OD 実験 ) / (average OD 陰性 -Average OD ブランク The inhibition rate of cell proliferation in each well was calculated using the formula "(inhibitor) × 100%", a cell proliferation inhibition rate curve was created using GraphPad Prism 7.0 software, and the half-percentage inhibitory concentration (IC50) of M1-c6v1 or rM1-c6 against tumor cells was calculated using the log(Inhibitor) vs. response--Variable slope(four parameters) analytical equation.

[0141] • Detection of cell activity by MTT Cells were inoculated at a rate of 20,000 cells / well into a 24-well plate (500 μl medium / well) and kept attached to the wall for 24 hours. Wild-type and mutant viruses were serially diluted 10-fold, and a viral load of 10 MOI was added to the 24-well plate. 72 hours after virus infection, 50 μL of MTT was added to each well, mixed uniformly, and left to stand in an incubator for another 2-4 hours. The supernatant was carefully aspirated, 500 μL of DMSO was added to each well to dissolve the blue-violet formazan crystals, and the mixture was shaken on a microwell plate oscillator to completely dissolve and uniformly mix the formazan. The absorbance was measured at a wavelength of 570 nm using a microplate reader. The experiment was repeated at least three times. The absorbance values ​​of each group were standardized using a blank control group, the relative cell viability of each treatment group was calculated, and cell viability curves were created.

[0142] • Statistical processing For all experimental results, the experiment was repeated at least twice independently. The experimental results are presented as mean ± standard deviation.

[0143] 4. Experimental Results As shown in Figure 2A, lesions were observed in cells infected with rM1-NS3M and rM1-E2M viruses under a microscope, indicating that single-site mutations can independently enhance the replication and oncolytic effects of M1 viruses in tumor cells. Compared to wild-type and single-site mutant viruses, infection rates reached over 90% and lesions developed in most cells after infection with the rM1-N3E2M bi-site mutant virus. Furthermore, the killing effects of rM1-WT, rM1-NS3M, rM1-E2M, and rM1-N3E2M on HCT 116 cells were compared using the MTT method. Consistent with microscopic observations, the dose-response curves for rM1-NS3M and rM1-E2M viruses shifted to the left, and the calculated EC50shift improvement was 80-fold and 60-fold, respectively. The dose-response curve of rM1-NS3E2M virus shifted more significantly to the left, with an EC50shift improvement reaching 7600-fold (Figure 2B), indicating that the double mutation site can synergistically enhance the oncolytic effect of the virus.

[0144] The oncolytic effects and safety of rM1-WT and three M1 virus variants—rM1-NS3M, rM1-E2M, and rM1-N3E2M—were compared using a larger number of tumor and normal cells. The results are shown in Tables 13 to 15. The results indicate that, compared to the rM1-WT virus, all three M1 virus variants enhanced the oncolytic effect to varying degrees while not exhibiting significant toxicity to normal cells.

[0145] [Table 14]

[0146] [Table 15]

[0147] [Table 16]

[0148] Similarly, the oncolytic effects and safety of M1-c6 and M1-c6v1 virus variants were compared using multiple strains of tumor cells and normal cells. The results are shown in Table 16. M1-c6v1 exhibits significant killing activity against most malignant tumor cells. M1-c6v1 exhibits similarly significant killing activity against most mouse-derived malignant tumor cells. Compared to the killing activity (IC50) of M1-c6, the IC50 of M1-c6v1 was 1.3 to 20.5 times lower than that of M1-c6, indicating that the oncolytic effect of M1-c6v1 is higher than that of M1-c6.

[0149] [Table 17]

[0150] 5. Conclusion of the experiment The mutant strains rM1-NS3M, rM1-E2M, rM1-N3E2M, and M1-c6v1 exhibit remarkable killing activity against most malignant tumor cells of human and mouse origin.

[0151] Example 3: Efficacy and safety study of rM1-N3E2M virus in the body material 1. rM1-N3E2M, rM1-WT virus 2. Colorectal cancer cell line HCT 116 3.30 female BALB / c-nu / nu nude mice, 6-8 weeks old.

[0152] method 1. Purchase female nude mice aged 1.4-6 weeks, culture a sufficient amount of HCT 116 cells in advance, digest them, resuspend them in sterile PBS, count them, and prepare cell suspensions as needed. Divide the HCT 116 cells into 5 × 10⁶ cells. 6 The sample was inoculated subcutaneously into the back of nude mice at a dose of 100 μL. The tumor size was approximately 50 mm. 3 When this occurred, the patients were randomly divided into groups and administered the drug for 6 consecutive days. The length and width of the tumor were measured every 3 days, and the tumor volume (volume = length × width) was measured. 2 The proliferative curve was created by calculating () / 2). On the third day after administration, tumor tissue was separated from normal organs such as the liver, heart, brain, and lungs, fixed with 4% paraformaldehyde, and used for immunohistochemical experiments.

[0153] 2. Immunohistochemistry Experiment Tumor tissue and normal organs were taken from nude mice, fixed with 4% paraformaldehyde, and the samples were mailed to Google Biotechnology Co., Ltd. for measurement of Cleaved-caspase 3 and Ki67 levels.

[0154] result 1. A subcutaneous tumor model was constructed using BALB / c nude mice, and the nude mice were administered the virus intravenously six times consecutively. The survival status of the nude mice was observed, and compared to the control group, the body weight of the administered nude mice did not change significantly (Figure 3B), and their mental state was good, indicating to some extent that the rM1-N3E2M mutant virus is safe. Compared to the control group, the tumor volume of the nude mice administered rM1-N3E2M was significantly smaller (Figure 3A), indicating that the rM1-N3E2M virus can reach tumor cells, kill them, and suppress tumor growth.

[0155] 2. To further investigate the inhibitory effect of rM1-N3E2M on tumor growth in the body, tumor tissue was dissected 3 days after administration and immunohistochemical staining was performed using shears of caspase-3 (Cl-casp3) and Ki67. Cl-casp3 is a marker of apoptosis, and its expression level indicates the degree of apoptosis in tumor cells. Ki67 is a marker of proliferation, and its expression level indicates the proliferative capacity of tumor cells. In Figure 4, brown indicates a positive signal. The rM1-N3E2M virus significantly upregulates Cl-casp3 expression and significantly downregulates Ki67 expression, indicating that it can induce apoptosis in tumor cells and suppress the malignant phenotype of rapid tumor cell proliferation.

[0156] 3. Safety of oncolytic virus therapy is extremely important. While the M1-N3E2M virus has been demonstrated to be safe in normal cell lines, further research is needed regarding its safety in vivo. In a nude mouse subcutaneous tumor formation model, major organs (including tissues such as heart, brain, liver, lungs, colon, and joints) of experimental animals in each group were isolated on day 3 after administration, and the expression of rM1-N3E2M viral protein was observed by immunohistochemistry. Since the rM1-N3E2M virus is obtained by continuous passage on colorectal cancer cell lines, attention was paid not only to important organs such as the heart and brain, but also to viral replication in mouse colon epithelial cells. Furthermore, since many alphaviruses have been reported to cause arthritis, we observed whether the two types of viruses used in the experiment replicated in the joints. As is clear from Figure 5, there was no viral replication, and there were no differences in the morphology of tissue cells in each group, indicating that the rM1-N3E2M virus is well safe in immunodeficient nude mice.

[0157] conclusion Animal studies have verified that the rM1-N3E2M virus effectively exerts antitumor effects in the body, suppressing tumor growth, and that rM1-N3E2M induces apoptosis in tumor tissue, thereby suppressing the malignant phenotype of tumor cells. During the experiments, there were no significant changes in the body weight of nude mice, and no viral replication was observed in normal tissues and organs, indicating good safety.

[0158] Example 4: Effective suppression of M1-c6v1 virus against tumor growth in the body material 1. M1-c6v1, M1-c6 viruses 2. Mouse melanoma B16-F10 cells 3.40 female C57 BL / 6 mice, 5-6 weeks old.

[0159] method Purchase 5-6 week old C57 BL / 6 female mice, culture a sufficient amount of B16-F10 cells in advance, digest them, resuspend them in sterile PBS, count them, and prepare cell suspensions as needed. Dilute the B16-F10 cells to 5 × 10⁶. 4 The sample was inoculated subcutaneously into the back of mice at a dose of 100 μL. The tumor size was approximately 60 mm. 3 When this occurred, the patients were randomly divided into groups and administered the drug for 7 consecutive days. The length and width of the tumor were measured every 3 days, and the tumor volume (volume = length × width) was measured. 2 We calculated () / 2) and created a growth curve.

[0160] result A subcutaneous tumor-bearing model was constructed using C57 BL / 6 mice. After intratumoral injection for seven consecutive days, the survival status of the mice was observed. Compared to the control group, the body weight of the treated mice did not change significantly (Figure 6B), and their mental state was good, indicating to some extent that the M1-c6v1 mutant virus is safe. Compared to the control group and the M1-c6 treated group, the tumor volume of the M1-c6v1 treated mice was significantly smaller (Figure 6A), indicating that the M1-c6v1 virus can kill tumor cells and suppress tumor growth.

[0161] conclusion Animal studies have demonstrated that the M1-c6v1 virus effectively exerts antitumor effects in the body, suppressing tumor growth. The lack of significant changes in the mice's body weight during the experiments indicates good safety.

[0162] Example 5: Construction, identification, and efficacy verification of M1 site-directed mutant virus strains The process of point mutation Based on the rM1-WT virus whose genome sequence is shown in Sequence ID No. 5, a series of site-directed mutations were performed (completed by Jinsrui Biotechnology Co., Ltd.), resulting in the pBR-M1-E2-4K, pBR-M1-E2-4L, pBR-M1-E2-4I, pBR-M1-E2-4V, pBR-M1-E2-4S, pBR-M1-E2-4C, pBR-M1-E2-4L, pBR-M1-E2-4D plasmids; and pBR-M1-NS3-358M, pBR-M1-NS3-358G, pBR-M1-NS3-358A, pBR-M1 -NS3-358L, pBR-M1-NS3-358I, pBR-M1-NS3-358V, pBR-M1-NS3-358P, pBR-M1-NS3-358S, pBR-M1-NS3-358Q, pBR-M1-NS3-358T, pBR-M1-NS3-358C, pBR-M1-NS3-358N, pBR-M1-NS3-358F, pBR-M1-NS3-358Y, pBR-M1-NS3-358D, pBR-M1-NS3-358K, pBR-M1-NS3-358R, and pBR-M1-NS3-358H were obtained.

[0163] 5.1 Reagents and Materials Main chemicals and reagents • DMEM / F12 (Gibco, 11320-033) MEM (Gibco, C11095500BT) DMEM (Corning, 10-013-CVRC) • Newborn calf serum (Hangzhou Tianhang Biological Journal, 22011-8615) • Fetal bovine serum (Corning, 35-081-CV) • Protease K (Tenkon, RT403) • Trypsin (Thermo, 25200-072) ·Lipofectamine Messenger MAX Transfection Reagent(Thermo,LMRNA003) ·Opti-MEM I Reduced Serum Medium(1X)(Thermo,31985-070) • Restriction enzyme XbaI (NEB, R0145S) ·Ribo m7G Cap Analog(Promega,P1711) • DNA purification and recovery kit (Tenkon, DP209) • PBS (Gibco, 2001-2027) · RiboMAX TM Large Scale RNA Production System(Promega,P1280) Goscript TM Reverse Transcription System(Promega,A5001) • Total RNA extraction kit (Promega, LS1040) ·Virus Production Serum Free Medium(VP-SFM)(Gibco,11681-020) ·GlutaMax I(100×)(Gibco,35050061) ·MEM NEAA(100×)(Gibco,11140050) • E2 sequencing primer (Kim Yu-ji, order number 80-423889537) • N3 sequencing primers (Kim Yu-ji, order numbers 80-429401706, 80-426066909) Preparation of main solutions • Preparation of VP-SFM solution 10 mL of 100×MEM NEAA and 20 mL of 100×GlutaMax I were added to 1 L VP-SFM, mixed uniformly, and stored at 4°C for later use.

[0164] 5.2 Equipment equipment [Table 18]

[0165] 5.3 Experimental Cells [Table 19]

[0166] 5.4 Test Steps 5.4.1 Primary Seed Lot Viral Construction Plasmid linearization [Table 20]

[0167] A. Plasmid linearization systems were prepared in PCR tubes according to Table 19, and then incubated in a PCR machine at 37°C for 1 hour. B. 5 μL of Protease K was added to each tube, and the tubes were then placed in a PCR machine and incubated at 50°C for 30 minutes.

[0168] (2) Purification and recovery of DNA A. 500 μL of BL was added to the adsorption column and centrifuged at 12000 rpm for 1 minute. B. The linearized plasmid was transferred to a 1.5 mL EP tube, 500 μL of PB was added, and it was thoroughly mixed. C. The mixture was transferred to an adsorption column, allowed to stand at room temperature for 2 minutes, centrifuged at 12000 rpm for 1 minute, and the filtrate was removed. D. 600 μL of PW was added, allowed to stand for 2 minutes, then centrifuged at 12000 rpm for 1 minute, and the filtrate was removed. E. Step D was repeated. F. Remove the filtrate, centrifuge at 12000 rpm for 2 minutes, open the lid, and let stand at room temperature for 5 minutes. G. 30 μL of sterile DEPC water was added, allowed to stand for 1 minute, eluted twice, and the filtrate was collected. H.DNA quantification

[0169] (3) In vitro transfer [Table 21] A. After preparing the in vitro transcription system in PCR tubes according to Table 20, the system was incubated in a PCR machine at 37°C for 2 hours. B.RQ1 RNase-Free DNase (1 μL / 1 μg DNA) was added, and the mixture was then incubated in a PCR machine at 37°C for 15 minutes.

[0170] (4) RNA transfection A. Cell inoculation Cells were subcultured and amplified under appropriate culture conditions according to the instructions for each cell type, using the subculturing ratios specified in the instructions. After the cells entered the logarithmic growth phase, they were digested, and Vero cells were inoculated into T25 culture flasks at a density of 4E05 / flask. The flasks were then placed in a cell incubator and cultured for 24 hours while remaining attached to the flask walls.

[0171] B. Transfection 250 μL of Opti-MEM medium and 7.5 μL of MessengerMAX were added to a 1.5 ml centrifuge tube, and 125 μL of Opti-MEM medium and all RNA were added to another centrifuge tube. The two mixtures were then homogeneously mixed, added to Vero cells, and cultured in a viral incubator.

[0172] (5) Virus collection Cellular status and the presence or absence of fluorescence were observed daily. When a significant increase in fluorescence was observed, it was photographed and recorded. After 90% of the cells floated to the surface, the Vero cell supernatant was collected and centrifuged at 4000 rpm for 5 minutes. The supernatant was stored in a cryopreservation tube and preserved in liquid nitrogen as a primary seed lot.

[0173] 5.4.2 Sequencing of Seed Lot Viruses (1) RNA extraction A. Seed lot virus samples stored under liquid nitrogen were removed and thawed. B. A 100 μL sample was aspirated and placed in a 1.5 mL centrifuge tube. 300 μL of the dissolving solution was added, the mixture was swirled, and after a short period of centrifugation, it was allowed to stand at room temperature for 5 minutes. D. 300 μL of diluent was added, the mixture was swirled and centrifuged briefly, and then allowed to stand at room temperature for 5 minutes. E. 350 μL of anhydrous ethanol was added, the mixture was swirled and centrifuged briefly, and then allowed to stand at room temperature for 5 minutes. F. The mixture was transferred to a centrifugation column (included in the kit), and the sample was loaded into the column in two steps. After centrifugation at 12000 rpm for 1 minute, the filtrate was discarded. G. 600 μL of RNA wash was added, and the mixture was centrifuged at 12000 rpm for 1 minute, after which the filtrate was discarded. H. 50 μL of DNASE I incubation solution (Table 21) was added to the center of the adsorption membrane and left to stand at room temperature for 15 minutes. [Table 22] 1. Add 600 μL of RNA wash solution, centrifuge at 12000 rpm for 1 minute, discard the filtrate, repeat the process twice, and discard the filtrate each time. Place a new centrifugation column in the collection tube and centrifuge at 12000 rpm for 2 minutes. The J. centrifugation column was transferred to an elution tube, 50 μL of nuclease-free water was added to the center of the centrifugation column membrane, and the column was allowed to stand at room temperature for 2 minutes. The RNA was then centrifuged at 12000 rpm for 1 minute and stored at -80°C.

[0174] (2) Reverse transcription (Table 22) [Table 23]

[0175] (3) PCR amplification A. The obtained cDNA was used as a template for PCR of the fragments. The reaction system is shown in Table 23. [Table 24] The reaction conditions are shown in Table 24. [Table 25]

[0176] (4) Sequence determination The sequencing results are shown in Figure 7. Each mutant virus underwent site-directed mutations as expected.

[0177] 5.4.3 Building a Virus Operation Library (1) Cell inoculation, virus collection, and sample collection The cells were subcultured and amplified under appropriate culture conditions according to the cell instructions, using the subculturing ratio specified in the instructions. After the cells entered the logarithmic growth phase, the cells were digested and 6 × 10⁶ cells were subcultured. 5 The cells were inoculated into a T25 cell culture flask and cultured for 24 hours while remaining attached to the flask wall. After 24 hours of adhesion to the cell wall, the original culture medium was aspirated and removed. VP-SFM virus medium containing 20 μL of seed lot virus was added, and the cells were placed in a virus incubator until they produced the virus. Cell morphology and the presence or absence of fluorescence were observed every 24 hours. When more than 90% of the cells floated to the surface, the images were taken and recorded. The Vero cell supernatant was collected and centrifuged at 4000 rpm for 5 minutes. 100 μL of the supernatant was taken and placed in a 1.5 mL centrifuge tube as a sample for viral titer measurement. The remaining supernatant was placed in a freezer tube (approximately 1.5 mL / tube) and stored under liquid nitrogen.

[0178] (2) Measurement of viral titer 1. BHK-21 cells were inoculated into 96-well plates at a rate of 1500 cells / well and cultured at 37°C under 5% CO2 conditions. The cells were used within 12–36 hours after inoculation. 2. An appropriate amount of MEM basal medium was placed in a preparation tank, and the medium was placed in a 96-well plate at 180 μL / well to prepare the diluent. The MEM diluent was prepared according to the number of samples to be measured, and the process was repeated twice for each sample and four times for the reference sample. 3. The sample to be measured was shaken thoroughly on a vortex oscillator for 15-30 seconds. 20 μL of the sample to be measured was taken and added to the first row of wells as described in "2" above. After homogeneous mixing, 20 μL was drawn up and added to the next row of wells. Dilution was carried out sequentially up to the eighth row of wells. Note that the pipette must be changed for each row. The diluted sample to be measured was fixed in a microwell plate mixer and shaken at approximately 3000 rpm for 3 minutes. 4. Add the diluted virus solution to the cells, 20 μL / well, and 10 -3 -10 -8 The virus solution was added to cells in a 96-well plate, and two samples were measured from each plate. 5. After adding the sample, the cells were kept at 37°C under 5% CO2 conditions for 5 days (the day the virus was added was considered day 1). After 6.5 days, the cells were observed and recorded under a microscope to determine if cytopathic effects (CPE) had occurred. 7. The CCID50 value was calculated using the Spearman-Karber method. lg(CCID50×20 / 1000)=LD(S-0.5), where L: logarithm of maximum dilution, -1; D: difference between logarithms of dilution, 1; S - sum of positive well ratios (sum of CPE numbers / 8).

[0179] Table 25 shows the results of measuring the virus titer of the working batch. [Table 26]

[0180] 5.4.4 Viral killing effect on HCT116 cells (1) Cell inoculation and administration Cells were subcultured and amplified according to the culture conditions and subculturing ratios described in the instructions for each cell type. After the cells entered the logarithmic growth phase, they were digested and inoculated into 48-well plates at a density of 10,000 cells / well, with a total well volume of 200 μL. Blank control group, negative control group, and multiple viral gradients were observed: Set up an experimental group of JPEG0007894402000029.jpg7170 (all repeated 3 times (Table 26). A - H in the table represent different viruses), inoculate no cells in the blank control group, and in the negative control group, administer the same volume of VP - SFM as the virus inoculation amount instead of the virus.

[0181]

Table 27

[0182] After administration, continue culturing for 72 h, observe the cell state under a microscope at the end point, photograph and record it, detect cell viability, and calculate the IC50 of each virus on the detected cells.

[0183] (2) Measurement of cell viability by CCK - 8 method Remove the original culture medium in the 48 - well plate, slowly add the coloring solution (90% complete medium + 10% CCK - 8 solution) at 200 μL / well along the wall, continue culturing at 37 °C for 0.5 - 3 h, and then measure the absorbance value of each well at a wavelength of 450 nm using an absorbance microplate reader.

[0184] (3) Calculation of the half - inhibitory concentration (IC50) of the virus against each cell From the obtained absorbance values of each well, calculate the growth inhibition rate of the cells in each well according to the formula: cell growth inhibition rate (IR) = (average OD solvent - OD experiment) / (average OD solvent - average OD blank)×100%, create a cell growth inhibition rate curve using GraphPad Prism 8.0 software, and calculate the value of the half - inhibitory concentration (IC50) of each virus against tumor cells according to the log(Inhibitor) vs. response - Variable slope (four parameters) analysis equation.

[0185] The killing curves of each site - specific mutant of M1 virus against HCT116 are shown in Figures 8A - 8Z (Note: Each curve in the figure represents one independent experiment, the horizontal axis represents the virus infection MOI, and the vertical axis represents the inhibition rate). <References> 1. Greenman C, Stephens P, Smith R, et al: Patterns of somatic mutation in human cancer genomes. Nature 446: 153-158, 2007. 2. Wood LD, Parsons DW, Jones S, et al: The genomic landscapes of human breast and colorectal cancers. Science 318: 1108-1113, 2007. 3. Pavet V, Portal MM, Moulin JC, Herbrecht R and Gronemeyer H: Towards novel paradigms for cancer therapy. Oncogene 30: 1-20, 2011. 4. Workenhe ST and Mossman KL: Oncolytic virotherapy and immunogenic cancer cell death: sharpening the sword for improved cancer treatment strategies. Mol Ther 22: 251-256, 2014. 5. Sun Q1, Barz M2, De Geest BG3,et al:Nanomedicine and macroscale materials in immuno- oncology. chemical society 2018 Nov 22. 6. Tran E1, Robbins PF1, Rosenberg SA :‘Final common pathway’ of human cancer immun- otherapy: targeting random somaticmutations. Nature immunology 2017 Feb 15;18(3):255-262. 7. Das T, Jaffar-Bandjee MC, Hoarau JJ, et al: Chikungunya fever: CNS infection and pathologies of a re-emerging arbovirus. Prog Neurobiol 91: 121-129, 2010. 8. Kelvin AA: Outbreak of Chikungunya in the Republic of Congo and the global picture. J Infect Dev Ctries 5: 441-444, 2011. 9. Moran TP, Burgents JE, Long B, et al: Alphaviral vector-transduced dendritic cells are successful therapeutic vaccines against neu-overexpressing tumors in wild-type mice. Vaccine 25: 6604-6612, 2007. 10.Weaver SC, Salas R, Rico-Hesse R, et al: Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. VEE Study Group. Lancet 348: 436-440, 1996. 11.Li XD, Qiu FX, Yang H, Rao YN and Calisher CH: Isolation of Getah virus from mosquitos collected on Hainan Island, China, and results of a serosurvey. Southeast Asian J Trop Med Public Health 23: 730-734, 1992. 12.Wen JS, Zhao WZ, Liu JW, et al: Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses. Virus Genes 35: 597-603, 2007. 13.Zhai YG, Wang HY, Sun XH, et al: Complete sequence characterization of isolates of Getah virus (genus Alphavirus, family Togaviridae) from China. J Gen Virol 89: 1446-1456, 2008. 14.Charlotte Rafaluk, Gunther Jansen, Hinrich Schulenburg, and Gerrit Joop: When experimental selection for virulence leads to loss of virulence Trends Parasitol. 2015 Sep;31(9):426-34. 15.Jose Luis Marti´nez, Fernando Baquero and Dan I Andersson: Beyond serial passages: new methods for predicting the emergence of resistance to novel antibiotics. Current Opinion in Pharmacology 2011, 11:439-445.

Claims

1. The isolated protein is either the NS3 protein or the E2 protein of the M1 mutant virus. The NS3 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 18, and includes an amino acid sequence in which amino acid residue M at the position corresponding to position 358 of SEQ ID NO: 8 or SEQ ID NO: 18 is substituted with G, A, L, I, V, P, S, Q, T, C, N, F, Y, D, K, R, or H. The envelope protein E2 has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 31, and includes an amino acid sequence in which amino acid residue K at the position corresponding to the 4th position of SEQ ID NO: 12 or amino acid residue E at the position corresponding to the 4th position of SEQ ID NO: 31 is substituted with M, L, I, V, S, C, N, or D. The aforementioned protein enhances the oncolytic effect of the M1 mutant virus compared to the wild-type M1 virus. Isolated proteins.

2. It is a viral vector, The viral vector comprises nucleic acids encoding the E2 protein and / or NS3 protein of the M1 mutant virus, The NS3 protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 18, and includes an amino acid sequence in which amino acid residue M at the position corresponding to position 358 of SEQ ID NO: 8 or SEQ ID NO: 18 is substituted with G, A, L, I, V, P, S, Q, T, C, N, F, Y, D, K, R, or H, and / or The envelope protein E2 has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 31, and includes an amino acid sequence in which amino acid residue K at the position corresponding to the 4th position of SEQ ID NO: 12 or amino acid residue E at the position corresponding to the 4th position of SEQ ID NO: 31 is substituted with M, L, I, V, S, C, N, or D; The protein is a viral vector that enhances the oncolytic effect of a mutant M1 virus containing the protein compared to a wild-type M1 virus.

3. The viral vector according to claim 2, wherein the vector is selected from retrovirus, Newcastle disease virus, rabies virus, vesicular stomatitis virus, maraba virus, alphavirus, reovirus, adenovirus, adeno-associated virus, herpes simplex virus, cowpox virus, or measles virus.

4. It is a viral vector, The aforementioned virus is an M1 mutant virus that exhibits oncolytic properties. The aforementioned M1 mutant virus is The NS3 protein comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 18, and in which amino acid residue M at the position corresponding to position 358 of SEQ ID NO: 8 or SEQ ID NO: 18 is substituted with G, A, L, I, V, P, S, Q, T, C, N, F, Y, D, K, R, or H, and / or A viral vector comprising an envelope protein E2 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 31, and comprising an amino acid sequence in which amino acid residue K at the position corresponding to the 4th position of SEQ ID NO: 12 or amino acid residue E at the position corresponding to the 4th position of SEQ ID NO: 31 is substituted with M, L, I, V, S, C, N, or D.

5. The viral vector according to any one of claims 2 to 4, further comprising an exogenous gene for the M1 mutant virus.

6. The viral vector according to claim 5, wherein the foreign gene expresses an antitumor-related molecule.

7. A composition comprising an effective amount of M1 mutant virus having oncolytic properties, The aforementioned M1 mutant virus is An NS3 protein comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 18, and wherein the amino acid residue M at the position corresponding to position 358 of SEQ ID NO: 8 or SEQ ID NO: 18 is substituted with G, A, L, I, V, P, S, Q, T, C, N, F, Y, D, K, R, or H, and / or Envelope protein E2 comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 31, and wherein amino acid residue K at the position corresponding to the 4th position of SEQ ID NO: 12 or amino acid residue E at the position corresponding to the 4th position of SEQ ID NO: 31 is substituted with M, L, I, V, S, C, N, or D. The composition, at least 10 １ A composition comprising individual virus particles or PFUs.

8. An effective amount of the isolated protein described in claim 1, or the vector described in any one of claims 2 to 4, and Pharmacologically acceptable carriers A composition containing the following:

9. The composition, at least 10 １ The composition according to claim 7, comprising individual virus particles or PFUs.

10. The composition is 10 １ 、10 ２ 、10 ３ 、10 ４ 、10 ５ 、10 ６ 、10 ７ 、10 ８ 、10 ９ 、10 １０ 、10 １１ 、10 １２ 、10 １３ 、10 １４ 、10 １５ 、10 １６ 、10 １７ 、10 １８ 、10 １９ 、10 ２０ 、10 ２１ 、or 10 ２２ virus particles or PFU, the composition according to claim 7.

11. The composition according to claim 7 or 8, wherein the dosage form is selected from injections, tablets, capsules, kits, and patches.

12. The above composition is used for antitumor purposes. The tumor is selected from solid tumors or hematological malignancies. The solid tumors mentioned above include liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, stomach cancer, adrenocortical cancer, accessory cortical cancer, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germ cell tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, and Lange's sarcoma. One or more of the following are selected from: Luhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia, mycosis fungoides, nasal and paranasal sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papilloma, paraganglioma, paranasal and paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal and laryngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, César syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, hemangioma, vulvar cancer, and solitary myeloma. The composition according to claim 7 or 8, wherein the hematological malignancy is one or more selected from B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia, and preleukemia.

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