US-20260125657-A1 - M1 VIRUS MUTANT AND USE THEREOF

Abstract

Provided is an M1 virus. Further provided are a series of uses of said virus. The uses include, but are not limited to, viral vectors, anti-tumor agents, and pharmaceutical compositions. Said 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. Said virus can be administrated by means of intravenous injection, having operational convenience.

Inventors

• Guangmei Yan
• Yuan Lin
• Li Guo
• Ziqing Lin
• Guang'en Wu

Assignees

• GUANGZHOU VIROTECH PHARMACEUTICAL CO., LTD.

Dates

Publication Date

20260507

Application Date

20251222

Priority Date

20190531

Claims (20)

1. 1 . An M1 virus, wherein an amino acid residue corresponding to the 358th site of the NS3 protein of the M1 virus is not M; and/or an amino acid residue corresponding to the 4th site of the envelope protein E2 is not E or K.
2. 2 . The M1 virus according to claim 1 , wherein the amino acid residue corresponding to the 358th site of the NS3 protein 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 corresponding to the 4th site of the envelope protein E2 is: M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R or H.
3. 3 . The M1 virus according to claim 1 , wherein an amino acid sequence of the NS3 protein comprised in the M1 virus has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 8 or SEQ ID NO: 18; and/or an amino acid sequence of the E2 protein comprised in the M1 virus has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 12 or SEQ ID NO: 31.
4. 4 . An M1 virus, wherein an amino acid residue corresponding to the 358th site of the NS3 protein encoded by a nucleotide sequence of the M1 virus is not M; and/or an amino acid residue corresponding to the 4th site of the E2 protein is not E or K; preferably, the nucleotide sequence of the M1 virus has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an M1 sequence shown as SEQ ID NO: 5 or SEQ ID NO: 15 or GenBank Accession No. EU015061.1 or GenBank Accession No. EF011023.1 or CCTCC V201423; preferably, the amino acid residue corresponding to the 358th site 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 corresponding to the 4th site of the E2 protein is: M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R or H; preferably, the M1 virus has a mutation of the amino acid site relative to a wild-type M1 virus or a pseudo-wild-type M1 virus; preferably, the M1 virus has a mutation of the amino acid site relative to an M1 virus having a sequence shown as SEQ ID NO: 5 or an M1 virus having a sequence shown as SEQ ID NO: 15; preferably, the mutation is: M358G, M358A, M358L, M358I, M358V, M358P, M358S, M358Q, M358T, M358C, M358N, M358F, M358Y, M358W, M358D, M358E, M358K, M358R or M358H on the NS3 protein; and/or 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 on the E2 protein; preferably, the M1 virus is obtained by a mutation of an amino acid residue M corresponding to the 358th site of the NS3 protein of the M1 virus having the sequence shown as SEQ ID NO: 5 into: G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, E, K, R or H; and/or a mutation of an amino acid residue K at the 4th site of the E2 protein into: M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R or H; and preferably, the amino acid residue M corresponding to the 358th site of the NS3 protein of the M1 virus mutates into L relative to the M1 virus having the sequence shown as SEQ ID NO: 15; and/or the amino acid residue E corresponding to the 4th site of the E2 protein mutates into D.
5. 5 . An amino acid sequence corresponding to the NS3 protein of an M1 virus according to claim 1 , wherein an amino acid residue corresponding to the 358th site of the amino acid sequence is not M; preferably, the amino acid residue corresponding to the 358th site is: G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, E, K, R or H; and preferably, the amino acid sequence corresponding to the NS3 protein of the M1 virus has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 8 or SEQ ID NO: 18.
6. 6 . An amino acid sequence corresponding to the E2 protein of an M1 virus according to claim 1 , wherein an amino acid residue corresponding to the 4th site of the amino acid sequence is not E or K; preferably, the amino acid residue at the 4th site is M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R or H; and preferably, the amino acid sequence corresponding to the E2 protein of the M1 virus has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 12 or SEQ ID NO: 31.
7. 7 . A nucleotide sequence for encoding an amino acid sequence according to claim 5 .
8. 8 . A nucleotide sequence for encoding an amino acid sequence according to claim 6 .
9. 9 . A vector, comprising a nucleic acid for encoding the E2 protein of an M1 virus, and/or the NS3 protein, wherein an amino acid residue corresponding to the 358th site of the NS3 protein is not M; and an amino acid residue corresponding to the 4th site of the E2 protein is not E or K; preferably, the amino acid residue corresponding to the 358th site 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; preferably, the amino acid residue corresponding to the 4th site of the E2 protein is: M, G, A, L, I, V, P, S, Q, T, C, N, F, Y, W, D, R or H; preferably, an amino acid sequence of the NS3 protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 8 or SEQ ID NO: 18; preferably, an amino acid sequence of the E2 protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 12 or SEQ ID NO: 31; preferably, the vector further comprises a coding sequence of the NS1 protein, the NS2 protein, the NS4 protein, the C protein, the E3 protein, the 6K protein, and/or the E1 protein of the M1 virus; preferably, an amino acid sequence of the NS1 protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 6 or SEQ ID NO: 16; preferably, an amino acid sequence of the NS2 protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 7 or SEQ ID NO: 17; preferably, an amino acid sequence of the NS4 protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 9 or SEQ ID NO: 19; preferably, an amino acid sequence of the C protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 10 or SEQ ID NO: 20; preferably, an amino acid sequence of the E3 protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 11 or SEQ ID NO: 30; preferably, an amino acid sequence of the 6K protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 13 or SEQ ID NO: 32; preferably, an amino acid sequence of the E1 protein has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9% or 100% sequence identity with an amino acid sequence shown as SEQ ID NO: 14 or SEQ ID NO: 33; preferably, the vector further comprises exogenous genes relative to the M1 virus; preferably, the exogenous genes express anti-tumor-associated molecules; preferably, the vector is selected from viruses; preferably, the vector is selected from a retrovirus, a Newcastle disease virus, a rabies virus, a vesicular stomatitis virus, a Maraba virus, an alphavirus, a Newcastle disease virus, a reovirus, an adenovirus, an adeno-associated virus, a herpes simplex virus, a vaccinia virus, or a measles virus; and preferably, the vector is selected from plasmids.
10. 10 . A nucleotide sequence comprising an M1 virus according to claim 1 .
11. 11 . A vector, comprising a nucleotide sequence according to claim 10 , wherein preferably, the vector is selected from plasmids.
12. 12 . A virus vector, wherein the virus is an M1 virus according to claim 1 ; preferably, the vector is inserted with exogenous genes; and preferably, the exogenous genes express anti-tumor-associated molecules.
13. 13 . A method for treating a tumor in a subject in need thereof, comprising: administering to the subject an effective amount of M1 virus according to claim 1 .
14. 14 . The method according to claim 13 , wherein the tumor is selected from a solid tumor and a hematological tumor; preferably, the solid tumor is selected from one or more of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, a glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, stomach cancer, adrenocortical carcinoma, accessory renal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, a rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, a brain tumor, a bronchial tumor, Burkitt lymphoma, a carcinoid tumor, a heart tumor, cholangiocarcinoma, chordoma, carcinoma of large intestine, craniopharyngioma, ductal carcinoma in situ, a germ tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, a germ Cell tumor, an extragonadal germ cell tumor, retinoblastoma, carcinoma of fallopian tube, carcinoma of gallbladder, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, renal carcinoma, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, carcinoma of nasal cavity and nasal sinuses, neuroblastoma, non-small cell lung cancer, ovarian cancer, a pancreatic neuroendocrine tumor, an islet cell tumor, papillomatosis, paraganglioma, carcinoma of nasal sinuses and nasal cavity, parathyroid carcinoma, carcinoma of penis, throat cancer, a pituitary tumor, pleuropulmonary blastoma, primary peritoneal carcinoma, retinoblastoma, a salivary gland tumor, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, carcinoma of small intestine, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrium and uterine sarcoma, vaginal cancer, a vascular tumor, vulvar cancer, and solitary myeloma; preferably, the hematological tumor is selected from one or more of B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), chronic myelogenous 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 pre-leukemia; and more preferably, the tumor is selected from one or more of liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, a glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, and stomach cancer.
15. 15 . A method for treating a tumor in a subject in need thereof, comprising: administering to the subject an effective amount of the M1 virus according to claim 4 .
16. 16 . A method for treating a tumor in a subject in need thereof, comprising: administering to the subject an effective amount of nucleotide sequence according to claim 10 .
17. 17 . A method for treating a tumor in a subject in need thereof, comprising: administering to the subject an effective amount of amino acid sequence of the NS3 protein according to claim 5 .
18. 18 . A method for treating a tumor in a subject in need thereof, comprising: administering to the subject an effective amount of amino acid sequence of the E2 protein according to claim 6 .
19. 19 . A method for treating a tumor in a subject in need thereof, comprising: administering to the subject an effective amount of nucleotide sequence according to claim 7 .
20. 20 . A method for treating a tumor in a subject in need thereof, comprising: administering to the subject an effective amount of nucleotide sequence according to claim 8 .

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 17/595,889, filed Nov. 29, 2021, which is a national stage application of International Appl. PCT/CN2020/093642, filed May 31, 2020, and which claims foreign priority from Chinese Appl. No. 201910472439.7, filed May 31, 2019, all of which are hereby incorporated by reference. TECHNICAL FIELD The present disclosure belongs to the field of biological medicines, and particularly relates to an M1 virus mutant and a use thereof. BACKGROUND ART Genic and epigenetic accumulated changes in normal cells drive normal cells to change into malignant tumor cells. This complex pathological process determines the diversity of mechanisms in the formation, maintenance, and metastasis of different tumors (1-3). Surgical excision, chemotherapy, and radiotherapy are conventional methods for treating tumors, the surgical excision easily leads to tumor recurrence, and the chemotherapy and the radiotherapy induce great toxic and side effects (4). Targeted therapies and tumor immunotherapy, including IL-2 control, adoptive cell therapy, and regulation of immune checkpoints such as PD-1, that have emerged in recent years have achieved certain effects in clinical treatment. However, the targeted therapies are prone to drug resistance, and the immunotherapy has a low response rate and may lead to serious immune-associated adverse events (5, 6). Therefore, it is urgent to develop more novel anticancer therapies which have low toxicity and high efficacy and difficultly cause drug resistance. Oncolytic viruses not only kill tumor cells, but also alert a host's immune system of the presence of cancer. Virotherapy is a therapy based on characteristics of the oncolytic viruses that are more likely to attack cancer tissues rather than healthy tissues. In 2005, the China Food and Drug Administration approved the marketing of the first oncolytic virotherapy with a trade name of Oncorine. This is a genetically modified virus that preferentially attack tumor cells and has been used to treat head and neck cancers. In 2015, the U.S. Food and Drug Administration (FDA) approved a melanoma therapy T-VEC that uses genetically modified herpes viruses, and was approved in Australia and the European Union in the following year. In addition, there are also some oncolytic viruses under research. Although, in recent decades, the virotherapy has made great progress, it still faces numerous difficulties. First, the efficacy is concerned. The oncolytic viruses have a limited anti-tumor effect or anti-tumor spectrum, many oncolytic viruses cannot well inhibit or kill tumor cells, and need to be used in combination with other chemotherapeutic drugs or immune checkpoint inhibitors, or used as a supplement to radiotherapy. For example, an M1 virus disclosed in a Chinese invention patent application No. 201410425510.3, when used as an anti-tumor drug, has a significantly effect on colorectal cancer, liver cancer, bladder cancer, and breast cancer, has a lower effect on pancreatic cancer, nasopharyngeal carcinoma, prostate cancer, and melanoma, has a much lower effect on a glioma, cervical cancer, and lung cancer, and has the lowest effect on stomach cancer. Second, the safety is concerned. Certain viruses themselves are dangerous to human bodies, and the dangerous viruses need to be modified and attenuated before being used in the virotherapy. Even if the oncolytic viruses are modified and attenuated, they still can become “escaping viruses”, i.e. viruses that change again or are bound to existing pathogens in a patient's body after release to rapidly infect healthy tissues. Furthermore, the delivery of viruses is concerned. That is, how to deliver viruses to a lesion. In most of the existing oncolytic virotherapies such as the melanoma therapy T-VEC approved by the U.S. Food and Drug Administration (FDA), the oncolytic viruses are injected into a tumor tissue. However, lesions and micrometastases of many solid tumors cannot be injected directly, or non-solid tumors such as hematological tumors are distributed throughout a body without a fixed injection site. It is difficult to adopt the existing virotherapies to treat these types of tumors. Therefore, the development of oncolytic viruses still poses great challenges. Alphaviruses belong to the Togaviridae family, which are a class of single positive-stranded RNA viruses with an envelope structure. It is reported in literatures that a Chikungunya virus belonging to the Alphavirus is a pathogenic virus for humans and highly toxic, and causes fever, rash, arthritis, and even fatal encephalitis after infection (7, 8). It is reported that another virus, i.e. a Venezuelan equine encephalitis virus, belonging to the Alphavirus can be used as a vector to transduce dendritic cells so as to treat tumors (9), but this encephalitis virus has caused fever, convulsions, abortion, and even death in humans (10). M1 virus (Alph