Application of STT3A in evaluation of curative effect of oncolytic virus treatment

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of STT3A in evaluation of treatment effect of oncolytic virus treatment. Based on the fact that STT3A can promote N-glycosylation, infectivity and inhibition of the oncolytic virus in tumor cells, the invention provides application of the oncolytic virus in preparation of STT3A high-expression tumor resistant drugs and application of an STT3A promoter in preparation of an oncolytic virus anti-tumor synergist or a drug resistance reversal agent for the first time. On the basis, the application of a substance for detecting the STT3A in preparation of products for oncolytic virus anti-tumor curative effect evaluation, oncolytic virus anti-tumor prognosis evaluation, treatment scheme selection and / or tumor diagnosis or prognosis evaluation is provided for the first time; meanwhile, it is found that the antitumor effect can be remarkably improved by combining the oncolytic virus and the STT3A accelerant.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of STT3A in the evaluation of the therapeutic efficacy of oncolytic virus therapy. Background Art

[0003] Traditional anti-cancer treatments are mainly chemotherapy, radiotherapy, and surgery. However, chemotherapy and radiotherapy have serious toxic side effects in actual application. Surgery not only has strict surgical standards, but is also prone to tumor recurrence after surgery. Obviously, traditional anti-cancer treatments have many limitations in clinical practice. In recent years, with the multi-point development of anti-tumor treatment research, cancer treatment has entered a new era.

[0004] Oncolytic virotherapy (OV therapy) is a new type of tumor targeted therapy. Oncolytic viruses often show excellent tumor selectivity, that is, they can specifically recognize tumor cells and exert a direct oncolytic effect without causing changes in the physiological functions of normal cells. Therefore, the emergence of oncolytic virotherapy provides more possibilities for precision anti-tumor treatment. However, the fact that patients benefit differently from OV treatment suggests that in order to successfully transform OV therapy into clinical practice, we also need to find predictive biomarkers that have a profound impact on OV replication and propagation.

[0005] Oncolytic virus M1 (OVM) is an enveloped positive-strand RNA alphavirus isolated from Culex mosquitoes in Hainan Island, China. Due to its good safety and oncolytic effect, OVM's Phase I clinical trial has been approved in Japan for the treatment of advanced solid tumors. Not long ago, the US FDA also approved it as an orphan drug for the treatment of liver cancer and malignant gliomas. The applicant has identified matrix remodeling-associated protein 8 (MXRA 8) as a receptor for OVM, and confirmed MXRA 8 as one of the biomarkers indicating the efficient oncolytic effect of OVM. Further discovery of more biomarkers will help establish more precise individual treatment plans. Summary of the invention

[0006] The purpose of the first aspect of the present invention is to provide the use of an oncolytic virus in the preparation of an anti-tumor drug, wherein the tumor is a tumor with high expression of STT3A.

[0007] The purpose of the second aspect of the present invention is to provide the use of STT3A promoter in the preparation of oncolytic virus antitumor enhancer or drug resistance reversal agent.

[0008] The third aspect of the present invention aims to provide the use of oncolytic viruses and STT3A promoters in the preparation of anti-tumor drugs.

[0009] The fourth aspect of the present invention aims to provide an anti-tumor composition.

[0010] The fifth aspect of the present invention aims to provide an anti-tumor drug kit.

[0011] The sixth aspect of the present invention aims to provide an anti-tumor medication system.

[0012] The purpose of the seventh aspect of the present invention is to provide the use of a substance for detecting STT3A in the preparation of a product for evaluating the anti-tumor efficacy of oncolytic viruses, evaluating the anti-tumor prognosis of oncolytic viruses, selecting a treatment regimen, and / or diagnosing or evaluating tumors.

[0013] The eighth aspect of the present invention aims to provide a method.

[0014] The purpose of the ninth aspect of the present invention is to provide a method for improving the anti-tumor efficacy of oncolytic viruses.

[0015] The tenth aspect of the present invention aims to provide a method for treating tumors.

[0016] The purpose of the eleventh aspect of the present invention is to provide a method for constructing a model with good anti-tumor efficacy of oncolytic virus.

[0017] The purpose of the twelfth aspect of the present invention is to provide a method for constructing a model in which oncolytic virus has poor anti-tumor efficacy.

[0018] In order to achieve the above object, the technical solution adopted by the present invention is:

[0019] In a first aspect of the present invention, there is provided use of an oncolytic virus in the preparation of an anti-tumor drug, wherein the tumor is a tumor with high expression of STT3A.

[0020] The level of STT3A expression can be preferably judged by, but not limited to, the following methods: low or high expression of STT3A refers to the conclusion drawn after comparing the amount of STT3A mRNA or protein between two groups (individuals) of samples. If one group (individuals) of samples has less or more STT3A mRNA or protein than another group (individuals), it is called low or high expression of STT3A in the sample. The samples used for the quantitative comparison of STT3A mRNA and protein can be tumor cells and normal cells, tumor tissue and adjacent non-tumor tissue, or tumors that are effectively and ineffectively treated with oncolytic viruses. Preferably, the samples used for the quantitative comparison of STT3A mRNA and protein are tumors that are effectively and ineffectively treated with oncolytic viruses.

[0021] Preferably, the drug further comprises other anti-tumor drugs.

[0022] Preferably, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0023] Preferably, the drug is in a form suitable for administration by one or more of the following methods: oral, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0024] Preferably, the dosage form of the drug comprises at least one of capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, pellets, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

[0025] The second aspect of the present invention provides the use of an STT3A promoter in the preparation of an oncolytic virus antitumor enhancer or drug resistance reversal agent.

[0026] Preferably, the oncolytic virus anti-tumor enhancer or drug resistance reversal agent further comprises other substances that enhance the oncolytic virus anti-tumor efficacy or reverse drug resistance.

[0027] Resistance reversal agents refer to the situation that when some oncolytic viruses are used as anti-tumor drugs to treat tumors, there are some tumors that are not very sensitive to oncolytic viruses, or these tumors are resistant to oncolytic viruses. At this time, oncolytic viruses can be used in combination with STT3A promoters (as resistance reversal agents) to reverse the tumor's resistance to the oncolytic viruses.

[0028] Preferably, the oncolytic virus antitumor enhancer or drug resistance reversal agent is in a form suitable for administration by one or more methods selected from the following groups: oral, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0029] Preferably, the dosage form of the oncolytic virus anti-tumor enhancer or drug resistance reversal agent comprises at least one of capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

[0030] The third aspect of the present invention provides the use of oncolytic viruses and STT3A promoters in the preparation of anti-tumor drugs.

[0031] Preferably, the drug further comprises other anti-tumor drugs.

[0032] Preferably, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0033] Preferably, the drug is in a form suitable for administration by one or more of the following methods: oral, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0034] Preferably, the dosage form of the drug comprises at least one of capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, pellets, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

[0035] A fourth aspect of the present invention provides an anti-tumor composition comprising: an oncolytic virus and an STT3A promoter.

[0036] Preferably, the anti-tumor composition further comprises a pharmaceutically acceptable carrier or excipient.

[0037] Preferably, the anti-tumor composition further comprises other anti-tumor drugs.

[0038] Preferably, the anti-tumor composition is in a form suitable for administration by one or more methods selected from the following groups: oral, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0039] Preferably, the dosage form of the anti-tumor composition comprises at least one of capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, pellets, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

[0040] Preferably, the anti-tumor composition further comprises instructions.

[0041] Preferably, the instructions record the number of administrations, dosage and administration method.

[0042] A fifth aspect of the present invention provides an anti-tumor drug kit, comprising: an oncolytic virus and an STT3A promoter; the oncolytic virus and the STT3A promoter exist independently.

[0043] Preferably, the oncolytic virus and the STT3A promoter each independently contain a pharmaceutically acceptable carrier or excipient.

[0044] Preferably, the oncolytic virus and STT3A promoter are independently in a form suitable for administration by one or more methods selected from the following groups: oral, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0045] Preferably, the dosage forms of the oncolytic virus and the STT3A promoter each independently comprise at least one of capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, pellets, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

[0046] Preferably, the oncolytic virus and the STT3A promoter are administered simultaneously or in any order, for example, the STT3A promoter is administered before the oncolytic virus, or the STT3A promoter is administered after the oncolytic virus, or both are administered simultaneously.

[0047] Preferably, the anti-tumor drug set further comprises instructions.

[0048] Preferably, the instructions record the number of administrations, dosage and administration method.

[0049] A sixth aspect of the present invention provides an anti-tumor drug system, comprising: a substance for detecting STT3A and an oncolytic virus.

[0050] In order to further improve the effectiveness of oncolytic viruses as anti-tumor drug treatment, in the selection of treatment plans, the expression of STT3A in patients (preferably tumors) can be first determined, and then the treatment plan of oncolytic viruses can be given in a targeted manner to improve the effectiveness of the treatment plan and avoid time delays and drug abuse caused by ineffective administration. For example, the expression of STT3A in patients is measured before administration. If the patient is a patient with high expression of STT3A (preferably a tumor), oncolytic virus treatment can be given directly; if the patient is a patient with normal expression of STT3A / low expression of STT3A (preferably a tumor), an STT3A promoter can be given before or at the same time as the oncolytic virus administration to increase the sensitivity of the tumor to oncolytic viruses and improve the effectiveness of treatment. The level of STT3A expression in patients (preferably tumors) directly affects the effectiveness of oncolytic virus treatment. The higher the expression of STT3A in patients (preferably tumors), the more conducive to the therapeutic effect of oncolytic viruses. To determine whether an individual / tumor is suitable for treatment with oncolytic viruses, the expression level of STT3A in patients (preferably tumors) can be detected first. The determination of the level of STT3A expression can be preferably but not limited to the following methods: low or high expression of STT3A refers to the conclusion drawn after comparing the amount of STT3A mRNA or protein between two groups (individuals) of samples. If one group (individuals) of samples is less or more than the amount of STT3A mRNA or protein in another group (individuals), it is called that the sample STT3A is low or high expression. The samples used for the quantitative comparison of STT3A mRNA and protein can be tumor cells and normal cells, tumor tissue and adjacent non-tumor tissue, or tumors or tumor patients with effective and ineffective oncolytic virus treatment. Preferably, the samples used for the quantitative comparison of STT3A mRNA and protein are tumors or tumor patients with effective and ineffective oncolytic virus treatment.

[0051] Preferably, the anti-tumor drug system further comprises an STT3A promoter.

[0052] A seventh aspect of the present invention provides use of a substance for detecting STT3A in preparing a product, wherein the product is used for at least one of (a1) to (a4):

[0053] (a1) Evaluation of the anti-tumor efficacy of oncolytic viruses;

[0054] (a2) Prognostic evaluation of oncolytic virus anti-tumor effects;

[0055] (a3) tumor treatment options;

[0056] (a4) Tumor diagnosis.

[0057] Preferably, the product comprises at least one of a reagent, a kit, a test paper, a chip, and a system.

[0058] Preferably, the test sample of the product is selected from at least one of body fluids, tissues, cells and excrement of the subject to be tested.

[0059] Preferably, the body fluid comprises at least one of blood and lymph.

[0060] Preferably, the blood comprises at least one of serum, plasma, dried blood spots and whole blood.

[0061] Preferably, the tissue comprises tumor tissue.

[0062] Preferably, the excrement includes at least one of urine, feces and tears.

[0063] Preferably, the subject to be tested includes mammals, such as humans, non-human primates (such as gorillas, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), and livestock (such as horses, cows, sheep, pigs, rabbits).

[0064] Preferably, the subject to be tested includes humans.

[0065] Preferably, the detected STT3A level is positively correlated with the oncolytic virus anti-tumor efficacy evaluation and the oncolytic virus anti-tumor therapy prognosis evaluation, that is, the higher the detected STT3A level, the better the oncolytic virus anti-tumor efficacy evaluation and the oncolytic virus anti-tumor therapy prognosis.

[0066] Preferably, the higher the detected STT3A level, the less the need for the combined use of an STT3A promoter.

[0067] Preferably, the detected STT3A level is negatively correlated with the diagnosis of tumor: a high expression level of STT3A is diagnosed as a tumor.

[0068] Preferably, the product also contains substances for detecting other markers used for evaluating the anti-tumor efficacy of oncolytic viruses, evaluating the prognosis of oncolytic viruses, selecting treatment options, and / or tumor diagnosis.

[0069] In an eighth aspect of the present invention, a method is provided for detecting the content and / or expression level of STT3A in a test sample; the method is used for at least one of (a1) to (a4):

[0070] (a1) Evaluation of the anti-tumor efficacy of oncolytic viruses;

[0071] (a2) Prognostic evaluation of oncolytic virus anti-tumor effects;

[0072] (a3) tumor treatment options;

[0073] (a4) Tumor diagnosis.

[0074] Preferably, the detection of the content and / or expression level of STT3A in the test sample includes gene level and / or protein level.

[0075] Preferably, the detecting the content and / or expression level of STT3A in the test sample comprises detecting the content and / or expression level of STT3A in the test sample at the gene level and / or protein level.

[0076] Preferably, the method for detecting the content and / or expression of STT3A in the test sample comprises one or more of the following groups: immunohistochemistry, Western blotting, Northern blotting, PCR, biochip, nucleic acid sequencing, and amino acid sequencing.

[0077] Preferably, the immunohistochemistry method is selected from at least one of: immunofluorescence method, enzyme-linked immunosorbent assay (ELISA) and immunocolloidal gold method.

[0078] Preferably, the test sample is selected from at least one of body fluids, tissues, cells and excrement of the subject to be tested.

[0079] Preferably, the body fluid comprises at least one of blood and lymph.

[0080] Preferably, the blood comprises at least one of serum, plasma, dried blood spots and whole blood.

[0081] Preferably, the tissue comprises tumor tissue.

[0082] Preferably, the excrement includes at least one of urine, feces and tears.

[0083] Preferably, the subject to be tested includes mammals, such as humans, non-human primates (such as gorillas, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), and livestock (such as horses, cows, sheep, pigs, rabbits).

[0084] Preferably, the subject to be tested includes humans.

[0085] Preferably, the detected STT3A level is positively correlated with the oncolytic virus anti-tumor efficacy evaluation and the oncolytic virus anti-tumor therapy prognosis evaluation, that is, the higher the detected STT3A level, the better the oncolytic virus anti-tumor efficacy evaluation and the oncolytic virus anti-tumor therapy prognosis.

[0086] Preferably, the higher the detected STT3A level, the less the need for the combined use of an STT3A promoter.

[0087] Preferably, the detected STT3A level is negatively correlated with the diagnosis of tumor: a high expression level of STT3A is diagnosed as a tumor.

[0088] Preferably, the method further comprises detecting the content and / or expression of other markers in the test sample for evaluating the anti-tumor efficacy of oncolytic virus, evaluating the prognosis of oncolytic virus anti-tumor, selecting a treatment regimen, and / or diagnosing tumors.

[0089] The ninth aspect of the present invention provides a method for improving the anti-tumor efficacy of oncolytic viruses, thereby increasing the content and / or activity of STT3A in an individual treated with oncolytic viruses for anti-tumor purposes.

[0090] Preferably, the method for increasing the content and / or activity of STT3A in an individual undergoing oncolytic virus anti-tumor therapy is to administer an effective amount of an STT3A promoter to the individual.

[0091] Preferably, the subject comprises a mammal, such as a human, non-human primate (e.g., gorilla, ape), rodent (e.g., rat, mouse, guinea pig), pet (e.g., cat, dog), livestock (e.g., horse, cow, sheep, pig, rabbit).

[0092] Preferably, the individual comprises a human.

[0093] The tenth aspect of the present invention provides a method for treating tumors, comprising administering an effective amount of the anti-tumor composition of the fourth aspect of the present invention and / or the anti-tumor drug kit of the fifth aspect of the present invention to an individual.

[0094] A method for treating tumors, comprising detecting the content and / or expression of STT3A in an individual;

[0095] e1) if STT3A is highly expressed in the individual, administering to the individual an effective amount of oncolytic virus, the anti-tumor composition of the fourth aspect of the present invention, and / or the anti-tumor drug set of the fifth aspect of the present invention;

[0096] e2) If STT3A is lowly expressed in the individual, administering an effective amount of the anti-tumor composition of the fourth aspect of the present invention and / or the anti-tumor drug set of the fifth aspect of the present invention to the individual.

[0097] Preferably, determining the STT3A expression level is the same as the first aspect of the present invention.

[0098] Preferably, the method for detecting the content and / or expression level of STT3A in an individual is the same as the method for detecting the content and / or expression level of STT3A in a test sample in the eighth aspect of the present invention.

[0099] Preferably, the subject comprises a mammal, such as a human, non-human primate (e.g., gorilla, ape), rodent (e.g., rat, mouse, guinea pig), pet (e.g., cat, dog), livestock (e.g., horse, cow, sheep, pig, rabbit).

[0100] Preferably, the individual comprises a human.

[0101] The eleventh aspect of the present invention provides a method for constructing a model with good anti-tumor efficacy of oncolytic virus, thereby increasing the content and / or activity of STT3A in the model.

[0102] Preferably, the method for increasing the content and / or activity of STT3A in the model is to administer an effective amount of an STT3A promoter to the model.

[0103] Preferably, the model comprises an animal model and / or a cell model.

[0104] Preferably, the cells are tumor cells.

[0105] Preferably, the animal comprises at least one of rats and mice.

[0106] Preferably, the good anti-tumor efficacy of the oncolytic virus includes a response to oncolytic virus anti-tumor treatment.

[0107] The twelfth aspect of the present invention provides a method for constructing a model with poor anti-tumor efficacy of oncolytic virus, thereby reducing the content and / or activity of STT3A in the model.

[0108] Preferably, the method for reducing the content and / or activity of STT3A in the model is to knock out the STT3A gene in the model.

[0109] Preferably, the model comprises an animal model and / or a cell model.

[0110] Preferably, the cells are tumor cells.

[0111] Preferably, the animal comprises at least one of rats and mice.

[0112] Preferably, the poor anti-tumor efficacy of the oncolytic virus comprises no response to oncolytic virus anti-tumor treatment.

[0113] In the above aspects of the present invention:

[0114] The STT3A is a catalytic subunit of mammalian oligosaccharyltransferase (OST), where oligosaccharyltransferase is a multi-subunit enzyme complex whose catalytic subunit is encoded by one of two independent homologs, STT3A or STT3B. STT3A is considered to be responsible for most of the N-glycosylation of proteins, while STT3B-dependent N-glycosylation modifications are mainly targeted at protein sites skipped by STT3A, such as the C-terminal site.

[0115] Preferably, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, coxsackievirus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, poliovirus, influenza virus, mumps virus and parvovirus; further preferably, the oncolytic virus is selected from alphavirus.

[0116] Preferably, the alpha virus comprises at least one of M1 virus and Getavirus (Getavirus is a virus with a homology of up to 97.8% (Wen et al. Virus Genes. 2007; 35(3): 597-603) with the M1 virus. The two have a high identity, and the M1 virus is also classified as a Getavirus-like virus by some literature. Therefore, it can be expected that Getavirus has similar effects to alpha virus); and further comprises M1 virus.

[0117] Preferably, the tumor comprises at least one of a solid tumor and a hematological tumor; and further comprises a solid tumor.

[0118] Preferably, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonic 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, renal cancer, Langerhans cell tissue cell At least one of: leukocyte proliferation, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, sinonasal cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papillomatosis, paraganglioma, sinonasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sezary 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, vascular tumor, vulvar cancer and single myeloma.

[0119] Preferably, the blood tumor is selected from at least one of B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's 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's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia and preleukemia.

[0120] Preferably, the STT3A promoter comprises at least one of a substance that increases the content or expression level of STT3A, a substance that enhances the activity of STT3A, and a substance that delays the metabolism of STT3A.

[0121] Preferably, the STT3A promoter comprises at least one of a natural purified substance, a modified natural purified substance, a semi-synthetic substance, and a chemically synthesized substance.

[0122] Preferably, the STT3A promoter comprises at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs.

[0123] Preferably, the STT3A promoter comprises: at least one of: biological materials related to STT3A, exogenous STT3A, nanoparticles carrying genes (STT3A genes), PEG-modified proteins (STT3A proteins), protein microspheres (STT3A proteins), liposomes encapsulating target genes (STT3A genes) or proteins (STT3A proteins), extracellular vesicles carrying target genes (STT3A genes) or proteins (STT3A proteins), and STT3A precursor proteins or conjugates or complexes that can be converted into STT3A in vivo.

[0124] Preferably, the biological material related to STT3A comprises at least one of b1) to b16):

[0125] b1) a nucleic acid molecule encoding STT3A;

[0126] b2) an expression cassette comprising the nucleic acid molecule described in b1);

[0127] b3) a vector comprising the nucleic acid molecule described in b1);

[0128] b4) a vector comprising the expression cassette described in b2);

[0129] b5) a transgenic cell line comprising the nucleic acid molecule described in b1);

[0130] b6) a transgenic cell line comprising the expression cassette described in b2);

[0131] b7) a transgenic cell line comprising the vector described in b3);

[0132] b8) a transgenic cell line comprising the vector described in b4);

[0133] b9) a microorganism comprising the nucleic acid molecule described in b1);

[0134] b10) a microorganism comprising the expression cassette described in b2);

[0135] b11) a microorganism comprising the carrier described in b3);

[0136] b12) a microorganism comprising the carrier described in b4);

[0137] b13) A virus comprising the nucleic acid molecule described in b1);

[0138] b14) A virus comprising the expression cassette described in b2);

[0139] b15) A virus comprising the vector described in b3);

[0140] b16) A virus comprising the vector described in b4).

[0141] Preferably, the STT3A promoter is specifically administered or targeted to tumor tissue, that is, a tumor-targeted STT3A promoter.

[0142] Preferably, the substance for detecting STT3A comprises a substance for quantitatively detecting STT3A.

[0143] Preferably, the substance for detecting STT3A comprises a substance for detecting STT3A at the gene level and / or protein level.

[0144] Preferably, the substance comprises a substance for one or more detection techniques or methods selected from the group consisting of immunohistochemistry, Western blotting, Northern blotting, PCR, biochip, nucleic acid sequencing, and amino acid sequencing.

[0145] Preferably, the immunohistochemistry method is selected from at least one of: immunofluorescence method, enzyme-linked immunosorbent assay (ELISA) and immunocolloidal gold method.

[0146] Preferably, the substance for detecting STT3A is selected from: one or more of a substance specific to STT3A, a probe specific to STT3A, a gene chip, and a PCR primer.

[0147] Preferably, the substance specific for STT3A comprises any one of (c1) to (c3):

[0148] (c1) an antibody that specifically binds to STT3A;

[0149] (c2) a ligand protein or polypeptide that specifically binds to STT3A;

[0150] (c3) Non-protein compounds that specifically recognize STT3A.

[0151] Preferably, the antibody comprises at least one of a polyclonal antibody, a monoclonal antibody, a single-chain antibody, a functional antibody fragment, an antibody Fab region, a nanobody, a chimeric antibody, and a multispecific antibody.

[0152] Preferably, the detected STT3A level is positively correlated with the oncolytic virus anti-tumor efficacy evaluation and the oncolytic virus anti-tumor therapy prognosis evaluation, that is, the higher the detected STT3A level, the better the oncolytic virus anti-tumor efficacy evaluation and the oncolytic virus anti-tumor therapy prognosis.

[0153] Preferably, the higher the detected STT3A level, the less the need for the combined use of an STT3A promoter.

[0154] Preferably, the detected STT3A level is negatively correlated with the diagnosis of tumor: a high expression level of STT3A is diagnosed as a tumor.

[0155] Preferably, the evaluation of the anti-tumor efficacy of oncolytic viruses refers to judging the anti-tumor effect of oncolytic viruses (whether there is a response to tumor treatment) based on the level of STT3A.

[0156] Preferably, the oncolytic virus anti-tumor prognosis assessment refers to judging the prognosis (good prognosis or poor prognosis) based on the level of STT3A.

[0157] Preferably, the treatment regimen selection refers to selecting whether to use an STT3A promoter (oncolytic virus therapy, or oncolytic virus combined with an STT3A promoter) according to the level of STT3A.

[0158] The beneficial effects of the present invention are:

[0159] The present invention is based on the inventors' discovery that STT3A can promote the N-glycosylation, infectivity and inhibition of oncolytic viruses in tumor cells, and thus proposes for the first time the use of oncolytic viruses in the preparation of anti-STT3A high-expressing tumor drugs, as well as the use of STT3A promoters in the preparation of oncolytic virus anti-tumor enhancers or resistance reversal agents. Based on this, it is proposed for the first time that a substance for detecting STT3A is used in the preparation of products for oncolytic virus anti-tumor efficacy evaluation, oncolytic virus anti-tumor prognosis evaluation, tumor treatment plan selection, and / or tumor diagnosis or prognosis evaluation; at the same time, it is proved that the combined use of oncolytic viruses and STT3A promoters can significantly improve the anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0160] Figure 1 It is a result diagram of the effect of the deglycosylation mutation of OVM on the binding of OVM to the receptor in Example 1: wherein A is a result diagram of the biolayer interferometry binding curve of the OVM virus particles (1 μM) and the biotinylated-MXRA8 bound to the streptavidin biosensor in Example 1: the results show that the binding affinity of the virus to the receptor decreases after the mutation of the N-glycosylation site, and one of the three representative experiments is shown in the figure; B is a result diagram of the OVM virus adhesion experiment in Example 1; "*" indicates P < 0.05, "***" indicates P < 0.001, and "ns" indicates P > 0.05.

[0161] Figure 2 The results of the deglycosylation mutation in Example 2 that impairs the infectivity and oncolytic effect of OVM are shown in Figure 2: A is a fluorescence microscope image of Vero cells inoculated with OVM-GFP, OVM-N141Q, OVM-N200Q and OVM-N262Q at MOI = 0.01; B is a fluorescence microscope image of Vero cells inoculated with OVM-GFP, OVM-N141Q, OVM-N200Q and OVM-N262Q at MOI = 0.01 recorded and analyzed by real-time fluorescence microscopy in Example 2 1 is a graph showing the relative total fluorescence intensity of Vero cells; C is a graph showing the results of the infectivity test of the OVM deglycosylation mutant on tumor cells with high expression of MXRA8 in Example 2; D is a schematic diagram showing the animal experiment scheme in Example 2 showing that the deglycosylation mutation impairs the oncolytic effect of OVM; E is a graph showing the growth curve of mouse tumors in the animal experiment in Example 2 showing that the deglycosylation mutation impairs the oncolytic effect of OVM; F is a graph showing the growth curve of HT-29 on day 30 in the animal experiment in Example 2 showing that the deglycosylation mutation impairs the oncolytic effect of OVM MXRA8+ Photographs of tumors; “*” indicates P < 0.05, “***” indicates P < 0.001, and “ns” indicates P > 0.05.

[0162] Figure 3The figures are the results of OST inhibitor blocking N-glycosylation and infectivity of OVM in tumor cells in Example 3: wherein A is a simplified schematic diagram of N-linked oligosaccharide synthesis and modification; B is a representative protein blot of E1 and E2 proteins in HepG2 cells treated with specific inhibitors and infected with OVM at MOI=3 in Example 3; C is a protein blot of E1 protein in HepG2 cells treated with OVM or OVM-NGI-1 in Example 3; D is a result of the content of E1 without N-glycosylation in HepG2 cells treated with specific inhibitors and infected with OVM at MOI=3 in Example 3; E is a result of the infection rate after HepG2 cells treated with specific inhibitors were infected with OVM at MOI=0.05 in Example 3; "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "ns" indicates P>0.05.

[0163] Figure 4 The figures are result graphs showing that STT3A promotes N-glycosylation and infectivity of OVM in tumor cells in Example 4: wherein A is the result graph showing the infection rate after HepG2 cells with STT3A or STT3B knockdown were infected with OVM at MOI=0.05 in Example 4; B is the result graph showing the infection rate after HepG2 cells overexpressing STT3A were infected with OVM at MOI=0.05 in Example 4; C is the representative protein blotting graph of E1 protein in HepG2 cells overexpressing STT3A infected with OVM at MOI=0.05 in Example 4; D is the result graph showing the inhibition rate of OVM in tumor cell lines with high and low expression of STT3A in Example 4; E is the result graph showing the correlation between the inhibition rate of OVM in tumor cell lines with high MXRA8 expression and the expression level of STT3A in Example 4; "*" indicates P<0.05, "***" indicates P<0.001, and "ns" indicates P>0.05.

[0164] Figure 5The figures are the results of the specific high expression of STT3A in various human cancers in Example 5: wherein A is an overview of the expression of STT3A in human cancers (RNA-seq data were downloaded from the TCGA database); B is a result of a one-way ANOVA analysis of the mRNA levels of STT3A in tumors and normal tissues; C is the proportion of tumor types in which STT3A is upregulated or downregulated in 26 human cancers in the TCGA and GTEx databases; D is a graph of the mRNA levels of STT3A in tumor tissues and normal tissues (unpaired t-test); E is a representative graph of STT3A immunostaining in the TMA of LIHC (E), where T represents tumor tissue and N represents normal tissue corresponding to the same patient; F is a representative graph of STT3A immunostaining in the TMA of COAD (F), where T represents tumor tissue and N represents normal tissue corresponding to the same patient; G is a graph of the analysis results of the average intensity of STT3A staining in LIHC (E) or COAD (F). DETAILED DESCRIPTION

[0165] The present invention is further described in detail below through specific examples.

[0166] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0167] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0168] Example 1 OVM deglycosylation mutation affects the binding of OVM to receptors

[0169] (1) Methods

[0170] 1) Generation of OVM deglycosylation mutants

[0171] To obtain the deglycosylated mutants of OVM virus, site-directed mutagenesis was used to integrate the mutants into the OVM-GFP genome on the pBR322 plasmid; green fluorescent protein (GFP) was used as a separate reporter gene for the expression of viral structural proteins, which did not affect the viral structure. The RiboMAXTM Large-Scale RNA Production System SP6 (Promega) was then used to produce RNA of the OVM-GFP mutants: OVM-N141Q, OVM-N200Q, OVM-N262Q, and OVM-N141-200-262Q (Original genome GenBank accession number: EF011023.1, OVM-N141Q means the 141st amino acid of E1 protein mutated from N to Q, i.e. the 421st base mutated from A to C, and the 423rd base mutated from T to G; OVM-N200Q means the 200th amino acid of E2 protein mutated from N to Q, i.e. the 598th base mutated from A to C; OVM-N262Q means the 262nd amino acid of E2 protein mutated from N to Q, i.e. the 784th base mutated from A to C), and the RNA of the mutant strain was further transfected into Vero cells using Lipofectamine MessengerMax reagent (ThermoFisher), and when 90% of the Vero cells showed GFP expression, the supernatant containing virus particles was collected.

[0172] In order to verify the mutation effect of the deglycosylation mutant, Viral RNA was extracted using the Spuer total RNA extraction kit (Promega) and reverse transcribed into cDNA using the GoscriptTM reverse transcription system (Promega). The cDNA was further amplified using specific primers containing the mutation site, and the success of the deglycosylation mutation was finally evaluated by Sanger sequencing analysis.

[0173] 2) Test of the binding ability of OVM deglycosylation mutant to receptor MXRA8

[0174] hMXRA8 protein production: To produce hMXRA8 protein, cDNA encoding the extracellular domain of human MXRA8 (AA residues 20-310, NM 001282585) was inserted into the pET-21a vector and a his tag was inserted at the C-terminus for purification. The purified plasmid was transfected into BL21 (Takara) cells, and the supernatant of the transfected cells was purified by affinity chromatography (Qiagen) using a Ni-NTA column (20 ml) and further verified by SDS-PAGE.

[0175] Virus adhesion assay: To detect the binding ability of OVM-N141Q, OVM-N200Q, and OVM-N262Q, OVM-N141Q, OVM-N200Q, and OVM-N262Q were normalized to the same number of OVM-GFP virus particles by RT-qPCR. HeLa and HT-29 cells overexpressing MXRA8 purchased from Guangzhou Funeng Gene Co., Ltd. (with HeLa and HT-29 cells not overexpressing MXRA8 as controls) were inoculated in 12-well plates at 3E5 / well in advance overnight, and OVM-GFP (MOI of 20) was used as a control. The deglycosylated mutant was inoculated into the cells with the same number of particles and incubated at 4°C for 45 min. After washing with PBS five times, viral RNA was extracted using a 5-min fast RNA extraction kit (HaiGene), and the number of virus particles adhering to the cells was quantified using RT-qPCR. Each treatment was repeated three times.

[0176] Biolayer interferometry receptor binding assay (BLI): After the virus supernatant was purified by ultracentrifugation, the copy number per microgram of RNA was determined by RT-qPCR. hMXRA8 protein and biotin were mixed at a mass ratio of 1:1 at room temperature for 30 minutes, and then the unreacted biotin was removed using a desalting column (Genemore). Biotinylated-MXRA8 was added at 5ug.mL- 1 Loaded onto a streptavidin biosensor (ForteBio), placed in PBS, pH 7.4, and 0.005% Tween 20 for 5 min, then 1 μM of OVM-GFP or its deglycosylated mutant was added in the same buffer, binding was measured over 30 min, and finally dissociated for 10 min at 25° C. Each treatment was repeated 3 times.

[0177] (2) Results

[0178] The results of BLI showed that the affinity of OVM-N262Q (~4-fold) or OVM-N200Q (~8-fold) for hMXRA8 was reduced compared with OVM-GFP ( Figure 1 However, the OVM-N141Q virus could not bind to hMXRA8 under the same experimental conditions ( Figure 1 (A).

[0179] Consistent with the BLI assay, the virus adhesion assay showed that overexpression of MXRA8 significantly promoted the binding of OVM-GFP to HeLa and HT-29 cells ( Figure 1 B), but only a slight increase in OVM-N262Q binding to both cells was observed, and binding to the above cells was not significantly affected by MXRA8 expression for either virus, OVM-N200Q or OVM-N141Q ( Figure 1 (middle B).

[0180] (3) Conclusion

[0181] The N-glycosylation group of OVM plays a key role in the virus-receptor binding process. Deglycosylation mutation of the virus will affect its binding to the MXRA8 receptor.

[0182] Example 2 Deglycosylation mutations impair the infectivity and oncolysis of OVM

[0183] (1) Methods

[0184] ① Test of the infectivity of OVM deglycosylation mutants on Vero cells

[0185] Vero cells were seeded into six-well plates at 3E5 / well overnight. After the cells adhered, OVM-GFP, OVM-N141Q, OVM-N200Q, and OVM-N262Q prepared in Example 1 were inoculated into the cells at a dose of MOI = 0.01, and then the infection of OVM-GFP and its mutants was recorded by real-time fluorescence microscopy. Each treatment was repeated 3 times.

[0186] ② Test of the infectivity of OVM deglycosylation mutants against tumor cells with high expression of MXRA8

[0187] The following MXRA8-highly expressing tumor cells were seeded into 12-well plates at 1E5 / well overnight: HepG2 (ATCC, hepatocellular carcinoma, Homo sapiens), HT-29 MXRA+ (Guangzhou FuNeng Gene, colon cancer, Homo sapiens), U87-MG (ATCC, glioma, Homo sapiens), Hs578T cells (ATCC, breast cancer, Homo sapiens), CT26 (ATCC, colorectal cancer, Mus musculus) and B16F10 (Guangzhou SaiKu Cell Technology, melanoma, mouse), after the cells adhered to the wall, OVM-GFP, OVM-N141Q, OVM-N200Q, OVM-N262Q were inoculated into the cells at a dose of MOI=0.05, and then the GFP positive rate of the cells was recorded by flow cytometry to reflect the infection of OVM and its mutants. Each treatment was repeated 3 times.

[0188] ③ Construction and treatment of tumor-bearing mice

[0189] Take HT-29 in the logarithmic growth phase MXRA+After trypsin digestion of cells, digestion was terminated with complete medium, centrifuged at 4°C 500×g for 5 minutes, supernatant discarded, resuspended with DPBS, pipetted evenly, centrifuged at 4°C 500×g for 5 minutes, supernatant discarded, resuspended with DPBS, counted, and the concentration of cell suspension was adjusted to 1E7 cells / mL, and stored on ice. The skin on the right rear side of the mouse was depilated in advance, and 0.1mL of cell suspension was subcutaneously injected into the depilated area with a 1mL syringe. After two weeks of tumor bearing, the tumor volume of the tumor-bearing mice grew to about 50-100mm 3 The mice were randomly divided into groups and given corresponding medications: a. Vehicle control group: 300 μL PBS was injected through the tail vein for three consecutive days; b. OVM-GFP group: 300 μL OVM-GFP (1×10 7 c. OVM-N141Q group: 300 μL OVM-N141Q (1×10 7 pfu); d. OVM-N200Q group: 300 μL OVM-N200Q (1×10 7 e. OVM-N262Q group: 300 μL OVM-N262Q (1×10 7 pfu). Each treatment was repeated 5 times.

[0190] (2) Results

[0191] The infection process recorded by real-time fluorescence microscopy showed that the infectivity of the three deglycosylated mutants was significantly reduced compared with OVM-GFP, especially OVM-N141Q, indicating that the loss of a single N-glycosylation site severely impairs the infectivity of OVM ( Figure 2 (A, B).

[0192] Furthermore, similar results were observed in other cancer cells with high MXRA8 expression, namely, OVM-N200Q and OVM-N141Q were significantly reduced compared with OVM-GFP ( Figure 2 C), OVM-N262Q infectivity was also impaired in most cancer cell lines ( Figure 2 To evaluate whether deglycosylation would affect the oncolytic effect of OVM in vivo, OVM-GFP and its deglycosylated mutants were injected into tumor-bearing mice via the tail vein ( Figure 2 Middle D), from the tumor growth curve and tumor size, it can be seen that the therapeutic effects of several deglycosylation mutants in tumor-bearing mice are weaker than OVM-GFP ( Figure 2 E and F).

[0193] (3) Conclusion

[0194] Deglycosylation mutation of OVM will affect its oncolytic effect in vivo and in vitro.

[0195] Example 3 OST inhibitors block N-glycosylation and infectivity of OVM in tumor cells

[0196] (1) Methods

[0197] ①Small molecule inhibitor screening experiment

[0198] Intracellular viral protein collection and detection method: Tumor cells (HepG2 cells) were seeded in a six-well plate at 3E5 cells / well overnight. After the cells adhered to the wall, small molecule inhibitors that affect N-glycoprotein biosynthesis enzymes (NGI-1 for OST, Castanospermine, 1-Deoxynojirimycin, Celgosivir for α-glucosidase, Kifunensine for α-mannosidase I, and Swainsonine for α-mannosidase II) were added. Figure 3 A) Added to a six-well plate according to the dosage in the instructions, the inhibitor and cells were co-incubated for 6 hours and then inoculated with OVM-GFP in Example 1 at a dose of MOI=3. After 24 hours of infection with OVM-GFP, the cell protein was collected using a protein lysate, and the electrophoretic mobility changes of the intracellular viral E1 and E2 proteins were detected by western blot. The loss of N-sugar on the viral envelope protein may lead to faster electrophoretic mobility, which can determine the effect of small molecule inhibitors on the N-glycosylation of OVM structural proteins. The cleavage site of PNGase F is the amide bond between the inner N-acetylglucosamine (GlcNAc) and the asparagine residue of the glycoprotein, so the virus sample treated with PNGase enzyme was used as a protein control lacking N-sugar. Each treatment was repeated 3 times.

[0199] Test of the effect of small molecule inhibitors on the infectivity of OVM: Tumor cells (HepG2 cells) were inoculated in a twelve-well plate at 1E5 cells / well overnight. After the cells adhered, small molecule inhibitors that affect N-glycoprotein biosynthesis enzymes were added to the twelve-well plate according to the dosage in the instructions. The inhibitors were co-incubated with the cells for 6 hours and then OVM-GFP was inoculated at a dose of MOI = 0.05. After OVM-GFP was infected for 48 hours, the GFP-positive rate of the cells was recorded by flow cytometry to reflect the blocking effect of different small molecule inhibitors on the infectivity of OVM. Each treatment was repeated 3 times.

[0200] (2) Results

[0201] The six inhibitors inhibit different steps in the synthesis of N-glycoproteins ( Figure 3Western blot results showed that among these inhibitors, the oligosaccharide transferase (OST) inhibitor NGI-1 had the strongest blocking effect on the N-glycosylation of the structural proteins E1 and E2 on OVM, resulting in more OVMs with insufficient E1 protein glycosylation in the progeny viruses ( Figure 3 Consistent with the western blot results, tumor cells treated with the oligosaccharide transferase (OST) inhibitor NGI-1 showed a significantly reduced infection rate when infected with OVM, and NGI-1 almost completely blocked the infection of OVM ( Figure 3 Middle E).

[0202] (3) Conclusion

[0203] OST inhibitors can significantly inhibit the glycosylation process of OVM in tumor cells and affect its infection in tumor cells.

[0204] Example 4 STT3A promotes N-glycosylation, infectivity and inhibition of OVM in tumor cells

[0205] (1) Methods

[0206] ① OVM infectivity test on tumor cells with knockdown of STT3A / STT3B

[0207] Because NGl-1 has been shown to inhibit the catalytic subunits STT3A and STT3B of the OST complex, the inventors next attempted to determine which subunit is more important for OVM infectivity. Tumor cells (HepG2 cells) were seeded in six-well plates overnight at 2E5 cells / well, and after the cells adhered, siRNA (50nM negative control, 50nM STT3A and 50nM STT3B were purchased from Ruibo) and Lipofectamine RNAiMAX reagent were mixed and transfected into the cells using the manufacturer's protocol. OVM-GFP (OVM) infection (MOI = 0.05) was performed 48 hours after siRNA transfection, and intracellular viral proteins were collected 24 hours later as described above. Each treatment was repeated 3 times.

[0208] ②OVM infectivity test on tumor cells overexpressing STT3A

[0209] Packaging of STT3A-overexpressing lentivirus: Human STT3A transcripts (NM_001278503) with a Flag tag at the C-terminus were synthesized and integrated into the pEZ-Lv216 vector (GeneCopoeia). The lentivirus was co-transfected with psPAX2 (Addgene #12260) and pCAGGs vector expressing vesicular stomatitis virus (VSV) G70 at a ratio of 3:2:1 using Lipofectamine 3000 (Invitrogen) to package the lentivirus in HEK 293T cells. The viral supernatant was collected for 48 hours and filtered through a 0.45 μm filter.

[0210] Construction of tumor cell lines overexpressing STT3A: Tumor cells (HepG2 cells) were seeded in a six-well plate at 3E5 cells / well overnight, and then the STT3A lentivirus obtained above was inoculated. After 72 hours, puromycin (1 μg.ml -1 ) The cell population with successful transfection was screened, and the ectopic expression of STT3A was detected by western blot. The tumor cells overexpressing STT3A (with tumor cells not overexpressing STT3A as control, the construction method was the same as that of tumor cells overexpressing STT3A) were infected with OVM-GFP (OVM) according to the above method, and the changes in intracellular virus and the changes in viral infectivity were tested respectively.

[0211] ③Inhibitory test of OVM in tumor cells with abundant MXRA8 expression

[0212] Cell selection: To reduce the promotion effect caused by the difference in MXRA8 expression in tumors, the inventors selected tumor cells with rich MXRA8 expression in the laboratory cell bank (the top ten cells selected based on the MXRA8 transcriptome data of the CCLE database) for further correlation analysis. To clarify the relationship between the host factor STT3A and OVM-GFP (OVM)-induced oncolysis, the inventors performed standardized tests on the oncolytic effect of these selected cells.

[0213] Cell classification: Among the ten cell lines with rich MXRA8 expression selected by the inventors, the cells with the top five STT3A expression levels were defined as tumor cells with high STT3A expression, namely: PK59 (Guangzhou Saiku cells, pancreatic cancer, Homo sapiens), HT1197 (ATCC, bladder cancer, Homo sapiens), HS578T (ATCC, breast cancer, Homo sapiens), LNCaP clone FGC (ATCC, prostate cancer, Homo sapiens), T47D (ATCC, breast cancer, Homo sapiens); the cells with the bottom five STT3A expression levels were defined as tumor cells with low STT3A expression, namely CALU1 (ATCC, lung cancer, Homo sapiens), SW1710 (Punosai Life Science, bladder cancer, Homo sapiens), HepG2 (ATCC, liver cancer, Homo sapiens), CFPAC1 (ATCC, pancreatic cancer, Homo sapiens), and HPAFII (ATCC, pancreatic cancer, Homo sapiens).

[0214] Cell viability assay: The above tumor cells were inoculated in 48-well plates at 10,000 cells per well and cultured overnight. After inoculation with OVM-GFP (OVM) at MOI = 0.05 for 72 hours, the medium was replaced with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg / mL, Sigma) and incubated at 37°C for another 3 hours. The medium was discarded and the MTT precipitate was dissolved in 100uL of DMSO. The absorbance was measured at 490nm using a microplate reader (BioTek, Synergy H1).

[0215] ④ Test of the inhibitory effect of OVM on tumor cells overexpressing STT3A

[0216] Construction of tumor cell lines overexpressing STT3A: Tumor cells (glioblastoma cells (e.g., U87-MG), prostate cancer cells (e.g., LNCaP clone FGC), pancreatic cancer cells (e.g., SW1990, CFPAC, HPAFII), lung cancer cells (e.g., CALU1), bladder cancer cells (e.g., SW1710), liver cancer cells (e.g., HepG2), colorectal cancer cells (e.g., LoVo, HCT-8, SW620, SW480), gastric cancer cells (e.g., NCI-N87, HGC) and other tumor cells) were inoculated in a six-well plate at 3E5 cells / well overnight, and then the STT3A lentivirus obtained in ② above was inoculated. After 72 hours, puromycin (1 μg.mL -1) The cell population with successful transfection was screened, and the ectopic expression of STT3A was detected by western blot. The tumor cells overexpressing STT3A (with tumor cells not overexpressing STT3A as control, the construction method was the same as that of tumor cells overexpressing STT3A) were infected with OVM-GFP (OVM) according to the above method, and the changes in intracellular virus and the changes in viral infectivity were tested respectively.

[0217] Cell viability assay: The above tumor cells were inoculated in a 48-well plate at 10,000 cells per well and cultured overnight. After inoculating OVM-GFP in Example 1 at MOI = 0.05 for 72 hours, the culture medium was replaced with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg / mL, Sigma) and incubated at 37°C for another 3 hours. The culture medium was discarded and the MTT precipitate was dissolved in 100uL of DMSO. The absorbance was measured at 490nm using an ELISA reader (BioTek, Synergy H1). The inhibition rate of tumor cells overexpressing STT3A and the inhibition rate of ordinary tumor cells (i.e., tumor cells not overexpressed) were statistically analyzed by GraphPad Prism 9.

[0218] (2) Results

[0219] The results of knockdown of the expression of the catalytic subunits of the OST complex, STT3A and STT3B, in HepG2 cells by specific siRNA showed that only cells treated with STT3A, but not STT3B, siRNA showed reduced OVM-GFP (OVM) infection ( Figure 4 (A).

[0220] Next, higher OVM-GFP (OVM) infection was also observed in HepG2 cells overexpressing STT3A, and these cells also produced viral progeny with more fully N-glycosylated envelope proteins ( Figure 4 (B, C).

[0221] The results of comparing the inhibition rate of OVM on tumor cells with different STT3A expression levels showed that the higher the expression level of STT3A, the more obvious the inhibitory effect of OVM on the tumor cells. The expression level of STT3A was positively correlated with the inhibition rate of OVM-GFP (OVM) in tumor cells ( Figure 4 (D, E).

[0222] Higher OVM-GFP (OVM) infection was observed in other types of tumor cells overexpressing STT3A. At the same time, the inhibition rate of OVM-GFP (OVM) on tumor cells can be increased by overexpressing STT3A in tumor cells, that is, the therapeutic effect of OVM-GFP (OVM) on tumor cells can be improved by increasing the activity and / or content of STT3A in tumor cells.

[0223] (3) Conclusion

[0224] The expression of STT3A in tumor cells promotes the N-glycosylation, infectivity, and inhibition of OVM in tumor cells; that is, the anti-tumor efficacy of oncolytic viruses can be improved by increasing the activity and / or content of STT3A in tumor cells.

[0225] Example 5 STT3A is widely overexpressed in various human cancers

[0226] (1) Methods

[0227] ① Analysis of differential expression of STT3A from cancer tissues and adjacent tissues of patients

[0228] Tumor tissue microarrays derived from tumor patients were purchased from a biological company, and 5 μm sections of the tissue were subjected to IHC staining to evaluate cytoplasmic STT3A expression. The slides were scanned using an Aperio slide scanner and analyzed using ImageScope software (Aperio).

[0229] ② Analysis of differential expression of STT3A between tumor patients and non-tumor patients

[0230] The analysis included cancer patients and non-cancer patients who visited the affiliated hospital. After obtaining informed consent from the patients, the basic information of the patients was collected, and tumor tissue or other samples (such as blood) were sampled from the patients for analysis of the expression of the marker (STT3A).

[0231] ③Analysis of the correlation between the efficacy of OVM treatment in clinical tumor patients and the expression of STT3A in patients

[0232] The analysis included tumor patients (liver cancer, colorectal cancer, brain glioma, lung cancer, gastric cancer, etc.) who visited the affiliated hospital and were treated with OVM. After obtaining the informed consent of the patients, the basic information of the patients (including patient age, gender, disease stage, pathological type, etc.) was collected, and the tumor tissue or other samples (such as blood, etc.) of the patients were sampled for analyzing the expression of markers.

[0233] Tumor measurement uses the sum of the longest diameters of the tumor, and the tumor is measured by (total) length. The response evaluation criteria in solid tumors (RECIST) were used to evaluate the treatment efficacy:

[0234] Complete remission (CR): All target lesions, except nodular disease, completely disappear. All target nodules must shrink to normal size (short axis <10 mm). All target lesions must be evaluated;

[0235] Partial response (PR): The sum of the diameters of all measurable target lesions is ≥30% lower than baseline. The sum of target nodules uses the short diameter, while the sum of all other target lesions uses the longest diameter. All target lesions must be evaluated;

[0236] Disease progression (PD): The minimum value of the sum of all target lesion diameters measured during the entire experimental study is used as a reference, and the relative increase in the sum of diameters is at least 20% (if the baseline measurement value is the minimum, the baseline value is used as a reference); in addition, the absolute value of the sum of diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered disease progression);

[0237] Stable disease (SD): The target lesions have not decreased to the level of PR, nor have they increased to the level of PD, but are somewhere in between. The minimum value of the sum of diameters can be used as a reference for research.

[0238] The expression of STT3A in cancer tissues was quantified by extracting RNA from the patient's tumor tissue. The patients were followed up and the efficacy of OVM was classified using the above evaluation criteria. The results were statistically analyzed using GraphPad Prism 9 and SPSS26.0.

[0239] (2) Results

[0240] Pan-cancer analysis of the Cancer Genome Atlas (TCGA) database showed that STT3A is ubiquitous and highly expressed in almost all types of human cancers ( Figure 5 Although STT3A, an important enzyme in the N-glycosylation pathway, is also expressed in normal human tissues, its mRNA is significantly upregulated in 16 human cancers, covering a large proportion of tumor types ( Figure 5 In order to verify that the upregulation of STT3A is specific in tumor tissues, we selected colon cancer and liver cancer for further study ( Figure 5STT3A immunohistochemistry (IHC) analysis was performed on tissue microarrays of tumors and adjacent normal tissues from the colon (n = 90) or liver (n = 87). Consistent with mRNA expression, STT3A staining was observed in both tumor and normal tissues, but most tumor tissues showed a higher degree of immunostaining compared with their respective normal tissues ( Figure 5 (EG).

[0241] At the same time, ROC curve analysis was performed based on the above results, and the results showed that STT3A can very effectively distinguish tumor tissue from non-tumor tissue (AUC>0.6, strong specificity and high sensitivity), indicating that STT3A can be used as a diagnostic marker for tumors (colorectal cancer, liver cancer).

[0242] Furthermore, ROC curve analysis was performed on the expression results of the marker (STT3A) in tumor patients and non-tumor patients. The results showed that STT3A can very effectively distinguish tumor patients from non-tumor patients (AUC>0.6, strong specificity and high sensitivity), further indicating that STT3A can be used as a diagnostic marker for tumors.

[0243] The ROC curve analysis of the efficacy of OVM treatment in tumor patients and the expression of STT3A in patients showed that STT3A could very effectively distinguish the efficacy of OVM treatment (AUC>0.6, strong specificity and high sensitivity), indicating that STT3A can be used as a marker to predict the anti-tumor efficacy of oncolytic viruses.

[0244] (3) Conclusion

[0245] STT3A can be used as a diagnostic marker for tumors and a marker for predicting the anti-tumor efficacy of oncolytic viruses.

[0246] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of oncolytic virus in the preparation of anti-tumor drugs, wherein the tumor is a tumor with high expression of STT3A.

2. Application of STT3A promoter in the preparation of oncolytic virus anti-tumor enhancers or drug resistance reversal agents.

3. The use according to claim 2, characterized in that: The oncolytic virus anti-tumor synergist or drug resistance reversal agent also includes other substances that enhance the anti-tumor efficacy of oncolytic virus or reverse drug resistance; Preferably, the oncolytic virus antitumor synergist or drug resistance reversal agent is in a form suitable for administration by one or more methods selected from the following group: oral administration, injection, gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal administration; Preferably, the dosage form of the oncolytic virus anti-tumor enhancer or drug resistance reversal agent comprises at least one of capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

4. Application of oncolytic viruses and STT3A promoters in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that: The medicine also contains other anti-tumor drugs; Preferably, the drug is in a form suitable for administration by one or more methods selected from the group consisting of oral administration, injection, gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration; Preferably, the dosage form of the drug comprises at least one of capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, pellets, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.

6. An anti-tumor composition comprising: an oncolytic virus and an STT3A promoter.

7. The antitumor composition according to claim 6, characterized in that: The anti-tumor composition further comprises a pharmaceutically acceptable carrier or excipient; Preferably, the anti-tumor composition further comprises other anti-tumor drugs; Preferably, the antitumor composition is in a form suitable for administration by one or more methods selected from the group consisting of oral administration, injection, gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration; Preferably, the dosage form of the anti-tumor composition comprises at least one of capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, dripping pills, honey pills, pills, granules, honey paste, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets; Preferably, the anti-tumor composition further comprises instructions.

8. An anti-tumor drug kit, comprising: an oncolytic virus and an STT3A promoter; the oncolytic virus and the STT3A promoter exist independently.

9. The anti-tumor drug set according to claim 8, characterized in that: The oncolytic virus and the STT3A promoter each independently contain a pharmaceutically acceptable carrier or excipient; Preferably, the oncolytic virus and the STT3A promoter are independently in a form suitable for administration by one or more methods selected from the following group: oral administration, injection, gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal administration; Preferably, the dosage forms of the oncolytic virus and the STT3A promoter each independently comprise at least one of capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honey pills, pills, granules, honey pastes, sustained-release preparations, oral liquid preparations, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets; Preferably, the oncolytic virus and the STT3A promoter are administered simultaneously or in any order; Preferably, the anti-tumor drug set further comprises instructions; Preferably, the anti-tumor drug set further comprises other anti-tumor drugs.

10. An anti-tumor drug system, comprising: a substance for detecting STT3A and an oncolytic virus.

11. Use of a substance for detecting STT3A in the preparation of a product, wherein the product is used for at least one of (a1) to (a4): (a1) Evaluation of the anti-tumor efficacy of oncolytic viruses; (a2) Prognostic evaluation of oncolytic virus anti-tumor effects; (a3) tumor treatment options; (a4) Tumor diagnosis.

12. The use according to claim 11, characterized in that: The product also includes substances for detecting other markers used for evaluating the anti-tumor efficacy of oncolytic viruses, evaluating the prognosis of oncolytic viruses, selecting treatment options, and / or diagnosing tumors; Preferably, the product comprises at least one of a reagent, a kit, a test paper, a chip, and a system; Preferably, the test sample of the product is selected from at least one of body fluids, tissues, cells, and excrement of the subject to be tested; Preferably, the body fluid comprises at least one of blood and lymph; Preferably, the blood comprises at least one of serum, plasma, dried blood spots, and whole blood; Preferably, the tissue comprises tumor tissue; Preferably, the excreta comprises at least one of urine, feces, and tears; Preferably, the subject to be tested comprises a mammal; Preferably, the subject to be tested includes humans.

13. A method for constructing a model with good anti-tumor efficacy of oncolytic virus, which increases the content and / or activity of STT3A in the model.

14. A method for constructing a model with poor anti-tumor efficacy of oncolytic virus, reducing the content and / or activity of STT3A in the model.

15. The use, antitumor composition, antitumor drug set, antitumor drug system, or construction method according to any one of claims 1 to 14, characterized in that: The oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, polio virus, influenza virus, mumps virus and parvovirus; Preferably, the oncolytic virus is selected from alphavirus; Preferably, the tumor comprises at least one of a solid tumor and a hematological tumor; Preferably, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonic 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, renal cancer, Langerhans cell tissue cell At least one of: leukocyte proliferation, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, sinonasal cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papillomatosis, paraganglioma, sinonasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sezary 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, vascular tumor, vulvar cancer and single myeloma; Preferably, the blood tumor is selected from at least one of B-cell acute lymphoid leukemia, T-cell acute lymphoid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's 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's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia and preleukemia.

16. The use, antitumor composition, or antitumor drug set according to any one of claims 2 to 9, characterized in that: The STT3A promoter comprises at least one of a substance that increases the content or expression level of STT3A, a substance that enhances the activity of STT3A, and a substance that delays the metabolism of STT3A; Preferably, the STT3A promoter comprises: at least one of a natural purified substance, a modified natural purified substance, a semi-synthetic substance, and a chemically synthesized substance; Preferably, the STT3A promoter comprises: at least one of chemical drugs, polypeptide drugs, protein drugs and gene drugs; Preferably, the STT3A promoter comprises: at least one of: biological materials related to STT3A, exogenous STT3A, nanoparticles carrying STT3A genes, PEG-modified STT3A proteins, STT3A protein microspheres, liposomes encapsulating STT3A genes or proteins, extracellular vesicles carrying STT3A genes or proteins, STT3A precursor proteins or conjugates or complexes that can be converted into STT3A in vivo; Preferably, the biological material related to STT3A comprises at least one of b1) to b16): b1) a nucleic acid molecule encoding STT3A; b2) an expression cassette comprising the nucleic acid molecule described in b1); b3) a vector comprising the nucleic acid molecule described in b1); b4) a vector comprising the expression cassette described in b2); b5) a transgenic cell line comprising the nucleic acid molecule described in b1); b6) a transgenic cell line comprising the expression cassette described in b2); b7) a transgenic cell line comprising the vector described in b3); b8) a transgenic cell line comprising the vector described in b4); b9) a microorganism comprising the nucleic acid molecule described in b1); b10) a microorganism comprising the expression cassette described in b2); b11) a microorganism comprising the carrier described in b3); b12) a microorganism comprising the carrier described in b4); b13) A virus comprising the nucleic acid molecule described in b1); b14) A virus comprising the expression cassette described in b2); b15) A virus comprising the vector described in b3); b16) A virus comprising the vector described in b4); Preferably, the STT3A promoter is a tumor-targeted STT3A promoter.

17. The anti-tumor drug administration system or use according to any one of claims 10 to 13, characterized in that: The substance for detecting STT3A comprises a substance for quantitatively detecting STT3A; Preferably, the substance for detecting STT3A comprises a substance for detecting STT3A at the gene level and / or protein level; preferably, the substance comprises a substance for one or more detection techniques or methods selected from the group consisting of: immunohistochemistry, Western blotting, Northern blotting, PCR, biochip, nucleic acid sequencing, amino acid sequencing; Preferably, the immunohistochemistry method is selected from at least one of immunofluorescence method, immunoenzyme labeling method and immunocolloidal gold method.

18. The anti-tumor drug administration system or use according to claim 17, characterized in that: The substance for detecting STT3A is selected from: one or more of a substance specific to STT3A, a probe specific to STT3A, a gene chip, and a PCR primer; Preferably, the substance specific for STT3A comprises any one of (c1) to (c3): (c1) an antibody that specifically binds to STT3A; (c2) a ligand protein or polypeptide that specifically binds to STT3A; (c3) non-protein compounds that specifically recognize STT3A; Preferably, the antibody comprises at least one of a polyclonal antibody, a monoclonal antibody, a single-chain antibody, a functional antibody fragment, an antibody Fab region, a nanobody, a chimeric antibody, and a multispecific antibody.

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