Application of TPRA1 in anti-tumor effect of oncolytic virus

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of TPRA1 in the anti-tumor effect of oncolytic viruses. The invention provides the application of the oncolytic virus in preparation of the TPRA1 high-expression tumor medicine and the application of the TPRA1 accelerant in preparation of the oncolytic virus anti-tumor synergist or the drug resistance reversal agent for the first time. On the basis, the application of a substance for detecting the TPRA1 in preparation of products for oncolytic virus anti-tumor curative effect evaluation, oncolytic virus anti-tumor prognosis evaluation, tumor treatment scheme selection and / or tumor diagnosis or prognosis evaluation is provided for the first time; meanwhile, it is proved that the antitumor effect can be remarkably improved through combined medication of the oncolytic virus and the TPRA1 accelerant.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of TPRA1 in the antitumor effect of oncolytic viruses. Background Technology

[0002] Oncolytic virus therapy is a novel anti-tumor immunotherapy combining targeted therapy, immunotherapy, and gene therapy. It selectively infects and directly kills tumor cells, thereby activating an anti-tumor immune response through mechanisms such as exposing tumor / virus antigens and releasing cytokines, thus exerting a direct or indirect tumor-suppressive effect. Furthermore, oncolytic viruses themselves can act as vectors carrying suicide genes, immunomodulatory genes, pro-apoptotic genes, and anti-angiogenic genes to further regulate the tumor microenvironment and enhance the efficacy of anti-tumor drugs. Compared with conventional treatments currently used in clinical practice, oncolytic virus therapy has advantages such as strong killing effect, high safety, and lower cost. Currently, dozens of oncolytic viruses are in clinical trials. In 2015, the oncolytic virus T-Vec was the first to be approved by the US FDA for the treatment of melanoma. In 2021, the oncolytic virus Delytact was approved in Japan for the treatment of malignant glioma. The naturally occurring alphavirus M1, a novel oncolytic virus originally discovered in my country (Lin et al., 2014), has good in vitro and in vivo anti-tumor activity and safety, and is currently undergoing clinical trials in China and Japan.

[0003] The primary prerequisite for oncolytic viruses to exert their anticancer effects is the precise targeting and infection of tumor cells. A crucial first step in viral infection of tumor cells is receptor-mediated viral entry. Receptor expression determines the tissue tropism of the virus. For example, the oncolytic virus T-Vec, developed based on HSV-1, has its receptors HVEM and Nectin highly expressed in melanoma; therefore, melanoma is the primary clinical indication for T-Vec. In conclusion, identifying novel receptors for oncolytic virus M1 will further elucidate the pharmacological mechanism of oncolytic virus M1 targeting tumors and provide predictive biomarkers for precision medicine, possessing significant scientific and practical value.

[0004] TPRA1 (Transmembrane protein adipocyte-associated 1), also known as TPRA40, GPR175, and TMEM227, is a membrane protein with a seven-transmembrane helix structure and belongs to the orphan G protein-coupled receptor family. TPRA1 was first cloned in 1999, but its detailed physiological functions remain unknown. Reports indicate that TPRA1 can inhibit cell division in mouse embryos and promote the Hedgehog signaling pathway. To date, there are no reported studies on the association between TPRA1 and viral infection. Summary of the Invention

[0005] The first aspect of this invention aims to provide the application of oncolytic viruses in the preparation of antitumor drugs, wherein the tumor is a TPRA1-highly expressed tumor.

[0006] A second aspect of the present invention aims to provide the application of TPRA1 promoter in the preparation of oncolytic virus antitumor synergists or drug resistance reversal agents.

[0007] The third aspect of this invention aims to provide the use of oncolytic viruses and TPRA1 promoters in the preparation of antitumor drugs.

[0008] A fourth aspect of the present invention is to provide an antitumor composition.

[0009] The fifth aspect of the present invention is to provide an anti-tumor drug kit.

[0010] The sixth aspect of the present invention is to provide an antitumor drug delivery system.

[0011] The seventh aspect of this invention aims to provide the application of a substance for detecting TPRA1 in the preparation of products.

[0012] The object of the eighth aspect of the present invention is to provide a method.

[0013] The ninth aspect of this invention aims to provide a method for improving the antitumor effect of oncolytic viruses.

[0014] The tenth aspect of this invention is to provide a method for treating tumors.

[0015] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0016] In a first aspect, the invention provides the use of oncolytic viruses in the preparation of antitumor drugs, wherein the tumor is a TPRA1-highly expressed tumor.

[0017] The preferred, but not limited to, method for determining the level of TPRA1 expression is as follows: Low or high TPRA1 expression refers to a conclusion drawn from a comparison of the amount of TPRA1 mRNA or protein between two groups (samples). If one group (sample) has less or more TPRA1 mRNA or protein than another group (sample), it is considered that the sample has low or high TPRA1 expression. Samples used for comparing the amount of TPRA1 mRNA and protein can be tumor cells and normal cells, tumor tissue and adjacent non-tumor tissue, or tumors that are effective and ineffective with oncolytic virus treatment. Preferably, the samples used for comparing the amount of TPRA1 mRNA and protein are tumors that are effective and ineffective with oncolytic virus treatment.

[0018] Preferably, the drug further comprises other antitumor drugs.

[0019] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0020] Preferably, the form of the drug is 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, plasmid DNA carried by reproduction-deficient bacteria, target DNA carried by replication-deficient adenovirus, or protein encoded by the target gene, via electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, or transdermal administration.

[0021] Preferably, the dosage form of the drug includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0022] A second aspect of the present invention provides the use of TPRA1 promoter in the preparation of oncolytic virus antitumor synergists or drug resistance reversal agents.

[0023] Preferably, the oncolytic virus antitumor synergist or drug resistance reversal agent further comprises other substances that enhance the antitumor efficacy of oncolytic viruses or reverse drug resistance.

[0024] Drug resistance reversal agents refer to the use of oncolytic viruses as anti-tumor drugs to treat tumors. Some tumors are not very sensitive to oncolytic viruses, or in other words, these tumors are resistant to oncolytic viruses. In this case, oncolytic viruses can be used in combination with TPRA1 promoters (as drug resistance reversal agents) to reverse the tumor's resistance to the oncolytic virus.

[0025] Preferably, the oncolytic virus antitumor potentiator or drug resistance reversal agent is in a form suitable for administration by one or more of the following methods: oral, injection, gold-coated gene gun bombardment, reproduction-deficient bacteria carrying plasmid DNA, replication-deficient adenovirus carrying target DNA or protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, or transdermal administration.

[0026] Preferably, the dosage form of the oncolytic virus antitumor synergist or drug resistance reversal agent includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0027] A third aspect of the invention provides the use of oncolytic viruses and TPRA1 promoters in the preparation of antitumor drugs.

[0028] Preferably, the drug further comprises other antitumor drugs.

[0029] Preferably, the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0030] Preferably, the form of the drug is 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, plasmid DNA carried by reproduction-deficient bacteria, target DNA carried by replication-deficient adenovirus, or protein encoded by the target gene, via electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, or transdermal administration.

[0031] Preferably, the dosage form of the drug includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0032] A fourth aspect of the invention provides an antitumor composition comprising: an oncolytic virus and a TPRA1 promoter.

[0033] Preferably, the antitumor composition further comprises a pharmaceutically acceptable carrier or excipient.

[0034] Preferably, the antitumor composition further comprises other antitumor drugs.

[0035] Preferably, the form of the antitumor composition is 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, propagation-deficient bacteria carrying plasmid DNA, replication-deficient adenovirus carrying target DNA or protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, or transdermal administration.

[0036] Preferably, the dosage form of the antitumor composition includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0037] Preferably, the antitumor composition further includes instructions for use.

[0038] Preferably, the instruction manual specifies the number of administrations, the dosage, and the method of administration.

[0039] A fifth aspect of the present invention provides an antitumor drug kit comprising: an oncolytic virus and a TPRA1 promoter; wherein the oncolytic virus and the TPRA1 promoter exist independently.

[0040] Preferably, the oncolytic virus and the TPRA1 promoter each independently comprise a pharmaceutically acceptable carrier or excipient.

[0041] Preferably, the oncolytic virus and the TPRA1 promoter are each in a form that makes them 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, reproduction-deficient bacteria carrying plasmid DNA, replication-deficient adenovirus carrying target DNA or protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, or transdermal administration.

[0042] Preferably, the dosage forms of the oncolytic virus and the TPRA1 promoter each independently include at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

[0043] Preferably, the oncolytic virus and the TPRA1 promoter are applied simultaneously or in any order, such as applying the TPRA1 promoter before the oncolytic virus, or applying the TPRA1 promoter after the oncolytic virus, or applying both simultaneously.

[0044] Preferably, the antitumor drug kit also includes an instruction manual.

[0045] Preferably, the instruction manual specifies the number of administrations, the dosage, and the method of administration.

[0046] A sixth aspect of the present invention provides an antitumor drug delivery system comprising: a substance for detecting PRTA1 and an oncolytic virus.

[0047] To further improve the efficacy of oncolytic viruses as anti-tumor drugs, the expression level of TPRA1 in the patient (preferably a tumor) can be determined first during treatment selection. Then, the oncolytic virus treatment regimen can be administered specifically to improve the effectiveness of the treatment and avoid delays and drug abuse caused by ineffective administration. For example, the TPRA1 expression level of the patient can be measured before administration. If the patient has high TPRA1 expression (preferably a tumor), oncolytic virus treatment can be administered directly. If the patient has normal or low TPRA1 expression (preferably a tumor), a TPRA1 promoter can be administered before or simultaneously with oncolytic virus administration to increase the tumor's sensitivity to the oncolytic virus and improve treatment effectiveness. The level of TPRA1 expression in the patient (preferably a tumor) directly affects the effectiveness of oncolytic virus treatment. Patients with higher TPRA1 expression levels (preferably tumors) are more likely to benefit from oncolytic virus treatment. To determine whether an individual / tumor is suitable for oncolytic virus treatment, the expression level of TPRA1 in the patient (preferably a tumor) can be measured first. Determining TPRA1 expression levels can be achieved, but is not limited to, the following methods: Low or high TPRA1 expression refers to a conclusion drawn from comparing the amount of TPRA1 mRNA or protein between two groups of samples. If one group of samples has less or more TPRA1 mRNA or protein than another group, that sample is considered to have low or high TPRA1 expression. Samples used for comparing the amount of TPRA1 mRNA and protein can be tumor cells and normal cells, tumor tissue and adjacent non-tumor tissue, or tumors or tumor patients who respond to or do not respond to oncolytic virus therapy. Preferably, samples used for comparing the amount of TPRA1 mRNA and protein are tumors or tumor patients who respond to or do not respond to oncolytic virus therapy.

[0048] Preferably, the antitumor drug system further includes a TPRA1 promoter.

[0049] A seventh aspect of the invention provides the use of a substance for detecting TPRA1 in the preparation of a product, said product being used in at least one of a1) to a4):

[0050] a1) Evaluation of the antitumor efficacy of oncolytic viruses;

[0051] a2) Prognostic assessment of oncolytic virus antitumor activity;

[0052] a3) Selection of tumor treatment options;

[0053] a4) Tumor diagnosis;

[0054] Preferably, the substance for detecting TPRA1 includes a substance for quantitative detection of TPRA1.

[0055] Preferably, the substance for detecting TPRA1 includes a substance for detecting TPRA1 at the gene level and / or protein level.

[0056] Preferably, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: immunohistochemistry, Western blotting, Northern blotting, PCR, microarray, nucleic acid sequencing, and amino acid sequencing.

[0057] Preferably, the immunohistochemical method is selected from at least one of immunofluorescence, immunoenzyme labeling, and immunogold assay.

[0058] Preferably, the substance used to detect TPRA1 is selected from one or more of the following: substances specific to TPRA1, TPRA1-specific probes, gene chips, and PCR primers.

[0059] Preferably, the substance specific to TPRA1 includes any one of b1) to b3):

[0060] b1) Antibodies that specifically bind to TPRA1;

[0061] b2) A ligand protein or polypeptide that specifically binds to TPRA1;

[0062] b3) Specifically recognizes non-protein compounds of TPRA1;

[0063] Preferably, the antibody comprises at least one of polyclonal antibody, monoclonal antibody, single-chain antibody, functional antibody fragment, antibody Fab region, nanobody, chimeric antibody, and multispecific antibody.

[0064] Preferably, the product further includes substances that detect other biomarkers used for evaluating the efficacy of oncolytic virus antitumor therapy, assessing the prognosis of oncolytic virus antitumor therapy, selecting treatment regimens, and / or diagnosing tumors.

[0065] Preferably, the product comprises at least one of reagents, reagent kits, test strips, chips, and systems.

[0066] Preferably, the test sample of the product is selected from at least one of the body fluids, tissues, cells, and excretions of the subject to be tested.

[0067] Preferably, the body fluid includes at least one of blood and lymph.

[0068] Preferably, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0069] Preferably, the tissue comprises tumor tissue.

[0070] Preferably, the excrement contains at least one of urine, feces, and tears.

[0071] Preferably, the object to be tested comprises a mammal.

[0072] Preferably, the object to be tested includes humans.

[0073] Preferably, the detected TPRA1 level is positively correlated with the evaluation of the efficacy of oncolytic virus antitumor therapy and the prognosis of oncolytic virus antitumor therapy, that is, the higher the detected TPRA1 level, the better the evaluation of the efficacy of oncolytic virus antitumor therapy and the prognosis of oncolytic virus antitumor therapy.

[0074] Preferably, the higher the detected TPRA1 level, the less likely it is to require the use of a TPRA1 promoter.

[0075] Preferably, the detected TPRA1 level is negatively correlated with the diagnosis of tumor: high TPRA1 expression indicates a tumor diagnosis.

[0076] Preferably, the product further comprises substances for detecting other biomarkers used in evaluating the efficacy of oncolytic virus antitumor therapy, assessing the prognosis of oncolytic virus antitumor therapy, selecting treatment regimens, and / or diagnosing tumors.

[0077] An eighth aspect of the present invention provides the method described in any one of c1) to c2):

[0078] c1) A method for constructing a model with good anti-tumor efficacy of oncolytic virus, thereby increasing the content and / or activity of TPRA1 in the model;

[0079] c2) A method for constructing a model of poor antitumor efficacy of oncolytic viruses, by reducing the content and / or activity of TPRA1 in the model.

[0080] Preferably, the model comprises an animal model and / or a cell model.

[0081] Preferably, the cells are tumor cells.

[0082] Preferably, the animal comprises at least one of rats and mice.

[0083] Preferably, the method for increasing the content and / or activity of TPRA1 in the model is to apply an effective amount of TPRA1 promoter to the model.

[0084] Preferably, the oncolytic virus having good anti-tumor efficacy includes responding to oncolytic virus anti-tumor treatment.

[0085] Preferably, the method for reducing the content and / or activity of TPRA1 in the model is to knock out the TPRA1 gene in the model.

[0086] Preferably, the poor efficacy of oncolytic virus antitumor therapy includes no response to oncolytic virus antitumor treatment.

[0087] The model is used for screening, identifying, and validating candidate drugs, and can be used for drug development and identification.

[0088] A ninth aspect of the present invention provides a method for improving the antitumor effect of oncolytic viruses by increasing the content and / or activity of TPRA1 in individuals receiving oncolytic virus antitumor treatment.

[0089] Preferably, the method for increasing the content and / or activity of TPRA1 in individuals undergoing oncolytic virus antitumor therapy is to administer an effective amount of TPRA1 promoter to the individual.

[0090] Preferably, the individuals include mammals, such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0091] Preferably, the individual includes humans.

[0092] A tenth aspect of the present invention provides a method for treating tumors.

[0093] The method for treating tumors includes, but is not limited to, one of a1) to a4):

[0094] a1) Evaluation of the antitumor efficacy of oncolytic viruses;

[0095] a2) Prognostic assessment of oncolytic virus antitumor activity;

[0096] a3) Selection of tumor treatment options;

[0097] a4) Tumor diagnosis.

[0098] Preferably, the method for treating tumors includes detecting the content and / or expression level of TPRA1 in the test sample.

[0099] Preferably, the detection of TPRA1 content and / or expression level in the test sample includes gene level and / or protein level.

[0100] Preferably, the detection of TPRA1 content and / or expression level in the test sample includes detecting TPRA1 content and / or expression level in the test sample at the gene level and / or protein level.

[0101] Preferably, the method for detecting the content and / or expression level of TPRA1 in the test sample includes one or more of the following: immunohistochemistry, Western blotting, Northern blotting, PCR, microarray, nucleic acid sequencing, and amino acid sequencing.

[0102] Preferably, the immunohistochemical method is selected from at least one of the following: immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), and immunogold assay.

[0103] Preferably, the detected TPRA1 level is positively correlated with the evaluation of the efficacy of oncolytic virus antitumor therapy and the prognosis of oncolytic virus antitumor therapy, that is, the higher the detected TPRA1 level, the better the evaluation of the efficacy of oncolytic virus antitumor therapy and the prognosis of oncolytic virus antitumor therapy.

[0104] Preferably, the higher the detected TPRA1 level, the less likely it is to require the use of a TPRA1 promoter.

[0105] Preferably, the detected TPRA1 level is negatively correlated with the diagnosis of tumor: high TPRA1 expression indicates a tumor diagnosis.

[0106] Preferably, the method further includes detecting the content and / or expression level of other biomarkers in the test sample used for evaluating the efficacy of oncolytic virus antitumor therapy, assessing the prognosis of oncolytic virus antitumor therapy, selecting treatment regimens, and / or diagnosing tumors.

[0107] Preferably, the method of treating tumors includes administering an effective amount of the antitumor composition of the fourth aspect of the present invention and / or the antitumor drug kit of the fifth aspect of the present invention to an individual after detecting the TPRA1 level of the sample.

[0108] The preferred option is as follows:

[0109] e1) If TPRA1 is highly expressed in an individual, then administer an effective amount of oncolytic virus, the antitumor composition of the fourth aspect of the present invention, and / or the antitumor drug kit of the fifth aspect of the present invention to the individual.

[0110] e2) If TPRA1 is expressed at low levels in an individual, then administer an effective amount of the antitumor composition of the fourth aspect of the present invention and / or the antitumor drug kit of the fifth aspect of the present invention to the individual.

[0111] Preferably, the determination of the TPRA1 expression level is the same as in the first aspect of the present invention.

[0112] Preferably, the method for detecting the content and / or expression level of TPRA1 in an individual is the same as the method for detecting the content and / or expression level of TPRA1 in a test sample in the eighth aspect of the present invention.

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

[0114] The oncolytic virus includes at least one of the following: alpha virus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, Coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, poliovirus, influenza virus, mumps virus, and parvovirus.

[0115] Preferably, the oncolytic virus is selected from alphaviruses.

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

[0117] Preferably, the tumor comprises at least one of a solid tumor and a hematoma; more preferably, it comprises a solid tumor.

[0118] Preferably, the solid tumors include liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, adrenocortical carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, rhabdomyosarcoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germinal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germinal tumor, gonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, and Langerhans cell histiocytoma. Cellular hyperplasia, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal cavity and sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papilloma, paraganglioma, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Cézare syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and single myeloma.

[0119] Preferably, the hematologic malignancy is selected from at least one of B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphoblastic 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's macroglobulinemia, and preleukemia.

[0120] Preferably, the tumor is a TPRA1-highly expressed tumor.

[0121] Preferably, the tumors with high TPRA1 expression include, but are not limited to, glioblastoma, bladder cancer, prostate cancer, glioma, lung cancer, colorectal cancer, rectal cancer, liver cancer, breast cancer, cervical cancer, melanoma, pancreatic cancer, nasopharyngeal carcinoma, gastric cancer, bladder urothelial carcinoma, acute myeloid leukemia, head and neck squamous cell carcinoma, and endometrial cancer.

[0122] Preferably, the TPRA1 promoter includes at least one of the following: substances that increase TPRA1 content or expression level, substances that enhance TPRA1 activity, and substances that delay TPRA1 metabolism.

[0123] Preferably, the TPRA1 promoter comprises at least one of the following: a naturally purified substance, a modified naturally purified substance, a semi-synthetic substance, and a chemically synthesized substance.

[0124] Preferably, the TPRA1 promoter comprises at least one of a chemical drug, a peptide drug, a protein drug, and a gene drug.

[0125] Preferably, the TPRA1 promoter comprises at least one of the following: TPRA1-related biomaterials, exogenous TPRA1, nanoparticles carrying the gene (TPRA1 gene), PEG-modified protein (TPRA1 protein), protein microspheres (TPRA1 protein), liposomes encapsulating the target gene (TPRA1 gene) or protein (TPRA1 protein), extracellular vesicles carrying the target gene (TPRA1 gene) or protein (TPRA1 protein), and TPRA1 precursor protein, conjugate, or complex that can be converted into TPRA1 in vivo.

[0126] Preferably, the biomaterial associated with TPRA1 comprises at least one of d1) to d16):

[0127] d1) Nucleic acid molecules encoding TPRA1;

[0128] d2) An expression cassette containing the nucleic acid molecules described in d1);

[0129] d3) A carrier containing the nucleic acid molecules described in d1);

[0130] d4) A carrier containing the expression box described in d2);

[0131] d5) Transgenic cell lines containing the nucleic acid molecules described in d1);

[0132] d6) Transgenic cell lines containing the expression cassette described in d2);

[0133] d7) Transgenic cell lines containing the vector described in d3);

[0134] d8) Transgenic cell lines containing the vector described in d4);

[0135] d9) Microorganisms containing the nucleic acid molecules described in d1);

[0136] d10) Microorganisms containing the expression cassette described in d2);

[0137] d11) Microorganisms containing the carrier described in d3);

[0138] d12) Microorganisms containing the carrier described in d4);

[0139] d13) Viruses containing the nucleic acid molecules described in d1);

[0140] d14) A virus containing the expression box described in d2);

[0141] d15) A virus containing the vector described in d3);

[0142] d16) is a virus containing the vector described in d4).

[0143] Preferably, the TPRA1 promoter is a tumor-targeting TPRA1 promoter.

[0144] Preferably, the TPRA1 is numbered NM_001136053.4 in the NCBI database.

[0145] Those skilled in the art should know that TPRA1 is a gene name, which has multiple transcripts. This invention uses one of these transcripts for experimental testing. Due to the high cost and long duration of biological experiments, those skilled in the art typically use only one transcript of a gene for testing, but this does not mean that other transcripts of the same gene cannot achieve the corresponding technical effect. For example, NM_001142646.4, NM_001353001.2, NM_001353002.2, NM_001353003.2, NM_001353004.2, NM_001353005.2, NM_001353006.2, and NM_001353007.2 can also achieve the technical effect of this solution.

[0146] Preferably, TPRA1 is a protein sequence having 95%, 96%, 97%, 98%, 99%, and 100% homology with the protein sequence shown in SEQ ID NO: 1, and the protein sequence has a function similar to TPRA1.

[0147] Preferably, TPRA1 means that the given amino acid sequence shares at least 95% identity with a reference sequence (such as SEQ ID NO: 1), for example, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. Alternatively, it means that the given amino acid sequence differs from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. For peptides, this difference is preferably an amino acid substitution or deletion.

[0148] Preferably, substantially similar sequences also retain the unique activity of the peptide, enabling it to detect endogenous antibodies with high efficiency. Substitutions are generally considered conserved, such as substitutions between aliphatic amino acids Ala, Val, Leu, and Ile; interchanges between hydroxyl residues Ser and Thr; exchange between acidic residues Asp and Glu; substitution between amide residues Asn and Gln; exchange between basic residues Lys and Arg; and substitution between aromatic residues Phe and Tyr.

[0149] The beneficial effects of this invention are:

[0150] This invention provides the application of TPRA1 in the antitumor effect of oncolytic viruses, and for the first time proposes the application of oncolytic viruses in the preparation of anti-TPRA1 highly expressed tumor drugs, as well as the application of TPRA1 promoters in the preparation of oncolytic virus antitumor synergists or drug resistance reversal agents. Based on this, it also proposes for the first time the application of substances that detect TPRA1 in the preparation of products for evaluating the efficacy of oncolytic virus antitumor therapy, the prognostic evaluation of oncolytic virus antitumor therapy, the selection of tumor treatment regimens, and / or tumor diagnosis or prognostic evaluation. At the same time, it demonstrates that the combined use of oncolytic viruses and TPRA1 promoters can significantly improve the antitumor effect.

[0151] This invention innovatively discovers that the G protein-coupled receptor TPRA1 is a novel receptor for oncolytic viruses. Through large-scale screening using CRISPR-Cas9 gene knockout, combined with verification through gene overexpression, knockdown, and knockout, it was found that TPRA1 can promote the infection of oncolytic virus M1. These studies identify TPRA1 as the receptor for oncolytic virus M1, and for the first time, demonstrate that the G protein-coupled receptor TPRA1 functions as a viral receptor. Furthermore, using replication-deficient pseudoviruses, it was demonstrated that TPRA1 can promote the invasion of the alphavirus Semliki Forest Virus (SFV) without promoting the unrelated vesicular stomatitis virus (VSV). This result strongly suggests the important role of TPRA1 in alphavirus infection and provides new insights into the biological research of alphaviruses.

[0152] Based on the discovery and identification of the novel receptor TPRA1 for oncolytic virus M1, this invention also demonstrates through in vitro and in vitro experiments that the expression of TPRA1 promotes the tumor-killing effect of oncolytic virus M1, and that the expression level of TPRA1 in cells is positively correlated with the sensitivity to oncolytic virus M1. This proves the important role of TPRA1 in the anti-tumor effect of oncolytic virus M1, provides a new biomarker for the precision treatment of oncolytic virus M1, further expands the indications for oncolytic virus M1, and has important application value. Attached Figure Description

[0153] Figure 1 To investigate the effect of TPRA1 overexpression on tumor cell infection with oncolytic virus M1, the following data were analyzed: A and E showed stable expression cell lines constructed by transducing empty vector control or HA-tagged TPRA1 into 22RV1 and UMUC3 cells with lentivirus, and Western blot analysis was performed. B and F showed the expression of green fluorescent protein observed by fluorescence microscopy one day after infecting cells overexpressing TPRA1 or empty vector control with 0.1 MOI M1-GFP virus. CD and GH showed the percentage of GFP-positive cells detected by flow cytometry. Scale bar: 100 μm. *** indicates p < 0.001, **** indicates p < 0.0001.

[0154] Figure 2To significantly inhibit the infection rate of oncolytic virus M1 after knocking down TPRA1 in tumor cells; A and D represent the knockdown of TPRA1 gene expression by siRNA in SW620 and 22RV1 cells, with mRNA knockdown efficiency detected by qPCR; BC and EF represent the proportion of GFP-positive cells detected by flow cytometry 24 hours after infection with 1 MOI of M1-GFP virus 48 h after siRNA transfection. * indicates p < 0.1; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0155] Figure 3 To determine the specificity of TPRA1 in promoting the invasion of pseudotype A virus into cells, the proportion of GFP-positive cells was detected by flow cytometry after infection with pseudotyped oncolytic virus M1 (A), pseudotyped Semliki Forest Virus (B), or pseudotyped vesicular stomatitis virus (C) overexpressing TPRA1 or empty vector UMUC3 cells for 4 days (A), 5 days (B), and 2 days (C). ns indicates no statistical significance; *** indicates p < 0.001.

[0156] Figure 4 Results of TPRA1 promoting the adhesion and internalization of oncolytic virus M1: A shows the results of infecting UMUC3 cells overexpressing TPRA1 or empty vector controls with 0.1 MOI M1-GFP virus, replacing the medium with agarose after 3 h, and then imaging the whole wells and performing quantitative analysis using a high-content imaging system after 48 h; B and C show the M1 viral RNA content detected by qPCR after incubating M1 cells with 22RV1 cells overexpressing TPRA1 or empty vector controls at 4℃ for 1 h; D and E show the intracellular M1 viral RNA content detected by qPCR after incubating M1 cells with UMUC3 cells overexpressing TPRA1 or empty vector controls at 4℃ for 1 h, transferring them to 37℃ for 0.5 h to digest the surface-adhered virus, and then using qPCR. ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0157] Figure 5 High expression of TPRA1 predicts better oncolytic virus M1 killing effect; where A represents 22RV1 WT and 22RV1 TPRA1 KO cells overexpressing TPRA1 or empty vector, respectively, and cell viability was detected by MTT assay 4 days after infection with M1 virus at 0.5 MOI (M1) or without virus treatment (ctrl); CD represents UMUC3 cells (C) or PC9 cells (D) overexpressing TPRA1 or empty vector infected with different concentrations (0, 0.2, 1 and 5 MOI) of oncolytic virus M1 2 days after infection, and cell viability was detected by MTT assay; ns indicates no statistical significance; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0158] Figure 6 This study investigated the upregulation of TPRA1 expression in various tumor tissues, including glioblastoma multiforme (GBM), glioma (GBMLGG), low-grade glioma of the brain (LGG), papillary renal cell carcinoma (KIRP), colorectal adenocarcinoma (COAD), colorectal cancer (COADREAD), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), hepatocellular carcinoma (LIHC), bladder urothelial carcinoma (BLCA), rectal adenocarcinoma (READ), pancreatic cancer (PAAD), acute myeloid leukemia (LAML), renal chromophobe carcinoma (KICH), and cholangiocarcinoma (CHOL), as well as their corresponding normal tissues. Tumor RNA sequencing data were obtained from the TCGA database, and normal tissue RNA sequencing data were obtained from the GTEx project. The expression differences between normal and tumor samples within each tumor were calculated using the Singerbox platform, and statistical significance analysis was performed using unpaired Wilcoxon Rank Sum and Signed Rank Tests. * indicates p < 0.05; ** indicates p < 0.01; **** indicates p < 0.0001.

[0159] Figure 7 The results show the in vivo tumor-killing effect of TPRA1 promoting oncolytic virus M1; where A is the animal experiment flowchart, a tumor-bearing mouse model was constructed in BALB / c-Nude mice using UMUC3 cells overexpressing TPRA1 or empty vector, and the tumor volume reached 50-150 mm. 3 M1 or solvent control mice were administered via tail vein injection for 7 consecutive days. On day 3 after the end of administration, the M1 viral load in the tumor was measured, and tumor volume was measured every other day until the ethical endpoint of 2000 mmHg was reached. 3 B represents the copy number of M1 virus in tumor tissue detected by absolute quantitative qPCR; C represents the growth curve of mouse tumor volume; D represents the Kaplan-Meier survival curve of tumor-bearing mice; ns indicates no statistical significance; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0160] Figure 8The expression level of TPRA1 in tumor cells is positively correlated with the killing effect of oncolytic virus M1. Among them, A is a schematic diagram for calculating AUC. According to the cell viability curve after infecting cells with different doses of oncolytic virus M1, the area under the curve (AUC) is calculated, that is, the area enclosed by the curve and the horizontal axis. The more sensitive the cells are to the M1 virus, the lower the AUC value. B shows that 17 breast cancer cell lines were infected with different doses (0, 0.1, 1, 10 MOI) of oncolytic virus M1, and the cell viability was detected by the MTT method 72 hours later. The AUC value was calculated according to the cell viability curve. The mRNA expression data of TPRA1 was downloaded from the CCLE database, and RPKM: Reads PerKilobase per Million mapped reads. C shows the correlation analysis between the mRNA expression level of TPRA1 and its AUC in each breast cancer cell line. r represents the Pearson correlation coefficient. P<0.05 indicates statistical significance.

[0161] Figure 9 It is the correlation analysis between the basal expression level of TPRA1 in the murine cell line control group and the efficacy of oncolytic virus. Specific implementation mode

[0162] The following will clearly and completely describe the concept of the present invention and the technical effects generated in combination with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0163] Cell source: Human bladder cancer cell line UMUC3 was purchased from the American Type Culture Collection (ATCC), human lung cancer cell line PC9 was purchased from Guangzhou Saiku Biotechnology Co., Ltd., and human prostate cancer cell line 22RV1 was preserved by the Neuroscience Research Center of Sun Yat-sen University.

[0164] Animal source: C57BL / 6 mice and BALB / c-Nude (nude mice), 5 weeks old, were purchased from Guangdong Medicilon Inc., and the animal production license number is SCXK (Guangdong) 2020-0054. They were raised in a SPF-level barrier environment.

[0165] Source of oncolytic virus M1: Aviral virus M1 was first isolated from Culex mosquitoes in Hainan Island, my country in 1964. The oncolytic virus M1 used in this experiment is stored at the Neuroscience Research Center of Sun Yat-sen University. Additionally, commercial oncolytic virus VRT106, batch number FJ20200915-1, produced and provided by Guangzhou Weirongte Pharmaceutical Technology Co., Ltd., was also used.

[0166] Statistical methods: Quantitative data conforming to a normal distribution are expressed as mean ± standard deviation. Statistical analysis was performed using t-tests, one-way ANOVA, two-way ANOVA, or log-rank tests. ns indicates no statistical significance; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0167] Mouse-derived cell lines: A20 (lymphoma), B16BL6 (melanoma), B16F10 (melanoma), CT26 (colorectal cancer), EMT-6 (breast cancer), H22 (liver cancer), Hepa1-6 (liver cancer), LL / 2 (lung cancer), MC38 (colon cancer), Pan02 (pancreatic cancer), Renca (kidney cancer), RM-1 (prostate cancer).

[0168] Table 1 Experimental cells and culture medium

[0169] Serial Number Cell Name Tumor categories Culture medium (v / v) 1 CT26 colon cancer RPMI 1640 + 10% FBS 2 H22 liver cancer RPMI 1640 + 10% FBS 3 MC38 colon cancer DMEM + 10% FBS 4 Pan02 pancreatic cancer RPMI 1640 + 10% FBS 5 LLC lung cancer DMEM + 10% FBS 6 A20 Lymphoma RPMI 1640 + 10% FBS 7 RM-1 Prostate cancer RPMI 1640 + 10% FBS 8 Renca Kidney cancer DMEM + 10% FBS 9 Hepa 1-6 liver cancer DMEM + 10% FBS 10 B16F10 Melanoma DMEM + 10% FBS 11 EMT-6 Breast cancer DMEM + 10% FBS 12 B16BL6 Melanoma RPMI 1640 + 10% FBS

[0170] Table 2 List of Experimental Instruments and Equipment

[0171]

[0172]

[0173] Table 3 Main reagents and consumables used in the experiment

[0174] Reagent Name Manufacturer / Source Item number M-PER Thermo 78505 T-PER Thermo 78510 Tris-MOPS-SDS electrophoresis powder Genscript M00138 SDS-PAGE protein loading buffer (5X) Biosharp BL502B PageRuler prestained protein molecular weight standards, 10 to 180 kDa Thermo 26616 SurePage precast adhesive Genscript M00660 PVDF immunoblotting membrane Roche 3010040001 eBlot L1 Transfer and Concentration Kit (PVDF Membrane Basic) Genscript L00726C-450 Isopropanol Aladdin M116115-500ml QuickBlock Western Sealing Solution Azure Sky P0252 Western primary antibody dilution Azure Sky P0023A Western bacterial secondary antibody dilution solution Azure Sky P0023D Western Blend detergent (10X) Azure Sky P0023C3 Developer Thermo 23227 SDS-PAGE protein loading buffer (5X) Biosharp BL502B TPRA1 Antibody Thermo Fisher PA5-33087 S-Actin(13B5) Rabbit mAb CST 4970S Goat anti-Rabbit IgG antibody(HRP) Sino Biological SSA004 BCA Protein Quantification Kit Thermofisher 23227

[0175] Example 1: Construction of a cell line overexpressing TPRA1

[0176] 1. Construction of lentiviral expression plasmids

[0177] The target fragment of TPRA1 (NM_001136053.4) was amplified by PCR using Q5 Hot Start High-Fidelity 2X Master Mix to acquire the HA tag. After purification, the fragment was cloned into a lentiviral expression vector and transformed into stbl3 competent cells. Plasmids were extracted according to the instructions of the Tiangen plasmid extraction kit.

[0178] The amino acid sequence of TPRA1 is as SEQ ID NO: 1 shown: MDTLEEVTWANGSTALPPPLAPNISVPHRCLLLLYEDIGTSRVRYWDLLLLIPNVLFLIFLLWKLPSARAKIRITSSPIFITFYILVFVVALVGIARAVVSMTVSTSNAATVADKILWEITRFFLLAIELSVIILGLAFGHLESKSSIKRVLAITTVLSLAYSVTQGTLEILYPDAHLSAEDF NIYGHGGRQFWLVSSCFFFLVYSLVVILPKTPLKERISLPSRRSFYVYAGILALLNLLQGLGSVLLCFDIIEGLCCVDATTFLYFSFFAPLIYVAFLRGFFGSEPKILFSYKCQVDETEEPDVHLPQPYAVARREGLEAAGAAGASAASYSSTQFDSAGGVAYLDDIASMPCHTGSINSTDSERWKAINA.

[0179] Forward primer: AAGACACCGACTCTAGAGGATCCATGGACACCCTGGAGGAGGT (SEQ ID NO: 2).

[0180] Reverse primer: CGCGTGGGTTTAAACCCCTGCAGCTAAGCGTAATCTGGAACATCGTATGGGTAGGCATTGATGGCCTTCCAGC (SEQ ID NO: 3).

[0181] 2. Construction of stable expression cell lines

[0182] HEK293T cells were seeded in 6-well plates and cultured in antibiotic-free medium. Transfection was performed the following day when the cell density was approximately 50%-60%. The 3000 transfection reagent instructions were followed to transfect the lentiviral packaging plasmid, envelope plasmid, and expression vector plasmid in a 5:1:5 ratio. 48 hours after transfection, the cell supernatant was collected, filtered, and used for later use.

[0183] Cells (UMUC3, PC9, 22RV1) were seeded into 12-well plates and cultured until the cell density reached approximately 50% on the second day. 500 μL of lentivirus solution was added for transduction. After 48 hours, the cells were passaged, and the medium was replaced with a medium containing selection antibiotics for selection. The medium was changed every two days, and the cells were passaged again when the cell density reached 90%. When all control cells not transduced with lentivirus died, the medium was replaced with normal medium for expansion culture.

[0184] Cells overexpressing UMUC3-TPRA1, PC9-TPRA1, and 22RV1-TPRA1 were obtained.

[0185] Plasmids expressing Cas9 protein and sgRNA were transfected into 22RV1 cells using CRISPR technology. Monoclonal cells were obtained by flow cytometry, and Western blotting confirmed the presence of TPRA1 knockout 22RV1 cells. Stable TPRA1-expressing 22RV1 cells were constructed using the above method.

[0186] Example 2: TPRA1-mediated infection of oncolytic virus M1

[0187] Method for detecting oncolytic virus M1 infection rate by flow cytometry: Cells infected with oncolytic virus M1 carrying the GFP reporter gene (Human Gene Therapy, 2021) were digested with trypsin, and digestion was stopped by adding FBS-containing medium. After centrifugation at 500g for 5 min, the supernatant was discarded, and the cells were washed with 1 mL PBS. After centrifugation again and discarding the supernatant, the cells were resuspended in 150 μL PBS. The percentage of GFP-positive cells at 510 nm was analyzed using the FITC channel of a flow cytometer.

[0188] In human prostate cancer cells 22RV1-TPRA1 and human bladder cancer cells UMUC3-TPRA1 prepared in Example 1, TPRA1 was overexpressed using lentivirus. Figure 1 Cells A and E were observed after infection with oncolytic virus M1 carrying the GFP reporter gene (M1-GFP). Compared with empty vector control cells, cells overexpressing TPRA1 showed more green fluorescence. Figure 1 (B and F); the proportion of GFP-positive cells, i.e., the infection rate of oncolytic virus M1, was detected by flow cytometry. It was found that the infection rate of oncolytic virus M1 was significantly increased in cells overexpressing TPRA1. Figure 1 (CD and GH). These results indicate that TPRA1 promotes infection with oncolytic virus M1.

[0189] Example 3: siRNA knockdown of TPRA1 significantly inhibited M1 virus infection.

[0190] To identify whether the endogenously expressed TPRA1 protein promotes the infection of cells by oncolytic virus M1, this study knocked down the expression of the TPRA1 gene in SW620 human colorectal adenocarcinoma cells and 22RV1 human prostate cancer cells using siRNA (Small interfering RNA), and verified the mRNA expression level of TPRA1 using qPCR.

[0191] siRNA_TPRA1_001:GAACGCTGCAACTGTTGCT (SEQ ID NO: 4).

[0192] siRNA_TPRA1_002:ATCCTCTTCTCCTACAAAT (SEQ ID NO: 5).

[0193] Forty-eight hours after siRNA transfection, oncolytic virus M1 was added for infection, and the difference in viral infection rate was detected. Results are as follows: Figure 2 As shown, knocking down TPRA1 significantly reduced the infection rate of oncolytic virus M1 in both SW620 and 22RV1 cells.

[0194] Example 4: TPRA1 specifically promotes the invasion of alpha nucleotides.

[0195] To further demonstrate that TPRA1 can promote the invasion of oncolytic virus M1 into cells, this embodiment constructed a pseudotyped virus. Pseudotyped viruses are viruses or viral vectors packaged with the envelope proteins of other viruses. After invading cells, they can only complete a single replication cycle and do not produce infectious progeny viruses. Therefore, pseudotyped viruses are often used to study viral invasion. Using the envelope protein of oncolytic virus M1, a lentiviral genome containing the GFP gene was coated to construct pseudotyped oncolytic virus M1. The results are as follows... Figure 3 As shown in Figure A, compared with the empty vector control cells, pseudo-oncolytic virus M1 had a higher infection rate in UMUC3 cells overexpressing TPRA1, indicating that TPRA1 can promote the invasion of pseudo-oncolytic virus M1 into cells.

[0196] Semliki Forest virus (SFV), belonging to the genus Alphavirus of the family Togaviridae, can cause severe neurological illness and death in rodents and other animals. Similarly, a pseudotyped Semliki Forest virus (SFV) was constructed by coating a lentiviral genome containing the GFP gene with the envelope protein of Semliki Forest virus. The results are as follows... Figure 3 As shown in Figure B, after cell infection, it was found that pseudotyped Semlikie forest virus had a higher infection rate on UMUC3 cells overexpressing TPRA1 than on empty control cells, indicating that TPRA1 can also promote the invasion and infection of Semlikie forest virus.

[0197] Vesicular stomatitis virus (VSV) is an enveloped, negative-sense RNA virus that can infect various mammalian and insect cells. Infection in humans can manifest as asymptomatic symptoms or cause mild fever. Similarly, a pseudotyped VSV was constructed by coating a lentiviral genome containing the GFP gene onto the envelope protein of VSV. The results are as follows... Figure 3 As shown in Figure C, the infection rate of pseudovirus did not change significantly after UMUC3 cells overexpressing TPRA1 and empty control cells were infected with pseudovesicular stomatitis virus, indicating that TPRA1 cannot promote VSV invasion.

[0198] The results above suggest that TPRA1 can specifically promote the invasion of alphaviruses, but not the invasion of other RNA viruses.

[0199] Example 5: TPRA1 promotes the adhesion and internalization of oncolytic virus M1

[0200] To investigate the specific stage of M1 virus infection promoted by TPRA1, this study used a plaque assay for preliminary exploration. A 2.5 wt% agarose solution was melted by heating, and the cell culture medium was preheated in an oven above 45°C. The cell culture medium was added to the agarose (the volume ratio of culture medium to agarose solution was approximately 3:1), bringing the final agarose concentration to 0.5-1 wt%. Three hours after infection with the oncolytic virus M1, the culture medium was aspirated, the cells were washed once with PBS, and 100 μL of agarose medium was added to cover the entire well. After rapid solidification at room temperature, the cells were returned to the cell culture incubator for further culture. Under solid-state culture conditions, progeny viruses produced after M1 infection do not easily spread in the culture system; infection is limited to the in situ and surrounding areas. Therefore, the number of GFP fluorescent spots represents the status of the first round of M1 virus infection. Forty-eight hours after infection, the infection status was observed, and the entire well was recorded using a microscope or a high-content imaging analysis system. Figure 4 (As shown in Figure A). The results showed that, compared with the empty vector control cells, the number of fluorescent spots in oncolytic virus M1-infected cells overexpressing TPRA1 was significantly increased. This indicates that TPRA1 already played a role in promoting M1 infection during the first round of infection.

[0201] The first step in viral infection of a host cell is entry. For alphaviruses, entry begins with binding to a receptor and attachment to the cell surface; subsequently, they internalize into the endosome via receptor-mediated endocytosis. As the endosome gradually acidifies, the viral envelope fuses with the endosome membrane, thereby delivering the genomic RNA into the cytoplasm.

[0202] To investigate whether TPRA1 can promote the adhesion and internalization of oncolytic virus M1, this study used UMUC3 cells overexpressing TPRA1 or with an empty vector control to perform virus adhesion and internalization experiments. In the virus adhesion experiment, test cells were uniformly seeded in multi-well cell culture plates. When the cell density reached 60-90%, oncolytic virus (10 MOI) was added, and the cells were incubated at 4°C for 1 hour to restrict cell membrane mobility. After washing three times with pre-cooled PBS, RNA was collected, and the content of the NS1 gene (M1_NS1) of oncolytic virus M1 was detected by RT-qPCR. The results showed that overexpression of TPRA1 in UMUC3 cells significantly increased the adhesion of oncolytic virus M1. Figure 4 (As shown in B and C). For the virus internalization experiment, cells were incubated at 4°C for 1 hour, then at 37°C for 30 minutes. After washing the cells once with pre-chilled PBS, they were digested with 500 ng / mL proteinase K or trypsin. After washing three more times with pre-chilled PBS, cells were collected for RNA extraction. The RNA content of the oncolytic virus M1 NS1 gene (M1_NS1) was detected by RT-qPCR. The results showed that overexpression of TPRA1 in UMUC3 cells significantly increased the internalization of oncolytic virus M1. Figure 4 (As shown in D and E).

[0203] The above results indicate that the cell membrane protein TPRA1 can promote the adhesion and internalization of oncolytic virus M1, thereby promoting the entry of M1 into tumor cells.

[0204] Example 6: The effect of TPRA1 in promoting the killing of tumor cells by oncolytic virus M1

[0205] To investigate whether overexpressing TPRA1 could enhance the cytotoxic effect of oncolytic virus M1, this study used oncolytic virus M1 to infect wild-type, TPRA1-overexpressing, and TPRA1-knockout 22RV1 cells. These cell types showed no significant difference in growth rate, but the tumor cell-killing effect of oncolytic virus M1 was significantly enhanced in TPRA1-overexpressing cells and significantly weakened in TPRA1-knockout cells. Furthermore, reintroducing TPRA1 expression into TPRA1-knockout 22RV1 cells restored the cytotoxic effect of oncolytic virus M1. Figure 5 (As shown in A).

[0206] To further investigate whether the expression level of TPRA1 in other tumor cells also affects the tumor cell killing ability of oncolytic virus M1, cells overexpressing TPRA1 or with an empty vector were constructed in UMUC3 and PC9 cells, and the killing effect of oncolytic virus M1 on these cells was detected. The results showed that overexpression of TPRA1 significantly enhanced the ability of oncolytic virus M1 to kill UMUC3 and PC9 tumor cells. In UMUC3 cells, the IC50 value of M1 virus killing tumor cells decreased from 5.109 MOI to 0.138 MOI; in PC9 cells, the IC50 value of M1 virus killing tumor cells decreased from 0.5284 MOI to 0.0567 MOI. Figure 5 (As shown in BC). The above results indicate that TPRA1 promotes the killing effect of oncolytic virus M1 on tumor cells.

[0207] Example 7: TPRA1 is highly expressed in various tumor tissues.

[0208] Because the M1 virus has tumor-targeting specificity, the mining results from the TCGA and GTEx databases in this embodiment are as follows: Figure 6 As shown, TPRA1 expression was significantly higher in various tumor tissues, including glioblastoma, colorectal cancer, liver cancer, and urothelial carcinoma of the bladder than in normal tissues.

[0209] Example 8: TPRA1 promotes the tumor-killing effect of oncolytic virus M1

[0210] Nude mouse subcutaneous tumor model:

[0211] Tumor cells for inoculation were cultured. After cell digestion, the cells were resuspended in serum-free medium and counted, adjusting the cell concentration to 1 × 10⁻⁶. 7 Tumor cells / ml; tumor cells were subcutaneously injected into the right back of five-week-old BALB / c-nu / nu mice. One to two weeks later, when the tumor reached 50-150 mm³, the mice were treated with oncolytic virus M1 via tail vein injection (1×10⁻⁶ cells / ml). 7 Mice were injected with 300 μL of pfu / ml for 7 days; tumor volume was recorded. The formula for calculating tumor volume is as follows:

[0212]

[0213] The mice were euthanized on a humanitarian basis when the tumor volume reached 2000 mm3. This animal experimental design was approved by the Animal Ethics Committee of Sun Yat-sen University School of Medicine.

[0214] To investigate whether upregulation of TPRA1 expression could increase the sensitivity of cells to oncolytic virus M1 killing, this example used UMUC3 cells overexpressing TPRA1 (prepared in Example 1) or empty vector cells for experiments. The results showed that, compared to control cells, M1 significantly enhanced the ability to kill cells overexpressing TPRA1. Figure 7 As shown in Figure A). The IC50 value of M1 killing tumor cells decreased from 5.109 MOI to 0.138 MOI. Figure 7 (As shown in Figure B). The above results indicate that TPRA1 promotes the killing effect of oncolytic virus M1 on tumor cells.

[0215] To investigate whether upregulation of TPRA1 expression in tumors promotes the antitumor effect of oncolytic virus M1 in vivo, this study constructed a subcutaneous tumor model in BALB / c-Nude nude mice using UMUC3 cells overexpressing TPRA1 or empty vector control, and treated them with continuous tail vein injection of M1. On day 3 after treatment, the level of M1 virus generated during tumor replication was measured. The results showed that the level of M1 virus in tumors overexpressing TPRA1 was significantly higher than that in the control group. Figure 7 As shown in Figure D), this indicates that TPRA1 expression can significantly increase M1 virus infection and replication in vivo.

[0216] Example 9: Correlation analysis between TPRA1 expression level and the antitumor effect of oncolytic virus M1

[0217] To investigate whether TPRA1 expression is positively correlated with the antitumor effect of oncolytic virus M1, this study infected 17 breast cancer cell lines with different doses (0, 0.1, 1, and 10 MOI) of oncolytic virus M1. Cell viability was assessed using the MTT assay after 72 hours, and the AUC value was calculated based on the cell viability curve. TPRA1 mRNA expression data were downloaded to the CCLE database (RPKM: Reads Per Kilobase Per Million Mapped Reads). AUC reflects the sensitivity of cells to oncolytic virus M1; a higher AUC indicates greater resistance to oncolytic virus M1, while a lower AUC indicates greater sensitivity. Figure 8 (As shown in Figure A). Correlation analysis results showed that the higher the expression level of TPRA1 in cells, the lower the AUC of oncolytic virus M1 killing (…). Figure 8 (As shown in C). The above results indicate that cells with higher TPRA1 expression levels are more sensitive to the killing effect of oncolytic virus M1.

[0218] Example 10: TPRA1 as a biomarker for evaluating the efficacy of oncolytic virus therapy

[0219] The oncolytic virus VRT106 was produced and supplied by Guangzhou Weirongte Pharmaceutical Technology Co., Ltd., batch number FJ20200915-1, with a concentration and titer of 1.37 × 10⁻⁶. 8 CCID 50 / mL. Take 13 mL of VRT106 and add it to 22.73 mL of PBS to prepare a titer of 5 × 10⁻⁶. 7 CCID 50 / mL, aliquot 1mL / tube, store at -80℃.

[0220] The modeling details of the homologous mouse tumor model are shown in Table 4. One hundred female BALB / C mice, 120 female C57BL / 6 mice, and 20 male C57BL / 6 mice (5-8 weeks old) were used in the experiment. The experimental animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. or Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and the animal husbandry and VRT106 efficacy experiments were conducted by Crown Bioscience.

[0221] Table 4 Mouse modeling information

[0222]

[0223] Pharmacodynamic evaluation of VRT106 in a homologous mouse tumor model:

[0224] Once the cells entered the logarithmic growth phase and reached a sufficient number, they were collected and subcutaneously inoculated with cell suspension in homologous mice (modeling conditions are shown in Table 4). After tumor formation, the tumors grew to an average volume of 80–120 mm. 3 The mice were randomly grouped and given medication based on tumor size and body weight.

[0225] Twelve types of mouse cell lines were cultured in different culture media (see Table 1). Mouse homologous cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0226] When the tumor grows to an average size of 80–120 mm 3 Based on tumor size and mouse weight, mice were randomly assigned to groups for drug administration using StudyDirector™ software. Specific grouping and drug administration treatments are shown in Table 5.

[0227] Table 5. Grouping and Dosing Information for Mice

[0228]

[0229] 1. Observation indicators and sample collection

[0230] (1) Clinical observation arrangements

[0231] General clinical observation: During the administration period, animals in each group were observed once a day, and the frequency could be increased as needed. Observation included, but was not limited to, death, onset of disease, respiration, secretions, feces, other symptoms, and diet and water intake.

[0232] (2)Weight

[0233] Mouse body weight was measured twice a week during tumor formation and administration.

[0234] (3) Tumor measurement

[0235] Tumor growth in mice was measured and recorded twice a week during tumor formation and drug administration.

[0236] (4) Sample collection

[0237] Tissue samples: At the experimental endpoint, samples were taken from the heart, liver, spleen, lungs, kidneys, brain, and tumors. Each tumor fragment was divided into five aliquots; one aliquot was used for pathological examination; the remaining four aliquots were flash-frozen in liquid nitrogen and stored at -80°C for protein Western blot analysis. See Tables 6 and 7 for the sample list.

[0238] Table 6. List of tumor control group samples

[0239] Serial Number cell Animal Number Serial Number cell Animal Number Serial Number cell Animal Number 1 A20 2158 21 EMT6 1765 41 MC38 47722 2 A20 2161 22 EMT6 1769 42 MC38 47697 3 A20 2213 23 EMT6 1773 43 MC38 47669 4 A20 2225 24 EMT6 1799 44 MC38 47723 5 A20 2234 25 EMT6 1806 45 MC38 47713 6 B16BL6 4844 26 H22 48383 46 Pan02 49244 7 B16BL6 4851 27 H22 48392 47 Pan02 49245 8 B16BL6 4862 28 H22 48407 48 Pan02 49248 9 B16BL6 4869 29 H22 48409 49 Pan02 49327 10 B16BL6 4902 30 H22 48457 50 Pan02 49335 11 B16F10 45836 31 Hepa 1 / 6 6542 51 Renca 7850 12 B16F10 45837 32 Hepa 1 / 6 6612 52 Renca 7855 13 B16F10 45876 33 Hepa 1 / 6 6513 53 Renca 7863 14 B16F10 45883 34 Hepa 1 / 6 6549 54 Renca 7875 15 B16F10 45806 35 Hepa 1 / 6 6619 55 Renca 7903 16 CT26 45258 36 LLC 47425 56 RM1 7684 17 CT26 45331 37 LLC 47426 57 RM1 7705 18 CT26 45268 38 LLC 47458 58 RM1 7722 19 CT26 45332 39 LLC 47463 59 RM1 7724 20 CT26 45298 40 LLC 47473 60 RM1 7734

[0240] Table 7. Sample list of tumor drug administration group

[0241]

[0242]

[0243] 2. Experimental Methods:

[0244] (1) Protein sample extraction

[0245] A. Prepare 1.5mL centrifuge tubes, and add 3 grinding beads and 300μL T-PER protein lysis buffer to each centrifuge tube.

[0246] B. Take about 30mg of tumor tissue and add it to a prepared 1.5mL centrifuge tube. Grind the tissue using a fully automated high-speed grinder at 60Hz for 60 seconds per cycle, for a total of 3 cycles.

[0247] C. Centrifuge the ground tissue lysis solution at 12,000 rpm for 5 minutes and collect the supernatant.

[0248] (2) Protein sample concentration determination and denaturation

[0249] Protein concentration was detected using the BCA assay kit (Thermo Fisher, 23227), and the procedure was performed according to the instructions.

[0250] (3) Western Blot

[0251] A. Prepare the electrophoresis buffer, remove the protective film from the pre-cast gel base, and correctly assemble the gel plates and electrophoresis tank. (The text abruptly ends here, likely due to an incomplete sentence or missing information.)

[0252] Inject electrophoresis buffer into the cavity, remove the comb, fill with buffer again, and let stand to observe for leakage; if there is no leakage, then place on the gel plate.

[0253] Add electrophoresis solution to half the height of the tank and assemble the electrophoresis tank;

[0254] B. Add 10 μL of sample to the precast gel according to the sample addition order, and add 4 μL and 2 μL of marker on both sides respectively.

[0255] C. After sample loading is complete, set a constant voltage of 120V and incubate for 60 minutes to begin electrophoresis. Observe the generation and direction of bubbles during the experiment.

[0256] D. Cut the PVDF membrane as needed and activate it by soaking it in methanol for 1 min; remove the PVDF membrane from the methanol and transfer it to the equilibration solution for 1 min; after electrophoresis is completed, disassemble the electrophoresis tank, remove the gel plate, carefully pry open the gel plate to remove the gel and place it in clean water; assemble the transfer insert in the order of cathode-sponge-gel-PVDF membrane-sponge-anodide and place it in the transfer apparatus for transfer.

[0257] E. After the transfer is complete, remove the insert, carefully remove the gel to avoid residue, rinse the membrane three times with purified water; absorb excess water, and place it in a rapid blocking solution for 20-30 minutes.

[0258] F. Based on the antibody preparation ratio, the TPRA1 antibody was diluted 1:1000, and the S-actin antibody was diluted 1:1000 to prepare the primary antibody working solution. After blocking, the bands were segmented according to the protein molecular weight and placed in the primary antibody working solution. The mixture was incubated overnight at 4°C on a shaker.

[0259] G. After incubation is complete, aspirate the primary antibody working solution, add TBST, and wash three times on a shaker for 5 minutes each time to remove excess antibody.

[0260] H. Prepare the working solution of the secondary antibody according to the type of primary antibody, remove excess TBST, and incubate on a shaker at room temperature for 1 hour.

[0261] I. After incubation is complete, aspirate the primary antibody working solution, add TBST, and wash three times on a shaker for 5 minutes each time to remove excess antibody.

[0262] J. Mix equal volumes of colorimetric solutions A and B, and add them evenly to the membrane; then perform development and imaging.

[0263] 3. Data Processing

[0264] According to the measurement data, the body weight gain curves and tumor volume growth curves of each group of mice were statistically analyzed and plotted.

[0265] The calculation formula for tumor volume (Tumor volume, TV) is: V = 1 / 2 × a × b 2 , where a and b represent the major axis and minor axis of the tumor length, respectively.

[0266] Relative tumor volume (Relative tumor volume, RTV) = V t / V0. Where V0 is the tumor volume measured before cage separation and drug administration, and V t is the tumor volume at each measurement.

[0267] Tumor growth inhibition rate (Tumor growth inhibition, TGI) = (1 - TmRTV / CmRTV) × 100%, where TmRTV and CmRTV are the average relative tumor volumes at a certain time point in the treatment group and the control group, respectively.

[0268] Pearson correlation analysis was performed on the expression level of TPRA1 and the anti-tumor efficacy of VRT1_{06}. A correlation coefficient r > 0 indicates a positive correlation, r < 0 indicates a negative correlation; |r| ≤ 0.3 indicates no linear correlation, 0.3 ≤ |r| ≤ 0.5 indicates a low-degree linear relationship, 0.5 ≤ |r| ≤ 0.8 indicates a significant linear relationship, |r| > 0.8 indicates a high-degree linear relationship; 0.05 < p < 0.1 indicates that the data has a correlation trend.

[0269] 4. Results:

[0270] (1) Results of the relative tumor growth inhibition rate (Tumor growth inhibition, TGI) of VRT1_{06}:

[0271] The best relative growth inhibition rate of VRT1_{06} on tumor cells was between -11.28% and 45.23%, and the specific values are shown in Table 9.

[0272] Table 8 Summary of the best TGI results of VRT1_{06} on murine cell lines

[0273] cell lines Optimal TGI (%) A20 -11.28 B16BL6 28.55 B16F10 31.97 CT26 30.80 EMT6 27.17 H22 23.47 Hepa 1-6 4.35 LLC 8.88 MC38 45.23 Pan02 24.78 Renca 43.74 RM1 23.59

[0274] (2) Protein expression level of TPRA1 in the control group (WB detection method)

[0275] The basal expression level of TPRA1 protein in the tissues of the control group of each cell was detected by the WB method, and the protein bands were scanned by gray scale using ImageJ software. The results are shown in Tables 10 and 11.

[0276] Table 9. TPRA1 expression levels in the control group of mouse cell lines.

[0277]

[0278]

[0279] Table 10 Summary of the mean gray values ​​of basal expression levels of TPRA1 protein in the control group after normalization.

[0280] control group TPRA1 grayscale mean (after homogenization) A20 0.19 B16BL6 0.33 B16F10 0.31 CT26 0.62 EMT6 0.55 H22 0.84 Hepa 1-6 0.31 LLC 0.35 MC38 0.80 Pan02 1.01 Renca 0.86 RM1 0.58

[0281] (3) Correlation analysis between the baseline expression level of TPRA1 in the control group (WB assay) and the best efficacy of VRT106

[0282] The basal expression level of TPRA1 in the control groups of 12 cell lines (WB assay) showed a significant linear relationship with the optimal therapeutic effect of VRT106 (r = 0.63, P = 0.03). Results are shown below. Figure 9 .

[0283] 5. Conclusion

[0284] The basal expression level of TPRA1 protein in the mouse cell line control group was significantly linearly positively correlated with the antitumor efficacy of VRT106. The higher the basal level of TPRA1 protein, the better the antitumor efficacy of VRT106.

Claims

1. Application of oncolytic viruses in the preparation of antitumor drugs, wherein the tumor is a TPRA1-highly expressed tumor.

2. Application of TPRA1 promoter in the preparation of oncolytic virus antitumor synergists or drug resistance reversal agents; Preferably, the oncolytic virus antitumor synergist or drug resistance reversal agent further comprises other substances that enhance the antitumor efficacy of oncolytic viruses or reverse drug resistance; Preferably, the form of the oncolytic virus antitumor potentiator or drug resistance reversal agent is suitable for administration by one or more of the following methods: oral, injection, gold-coated gene gun bombardment, reproduction-deficient bacteria carrying plasmid DNA, replication-deficient adenovirus carrying target DNA or protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, or transdermal administration. Preferably, the dosage form of the oncolytic virus antitumor synergist or drug resistance reversal agent includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets.

3. Application of oncolytic viruses and TPRA1 promoters in the preparation of antitumor drugs; Preferably, the drug further comprises other antitumor drugs.

4. An antitumor composition comprising an oncolytic virus and a TPRA1 promoter; Preferably, the antitumor composition further includes a pharmaceutically acceptable carrier or excipient; Preferably, the antitumor composition further includes other antitumor drugs; Preferably, the form of the antitumor composition is suitable for administration by one or more methods selected from the group consisting of: oral, injection, gold-coated gene gun bombardment, reproduction-deficient bacteria carrying plasmid DNA, replication-deficient adenovirus carrying target DNA or protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration. Preferably, the dosage form of the antitumor composition includes at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets. Preferably, the antitumor composition further includes instructions for use.

5. An antitumor drug kit comprising: an oncolytic virus and a TPRA1 promoter; wherein the oncolytic virus and the TPRA1 promoter exist independently; Preferably, the oncolytic virus and the TPRA1 promoter each independently comprise a pharmaceutically acceptable carrier or excipient; Preferably, the oncolytic virus and the TPRA1 promoter are each in a form that makes them suitable for administration by one or more of the following methods: oral, injection, gold-coated gene gun bombardment, reproduction-deficient bacteria carrying plasmid DNA, replication-deficient adenovirus carrying target DNA or protein encoded by the target gene, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, or transdermal administration. Preferably, the dosage forms of the oncolytic virus and the TPRA1 promoter each independently include at least one of the following: capsules, tablets, microcapsules, injections, suppositories, sprays, powders, soft capsules, drop pills, honey pills, pills, granules, honey-infused pastes, sustained-release preparations, oral liquid preparations, chewable tablets, oral tablets, transdermal patches, and effervescent tablets. Preferably, the oncolytic virus and the TPRA1 promoter are administered simultaneously or in any sequential order. Preferably, the antitumor drug kit also includes an instruction manual; Preferably, the antitumor drug kit also includes other antitumor drugs.

6. An antitumor drug delivery system, comprising: Detection of TPRA1 substances and oncolytic viruses.

7. The application of the substance used to detect TPRA1 in the preparation of a product, wherein the product is used in at least one of a1) to a4): a1) Evaluation of the antitumor efficacy of oncolytic viruses; a2) Prognostic assessment of oncolytic virus antitumor activity; a3) Selection of tumor treatment options; a4) Tumor diagnosis; Preferably, the substance for detecting TPRA1 includes a substance for quantitative detection of TPRA1; Preferably, the substance for detecting TPRA1 includes a substance for detecting TPRA1 at the gene level and / or protein level; Preferably, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: immunohistochemistry, Western blotting, Northern blotting, PCR, microarray, nucleic acid sequencing, and amino acid sequencing. Preferably, the immunohistochemical method is selected from at least one of the following: immunofluorescence assay, immunoenzyme labeling assay, and immunogold assay. Preferably, the substance used to detect TPRA1 is selected from one or more of the following: substances specific to TPRA1, TPRA1-specific probes, gene chips, and PCR primers; Preferably, the substance specific to TPRA1 includes any one of b1) to b3): b1) Antibodies that specifically bind to TPRA1; b2) A ligand protein or polypeptide that specifically binds to TPRA1; b3) Specifically recognizes non-protein compounds of TPRA1; Preferably, the antibody comprises at least one of polyclonal antibody, monoclonal antibody, single-chain antibody, functional antibody fragment, antibody Fab region, nanobody, chimeric antibody, and multispecific antibody; Preferably, the product further includes substances that detect other biomarkers used for evaluating the efficacy of oncolytic virus antitumor therapy, assessing the prognosis of oncolytic virus antitumor therapy, selecting treatment regimens, and / or diagnosing tumors; Preferably, the product comprises at least one of reagents, reagent kits, test strips, chips, and systems; Preferably, the test sample of the product is selected from at least one of the body fluids, tissues, cells, and excrement of the subject to be tested; Preferably, the body fluid includes at least one of blood and lymph. Preferably, the blood includes at least one of serum, plasma, dried blood spots, and whole blood; Preferably, the tissue comprises tumor tissue; Preferably, the excrement contains at least one of urine, feces, and tears; Preferably, the object to be tested includes mammals; Preferably, the object to be tested includes humans.

8. The method described in any one of c1) to c2): c1) A method for constructing a model with good anti-tumor efficacy of oncolytic virus, thereby increasing the content and / or activity of TPRA1 in the model; c2) A method for constructing a model of poor antitumor efficacy of oncolytic viruses, by reducing the content and / or activity of TPRA1 in the model.

9. The application according to any one of claims 1 to 3, the antitumor composition according to claim 4, the antitumor drug kit according to claim 5, the antitumor drug delivery system according to claim 6, the application according to claim 7, and / or the method according to claim 8, characterized in that: The oncolytic virus includes at least one of the following: alpha virus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, Coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, poliovirus, influenza virus, mumps virus, and parvovirus. Preferably, the alpha virus is selected from the M1 virus; Preferably, the tumor is a TPRA1-highly expressed tumor; Preferably, the tumors with high TPRA1 expression include, but are not limited to, glioblastoma, bladder cancer, prostate cancer, glioma, lung cancer, colorectal cancer, rectal cancer, liver cancer, breast cancer, cervical cancer, melanoma, pancreatic cancer, nasopharyngeal carcinoma, gastric cancer, bladder urothelial carcinoma, acute myeloid leukemia, head and neck squamous cell carcinoma, and endometrial cancer.

10. The application according to any one of claims 2 to 3, the antitumor composition according to claim 4, and / or the antitumor pharmaceutical kit according to claim 5, characterized in that: The TPRA1 promoter includes at least one of the following: substances that increase TPRA1 content or expression level, substances that enhance TPRA1 activity, and substances that delay TPRA1 metabolism. Preferably, the TPRA1 promoter comprises at least one of the following: a naturally purified substance, a modified naturally purified substance, a semi-synthetic substance, and a chemically synthesized substance; Preferably, the TPRA1 promoter comprises at least one of a chemical drug, a peptide drug, a protein drug, and a gene therapy drug; Preferably, the TPRA1 promoter comprises at least one of the following: TPRA1-related biomaterials, exogenous TPRA1, nanoparticles carrying the TPRA1 gene, PEG-modified TPRA1 protein, TPRA1 protein microspheres, liposomes encapsulating the TPRA1 gene or protein, extracellular vesicles carrying the TPRA1 gene or protein, and TPRA1 precursor protein or conjugate or complex that can be converted into TPRA1 in vivo. Preferably, the biomaterial associated with TPRA1 comprises at least one of d1) to d16): d1) Nucleic acid molecules encoding TPRA1; d2) An expression cassette containing the nucleic acid molecule described in d1); d3) A carrier containing the nucleic acid molecules described in d1); d4) A carrier containing the expression box described in d2); d5) Transgenic cell lines containing the nucleic acid molecules described in d1); d6) Transgenic cell lines containing the expression cassette described in d2); d7) Transgenic cell lines containing the vector described in d3); d8) Transgenic cell lines containing the vector described in d4); d9) Microorganisms containing the nucleic acid molecules described in d1); d10) Microorganisms containing the expression cassette described in d2); d11) Microorganisms containing the carrier described in d3); d12) Microorganisms containing the carrier described in d4); d13) Viruses containing the nucleic acid molecules described in d1); d14) A virus containing the expression box described in d2); d15) A virus containing the vector described in d3); d16) A virus containing the vector described in d4); Preferably, the TPRA1 promoter is a tumor-targeting TPRA1 promoter.

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