Use of coronavirus 3CL protease for preventing or treating tumors

Abstract

A use of a coronavirus 3CL protease or a nucleic acid molecule encoding the coronavirus 3CL protease in preparation of drugs for preventing and / or treating tumors.

Description

Use of 3CL protease of coronavirus for preventing or treating tumor

[0001] This application is based on and claims priority to the application CN application number 202311153380.8, filed on September 7, 2023, the disclosure of which is incorporated herein in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of medical biotechnology, and specifically relates to the use of 3CL protease of coronavirus or a nucleic acid molecule encoding the same in the preparation of a medicament for preventing and / or treating tumor. BACKGROUND

[0003] Cancer is a disease characterized by uncontrolled cell growth due to genetic mutations or abnormalities in regulation, and shows strong spatiotemporal heterogeneity according to the site of occurrence. Cancer treatment drugs not only need to overcome this heterogeneity, but also need to achieve the purpose of killing cancer cells selectively with minimal toxicity to normal cells. In the development of anti-cancer drugs, how to reduce the toxicity of current clinical drugs to the body while maintaining the broad spectrum of the drug's anti-cancer spectrum has always been a challenging problem. For different types of cancer, including solid tumors and non-solid tumors, current clinical treatment options usually include one or a combination of surgical operation, chemotherapy, radiotherapy, bone marrow / stem cell transplantation, hormone therapy, targeted therapy, and immunotherapy. These seven major types of anti-tumor therapies can achieve the purpose of eliminating tumors to some extent, but patients may develop drug resistance and relapse, and patients may experience pain or irreversible damage to the body during multiple treatments. New and effective anti-tumor treatment methods are urgently needed to be developed and applied.

[0004] Gene drug therapy and gene vaccination belong to the rapidly developing anti-tumor strategies in recent years, which provide specific and individualized options for the treatment and prevention of various tumors. At present, there are only a few gene drug therapies approved for the field of tumors worldwide, such as Gendicine for the treatment of head and neck squamous cell carcinoma, Imlygic for the treatment of melanoma lesions that cannot be completely resected by surgery, Kymriah for the treatment of relapsed or refractory acute lymphoblastic leukemia in patients aged 3-25 years, and Yescarta for the treatment of relapsed or refractory diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma in adults after two or more systemic treatments. These four treatment methods represent gene drug therapies that use viruses, modified immune cells or other delivery vectors to bring suicide genes, immune-regulating genes into cells and insert them into the existing genome, thereby eliminating the effects of harmful mutations in genes or stimulating the patient's anti-tumor immune response to achieve anti-tumor purposes. In addition to this way of introducing exogenous genes, oligonucleotides can also be delivered into tumor cells by delivery vectors to inhibit or interfere with endogenous genes to achieve therapeutic purposes.

[0005] In addition, researchers have developed a variety of DNA vaccines or RNA vaccines encoding tumor antigens for the prevention and treatment of tumors, which belong to the field of gene vaccines. Related studies have shown that messenger RNA vaccines have become a promising cancer immunotherapy strategy. After vaccination with naked vaccines or drug-loaded mRNA vaccines, tumor antigens will be expressed in antigen-presenting cells (APCs), thereby promoting the activation of APCs and innate / adaptive immunity. mRNA cancer vaccines have the advantages of high efficiency, safety, great development potential and low production cost, leading other conventional vaccine platforms. As an immunotherapy approach, tumor vaccines are highly dependent on the body's own immune system, but the immune system of cancer patients is often suppressed and has low defense function, so there are certain limitations in the application of tumor vaccines.

[0006] mRNA therapy in gene therapy has been explored as the most safe and effective method for treating tumors. mRNA is composed of four nucleotides, abbreviated as letters A, U, C and G. Cells interpret the order of these nucleotides, and if they cannot read the information encoded by mRNA, they cannot translate it into a protein with specific structure and function, and mRNA is eventually metabolized and degraded by cells, without producing the corresponding biological effects. If the opposite occurs, the body will only manufacture the proteins it needs at the right time and in the right place. Therefore, mRNA drug therapy is considered a precise and safe anti-tumor strategy.

[0007] Therefore, there is still a great medical need for anti-tumor mRNA drugs to improve patient survival rates and bring more therapeutic benefits to patients themselves, their families and society.

[0008] Content of the application

[0009] The present inventors surprisingly found that the 3CL protease of a coronavirus (e.g., SARS-CoV-2) has a broad-spectrum, significant tumor cell-killing ability. Based on this finding, the present inventors developed a method for preventing and / or treating tumors based on the 3CL protease or a nucleic acid molecule encoding the 3CL protease. The therapeutic drug (e.g., nucleic acid drug) based on the 3CL protease or a nucleic acid molecule encoding the 3CL protease provided herein is expected to expand the scope of benefits for cancer patients, improve patient survival rate, and bring more therapeutic benefits to the patients themselves, their families, and society.

[0010] The first aspect of the present application provides use of a 3CL protease of a coronavirus or a nucleic acid molecule encoding the 3CL protease of the coronavirus in the preparation of a medicament for preventing and / or treating tumors in a subject.

[0011] In certain embodiments, the coronavirus is SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0012] In certain embodiments, the coronavirus is SARS-CoV-2. In certain embodiments, the SARS-CoV-2 includes not only the original strain of SARS-CoV-2, but also its variants, such as the Alpha, Beta, Gamma, Delta, and Omicron variants.

[0013] As readily understood by those skilled in the art, the “3CL protease” described herein can be directly derived from the coronavirus (e.g., SARS-CoV, MERS-CoV, or SARS-CoV-2), or obtained by artificially modifying (e.g., introducing mutations or modifications) the 3CL protease directly derived from the coronavirus, as long as it retains the biological activity (e.g., tumor cell-killing activity, or oncolytic activity) of the 3CL protease from which it is derived. In addition, the 3CL protease described herein can be a full-length 3CL protease or an active fragment thereof (e.g., an active fragment having tumor cell-killing activity or oncolytic activity).

[0014] In certain embodiments, the 3CL protease is derived from SARS-CoV (e.g., SARS coronavirus BJ01 strain (Genbank: AY278488.2), SARS coronavirus BJ03 strain (Genbank: AY278490.3), SARS coronavirus Tor2 strain (Genbank: AY274119.3)). In certain embodiments, the 3CL protease is as set forth in any one of Uniprot: P0C6U8, P0C6X7, B8Q8V7.

[0015] In certain embodiments, the 3CL protease is derived from MERS-CoV (e.g., Middle East respiratory syndrome coronavirus isolate Bisha_1_2012 strain (Genbank: KF600620.1), Middle East respiratory syndrome-related coronavirus isolate MERS-CoV / dromedary camel / Egypt / NC282 / 2015 strain (Genbank: OP654178.1), Middle East respiratory syndrome coronavirus isolate Riyadh_2_2012 strain (Genbank: KF600652.1)). In certain embodiments, the 3CL protease is as set forth in any one of Uniprot: K9N638, K9N7C7, A0A023W112.

[0016] In certain embodiments, the 3CL protease is derived from SARS-CoV-2 (e.g., Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 strain (Genbank: NC_045512.2), Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2 / human / MEX / CMX_2019 / 2022 strain (Genbank: OR157051.1), Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2 / human / LBY / BTRC Libya SARS-CoV-2 WGS-33 / 2021 strain (Genbank: MZ054889.1)). In certain embodiments, the 3CL protease is as set forth in any one of Uniprot: P0DTC1, P0DTD1, A0A8B1JPK4.

[0017] As will be readily understood by one skilled in the art, the 3CL protease can further contain additional polypeptides for the purposes of, e.g., expression, purification, and / or tracking of the protein.

[0018] In certain embodiments, the additional polypeptide is optionally linked to the N- or C-terminus of the 3CL protease via a linker (e.g., a peptide linker or a small organic molecule linker). Such linkers (e.g., peptide linkers) are well known in the art, examples of which include, but are not limited to, a peptide linker comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., Gly or Ser). In certain embodiments, the peptide linker is flexible. In certain embodiments, a flexible peptide linker can be advantageous in that it is able to link two protein / polypeptide components and maintain their respective activities and functions. Such peptide linkers include, but are not limited to, (GGGGS) n .

[0019] In certain embodiments, the additional polypeptide is selected from a tag, a signal peptide or a leader peptide, a detectable label (e.g., luciferase (fluc), green fluorescent protein (GFP)), a polypeptide sequence encoding an antibody, a polypeptide sequence encoding a localization or bioactive protein (e.g., PD1, PD-L1, EGFR, etc.), or any combination thereof.

[0020] In certain embodiments, the 3CL protease has an amino acid sequence selected from the group consisting of:

[0021] i) a sequence as set forth in SEQ ID NO: 6, 10, or 13;

[0022] ii) a sequence having one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions compared to the sequence as set forth in SEQ ID NO: 6, 10, or 13; and

[0023] iii) a sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 6, 10, or 13.

[0024] In certain embodiments, the substitution in ii) is a conservative substitution.

[0025] The sequence as set forth in SEQ ID NO: 6, 10, or 13 comprises a start codon (e.g., AUG / ATG) encoded amino acid (e.g., methionine (Met)) at its N-terminus. Those skilled in the art understand that, during the process of producing a protein by genetic engineering, the first amino acid of the polypeptide chain produced is often the start codon encoded amino acid (e.g., Met) due to the action of the start codon. The 3CL protease of the present application encompasses not only the amino acid sequence comprising a start codon encoded amino acid (e.g., Met) at its N-terminus, but also the amino acid sequence not comprising a start codon encoded amino acid (e.g., Met) at its N-terminus. Therefore, the sequence not comprising a start codon encoded amino acid (e.g., Met) at the N-terminus of the above-mentioned amino acid sequence is also within the scope of the present application.

[0026] In certain embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0027] In certain embodiments, the nucleic acid molecule is single-stranded or double-stranded.

[0028] In certain embodiments, the nucleic acid molecule is an RNA molecule, preferably an mRNA molecule.

[0029] In certain embodiments, the nucleic acid molecule comprises a coding sequence of the 3CL protease.

[0030] In certain embodiments, the nucleic acid molecule further comprises one or more selected from the group consisting of 5’UTR, Kozak sequence, start codon, stop codon, 3’UTR, poly-A tail.

[0031] In certain embodiments, the nucleic acid molecule comprises, in order from the 5’ end to the 3’ end: a 5’ UTR, a Kozak sequence, a start codon, a coding sequence for the 3CL protease, a stop codon, a 3’ UTR, a poly-A tail.

[0032] In certain embodiments, the nucleic acid molecule consists of, in order from the 5’ end to the 3’ end: a 5’ UTR, a Kozak sequence, a start codon, a coding sequence for the 3CL protease, a stop codon, a 3’ UTR, a poly-A tail.

[0033] In certain embodiments, the coding sequence for the 3CL protease is subject to codon degeneracy. In certain embodiments, the coding sequence for the 3CL protease is codon optimized according to the codon bias of the host cell (e.g., human cell) or is unoptimized.

[0034] In certain embodiments, the coding sequence for the 3CL protease has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 8, or 11. In certain embodiments, the coding sequence for the 3CL protease has the sequence set forth in SEQ ID NO: 1, 8, or 11.

[0035] In certain embodiments, the 5’ end of the nucleic acid molecule is subject to a 5’ modification (e.g., CAP Type 0, CAP Type 1, CAP Type 2). In certain embodiments, the 5’ end of the nucleic acid molecule is subject to a 5’ Cap Type 1 modification;

[0036] In certain embodiments, the nucleic acid molecule contains one or more N1-methyl pseudouridine (m1 ψ) modifications. In certain embodiments, all or a portion of the uracil nucleotides in the nucleic acid molecule are replaced with N1-methyl-pseudouracil nucleotides.

[0037] In certain embodiments, the 3’ end of the coding sequence for the 3CL protease is subject to one or more stop codons.

[0038] In certain embodiments, the 5’ UTR is selected from the 5’ UTR derived from mRNA 1273 from Moderna, Inc.

[0039] In certain embodiments, the 3’ UTR is selected from the 3’ UTR derived from mRNA 1273 from Moderna.

[0040] In certain embodiments, the 5’ UTR has a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 2. In certain embodiments, the 5’ UTR has the nucleotide sequence set forth in SEQ ID NO: 2.

[0041] In certain embodiments, the 3’ UTR has a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 4. In certain embodiments, the 3’ UTR has the nucleotide sequence set forth in SEQ ID NO: 4.

[0042] In certain embodiments, the Kozak sequence has the sequence set forth in SEQ ID NO: 3.

[0043] In certain embodiments, the poly-A tail comprises one or more poly-adenylate sequences, each independently consisting of 20-120 consecutive adenylates; preferably, the poly-A tail comprises multiple poly-adenylate sequences, and the adjacent poly-adenylate sequences are connected by a spacer sequence comprising non-A. In certain embodiments, the poly-A tail has the sequence set forth in SEQ ID NO: 5.

[0044] In certain embodiments, the nucleic acid molecule has a nucleotide sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 7, 9, or 12. In certain embodiments, the nucleic acid molecule has the sequence set forth in SEQ ID NO: 7, 9, or 12.

[0045] The second aspect of the application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a 3CL protease, wherein the 3CL protease is as defined in the first aspect of the application.

[0046] In certain embodiments, the isolated nucleic acid molecule is a nucleic acid molecule as defined in the first aspect of the application. In certain embodiments, the isolated nucleic acid molecule encodes a nucleic acid molecule as defined in the first aspect of the application.

[0047] The third aspect of the application provides a vector comprising the isolated nucleic acid molecule of the second aspect of the application.

[0048] In certain embodiments, the vector of the application is, for example, a plasmid, a cosmid, a phage, a lentivirus, and the like.

[0049] In certain embodiments, the vector is a cloning vector or an expression vector.

[0050] In certain embodiments, the vector is capable of expressing the 3CL protease as defined in the first aspect in vivo in a subject.

[0051] In certain embodiments, the vector is a DNA vector or an RNA vector.

[0052] The fourth aspect of the application provides a delivery composition comprising a delivery vector, and one or more selected from the group consisting of the 3CL protease of the first aspect of the application, the isolated nucleic acid molecule of the second aspect of the application, the vector of the third aspect of the application.

[0053] In certain embodiments, the delivery vector is used to encapsulate, carry, or deliver the 3CL protease, the isolated nucleic acid molecule, or the vector.

[0054] In certain embodiments, the delivery vector comprises a non-viral vector, a viral vector, and a virosome (VLP) vector between the viral vector and the non-viral vector.

[0055] In certain embodiments, the non-viral vector comprises, but is not limited to, a cationic liposome, a lipid nanoparticle, a lipid polymer, an artificial microsphere, a micelle, a lipid-polymer hybrid system, an extracellular vesicle, a natural or engineered exosome, and the like. In certain embodiments, the viral vector comprises, but is not limited to, an adeno-associated virus, a lentivirus, an adenovirus, a retrovirus, a vaccinia virus, a measles virus, a herpes simplex virus, an alphavirus, a vesicular stomatitis virus, an influenza virus, and the like. In certain embodiments, the virosome vector comprises, but is not limited to, a VLP-mRNA delivery vector utilizing the principle of specific recognition of mRNA stem-loop structures by phage coat proteins.

[0056] In certain embodiments, the delivery vector is a lipid nanoparticle.

[0057] The fifth aspect of the present application provides a pharmaceutical composition comprising the 3CL protease of the first aspect of the present application, the isolated nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, and / or the delivery composition of the fourth aspect of the present application, and a pharmaceutically acceptable carrier and / or excipient.

[0058] In certain embodiments, the pharmaceutical composition further comprises other anti-tumor drugs.

[0059] In certain embodiments, the other anti-tumor drugs are selected from small molecule drugs, biological macromolecule drugs, chimeric antigen receptor drugs, and antibody conjugate drugs.

[0060] In certain embodiments, the pharmaceutical composition is administered orally, by spray inhalation, rectally, nasally, buccally, vaginally, topically, parenterally, such as by subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, endotracheally, by surgical implantation, transdermally, by local injection, by hyper-velocity injection / bombardment, or by means of an external reservoir. In certain embodiments, the pharmaceutical composition is administered orally, intraperitoneally, intrathoracically, or intravenously.

[0061] The sixth aspect of the present application provides a method of inhibiting tumor cells in vivo or in vitro, comprising contacting or delivering into the tumor cells the 3CL protease of the first aspect of the present application, the isolated nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, and / or the delivery composition of the fourth aspect of the present application, or the pharmaceutical composition of the fifth aspect of the present application.

[0062] The seventh aspect of the present application provides use of the isolated nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, the delivery composition of the fourth aspect of the present application, or the pharmaceutical composition of the fifth aspect of the present application, in the manufacture of a medicament for preventing and / or treating tumors.

[0063] The eighth aspect of the present application provides use of the 3CL protease of the first aspect of the present application, the isolated nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, the delivery composition of the fourth aspect of the present application, or the pharmaceutical composition of the fifth aspect of the present application, in preventing and / or treating tumors.

[0064] The ninth aspect of the present application provides a method of preventing and / or treating a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the 3CL protease of the first aspect of the present application, the isolated nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, the delivery composition of the fourth aspect of the present application, or the pharmaceutical composition of the fifth aspect of the present application. In certain embodiments, the method further comprises administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; alternatively, the second therapy can be applied simultaneously, separately, or sequentially.

[0065] The tenth aspect of the present application provides a medicament or composition for use in preventing and / or treating a tumor in a subject, comprising the 3CL protease of the first aspect of the present application, the isolated nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, the delivery composition of the fourth aspect of the present application, or the pharmaceutical composition of the fifth aspect of the present application.

[0066] The eleventh aspect of the present application provides the 3CL protease of the first aspect of the present application, the isolated nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, the delivery composition of the fourth aspect of the present application, or the pharmaceutical composition of the fifth aspect of the present application, for use in preventing and / or treating a tumor in a subject.

[0067] In certain embodiments, the tumor is a solid tumor or a hematological tumor (e.g., leukemia, lymphoma, myeloma).

[0068] In certain embodiments, the tumor is a solid tumor.

[0069] In certain embodiments, the tumor is selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, melanoma, intestinal cancer, kidney cancer, cervical cancer, osteosarcoma, prostate cancer, brain glioma, and lung cancer.

[0070] In certain embodiments, the tumor cell is a cell of a solid tumor or a hematological tumor (e.g., leukemia, lymphoma, myeloma).

[0071] In certain embodiments, the tumor cell is selected from the group consisting of a cell of pancreatic cancer, gastric cancer, liver cancer, melanoma, intestinal cancer, kidney cancer, cervical cancer, osteosarcoma, prostate cancer, brain glioma, and lung cancer.

[0072] In certain embodiments, the pancreatic cancer subtypes include, but are not limited to, acinar cell carcinoma, adenosquamous carcinoma, squamous cell carcinoma, signet ring cell carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with giant cells, ampullary carcinoma, pancreatic neuroendocrine tumor, etc. In certain embodiments, the gastric cancer subtypes include, but are not limited to, cardia cancer, corpus cancer, pyloric cancer. In certain embodiments, the liver cancer subtypes include, but are not limited to, hepatocellular carcinoma, hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, intrahepatic cholangiocarcinoma, angiosarcoma, hepatoblastoma, secondary liver cancer, benign liver tumor. In certain embodiments, the melanoma subtypes include, but are not limited to, local melanoma, local regional melanoma, distant metastatic melanoma. In certain embodiments, the intestinal cancer subtypes include, but are not limited to, colon cancer and rectal cancer, including, in particular, adenocarcinoma, undifferentiated carcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, carcinoid, mucinous carcinoma, etc. In certain embodiments, the kidney cancer subtypes include, but are not limited to, clear cell carcinoma, papillary cell carcinoma, chromophobe cell carcinoma, low potential for malignancy multilocular cystic renal cell tumor, collecting duct carcinoma, renal medulla carcinoma. In certain embodiments, the cervical cancer subtypes include, but are not limited to, squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma. In certain embodiments, the bone sarcoma subtypes include, but are not limited to, telangiectatic osteosarcoma, small round cell osteosarcoma, fibrohistiocytic osteosarcoma, intramedullary well-differentiated osteosarcoma, multicentric osteosarcoma, intracortical osteosarcoma, parosteal osteosarcoma, dedifferentiated parosteal osteosarcoma, periosteal osteosarcoma, highly malignant surface osteosarcoma. In certain embodiments, the prostate cancer subtypes include, but are not limited to, adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, adenosquamous carcinoma. In certain embodiments, the brain glioma subtypes include, but are not limited to, PAs, astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligoastrocytoma, glioblastoma. In certain embodiments, the lung cancer subtypes include, but are not limited to, small cell lung cancer and non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, carcinoid, etc.).

[0073] Further, according to the progress of each type of tumor, including but not limited to early, intermediate, advanced or I, II, III, IV stage tumors.

[0074] The pharmaceutical, composition, or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical, composition, or pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the pharmaceutical, composition, or pharmaceutical composition of the present application in the required amount in an appropriate solvent with one or more of the other ingredients, including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof, and filtering sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum or freeze-drying) for ease of storage and use. Such lyophilized powders can be reconstituted into sterile solutions prior to use.

[0075] In addition, the 3CL protease, nucleic acid molecule encoding the 3CL protease, vector, or delivery composition of the present application can be present in the pharmaceutical, composition, or pharmaceutical composition in unit dosage form to facilitate administration.

[0076] The pharmaceutical, composition, or pharmaceutical composition of the present application can be administered by any suitable route known in the art, including, but not limited to, oral, spray inhalation, rectal, nasal, buccal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, endotracheal installation, surgical implantation, transdermal delivery, local injection, hyper-velocity injection / bombardment, or by means of an ex-plant reservoir. For many therapeutic uses, the preferred route / mode of administration is oral, intraperitoneal, or intravenous. The skilled artisan will appreciate that the route and / or mode of administration will vary depending upon the intended goal.

[0077] The drug, composition, or pharmaceutical composition of the present application can include a "therapeutically effective amount" or a "prophylactically effective amount" of the 3CL protease, nucleic acid molecule encoding the 3CL protease, vector, or delivery composition of the present application. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to prevent, retard, or delay the development of a disease. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to cure or at least partially arrest the disease and its complications in an individual already suffering from a disease. The therapeutically effective amount of the 3CL protease, nucleic acid molecule encoding the 3CL protease, vector, or delivery composition of the present application can vary according to factors such as the disease severity, the overall state of the patient's own immune system, the patient's general condition such as age, body weight, and gender, the mode of administration of the drug, and other therapies being administered concurrently, and the like.

[0078] In the present application, the dosing regimen can be adjusted to achieve the best response for the intended purpose (e.g., therapeutic or prophylactic response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0079] In certain embodiments, the isolated nucleic acid molecule, e.g., RNA molecule, can be prepared by in vitro transcription methods, or can be prepared using one or more combinations of oligonucleotide synthesizers, host cell expression, or any other method known in the art.

[0080] Specifically, an in vitro transcription template plasmid can be constructed first, and in vitro transcription can be performed by mimicking the in vivo transcription environment.

[0081] The basic structure of the in vitro transcription template plasmid contains multiple polynucleotide functional regions. A promoter sequence can be included at the 5' end of the plasmid template to be responsible for the initiation of in vitro or in vivo transcription. One or more complete or incomplete 5' untranslated regions (UTRs) can be included at the 5' end of the plasmid template, such as native 5' UTRs and non-native 5' UTRs encoding mRNA, including but not limited to heterologous 5' UTRs or synthetic 5' UTRs. One or more signal sequence regions can also be included at the 5' end of the plasmid template. One or more complete or incomplete 3' UTRs can be included at the 3' end of the plasmid template, such as native 3' UTRs and non-native 3' UTRs encoding mRNA, including but not limited to heterologous 3' UTRs or synthetic 3' UTRs. An open reading frame (ORF) that can encode at least one target polypeptide or protein should be included between the 5' UTR and the 3' UTR of the plasmid template. A 3' tailing sequence can also be included at the 3' end of the plasmid template. The 3' tailing sequence can be, but is not limited to, a poly-A tail, a poly-A-G tetrad, and / or a stem-loop sequence. The regulatory features of the UTRs can be incorporated into the polynucleotides of the present application to enhance the stability of the molecules.

[0082] The 5' UTR, 3' UTR region nucleotide sequences provided herein are applied in some embodiments, but do not limit the scope of protection of the present application. Those skilled in the art will understand that, in certain embodiments, commonly used 5' UTR, 3' UTR sequence variants can be added to the in vitro transcription template plasmid, and then transcribed to obtain mRNA including the corresponding codon. One or more nucleotides in the 5' UTR, 3' UTR sequence variant can be added or removed to the end. Further, the 5' UTR, 3' UTR sequence variant can be placed in the same direction or position of the transcript selected from, so that the 5' UTR, 3' UTR sequence can be reversed, lengthened, shortened, and made with one or more other 5' UTR, 3' UTR sequences. The 5' UTR, 3' UTR sequence applied with different nucleotide combinations can achieve the purpose of the method for in vitro transcription and translation of the optimized mRNA molecule of the present application, and is within the scope of protection of the present application.

[0083] In other aspects, the 5' UTR region and the 3' UTR can be derived from different species. The UTR region can also include a translation enhancer element (TEE).

[0084] The in vitro transcription template plasmid containing the isolated nucleic acid molecule (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequence) described in the present application can comprise a promoter element, such as a T7 promoter. In certain embodiments, the T7 sequence is preferably 5'-TAATACGACTCACTATA-3'. As non-limiting examples, other promoter sequences that can replace the T7 promoter sequence and enable efficient transcription of functional RNA under the guidance of an RNA polymerase are within the scope of protection of the present application, such as a Sp6 promoter, a Syn5 promoter, or a promoter that can be used for synthesis of RNA of the present application.

[0085] The in vitro transcription template plasmid containing the isolated nucleic acid molecule (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequence) described in the present application and the isolated nucleic acid molecule can also comprise an element similar to or similar to the start codon region. In certain embodiments, the nucleotide sequence of the start codon is preferably ATG, and the corresponding start codon sequence in the RNA is AUG, which can achieve high translation efficiency. As non-limiting examples, translation of a polynucleotide can start from a codon that is not the start codon AUG, i.e., an alternative start codon. Therefore, the nucleotide sequence of the start codon can also be, but is not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, ATA / AUA, ATT / AUU, TTG / UUG.

[0086] The in vitro transcription template plasmids comprising the isolated nucleic acid molecules (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequences) described herein and the isolated nucleic acid molecules can further comprise at least one or two or a plurality of consecutive stop codon elements located immediately 3’ of the 3’ UTR. The nucleotide sequence of the stop codon can be selected from the group consisting of UGA, UAA, and UAG, and combinations thereof. In certain embodiments, the nucleotide sequence of the stop codon is preferably a UGA. In certain embodiments, the nucleotide sequence of the stop codon is preferably UGA UAA UAG.

[0087] The isolated nucleic acid molecules (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequences) described herein can further comprise a polyadenylation tailing sequence located immediately downstream of the 3’ UTR element to increase the stability of the nucleic acid molecule. The polyadenylation tail can be present or absent in the circular in vitro transcription template plasmid, can be transcribed with the template or can be added post-transcriptionally. The length of the tailing sequence is designed based on the length of the overall nucleic acid molecule or the length of a particular region. Such design can be based on the length of the coding region, the length of a particular feature or region, or the length of the final product of the polynucleotide expression. It is also understood that the 3’ tailing sequence can be, but is not limited to, a poly-A tail, a poly-A-G tetrad, and / or a stem-loop sequence. The design of the tailing sequence can be optimized by hand to achieve the goal of maximum protection of the nucleic acid molecule (e.g., RNA molecule) from degradation. In certain embodiments, the preferred length of the tailing sequence is 110 nt. The tailing sequence described herein is not limiting to the scope of the present application as non-limiting examples.

[0088] The in vitro transcription template plasmids comprising the isolated nucleic acid molecules (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequences) described herein and the isolated nucleic acid molecules can further comprise a tag element. Typically, the nucleotide sequence of the tag element is located immediately downstream of the gene coding region (CDS), and correspondingly in the nucleic acid molecule, the codon sequence of the tag element is immediately downstream of the codon of the coding region. Once the coding region codon is translated, the codon of the tag element is also translated. The tag element can be a fluorescent protein nucleotide sequence, including but not limited to the nucleotide sequence of green fluorescent protein, red fluorescent protein, firefly luciferase. The transcription and translation of the tag sequence facilitate the monitoring of the expression of the target nucleic acid molecule in cells and in organisms.

[0089] The in vitro transcription template plasmids comprising the isolated nucleic acid molecules (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease-encoding sequences) described herein and the isolated nucleic acid molecules can also comprise a signal peptide element. Typically, the nucleotide sequence of the signal peptide element is located between the 5’ UTR and the CDS region, and correspondingly in the RNA, the codon sequence of the signal peptide element is located between the 5’ UTR codon and the CDS codon. The nucleotide sequence of the signal peptide element can be encoded, which upon transcription and translation facilitates the intracellular or extracellular localization of the polypeptide sequence translated from the RNA.

[0090] The isolated nucleic acid molecules (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease-encoding sequences) described herein can also comprise a 5’ cap structure, which can bind to RNA cap binding proteins (CBPs), thereby increasing the stability of the RNA. The capping method can be post-transcriptional capping using a capping enzyme, or a co-transcriptional capping method by adding a cap analog in the in vitro transcription reaction system. The cap analog can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to the nucleotide sequence of the present application, for example, an anti-reverse cap analog (ARCA) cap, an mCap cap, or a Cap1. As a non-limiting example, the present application preferably adopts a co-transcriptional capping method to add a Cap1 cap in the RNA. Different cap structures can also be added in other ways in the isolated nucleic acid molecules (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease-encoding sequences) to achieve efficient translation of the isolated nucleic acid molecules.

[0091] The isolated nucleic acid molecules (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease-encoding sequences) described herein can also comprise the nucleotide sequence of one or more other heterologous polypeptides for improving the pharmacokinetic or pharmacodynamic properties of the isolated nucleic acid molecules and their translation products, for example, prolonging the half-life.

[0092] In certain embodiments, the isolated nucleic acid molecules can be synthesized with specific chemical modifications of the nucleotides, which can reduce the interaction of the target molecules with innate immune receptors, reduce their own immunogenicity, and also optimize the translation efficiency of the nucleic acid molecules to proteins.

[0093] In certain embodiments, one modified nucleotide, N1-methylpseudouridine (N1-me-Pseudo U, mlΨ), is employed in place of the natural uracil nucleoside, U, as one of the transcriptional feedstocks, but it is not intended that the protected mRNA sequences of the present application are limited to only this one nucleotide modification. Different modifications by replacing the A, C, G, U four natural nucleotide feedstocks can be made to produce the artificially optimized isolated nucleic acid molecules of the present application when prepared by in vitro transcription using the in vitro transcription template plasmids described herein, or using an oligonucleotide synthesizer, or using a host cell expression system, or any other method known in the art in combination with one or more of the above. For example, the specific nucleotide replacement can be, but is not limited to, N1-methyladenosine (mlA), N6-methyladenosine (m6A) in place of natural adenosine, 5-methylcytidine (m5C) in place of natural cytidine, 5-methyluridine (m5U), s2U, 5-methoxyuridine (5moU), pseudouridine (ψ), N1-methylpseudouridine (mlΨ), etc. in place of natural uridine.

[0094] Generally, a sequence-optimized nucleic acid is produced by at least one step that includes replacing codons in a reference sequence with synonymous codons (i.e., codons that encode the same amino acid).

[0095] A skilled artisan will appreciate that a T base in a codon map of the present application is present in DNA, and that the T base will be replaced by a U base in the corresponding RNA. For example, a T base of a codon-nucleotide sequence of the present application in DNA form (e.g., a vector or an in vitro translation (IVT) template) will be transcribed as a U base in its corresponding transcribed mRNA. In this regard, both a codon-optimized DNA sequence (comprising T) and its corresponding RNA sequence (comprising U) are considered to be a codon-optimized nucleotide sequence of the present application. For example, a DNA and / or RNA reference sequence provided herein can be optimized by replacing all codons that encode a certain amino acid with only one of the alternative codons provided in the codon map. A skilled artisan will also appreciate that an equivalent codon map can be produced by replacing one or more bases with a non-natural base. Thus, for example, a TTC codon (DNA map) will correspond to a UUC codon (RNA map), which in turn will correspond to a ΨΨC codon (RNA map in which the U has been replaced with pseudouridine).

[0096] Further, one or more codon optimization methods or combinations thereof can be used to produce the sequence-optimized nucleic acid molecules and in vitro transcription plasmid templates of the present application.

[0097] The isolated nucleic acid molecules described in this application (e.g., coronavirus 3CL protease-coding sequences, including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) can be circular or cyclic. A circular nucleic acid molecule means that the molecules are linked from the ends in some way, whether by linkage, covalent bonding, association with the same protein or other molecules or complexes, or hybridization.

[0098] The isolated nucleic acid molecules described in this application (e.g., coronavirus 3CL protease-coding sequences, including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) can be designed to conjugate with the following: other polynucleotides, dyes, intercalating agents. Agents (e.g., acridine), cross-linking agents (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphate esters, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins (e.g., glycoproteins) or peptides (e.g., molecules with specific affinity for coligands) or antibodies (e.g., antibodies that bind to specific cell types such as cancer cells, endothelial cells, or osteocytes), hormones and hormone receptors, non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, or drugs. Conjugation can produce increased stability and / or half-life and is particularly suitable for targeting polynucleotides to specific sites in cells, tissues or organisms.

[0099] Terminology Definition

[0100] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational procedures used herein, such as virology, cell culture, biochemistry, cell biology, and nucleic acid chemistry, are all routine procedures widely used in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below.

[0101] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0102] The terms "a" and "an," along with their equivalents, are intended to include both singular and plural forms, unless the context clearly indicates otherwise. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

[0103] As used herein, the term "and / or," when used in the context of two or more features or elements, should be interpreted as specific disclosure of each of the features or elements individually and, in combination with one another. Thus, the term "and / or" as used in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0104] As used herein, the term "coronavirus" is a single-stranded, positive-sense RNA virus with a crown-like appearance of spike proteins projecting from its envelope surface. The coronavirus includes, but is not limited to, MERS-CoV, SARS-CoV-2, SARS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, HCoV-NL63, or their variants, etc. The SARS-CoV-2 includes the original strain of SARS-CoV-2, as well as its variants, such as Alpha, Beta, Gamma, Delta, and Omicron variants.

[0105] As used herein, the term "identity" is used in the context of a match between sequences of two polypeptides or two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is the number of matching positions shared by the two sequences divided by the number of positions compared times 100. For example, if two sequences are 6 of 10 positions identical, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are identical). Generally, the comparison is made over the full length of the sequences being compared. Such a comparison can be conveniently performed by means of the algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443-453, using, for example, the computer program ALIGN available from DNAstar, Inc. The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0106] The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by their one- and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0107] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter essential properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue for an amino acid residue with similar side chains, e.g., substitutions that take place within a family of amino acid residues that have similar side chains e.g., residues that are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, conservative substitutions generally are ones in which the amino acid residue is replaced with another amino acid residue from the same side chain family. Methods of identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0108] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, pharmacologically and / or physiologically, with the subject and the active ingredient, are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, ionic strength enhancers, agents to maintain osmotic pressure, agents to retard absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity (e.g., oncolytic activity) of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactoserum, albumin or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like.

[0109] As used herein, the term "prevention" refers to a method implemented in order to stop or delay the occurrence of a disease or disorder or a symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0110] As used herein, the term "effective amount" refers to an amount that is effective for an intended purpose. For example, a therapeutically effective amount can be an amount that is effective for treating or curing a disease (e.g., a tumor), delaying the onset of symptoms of a disease (e.g., a tumor), and / or delaying the progression of a disease (e.g., a tumor). Such an effective amount can be readily determined by one of skill in the art or a physician, and can be related to the intended purpose (e.g., treatment), the general health condition, age, sex, weight, severity of the disease to be treated, complications, mode of administration, and the like of the subject. The determination of such an effective amount is well within the capabilities of one of skill in the art.

[0111] As used herein, the term "subject" refers to a mammal, such as a primate, e.g., a human. In certain embodiments, the subject (e.g., a human) has a tumor, or is at risk of having a tumor.

[0112] As used herein, the terms "cancer" and "tumor" are used interchangeably and refer to a large class of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division leads to the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream. Cancer includes benign and malignant cancers and dormant tumors or micrometastases. Cancer also includes hematological malignancies, such as lymphoma, leukemia, myeloma or lymphoid malignancies, as well as spleen and lymph node tumors. Beneficial effects

[0113] The 3CL protease of coronavirus (e.g., SARS-CoV-2) or nucleic acid molecule encoding the 3CL protease provided in the present application has broad-spectrum, significant tumor cell killing ability, and can be used for preventing and / or treating tumors. BRIEF DESCRIPTION OF DRAWINGS

[0114] Embodiments of the present application will be described in detail below with reference to the attached drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustration of the present application, and are not a limitation on the scope of the present application. According to the following detailed description of the drawings and preferred embodiments, various purposes and advantageous aspects of the present application will become apparent to those skilled in the art.

[0115] Figure 1 shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of various normal cell lines and tumor cell lines. Among them, A shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of normal cell line human embryonic kidney cells (HEK 293T); B shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human umbilical vein endothelial cell line HUVEC; C shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human pancreatic cancer cell line PANC-1; D shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human glioma cell line U87; E shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human glioma cell line A172; F shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human gastric cancer cell line AGS; G shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human hepatocellular carcinoma cell line Huh7; H shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human melanoma cell line CHL-1; I shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human colon cancer cell line HCT116; J shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human renal cancer cell line ACHN; K shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human cervical cancer cell line Hela; L shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human osteosarcoma cell line U2OS; M shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human prostate cancer cell line DU145; N shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human lung cancer cell line NCI-H1975; O shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human lung cancer cell line NCI-H1650; P shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth activity of human lung cancer cell line A549.

[0116] Sequence information

[0117] The information of part of the sequences involved in the present disclosure is shown in Table A below.

[0118] Table A Information of part of the sequences Note: According to the WIPO Standard ST.26, the symbol "u" cannot be used to represent uracil in an RNA molecule in the sequence listing, the symbol "t" is understood as uracil in an RNA sequence. DETAILED DESCRIPTION

[0119] The application will now be described with reference to the following examples, which are intended to be illustrative only and not limiting of the application. The following examples can include compilations of data collected at various times during the development and experimentation associated with the subject matter of the present disclosure.

[0120] Unless specifically indicated otherwise, the molecular biology experimental methods and immunological detection methods used in the present application are performed essentially as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995; the use of restriction enzymes is in accordance with the conditions recommended by the manufacturer. Where no specific conditions are indicated in the examples, the procedures are performed under conventional conditions or under conditions recommended by the manufacturer. Where no manufacturer is indicated for the reagents or instruments used, these are conventional products that are commercially available. Those skilled in the art will appreciate that the examples describe the present application by way of example only and are not intended to limit the scope of the application as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

[0121] Example 1 In vitro transcription synthesis of mRNA encoding SARS-CoV-2 3CL protease

[0122] Experimental materials: The pUC57 vector used in the experiment was purchased from Addgene (item number 54338), ATP was purchased from Thermo (item number: R0441), CTP was purchased from Thermo (item number: 18331017), GTP was purchased from Thermo (item number: 18332015), UTP was purchased from Thermo (item number: R1471), Nuclease-free water was purchased from Thermo (item number: 10977015), Top10 competent cells were purchased from Invitrogen (item number C404010), Gentamicin was purchased from Shengong Bioengineering Co., Ltd. (item number A100304), primers were synthesized by Shengong Bioengineering Co., Ltd., AccuPrime TMTaq high fidelity DNA polymerase was purchased from Invitrogen (Cat No. 12346086), Endotoxin-free plasmid extraction kit was purchased from Invitrogen (Cat No. K210004), T7 RNA polymerase was purchased from Thermo (Cat No: EP0111), Inorganic pyrophosphatase was purchased from Thermo (Cat No: EF0221), RNase inhibitor was purchased from Applied Biosystems (Cat No N8080119), Modified nucleotide (N1-methylpseudouridine) was purchased from Yixing Bio (Cat No 10651ES80), S-adenosyl methionine (SAM) was purchased from NEN (Cat No: B9003S), Cap analog (m7G(5')ppp(5')G) was purchased from Invitrogen (Cat No: AM8050), DNase I was purchased from Thermo (Cat No: 18047019). Gene amplifier manufacturer is Hangzhou Biheng Technology Co., Ltd. (Model: AUTO-96), ultramicro UV-Vis spectrophotometer Nanodrop manufacturer is Thermo (Cat No: 840-317400), constant temperature mixing instrument (refrigeration) manufacturer is its Linbeier instrument (Cat No: BE-3200), refrigerated centrifuge manufacturer is Germany / Eppendorf (Model: 5424R), dry thermostat manufacturer is Hangzhou Aosheng Instrument Co., Ltd. (Model: K30).

[0123] Step one, DNA template generation

[0124] The mRNA template sequence was designed by considering codon preference, GC content, and the thermodynamic stability of RNA secondary structure, etc. The in vitro transcription template plasmid with pUC57 as the backbone was constructed by de novo gene synthesis. The pUC57 vector template contains a T7 promoter, a 5'UTR, a Kozak, a 3'UTR sequence, and the gene coding region of SARS-CoV-2 3CL protease (the amino acid sequence is shown as SEQ ID NO: 6) is inserted between the UTRs. The synthesized plasmid was transformed into the host bacteria and plated on solid plates containing antibiotics. Positive clones were verified by colony PCR and Sanger sequencing method, and the plasmid sequencing file was confirmed to be the target gene sequence by alignment. The correct clone was amplified in liquid medium, and the plasmid was purified by endotoxin-free plasmid extraction kit. PCR amplification was performed using PCR primers with PolyA_110 and high-fidelity DNA polymerase, and the plasmid template was DNA-tailed and linearized. The PCR product was purified to remove enzymes and reaction components to obtain the linearized template. The product concentration and A260 / 280 were determined by Nanodrop, and the product quality was detected by agarose gel electrophoresis.

[0125] Step two, mRNA in vitro transcription

[0126] In vitro co-transcription reaction was performed using T7 transcriptase, inorganic pyrophosphatase, RNAse inhibitor, nucleotides, modified nucleotides (N1-methyl pseudouridine), SAM, cap analog, linearized template to generate mRNA with cap structure in one step; DNA template in the product was digested using DNase I; the transcription product was purified by column to obtain the target mRNA; the product concentration and A260 / 280 were determined by Nanodrop, and the product quality was detected by denaturing agarose gel electrophoresis, and finally the target mRNA product was obtained. The mRNA was stored in RNase-free water, the purity was ≥90%, and was stored at -80°C. The nucleotide sequence of the finally obtained mRNA molecule is shown in SEQ ID NO: 7, wherein the coding sequence of SARS-CoV-2 3CL protease is shown in SEQ ID NO: 1.

[0127] Referring to the above method, SARS-CoV 3CL protease mRNA and MERS-CoV 3CL protease mRNA were also synthesized in vitro, and the sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 12, respectively.

[0128] Example 2 Effect of SARS-CoV-2 3CL protease mRNA on the growth activity of various types of cell lines

[0129] This example was carried out according to the SARS-CoV-2 3CL mRNA prepared in Example 1.

[0130] Experimental materials: The tumor cell lines used in the experiment, such as human glioblastoma cell U87MG purchased from ATCC (item number: HTB-14), A172 purchased from ATCC (item number: CRL-1620), human osteosarcoma cell U2OS purchased from ATCC (item number: HTB-96), human melanoma cell CHL-1 purchased from ATCC (item number: CRL-9446), human prostate cancer cell DU145 purchased from ATCC (item number: HTB-81), human colon cancer cell HCT-116 purchased from ATCC (item number: CCL-247), human cervical cancer cell Hela purchased from ATCC (item number: CCL-2), human gastric adenocarcinoma cell AGS purchased from ATCC (item number: CRL-1739), human lung cancer cell A549 purchased from ATCC (item number: CCL-185), human non-small cell lung cancer cell NCI-H1975 purchased from ATCC (item number: CRL-5908), human bronchial alveolar carcinoma cell NCI-H1650 purchased from ATCC (item number: CRL-5883), human renal cancer cell ACHN purchased from ATCC (item number: CRL-1611), human hepatocellular carcinoma cell Huh7 purchased from the National Experimental Cell Resource Sharing Platform, human pancreatic cancer cell PANC-1 purchased from ATCC (item number: CRL-1469). The non-tumor cell lines used in the experiment, such as human intravenous endothelial cell HUVEC purchased from Zhejiang Meisen Cell Technology Co., Ltd. (item number: CTCC-0804-PC), human embryonic kidney epithelial cell 293T purchased from ATCC (item number CRL-3216).

[0131] The above tumor cells were cultured in a cell incubator at 37°C and 5% CO2, and the complete culture medium for cell growth was DMEM high glucose medium (purchased from Gibco Company, item number: 11995065), MEM medium (purchased from Gibco Company, item number: 10370021), McCoy's 5A medium (purchased from Gibco Company, item number: 16600082), F-12K medium (purchased from Gibco Company, item number: 21127022) or RPMI-1640 medium (purchased from Gibco Company, item number: 22400089) added with 10% fetal bovine serum (purchased from Gibco Company, item number: 16000044) and penicillin-streptomycin double antibody (purchased from Gibco Company, item number: 2321152).

[0132] The complete culture medium for HUVEC cells was endothelial cell-specific medium added with 10% fetal bovine serum and penicillin-streptomycin double antibody, purchased from Zhejiang Meisen Cell Technology Co., Ltd. (item number: M22SN3004).

[0133] The complete culture medium for 293T cells is DMEM high glucose medium (purchased from Gibco Company, item number: 11995065) added with 10% fetal bovine serum (purchased from Gibco Company, item number: 16000044) and penicillin-streptomycin double antibody (purchased from Gibco Company, item number: 2321152).

[0134] Cell-Titer Luminescent Cell Viability detection solution was purchased from Promega Company (item number: G7572). The microplate reader was purchased from Molecular Devices Company, model SpectraMax M5.

[0135] The experimental scheme is as follows:

[0136] The cells to be tested were inoculated in a white wall transparent bottom 96-well plate at a density of about 1.0 x 10 4 After 24 hours of culture at 37°C, 5% CO2, the SARS-CoV-2 3CL protease mRNA was gradient diluted with Opti-MEM and transfected into cells with Lipo2000 liposome. The final concentration of SARS-CoV-2 3CL protease mRNA transfected into 293T cells and HUVEC cells was: 10000 ng / ml, 5000 ng / ml, 2500 ng / ml, 1250 ng / ml, 625 ng / ml, 312.5 ng / ml, 156.25 ng / ml, 78.125 ng / ml, 39.06 ng / ml. The final concentration of ACHN cells, Hela cells, U2OS cells, DU145 cells, U87 MG cells, A172 cells, A549 cells, NCI-H1975 cells, NCI-H1650 cells was 1000 ng / ml, 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml, 15.65 ng / ml. The final concentration of PANC-1 cells, AGS cells, Huh7 cells, CHL-1 cells, HCT-116 cells was 2000 ng / ml, 1000 ng / ml, 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml. The final concentration of mRNA transfected in the control group was 0, and other conditions were completely consistent with the test group. After 48 hours of incubation, the supernatant was discarded, 100 μl of diluted Cell-Titer Luminescent Cell Viability detection solution was purchased from Promega Company (item number: G7572). The microplate reader was purchased from Molecular Devices Company, model SpectraMax M5.

[0137] Calculation formula: Cell viability (%) = experimental group / cell control group average value x 100%

[0138] Statistical analysis: S-type curve fitting of inhibition rate-concentration was performed using Origin 9.0 software, and the half maximal inhibitory concentration (IC 50 ) was calculated.

[0139] The experimental results are shown in Tables 1-2 and Figure 1, which is the S-type curve of cell viability and IC 50 values corresponding to Tables 1 and 2.

[0140] Table 1. Effect of SARS-CoV-2 3CL protease mRNA on the viability of non-tumor cell lines

[0141] Table 1 lists the effects of different concentrations of SARS-CoV-2 3CL protease mRNA on the growth viability of two human non-tumor cell lines. In combination with the S-type curves shown in Figures 1 A-B, it is shown that the mRNA has no significant inhibitory effect on the growth of the two normal cell lines. Specifically, as shown in Figure 1 A, the IC 50 value of SARS-CoV-2 3CL protease mRNA for normal human embryonic kidney cells 293T is greater than 10000 ng / ml; as shown in Figure 1 B, the IC 50 value for human umbilical vein endothelial cells HUVEC is greater than 5000 ng / ml. The above results show that the mRNA provided by the present application has no significant toxic effect on the two normal cell lines.

[0142] Table 2. Effect of SARS-CoV-2 3CL protease mRNA on the viability of tumor cell lines

[0143] Table 2 lists the effects of different concentrations of SARS-CoV-2 3CL protease mRNA on the growth viability of various human tumor cell lines. In combination with the S-type curves shown in Figures 1 C-P, it is shown that the mRNA provided by the present application has a significant killing effect on the growth of various tumor cell lines. Specifically, as shown in Figure 1 C, the IC 50 value of the mRNA for human pancreatic cancer cell line PANC-1 is 574.626 ± 52.61 ng / ml; as shown in Figure 1 D, the IC 50 value for malignant human glioma cell line U87 is 184.268 ± 25.76 ng / ml; as shown in Figure 1 E, the IC 50The IC value against human gastric cancer cell line AGS was 175.569 ± 35.83 ng / ml, as shown in F of FIG. 1. 50 The IC value against human hepatocarcinoma cell line Huh7 was 248.778 ± 14.17 ng / ml, as shown in G of FIG. 1. 50 The IC value against human melanoma cell line CHL-1 was 310.065 ± 20.67 ng / ml, as shown in H of FIG. 1. 50 The IC value against human colon cancer cell line HCT116 was 444.233 ± 57.54 ng / ml, as shown in I of FIG. 1. 50 The IC value against human renal cancer cell line ACHN was 976.261 ± 262.17 ng / ml, as shown in J of FIG. 1. 50 The IC value against human cervical cancer cell line Hela was 430.272 ± 107.46 ng / ml, as shown in K of FIG. 1. 50 The IC value against human osteosarcoma cell line U2OS was 258.346 ± 20.20 ng / ml, as shown in L of FIG. 1. 50 The IC value against human prostate cancer cell line DU145 was 122.175 ± 14.78 ng / ml, as shown in M of FIG. 1. 50 The IC value against human lung cancer cell line NCI-H1975 was 78.716 ± 8.20 ng / ml, as shown in N of FIG. 1. 50 The IC value against human lung cancer cell line NCI-H1650 was 274.938 ± 61.75 ng / ml, as shown in O of FIG. 1. 50 The IC value against human lung cancer cell line A549 was 158.56 ± 42.74 ng / ml, as shown in P of FIG. 1. 50 The IC value against human lung cancer cell line A549 was 158.56 ± 42.74 ng / ml, as shown in P of FIG. 1. 50 The IC values of the mRNA against all the tested human tumor cell lines were less than 1000 ng / ml, which were significantly lower than the IC values of the mRNA against the four non-tumor cell lines. 50 The IC values of the mRNA against all the tested human tumor cell lines were less than 1000 ng / ml, which were significantly lower than the IC values of the mRNA against the four non-tumor cell lines.

[0144] Table 3. Therapeutic index of SARS-CoV-2 3CL protease mRNA

[0145] Table 3 is the therapeutic index of SARS-CoV-2 3CL protease mRNA relative to each tumor cell line, with 293T cells as control cells. Formula: Therapeutic index = 293T maximum IC 50 value ÷ IC of each tumor cell line.50 The list results show that the SARS-CoV-2 3CL protease mRNA provided by the present application has good therapeutic safety.

[0146] Referring to the experimental method of Example 2, the effects of SARS-CoV 3CL protease mRNA and MERS-CoV 3CL protease mRNA on the growth activity of various types of cell lines were also investigated, wherein the types of cell lines tested were the same as in Example 2, and the results showed that the two types of mRNA also had broad-spectrum anti-tumor activity and good selectivity.

[0147] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the above examples do not limit the present application in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of the present application.

Claims

1. Use of a 3CL protease of a coronavirus or a nucleic acid molecule encoding the same in the manufacture of a medicament for the prevention and / or treatment of a tumor.

2. The use of claim 1, wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2, preferably SARS-CoV-2.

3. The use of claim 1 or 2, wherein the 3CL protease has an amino acid sequence selected from the group consisting of: i) a sequence as set forth in SEQ ID NO: 6, 10 or 13; ii) a sequence having one or several (e.g., 1, 2, 3, 4 or 5) amino acid substitutions, deletions or additions compared to the sequence as set forth in SEQ ID NO: 6, 10 or 13; and iii) a sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 6, 10 or 13; preferably, the substitution in ii) is a conservative substitution.

4. The use of any one of claims 1-3, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule; preferably, the nucleic acid molecule is single-stranded or double-stranded.

5. The use of claim 4, wherein the nucleic acid molecule is an RNA molecule, preferably an mRNA molecule; preferably, the nucleic acid molecule comprises a coding sequence of the 3CL protease; preferably, the nucleic acid molecule further comprises one or more selected from the group consisting of a 5’UTR, a Kozak sequence, a start codon, a stop codon, a 3’UTR, a poly-A tail; preferably, the nucleic acid molecule comprises, in the order from 5’ end to 3’ end: a 5’UTR, a Kozak sequence, a start codon, a coding sequence of the 3CL protease, a stop codon, a 3’UTR, a poly-A tail; preferably, the nucleic acid molecule consists of, in the order from 5’ end to 3’ end: a 5’UTR, a Kozak sequence, a start codon, a coding sequence of the 3CL protease, a stop codon, a 3’UTR, a poly-A tail.

6. The use of claim 5, having one or more features selected from the group consisting of: (1) the coding sequence of the 3CL protease is codon-optimized according to the codon bias of the host cell (e.g., a human cell) or is not optimized. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ (2) the coding sequence of the 3CL protease has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 8, or 11; preferably, the coding sequence of the 3CL protease has the sequence set forth in SEQ ID NO: 1, 8, or 11; (3) the 5’ end of the nucleic acid molecule is present with a 5’ modification (e.g., CAP0 type, CAP1 type, CAP2 type); preferably, the 5’ end of the nucleic acid molecule is present with a 5’ Cap 1 type modification; (4) the nucleic acid molecule contains one or more N1-methyl pseudouridine (m1y) modifications; preferably, all or a portion of the uracil nucleotides in the nucleic acid molecule are replaced with N1-methyl-pseudouracil nucleotides; (5) the 3’ end of the coding sequence of the 3CL protease is present with one or more stop codons; (6) the 5’ UTR has a nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2; preferably, the 5’ UTR has the nucleotide sequence set forth in SEQ ID NO: 2; (7) the 3’ UTR has a nucleotide sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4; preferably, the 3’ UTR has the nucleotide sequence set forth in SEQ ID NO: 4; (8) the Kozak sequence has the sequence set forth in SEQ ID NO: 3; (9) the poly-A tail comprises one or more poly-adenosine sequences, each of which independently consists of 20-120 consecutive adenosine; preferably, the poly-A tail comprises multiple poly-adenosine sequences, and the adjacent poly-adenosine sequences are connected by a spacer sequence comprising non-A; preferably, the poly-A tail has the sequence set forth in SEQ ID NO: 5; (9) the poly-A tail comprises one or more poly-adenosine sequences, each of which independently consists of 20-120 consecutive adenosine; preferably, the poly-A tail comprises multiple poly-adenosine sequences, and the adjacent poly-adenosine sequences are connected by a spacer sequence comprising non-A; preferably, the poly-A tail has the sequence set forth in SEQ ID NO: 5; (10) the nucleic acid molecule has a nucleotide sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 7, 9, or 12; preferably, the nucleic acid molecule has the sequence as set forth in SEQ ID NO: 7, 9, or 12.

7. The use of any one of claims 1-6, wherein the tumor is a solid tumor or a hematological tumor (e.g., leukemia, lymphoma, myeloma); Preferably, the tumor is a solid tumor; Preferably, the tumor is selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, melanoma, intestinal cancer, kidney cancer, cervical cancer, osteosarcoma, prostate cancer, brain glioma, and lung cancer; Preferably, the pancreatic cancer types include but are not limited to pancreatic acinar cell carcinoma, adenosquamous carcinoma, squamous cell carcinoma, signet ring cell carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with giant cells, ampullary carcinoma, pancreatic neuroendocrine tumor, etc.; the gastric cancer types include but are not limited to cardia cancer, body of stomach cancer, pyloric cancer; the liver cancer types include but are not limited to hepatocellular carcinoma, hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, intrahepatic bile duct carcinoma, angiosarcoma, hepatoblastoma, secondary liver cancer, benign liver tumor; the melanoma types include but are not limited to local melanoma, local regional melanoma, distant metastatic melanoma; the intestinal cancer types include but are not limited to colon cancer and rectal cancer, and specifically include adenocarcinoma, undifferentiated carcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, carcinoid, mucinous carcinoma, etc.; the kidney cancer types include but are not limited to clear cell carcinoma, papillary cell carcinoma, chromophobe cell carcinoma, low malignant potential multilocular cystic renal cell tumor, collecting duct carcinoma, renal medulla carcinoma; the cervical cancer types include but are not limited to squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma; the osteosarcoma types include but are not limited to telangiectatic osteosarcoma, small round cell osteosarcoma, fibrohistiocytic osteosarcoma, intramedullary well-differentiated osteosarcoma, multicentric osteosarcoma, intracortical osteosarcoma, parosteal osteosarcoma, dedifferentiated parosteal osteosarcoma, periosteal osteosarcoma, highly malignant surface osteosarcoma; the prostate cancer types include but are not limited to adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, adenosquamous carcinoma; the brain glioma types include but are not limited to PAs, astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligoastrocytoma, glioblastoma; the lung cancer types include but are not limited to small cell lung cancer and non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, carcinoid, etc.).

8. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a 3CL protease, wherein the 3CL protease is as defined in any one of claims 1-3. Preferably, the isolated nucleic acid molecule is a nucleic acid molecule as defined in any one of claims 4-6, or, the isolated nucleic acid molecule encodes a nucleic acid molecule as defined in any one of claims 4-6.

9. A vector comprising the isolated nucleic acid molecule of claim 8.

10. A delivery composition comprising a delivery vehicle, and one or more selected from the group consisting of: a 3CL protease, the isolated nucleic acid molecule of claim 8, the vector of claim 9, wherein the 3CL protease is as defined in any one of claims 1-3; Preferably, the delivery vehicle is used for encapsulating, carrying or delivering the 3CL protease, the isolated nucleic acid molecule or the vector; Preferably, the delivery vehicle comprises a non-viral vector, a viral vector and a virosome (VLP) vector therebetween; Preferably, the non-viral vector comprises, but is not limited to, a cationic liposome, a lipid nanoparticle, a lipid polymer, an artificial microsphere, a micelle, a lipid-polymer hybrid system, an extracellular vesicle, a natural or engineered exosome, etc., and the viral vector comprises, but is not limited to, an adeno-associated virus, a lentivirus, an adenovirus, a retrovirus, a vaccinia virus, a measles virus, a herpes simplex virus, an alphavirus, a vesicular stomatitis virus, an influenza virus, etc. Preferably, the delivery vehicle is a lipid nanoparticle.

11. A pharmaceutical composition comprising a 3CL protease, the isolated nucleic acid molecule of claim 8, the vector of claim 9, and / or the delivery composition of claim 10, and a pharmaceutically acceptable carrier and / or excipient, wherein, The 3CL protease is as defined in any one of claims 1-3; Preferably, the pharmaceutical composition further comprises other anti-tumor drugs; Preferably, the other anti-tumor drugs are selected from the group consisting of small molecule drugs, biological macromolecule drugs, chimeric antigen receptor cell drugs, antibody conjugate drugs; Preferably, the pharmaceutical composition is administered orally, by spray inhalation, rectally, nasally, buccally, vaginally, topically, parenterally, such as subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathoracically, intrathecally, intraventricularly, intrasternally and intracranially injection or infusion, endotracheally, surgically implanted, transdermally, locally, ultra-rapid injection / impact, or by means of an external implanted reservoir, Preferably, the drug is administered orally, intraperitoneally, intrathoracically or intravenously.

12. A method of inhibiting tumor cells in vivo or in vitro comprising: The 3CL protease, the isolated nucleic acid molecule of claim 8, the vector of claim 9, the delivery composition of claim 10 or the pharmaceutical composition of claim 11 is contacted with, or delivered into, the tumor cell, wherein the 3CL protease is as defined in any one of claims 1-3, Preferably, the tumor cell is a cell of a solid tumor or a hematological tumor (e.g., leukemia, lymphoma, myeloma); Preferably, the tumor cell is a cell selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, melanoma, intestinal cancer, kidney cancer, cervical cancer, osteosarcoma, prostate cancer, brain glioma and lung cancer; Preferably, the tumor cell is a cell selected from the group consisting of pancreatic cancer, gastric cancer, liver cancer, melanoma, intestinal cancer, kidney cancer, cervical cancer, osteosarcoma, prostate cancer, brain glioma and lung cancer; Preferably, the pancreatic cancer types include, but are not limited to, pancreatic acinar cell carcinoma, adenosquamous carcinoma, squamous cell carcinoma, signet ring cell carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with giant cells, ampullary carcinoma, pancreatic neuroendocrine tumor, etc.; the gastric cancer types include, but are not limited to, cardia cancer, body of stomach cancer, pyloric cancer; the liver cancer types include, but are not limited to, hepatocellular carcinoma, hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, intrahepatic cholangiocarcinoma, angiosarcoma, hepatoblastoma, secondary liver cancer, benign liver tumor; the melanoma types include, but are not limited to, local melanoma, local regional melanoma, distant metastatic melanoma; the intestinal cancer types include, but are not limited to, colon cancer and rectal cancer, and specifically include adenocarcinoma, undifferentiated carcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, carcinoid, mucinous carcinoma, etc.; the kidney cancer types include, but are not limited to, clear cell carcinoma, papillary cell carcinoma, chromophobe cell carcinoma, low malignant potential multilocular cystic renal cell tumor, collecting duct carcinoma, renal medullary carcinoma; the cervical cancer types include, but are not limited to, squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma; the osteosarcoma types include, but are not limited to, osteosarcoma with capillary expansion, small round cell osteosarcoma, fibrohistiocytic osteosarcoma, intramedullary well-differentiated osteosarcoma, multicentric osteosarcoma, intracortical osteosarcoma, parosteal osteosarcoma, dedifferentiated parosteal osteosarcoma, periosteal osteosarcoma, highly malignant surface osteosarcoma; the prostate cancer types include, but are not limited to, adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, adenosquamous carcinoma; the brain glioma types include, but are not limited to, PAs, astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligoastrocytoma, glioblastoma; the lung cancer types include, but are not limited to, small cell lung cancer and non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma, carcinoid, etc.).

13. Use of the isolated nucleic acid molecule of claim 9, the vector of claim 10, the delivery composition of claim 11 or 12, or the pharmaceutical composition of claim 13 for the manufacture of a medicament for the prevention and / or treatment of a tumor.

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