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 coronavirus 3CL protease for preventing or treating tumors

[0001] This application is based on the application with CN application number 202311153380.8 and application date September 7, 2023, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present application belongs to the field of medical biotechnology, and specifically relates to the use of coronavirus 3CL protease or nucleic acid molecules encoding the same in the preparation of drugs for preventing and / or treating tumors. Background Art

[0003] Cancer is a disease characterized by uncontrolled cell growth caused by genetic mutations or dysregulation, and exhibits significant spatiotemporal heterogeneity depending on the site of onset. The application of cancer therapeutics must not only overcome this heterogeneity but also minimize toxicity to normal cells and selectively kill cancer cells. In the development of anticancer drugs, reducing the toxicity of current clinically used drugs while maintaining a broad anticancer spectrum remains a challenging challenge. Current clinical treatment options for different cancer types, including solid and non-solid tumors, typically include surgery, chemotherapy, radiotherapy, bone marrow / stem cell transplantation, hormone therapy, targeted therapy, and immunotherapy, or a combination thereof. While these seven major categories of anticancer therapies can achieve tumor elimination to a certain extent, they can also lead to drug resistance and recurrence in patients, and multiple treatments can cause pain or irreversible damage to patients. The development and application of new and effective anticancer treatments is urgently needed.

[0004] Gene therapy and genetic vaccination are rapidly developing anti-cancer strategies in recent years, offering specific and personalized options for the treatment and prevention of various tumors. Currently, only a few gene therapy approaches have been approved for oncology use worldwide, including Gendicine for the treatment of head and neck squamous cell carcinoma, Imlygic for the treatment of inoperable melanoma lesions, Kymriah for the treatment of relapsed or refractory acute lymphoblastic leukemia in patients aged 3 to 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 therapies. These four treatment approaches, exemplified by gene therapy, involve the use of viruses, engineered immune cells, or other delivery vehicles to introduce suicide genes or immunomodulatory genes into cells and insert them into the existing genome, thereby eliminating the effects of deleterious mutations or stimulating the patient's anti-tumor immune response. In addition to this method of introducing exogenous genes, delivery vehicles can also be used to deliver oligonucleotides into tumor cells to inhibit or interfere with endogenous genes for 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 belongs 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 APC activation and innate / adaptive immunity. mRNA cancer vaccines have the advantages of high efficiency, safety, great development potential, and low production costs, and are ahead of other conventional vaccine platforms. As a means of immunotherapy, tumor vaccines are highly dependent on the body's own immune system, but the immune system of cancer patients is often suppressed and their defense function is low, so the application of tumor vaccines has certain limitations.

[0006] mRNA therapy, a form of gene therapy, has been explored as one of the safest and most effective methods for treating tumors. mRNA is composed of four nucleotides, abbreviated as the letters A, U, C, and G. Cells interpret the order in which these nucleotides are arranged. If the information encoded by the mRNA cannot be read, it cannot be translated into proteins with specific structure and function. The mRNA is ultimately metabolized and degraded by the cells, without producing the corresponding biological effect. If the opposite occurs, the body will only produce 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 huge medical need for anti-tumor mRNA drugs to improve patient survival rates and bring more therapeutic benefits to patients, their families and society.

[0008] Application Contents

[0009] The inventors of this application unexpectedly discovered that the 3CL protease of a coronavirus (e.g., SARS-CoV-2) has a broad spectrum and significant ability to kill tumor cells. Based on this discovery, the inventors of this application have developed methods for preventing and / or treating tumors based on the 3CL protease or nucleic acid molecules encoding the 3CL protease. The therapeutic drugs (e.g., nucleic acid drugs) based on the 3CL protease or nucleic acid molecules encoding the 3CL protease provided in this application are expected to expand the scope of benefits for cancer patients, improve patient survival rates, and bring more therapeutic benefits to patients, their families, and society.

[0010] The first aspect of the present application provides use of a coronavirus 3CL protease or a nucleic acid molecule encoding the coronavirus 3CL protease 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 both the original strain of SARS-CoV-2 and variants thereof, such as Alpha, Beta, Gamma, Delta, and Omicron variants.

[0013] It is easy for those skilled in the art to understand that 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 of the 3CL protease from which it is derived (e.g., tumor cell killing activity or oncolytic activity). 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 represented by any one of Uniprot: P0C6U8, P0C6X7, and 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 represented by any one of Uniprot: K9N638, K9N7C7, and 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 shown in any one of Uniprot: P0DTC1, P0DTD1, A0A8B1JPK4.

[0017] Those skilled in the art will readily appreciate that, in order to facilitate protein expression, purification and / or tracing, the 3CL protease may further contain another polypeptide.

[0018] In certain embodiments, the additional polypeptide is optionally connected to the N-terminus or C-terminus of the 3CL protease via a linker (e.g., a peptide linker or an organic small molecule linker). Such linkers (e.g., peptide linkers) are well known in the art, and examples include, but are not limited to, peptide linkers 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 may be advantageous in that it is able to connect two protein / polypeptide components while maintaining their respective activity and function. 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 marker (e.g., luciferase (fluc), green fluorescent protein (GFP)), a polypeptide sequence encoding an antibody, a polypeptide sequence encoding a localization or biologically active 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) the sequence shown in SEQ ID NO: 6, 10 or 13;

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

[0024] In certain embodiments, the substitutions described in ii) are conservative substitutions.

[0025] The sequence shown in SEQ ID NO: 6, 10, or 13 contains an amino acid (such as methionine (Met)) encoded by a start codon (such as AUG / ATG) at its N-terminus. Those skilled in the art will appreciate that, during protein production by genetic engineering, the first amino acid in the resulting polypeptide chain is often encoded by the start codon (such as Met) due to the effect of the start codon. The 3CL protease of the present application encompasses not only amino acid sequences containing an amino acid (such as Met) encoded by a start codon at its N-terminus, but also amino acid sequences that do not contain an amino acid (such as Met) encoded by a start codon at its N-terminus. Therefore, sequences that do not contain an amino acid (such as Met) encoded by a start codon at the N-terminus of the above amino acid sequences are also within the scope of protection 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 for the 3CL protease.

[0030] In certain embodiments, 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, and a poly-A tail.

[0031] In certain embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end, the following sequence: 5'UTR, Kozak sequence, start codon, coding sequence of the 3CL protease, stop codon, 3'UTR, and poly-A tail.

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

[0033] According to codon degeneracy in the art, in certain embodiments, the coding sequence of the 3CL protease can be replaced according to codon degeneracy. In certain embodiments, the coding sequence of the 3CL protease is codon-optimized or non-optimized according to the codon preference of the host cell (e.g., human cells).

[0034] In certain embodiments, 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 of SEQ ID NO: 1, 8, or 11. In certain embodiments, the coding sequence of 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 has 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 has a 5' Cap type 1 modification;

[0036] In certain embodiments, the nucleic acid molecule contains one or more N1-methylpseudouridine (m1ψ) modifications. In certain embodiments, all or part of the uracil nucleotides in the nucleic acid molecule are replaced with N1-methyl-pseudouridine nucleotides.

[0037] In certain embodiments, one or more stop codons are present 3' to the coding sequence of the 3CL protease.

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

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

[0040] In certain embodiments, the 5'UTR has a nucleotide sequence that has 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 shown in SEQ ID NO: 2. In certain embodiments, the 5'UTR has the nucleotide sequence shown in SEQ ID NO: 2.

[0041] In certain embodiments, the 3'UTR has a nucleotide sequence that has 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. 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 shown in SEQ ID NO:3.

[0043] In certain embodiments, the poly-A tail comprises one or more poly(A) sequences, each of which independently consists of 20-120 consecutive adenine nucleotides. Preferably, the poly-A tail comprises multiple poly(A) sequences, and adjacent poly(A) sequences are connected by a spacer sequence containing a non-A residue. In certain embodiments, the poly-A tail has the sequence shown in SEQ ID NO:5.

[0044] In certain embodiments, the nucleic acid molecule has a nucleotide sequence that 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: 7, 9, or 12. In certain embodiments, the nucleic acid molecule has a sequence as set forth in SEQ ID NO: 7, 9, or 12.

[0045] The second aspect of the present 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 present application.

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

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

[0048] In certain embodiments, the vector of the present application is, for example, a plasmid, a cosmid, a phage, a lentivirus, or 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 of the first aspect in a subject.

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

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

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

[0054] In certain embodiments, the delivery vectors include non-viral vectors, viral vectors, and virus-like particles (VLP) vectors that are intermediate between viral vectors and non-viral vectors.

[0055] In certain embodiments, the non-viral vectors include, but are not limited to, cationic liposomes, lipid nanoparticles, lipopolymers, artificial microspheres, micelles, lipid-polymer hybrid systems, extracellular vesicles, natural or engineered exosomes, etc. In certain embodiments, the viral vectors include, but are not limited to, adeno-associated viruses, lentiviruses, adenoviruses, retroviruses, vaccinia viruses, measles viruses, herpes simplex viruses, alphaviruses, vesicular stomatitis viruses, influenza viruses, etc. In certain embodiments, the virosome vectors include, but are not limited to, VLP-mRNA delivery vectors that utilize the principle of specific recognition between the mRNA stem-loop structure and the phage capsid protein.

[0056] In certain embodiments, the delivery vehicle 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 antibodies or proteins, chimeric antigen receptor cell drugs, and antibody-coupled drugs.

[0060] In certain embodiments, the pharmaceutical composition is administered orally, by inhalation spray, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, intratracheal installation, surgical implantation, transdermal delivery, local injection, ultra-fast injection / bombardment, or via an explanted reservoir. In certain embodiments, the drug is administered orally, intraperitoneally, intrathoracic or intravenously.

[0061] In a sixth aspect, the present application provides a method for inhibiting tumor cells in vivo or in vitro, comprising: contacting 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 with the tumor cells, or delivering them into the tumor cells.

[0062] The seventh aspect of the present application provides the 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 preparation of a drug for preventing and / or treating tumors.

[0063] In an eighth aspect, 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 for preventing and / or treating tumors.

[0064] In a ninth aspect, the present application provides a method for 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, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the delivery composition of the fourth aspect, or the pharmaceutical composition of the fifth aspect. In certain embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy may be applied simultaneously, separately, or sequentially.

[0065] The tenth aspect of the present application provides a drug or composition for preventing and / or treating tumors 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 tumors in a subject.

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

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

[0069] In certain embodiments, the tumor is selected from 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 (eg, leukemia, lymphoma, myeloma).

[0071] In certain embodiments, the tumor cells are selected from cells 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 classification includes but is 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. In certain embodiments, the gastric cancer classification includes but is not limited to cardia cancer, gastric body cancer, and pyloric cancer. In certain embodiments, the liver cancer classification includes but is not limited to hepatocellular carcinoma, hepatocellular carcinoma, fibrolamellar liver cancer, intrahepatic bile duct carcinoma, angiosarcoma, hepatoblastoma, secondary liver cancer, and benign liver tumors. In certain embodiments, the melanoma classification includes but is not limited to localized melanoma, locoregional melanoma, and distant metastatic melanoma. In certain embodiments, the intestinal cancer classification includes but is not limited to colon cancer and rectal cancer, specifically including adenocarcinoma, undifferentiated carcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, carcinoid, mucinous carcinoma, etc. In certain embodiments, the renal cancer classification includes but is not limited to clear cell carcinoma, papillary cell carcinoma, chromophobe cell carcinoma, multilocular cystic renal cell tumor of low malignant potential, collecting duct carcinoma, and renal medullary carcinoma. In certain embodiments, the cervical cancer classification includes but is not limited to squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma. In certain embodiments, the osteosarcoma classification includes but is not limited to telangiectatic osteosarcoma, small round cell osteosarcoma, fibrous histiocytic osteosarcoma, intramedullary well-differentiated osteosarcoma, multicentric osteosarcoma, intracortical osteosarcoma, parosteal osteosarcoma, dedifferentiated parosteal osteosarcoma, periosteal osteosarcoma, and highly malignant surface osteosarcoma. In certain embodiments, the prostate cancer classification includes but is not limited to adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, and adenosquamous carcinoma. In certain embodiments, the glioma classification includes but is not limited to PAs, astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligoastrocytoma, and glioblastoma. In certain embodiments, the lung cancer classification includes but is 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] Furthermore, according to the progression of each type of tumor, including but not limited to early, middle, late or stage I, II, III, IV tumors.

[0074] The drugs, compositions, or pharmaceutical compositions of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended route of administration and therapeutic use. The drugs, compositions, or pharmaceutical compositions of the present application should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the drugs, compositions, or pharmaceutical compositions of the present application into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before 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 a unit dosage form in a drug, composition or pharmaceutical composition for easy administration.

[0076] The medicine of the present application, composition or pharmaceutical composition can be used by any suitable approach known in the art, including but not limited to, oral, spray inhalation, rectal medication, nasal medication, buccal medication, vaginal medication, topical medication, parenteral medication such as subcutaneous, vein, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraventricular, intrasternal and intracranial injection or input, intratracheal installation, surgical implantation, transdermal delivery, local injection, hyperinjection / bombardment or by a kind of explantation reservoir medication.For many therapeutic uses, preferred route of administration / mode is oral, intraperitoneal or intravenous.Technician should understand that route of administration and / or mode will change according to intended purpose.

[0077] The medicaments, compositions, or pharmaceutical compositions of the present application may include a "therapeutically effective amount" or a "prophylactically effective amount" of the 3CL protease of the present application, a nucleic acid molecule encoding the 3CL protease, a vector, or a delivery composition. A "prophylactically effective amount" refers to an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the 3CL protease of the present application, a nucleic acid molecule encoding the 3CL protease, a vector, or a delivery composition may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered therapies.

[0078] In the present application, the dosage regimen can be adjusted to obtain the best intended response (e.g., therapeutic or preventive response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the treatment situation.

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

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

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

[0082] The application provides the 5'UTR and 3'UTR district nucleotide sequences used in some embodiments, but does not limit the protection scope of the application. It will be understood by those skilled in the art that, in certain embodiments, conventional 5'UTR and 3'UTR sequence variants can be added to the in vitro transcription template plasmid, and then the mRNA including the corresponding codon is transcribed. One or more nucleotides in the 5'UTR and 3'UTR sequence variants can be added or removed to the end. Further, the 5'UTR and 3'UTR sequence variants can be placed in the same direction or position as the transcript selected from them and changed. Therefore, the 5'UTR and 3'UTR sequences can be reversed, increased, shortened, and made together with one or more other 5'UTR and 3'UTR sequences. The 5'UTR and 3'UTR sequences using different nucleotide combinations can reach the method for the application to carry out in vitro transcription and translation of the described optimization mRNA molecule purpose, all within the protection scope of the application.

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

[0084] The in vitro transcription template plasmid comprising an isolated nucleic acid molecule (e.g., a coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequence) described herein may 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 efficiently transcribe functional RNA under the guidance of RNA polymerase are within the scope of protection of this application, such as the Sp6 promoter, the Syn5 promoter, or can be used for the synthesis of RNA in this application.

[0085] The in vitro transcription template plasmid comprising an isolated nucleic acid molecule (e.g., a 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 may also include an element similar to the start codon region or that functions similarly to the start codon region. In certain embodiments, the nucleotide sequence of the start codon is preferably ATG, and the start codon sequence corresponding to the RNA is AUG, which can achieve efficient translation efficiency. As a non-limiting example, the translation of the polynucleotide can start at 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 plasmid comprising an isolated nucleic acid molecule (e.g., a coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequence) and the isolated nucleic acid molecule described herein may further comprise at least one, two, or multiple consecutive stop codon elements located before the 3'UTR. The nucleotide sequence of the stop codon may be selected from UGA, UAA, and UAG, and combinations thereof. In certain embodiments, the nucleotide sequence of the stop codon is preferably UGA. In certain embodiments, the nucleotide sequence of the stop codon is preferably UGA.

[0087] The isolated nucleic acid molecules described in the present application (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequences) may also include a polyadenylation tailing sequence located in the immediate downstream region of the 3'UTR element to increase the stability of the nucleic acid molecule. The polyadenylation tail may be present or absent in the circular in vitro transcription template plasmid, and may be transcribed together with the template or added after transcription. The length of the tailing sequence is designed based on the length of the overall nucleic acid molecule or the length of a specific region. This design may be based on the length of the coding region, the length of a specific feature or region, or the length of the final product of polynucleotide expression. It should also be understood that the 3' tailing sequence may be, but is not limited to, a poly A tail, a poly AG quadruplex, and / or a stem-loop sequence, and the design of the tailing sequence may be artificially optimized to achieve the goal of protecting nucleic acid molecules (e.g., RNA molecules) from degradation to the greatest extent possible. In certain embodiments, the preferred length of the tailing sequence is 110nt. As a non-limiting example, the tailing sequence described herein does not limit the scope of protection of the present application.

[0088] The in vitro transcription template plasmid comprising an isolated nucleic acid molecule (e.g., a 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 may also comprise a tag element. Typically, the nucleotide sequence of the tag element is located immediately downstream of the gene coding region (CDS), and accordingly, in the nucleic acid molecule, the codon sequence of the tag element is immediately after the coding region codon. Once the coding region codon is translated, the codon of the tag element will also be 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, and firefly luciferase. Transcription and translation of the tag sequence are beneficial for monitoring the expression of the target nucleic acid molecule in cells and organisms.

[0089] The in vitro transcription template plasmid comprising an isolated nucleic acid molecule (e.g., a coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequence) and the isolated nucleic acid molecule described herein may further comprise a signal peptide element. Typically, the nucleotide sequence of the signal peptide element is located between the 5'UTR and the CDS region. Accordingly, in 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 may be encoded, and its transcription and translation facilitates providing a localization signal inside and outside the cell for the polypeptide sequence translated from the RNA.

[0090] The isolated nucleic acid molecules described in the present application (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequence) may further include a 5' cap structure that can bind to RNA cap binding protein (CBP), thereby increasing RNA stability. The capping method may be post-transcriptional capping using a capping enzyme, or a co-transcriptional capping method may be adopted by adding a cap analog to the in vitro transcription reaction system. The cap analog may 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 Cap1. As a non-limiting example, the present application preferably adopts a co-transcriptional capping method to add a Cap1 cap to the RNA. Different cap structures may also be added to the coding sequence of the isolated nucleic acid molecule (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease) in other ways to achieve efficient translation of the isolated nucleic acid molecule.

[0091] The isolated nucleic acid molecules described in the present application (e.g., coronavirus (including SARS-CoV, MERS-CoV, SARS-CoV-2, etc.) 3CL protease coding sequences) may also contain nucleotide sequences of one or more other heterologous polypeptides to improve the pharmacokinetic or pharmacodynamic properties of the isolated nucleic acid molecules and their translation products, such as extending half-life.

[0092] In certain embodiments, the isolated nucleic acid molecule can be synthesized using specifically chemically modified nucleotides, which can reduce the interaction of the target molecule with innate immune receptors, reduce its own immunogenicity, and optimize the translation efficiency of the nucleic acid molecule into protein.

[0093] In certain embodiments, a modified nucleotide N1-methylpseudouridine (N1-me-Pseudo U, m1Ψ) is used to replace the natural uridine nucleoside U as one of the transcription raw materials, but this does not mean that the mRNA sequence protected by the present application is limited to this nucleotide modification. When the isolated nucleic acid molecule described in the present application is prepared by in vitro transcription using the in vitro transcription template plasmid described in the present application, or using an oligonucleotide synthesizer, or using a host cell expression system, or one or more combinations of any other methods known in the art, the artificially optimized isolated nucleic acid molecule described in the present application can be obtained by replacing the four natural nucleotide substrate raw materials A, C, G, and U and performing different modifications. For example, specific nucleotide replacement methods can be, but are not limited to: replacing natural adenosine with N1-methyladenosine (m1A) or N6-methyladenosine (m6A), replacing natural cytidine with 5-methylcytidine (m5C), and replacing natural uridine with 5-methyluridine (m5U), s2U, 5-methoxyuridine (5moU), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), etc.

[0094] Generally, sequence-optimized nucleic acids are generated by at least one step involving replacing codons in a reference sequence with synonymous codons (ie, codons that encode the same amino acid).

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

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

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

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

[0099] Definition of terms

[0100] In this application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures used herein in virology, cell culture, biochemistry, cell biology, nucleic acid chemistry, and other fields are conventional procedures widely used in the corresponding fields. To facilitate a better understanding of this application, definitions and explanations of relevant terms are provided below.

[0101] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0102] The terms "a" and "an" and "the" and similar referents in the context of describing this application (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0103] As used herein, the term "and / or" should be considered as a specific disclosure of each of two or more specified features or elements, as well as any combination of two or more features or elements. Thus, the term "and / or" used in phrases 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" refers to a single-stranded, positive-stranded RNA virus with spikes extending from its envelope, forming a crown-like structure. Coronaviruses include, but are not limited to, MERS-CoV, SARS-CoV-2, SARS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-229E, HCoV-NL63, or variants thereof. SARS-CoV-2 includes both the original strain of SARS-CoV-2 and its variants, such as the Alpha, Beta, Gamma, Delta, and Omicron variants.

[0105] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent 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)), which has been incorporated 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 percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using 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 twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, 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 this application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in this application, amino acids are generally represented by single-letter and three-letter abbreviations 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 change the essential properties of the 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 in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions in which residues are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). 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 refer to replacing the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid 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 carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay 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). Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvant (e.g., complete Freund's adjuvant), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and are capable of stabilizing the desired activity (e.g., oncolytic activity) of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or degradation products thereof (e.g., lactalbumin hydrolyzate), etc.

[0109] As used herein, the term "prevention" refers to the method implemented in order to prevent or delay the occurrence of disease or illness or symptom (for example, tumor) in a subject. As used herein, the term "treatment" refers to the method implemented in order to obtain beneficial or required clinical results. For the purpose of the application, beneficial or required clinical results include but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (that is, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease and alleviating symptoms (no matter in part or in whole), no matter it is detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival (if not receiving treatment), extending the survival.

[0110] As used herein, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, a therapeutically effective amount can be an amount that is effective or sufficient to treat or cure a disease (e.g., a tumor), delay the onset of symptoms of a disease (e.g., a tumor), and / or delay the development of a disease (e.g., a tumor). Such an effective amount can be easily determined by those skilled in the art or a doctor, and can be related to the intended purpose (e.g., treatment), the general health condition of the subject, age, sex, weight, severity of the disease to be treated, complications, mode of administration, etc. The determination of such an effective amount is entirely within the capabilities of those skilled in the art.

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

[0112] As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division may lead to the formation of malignant tumors, or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies, such as lymphomas, leukemias, myelomas, or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Beneficial effects

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

[0114] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application and are not intended to limit the scope of the present application. Based on the following detailed description of the drawings and preferred embodiments, various objects and advantages 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 viability 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 viability 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 viability of human umbilical vein endothelial cell line HUVEC; C shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human pancreatic cancer cell line PANC-1; D shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human brain glioma cell line U87; E shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human brain glioma cell line A172; F shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human gastric cancer cell line AGS; G shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human liver cancer cell line Huh7; H shows the effect of mRNA encoding SARS-CoV-2 Effects of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human melanoma cell line CHL-1; I shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human colon cancer cell line HCT116; J shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human renal cancer cell line ACHN; K shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human cervical cancer cell line Hela; L shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human osteosarcoma cell line U2OS; M shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human prostate cancer cell line DU145; N shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human lung cancer cell line NCI-H1975; O shows the effect of mRNA encoding SARS-CoV-2 3CL protease on the growth viability of human lung cancer cell line NCI-H1650; P shows the effect of mRNA encoding SARS-CoV-2 Effect of 3CL proteinase mRNA on the growth activity of human lung cancer cell line A549.

[0116] Sequence information

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

[0118] Table A: Information on some sequences Note: In accordance with WIPO Standard ST.26, in sequence listings, the symbol "u" ​​cannot be used to represent uracil in RNA molecules. The symbol "t" in RNA sequences is understood to be uracil. DETAILED DESCRIPTION

[0119] The present application will now be described with reference to the following examples which are intended to illustrate the present application (but not to limit the present application).The following examples may include a compilation of data collected at various times during the development and testing process related to the presently disclosed subject matter.

[0120] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this application are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Compendium, 3rd edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases follows the conditions recommended by the product manufacturers. If specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, for which the manufacturer is not specified, are all conventional products that can be obtained commercially. Those skilled in the art will appreciate that the examples describe the present application by way of example and are not intended to limit the scope of protection claimed in this application. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

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

[0122] Experimental materials: The pUC57 vector used in the experiment was purchased from Addgene (catalog number 54338), ATP was purchased from Thermo (catalog number: R0441), CTP was purchased from Thermo (catalog number: 18331017), GTP was purchased from Thermo (catalog number: 18332015), UTP was purchased from Thermo (catalog number: R1471), Nuclease-free water was purchased from Thermo (catalog number: 10977015), Top10 competent cells were purchased from Invitrogen (catalog number C404010), gentamicin was purchased from Sangon Biotech Co., Ltd. (catalog number A100304), primers were synthesized by Sangon Biotech 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 Yisheng 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), and DNase I was purchased from Thermo (Cat. No. 18047019). The manufacturer of the gene thermal cycler is Hangzhou Baiheng Technology Co., Ltd. (model: AUTO-96), the manufacturer of the ultra-micro UV-Vis spectrophotometer Nanodrop is Thermo (product number: 840-317400), the manufacturer of the constant temperature mixer (refrigeration) is Linbel Instruments (product number: BE-3200), the manufacturer of the refrigerated centrifuge is Germany / Eppendorf (model: 5424R), and the manufacturer of the dry-bed thermostat is Hangzhou Aosheng Instrument Co., Ltd. (model: K30).

[0123] Step 1: DNA template generation

[0124] The mRNA template sequence was designed based on codon preference, GC content, and the thermodynamic stability of RNA secondary structure. A pUC57-based in vitro transcription template plasmid was constructed through de novo gene synthesis. The pUC57 vector template contains a T7 promoter, a 5' untranslated region (UTR), a Kozak sequence, and a 3' untranslated region (UTR). The gene coding region for the SARS-CoV-2 3CL protease (amino acid sequence shown in SEQ ID NO:6) was inserted between the UTRs. The synthesized plasmid was transformed into a host bacterium and plated on antibiotic-containing solid plates. Positive clones were verified by colony PCR and Sanger sequencing. The plasmid sequence file was then aligned to confirm the target gene sequence. Correct clones were amplified in liquid culture medium and purified using an endotoxin-free plasmid extraction kit. PCR amplification was performed using PCR primers containing PolyA_110 and a high-fidelity DNA polymerase. The plasmid template was DNA tailed and linearized. The PCR product was purified to remove the enzyme and reaction components to obtain a linearized template. The product concentration and A260 / 280 were measured by Nanodrop, and the product quality was tested by agarose gel electrophoresis.

[0125] Step 2: mRNA in vitro transcription

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

[0127] Referring to the above method, mRNA encoding SARS-CoV 3CL protease and mRNA encoding MERS-CoV 3CL protease were also transcribed and synthesized in vitro, with the sequences 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 viability of various cell lines

[0129] This example was carried out based on 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 cells U87MG, purchased from ATCC (catalog number: HTB-14), A172, purchased from ATCC (catalog number: CRL-1620), human osteosarcoma cells U2OS, purchased from ATCC (catalog number: HTB-96), human melanoma cells CHL-1, purchased from ATCC (catalog number: CRL-9446), human prostate cancer cells DU145, purchased from ATCC (catalog number: HTB-81), human colon cancer cells HCT-116, purchased from ATCC (catalog number: CCL-247), and human cervical cancer cells Hela, purchased from ATCC (catalog number: CCL- 2) Human gastric adenocarcinoma cells AGS were purchased from ATCC (catalog number: CRL-1739), human lung cancer cells A549 were purchased from ATCC (catalog number: CCL-185), human non-small cell lung cancer cells NCI-H1975 were purchased from ATCC (catalog number: CRL-5908), human bronchial alveolar carcinoma cells NCI-H1650 were purchased from ATCC (catalog number: CRL-5883), human renal carcinoma cells ACHN were purchased from ATCC (catalog number: CRL-1611), human hepatoma cells Huh7 were purchased from the National Laboratory Cell Resource Sharing Platform, and human pancreatic cancer cells PANC-1 were purchased from ATCC (catalog number: CRL-1469). Non-tumor cell lines used in the experiments, such as human intravenous endothelial cells HUVEC, were purchased from Zhejiang Meisen Cell Technology Co., Ltd. (catalog number: CTCC-0804-PC) and human embryonic kidney epithelial cells 293T were purchased from ATCC (catalog number: CRL-3216).

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

[0132] The complete culture medium used for HUVEC cells was an endothelial-specific culture medium supplemented with 10% fetal bovine serum and penicillin-streptomycin double antibody, purchased from Zhejiang Meisen Cell Technology Co., Ltd. (Cat. No.: M22SN3004).

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

[0134] Cell-Titer Luminescent Cell Viability Assay Solution was purchased from Promega (Cat. No. G7572). Microplate reader was purchased from Molecular Devices (model: SpectraMax M5).

[0135] The experimental plan is as follows:

[0136] The cells to be tested were placed at a density of approximately 1.0 × 10 4 Cells were seeded at a density of 100 μg / ml in white-walled, clear-bottomed 96-well plates and incubated at 37°C, 5% CO2 for 24 hours. SARS-CoV-2 3CL protease mRNA was then serially diluted with Opti-MEM and transfected into cells using Lipo2000 liposomes. The final concentrations of SARS-CoV-2 3CL protease mRNA transfected into 293T cells and HUVEC cells were: 10,000 ng / ml, 5,000 ng / ml, 2,500 ng / ml, 1,250 ng / ml, 625 ng / ml, 312.5 ng / ml, 156.25 ng / ml, 78.125 ng / ml, and 39.06 ng / ml. The final transfection concentrations for ACHN, Hela, U2OS, DU145, U87 MG, A172, A549, NCI-H1975, and NCI-H1650 cells were 1000 ng / ml, 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml, and 15.65 ng / ml, respectively. The final transfection concentrations for PANC-1, AGS, Huh7, CHL-1, and HCT-116 cells were 2000 ng / ml, 1000 ng / ml, 500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, and 31.25 ng / ml, respectively. The final mRNA concentration for the control group was zero; all other conditions were identical to those for the experimental groups. After incubation for 48 hours, discard the supernatant and add 100 μl diluted Cell-Titer Luminescent Cell Viability Assay Solution was added, lysed with shaking for 5 minutes in the dark, allowed to stand for 3 minutes, and the fluorescence value of each well was measured using a microplate reader.

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

[0138] Statistical analysis: Origin9.0 software was used to fit the inhibition rate-concentration S-shaped curve and calculate the half maximal inhibitory concentration (IC 50 ).

[0139] The experimental results are shown in Tables 1-2 and Figure 1. Figure 1 shows the S-shaped curve of cell viability and IC corresponding to Tables 1 and 2. 50 value.

[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. Combined with the S-shaped curves shown in Figure 1 AB, it shows 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 of SARS-CoV-2 3CL protease mRNA on normal human embryonic kidney cell 293T is 0. 50 The value is greater than 10000ng / ml; as shown in Figure 1B, the IC 50 The above results indicate 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 activity of various human tumor cell lines. Combined with the S-shaped curve shown in CP in Figure 1, it is shown that the mRNA provided by this application has a significant killing effect on the growth of various tumor cell lines. Specifically, as shown in Figure 1 C, the IC of the mRNA on the human pancreatic cancer cell line PANC-1 is 50 The value was 574.626±52.61ng / ml; As shown in Figure 1D, the IC 50 The value was 184.268±25.76ng / ml; as shown in Figure 1E, the IC 50The value was 175.569±35.83ng / ml; as shown in Figure 1F, the IC 50 The value was 248.778±14.17ng / ml; as shown in Figure 1G, the IC 50 The value was 310.065±20.67ng / ml; as shown in Figure 1H, the IC 50 The value was 444.233±57.54 ng / ml; as shown in Figure 1, IC 50 The value was 976.261±262.17 ng / ml; as shown in Figure 1 J, the IC 50 The value was 430.272±107.46ng / ml; as shown in Figure 1 K, the IC 50 The value was 258.346±20.20ng / ml; as shown in Figure 1 L, the IC 50 The value was 122.175±14.78ng / ml; as shown in Figure 1, IC 50 The value was 78.716±8.20ng / ml; as shown in Figure 1, IC 50 The value was 274.938±61.75ng / ml; as shown in Figure 1, IC 50 The value was 158.56±42.74ng / ml; as shown in Figure 1, the IC 50 The value was 106.69±24.75ng / ml. In summary, the mRNA measured had an IC of 106.69±24.75ng / ml for all tested human tumor cell lines. 50 The values ​​were all less than 1000 ng / ml, which were significantly lower than the IC values ​​of mRNA for four non-tumor cell lines. 50 The values ​​indicate that the mRNA provided by this application has broad-spectrum anti-tumor activity and good selectivity.

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

[0145] Table 3 shows the therapeutic index of SARS-CoV-2 3CL protease mRNA relative to various tumor cell lines using 293T cells as control cells. Formula: Therapeutic index = 293T maximum IC 50 Value ÷ IC of each tumor cell line50 The tabulated results indicate that the SARS-CoV-2 3CL protease mRNA provided in this 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 viability of various types of cell lines were also investigated. The types of cell lines tested were the same as those in Example 2. The results showed that these two mRNAs also had broad-spectrum anti-tumor activity and good selectivity.

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

Claims

1. Use of a coronavirus 3CL protease or a nucleic acid molecule encoding the same in the preparation of a drug for preventing and / or treating tumors.

2. The method 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 shown in SEQ ID NO: 6, 10 or 13; ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown 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 shown in SEQ ID NO: 6, 10 or 13; Preferably, the substitutions described in ii) are conservative substitutions.

4. The use according to any one of claims 1 to 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 according to claim 4, wherein the nucleic acid molecule is an RNA molecule, preferably an mRNA molecule; Preferably, the nucleic acid molecule comprises the coding sequence of the 3CL protease; Preferably, the nucleic acid molecule further comprises one or more selected from 5'UTR, Kozak sequence, start codon, stop codon, 3'UTR, poly-A tail; Preferably, the nucleic acid molecule comprises, from the 5' end to the 3' end, in sequence: 5'UTR, Kozak sequence, start codon, coding sequence of the 3CL protease, stop codon, 3'UTR, poly-A tail; Preferably, the nucleic acid molecule is composed of 5'UTR, Kozak sequence, start codon, coding sequence of 3CL protease, stop codon, 3'UTR, poly-A tail from 5' to 3' end. become.

6. The use according to claim 5, which has one or more characteristics selected from the following: (1) The coding sequence of the 3CL protease is codon-optimized or unoptimized according to the codon preference of the host cell (e.g., human cell); (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 with the nucleotide sequence shown in SEQ ID NO: 1, 8 or 11; preferably, the coding sequence of the 3CL protease has the sequence shown in SEQ ID NO: 1, 8 or 11; (3) There is a 5' modification (e.g., CAP0 type, CAP1 type, CAP2 type) at the 5' end of the nucleic acid molecule; preferably, there is a 5'Cap 1 type modification at the 5' end of the nucleic acid molecule; (4) The nucleic acid molecule contains one or more N1-methyl pseudouridine (m1ψ) modifications; preferably, all or part of the uracil nucleotides in the nucleic acid molecule are replaced by N1-methyl-pseudouracil nucleotides; (5) There are one or more stop codons at the 3' end of the coding sequence of the 3CL protease; (6) the 5'UTR has a nucleotide sequence that has 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 shown in SEQ ID NO: 2; preferably, the 5'UTR has the nucleotide sequence shown in SEQ ID NO: 2; (7) the 3'UTR has a nucleotide sequence that has 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 shown in SEQ ID NO:4; preferably, the 3'UTR has the nucleotide sequence shown in SEQ ID NO:4; (8) The Kozak sequence has the sequence shown in SEQ ID NO: 3; (9) The poly-A tail comprises one or more polyadenylic acid sequences, each of which independently consists of 20-120 consecutive adenylic acids; preferably, the poly-A tail comprises multiple polyadenylic acid sequences, and the adjacent polyadenylic acid sequences are connected by a non-A spacer sequence; preferably, the poly-A tail has a sequence as shown in SEQ ID NO: 5; (10) The nucleic acid molecule has a nucleotide sequence that 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 shown in SEQ ID NO:7, 9 or 12; preferably, the nucleic acid molecule has the sequence shown in SEQ ID NO:7, 9 or 12.

7. The use according to any one of claims 1 to 6, wherein the tumor is a solid tumor or a blood tumor (eg, leukemia, lymphoma, myeloma); Preferably, the tumor is a solid tumor; Preferably, the tumor is selected from 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 classification includes but is 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 classification includes but is not limited to cardia cancer, gastric body cancer, pyloric cancer; the liver cancer classification includes but is not limited to hepatocellular carcinoma, hepatocellular carcinoma, fibrolamellar liver cancer, intrahepatic bile duct carcinoma, angiosarcoma, hepatoblastoma, secondary liver cancer, benign liver tumor; the melanoma classification includes but is not limited to local melanoma, local regional melanoma, distant metastatic melanoma; the intestinal cancer classification includes but is not limited to colon cancer and rectal cancer, specifically including adenocarcinoma, undifferentiated carcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, carcinoid, mucinous carcinoma, etc.; the renal cancer classification includes but is not limited to clear cell carcinoma, papillary cell carcinoma, chromophobe cell carcinoma , multilocular cystic renal cell tumor of low malignant potential, collecting duct carcinoma, renal medullary carcinoma; the cervical cancer classification includes but is not limited to squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma; the osteosarcoma classification includes but is 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 classification includes but is not limited to adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, adenosquamous carcinoma; the glioma classification includes but is not limited to PAs, astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligoastrocytoma, glioblastoma; the lung cancer classification includes but is 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 to 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 vector, and one or more selected from the following: a 3CL protease, an isolated nucleic acid molecule according to claim 8, a vector according to claim 9, wherein the 3CL protease is as defined in any one of claims 1 to 3; Preferably, the delivery vehicle is used to encapsulate, carry or deliver the 3CL protease, isolated nucleic acid molecule or vector; Preferably, the delivery vector includes a non-viral vector, a viral vector, and a virus-like body (VLP) vector between a viral vector and a non-viral vector; Preferably, the non-viral vector includes but is not limited to cationic liposomes, lipid nanoparticles, lipid polymers, artificial microspheres, micelles, lipid-polymer hybrid systems, extracellular vesicles, natural or engineered exosomes, etc., and the viral vector includes but is not limited to adeno-associated virus, lentivirus, adenovirus, retrovirus, vaccinia virus, measles virus, herpes simplex virus, alphavirus, vesicular stomatitis virus, influenza virus, etc.; Preferably, the delivery vehicle is a lipid nanoparticle.

11. A pharmaceutical composition comprising a 3CL protease, an isolated nucleic acid molecule according to claim 8, a vector according to claim 9 and / or a delivery composition according to claim 10, and a pharmaceutically acceptable carrier and / or excipient, wherein: The 3CL protease is as defined in any one of claims 1 to 3; Preferably, the pharmaceutical composition further comprises other anti-tumor drugs; Preferably, the other anti-tumor drugs are selected from small molecule drugs, biomacromolecule antibodies or proteins, chimeric antigen receptor cell drugs, and antibody-coupled drugs; Preferably, the pharmaceutical composition is administered orally, by spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, intratracheal installation, surgical implantation, transdermal delivery, local injection, ultra-fast injection / bombardment, or administration via an explanted reservoir. Preferably, the drug is administered orally, intraperitoneally, intrathoracically or intravenously.

12. A method for inhibiting tumor cells in vivo or in vitro, comprising: contacting the tumor cell with a 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, or delivering the 3CL protease into the tumor cell, wherein the 3CL protease is as defined in any one of claims 1 to 3, Preferably, the tumor cell is a cell of a solid tumor or a blood tumor (e.g., leukemia, lymphoma, myeloma); Preferably, the tumor cells are selected from cells 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 classification includes but is 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 classification includes but is not limited to cardia cancer, gastric body cancer, pyloric cancer; the liver cancer classification includes but is not limited to hepatocellular carcinoma, hepatocellular carcinoma, fibrolamellar liver cancer, intrahepatic bile duct carcinoma, angiosarcoma, hepatoblastoma, secondary liver cancer, benign liver tumor; the melanoma classification includes but is not limited to local melanoma, local regional melanoma, distant metastatic melanoma; the intestinal cancer classification includes but is not limited to colon cancer and rectal cancer, specifically including adenocarcinoma, undifferentiated carcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, carcinoid, mucinous carcinoma, etc.; the renal cancer classification includes but is not limited to clear cell carcinoma, papillary cell carcinoma, chromophobe cell carcinoma, low malignant potential multifocal carcinoma Atriocystic renal cell tumor, collecting duct carcinoma, renal medullary carcinoma; the cervical cancer classification includes but is not limited to squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma; the osteosarcoma classification includes but is 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 classification includes but is not limited to adenocarcinoma (acinar adenocarcinoma), ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma, adenosquamous carcinoma; the glioma classification includes but is not limited to PAs, astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligoastrocytoma, glioblastoma; the lung cancer classification includes but is 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 in the preparation of a medicament for preventing and / or treating tumors.

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