Oncolytic virus and application thereof in preparation of tumor inhibition drugs

Abstract

The invention discloses an oncolytic virus and application of the oncolytic virus in preparation of tumor inhibition drugs. The oncolytic virus is a lentiviral vector, and the lentiviral vector comprises polynucleotide encoding p16 protein or a bioactive part of the p16 protein containing CDKN2A gene, and can effectively inhibit growth of cancer related to CDKN2A gene mutation, so that the problem that the existing oncolytic virus has biological safety risk in delivery of cancer suppressor genes is effectively solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to oncolytic viruses and their use in the preparation of tumor suppressor drugs. Background Art

[0002] TP16 (also known as CDKN2A or INK4A) is an important tumor suppressor gene. The protein it encodes, p16, plays a key role in cell cycle regulation. P16 is closely linked to cancer, and its dysfunction or loss is closely associated with the development and progression of various cancers. Loss of function or abnormal expression of P16 can lead to uncontrolled cell cycle activity, thereby promoting the development and progression of tumors. Mutations in the CDKN2A gene, which encodes the p16 protein, may affect the cell cycle, apoptosis, and other anti-tumor defense mechanisms, thereby indirectly affecting the immune environment of tumor cells.

[0003] Existing technologies typically use conventional, self-replicating oncolytic viruses to deliver tumor suppressor genes, which poses biosafety risks. Therefore, there is an unmet need for a drug that can safely and effectively inhibit the growth of tumors associated with CDKN2A gene mutations, which remains a major challenge for drug developers. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an oncolytic virus and its use in the preparation of tumor suppressor drugs, which solves the problem of biosafety risks in the delivery of tumor suppressor genes by existing oncolytic viruses.

[0005] In order to achieve the above object, the technical solution of the present invention is implemented as follows: an oncolytic virus, wherein the oncolytic virus is a lentiviral vector, and the lentiviral vector comprises a polynucleotide encoding the p16 protein or a biologically active portion thereof comprising the CDKN2A gene.

[0006] The application described in the above technical solution, wherein: the amino acid sequence of the p16 protein is as shown in SEQ ID NO: 1; the amino acid sequence of the p16 protein has at least 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 1.

[0007] The application described in the above technical solution, wherein: the nucleic acid sequence of the polynucleotide comprising the CDKN2A gene is as shown in SEQ ID NO: 2; the nucleic acid sequence of the polynucleotide has at least 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2.

[0008] The application described in the above technical solution, wherein: it also includes a polynucleotide encoding an anti-PD-1 antibody.

[0009] The application described in the above technical solution, wherein: the anti-PD-1 antibody encodes a recombinant anti-PD-1 antibody, and the polynucleotide encoding the recombinant anti-PD-1 antibody sequentially encodes from the 5' end to the 3' end: a secretory protein signal peptide, an anti-PD-1 antibody heavy chain, a 2A peptide, a secretory protein signal peptide, and an anti-PD-1 antibody light chain.

[0010] The application described in the above technical solution, wherein: the amino acid sequence of the HCDR1 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 3; the amino acid sequence of the HCDR2 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 4; the amino acid sequence of the HCDR3 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 5;

[0011] The amino acid sequence of the LCDR1 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 6; the amino acid sequence of the LCDR2 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 7; and the amino acid sequence of the LCDR3 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 8.

[0012] The application described in the above technical solution, wherein: the amino acid sequence of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 9; the amino acid sequence of the anti-PD-1 antibody heavy chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9; the amino acid sequence of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 10; the amino acid sequence of the anti-PD-1 antibody light chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10.

[0013] The application described in the above technical solution, wherein: the secretory protein signal peptide is IL-10 signal peptide; and the 2A peptide is FT2A peptide.

[0014] The application described in the above technical solution, wherein: the amino acid sequence of the IL-10 signal peptide is shown in SEQ ID NO:11; the amino acid sequence of the IL-10 signal peptide is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11; the amino acid sequence of the FT2A peptide is shown in SEQ ID NO:12; the amino acid sequence of the FT2A peptide is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:12.

[0015] The application described in the above technical solution, wherein: the viral glycoprotein of the lentiviral vector is VSV-G; the amino acid sequence of the VSV-G is shown in SEQ ID NO: 13; the amino acid sequence of the VSV-G has at least 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 13.

[0016] The second technical solution of the present invention is achieved as follows: a pharmaceutical composition comprising the above-mentioned oncolytic virus and a pharmaceutically acceptable excipient or carrier.

[0017] The third technical solution of the present invention is achieved as follows: use of oncolytic virus or the above-mentioned pharmaceutical composition in the preparation of tumor suppressor drugs.

[0018] The application of the above technical solution, wherein: the tumor is LDL-R with CDKN2A gene mutation + Tumor; LDL-R with CDKN2A gene mutation + Tumors include liver cancer, glioblastoma, pancreatic ductal adenocarcinoma, pancreatic cancer, colorectal cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, non-small cell lung cancer, and melanoma.

[0019] Compared with the existing technology, the oncolytic virus of the present invention is a lentiviral vector, and the lentiviral vector contains the CDKN2A gene, which can effectively inhibit the growth of cancers related to CDKN2A gene mutations, thereby effectively solving the problem of biosafety risks in the delivery of tumor suppressor genes by existing oncolytic viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the shuttle gene contained in the transfer plasmid 2 in Example 1 of the present invention;

[0021] Figure 2 Schematic diagram of the test results of the effect of adding or not adding LVV2 on the efficiency of PBMCs in killing PANC-1 tumor cells in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] All technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] The terms "a" and "an" refer to one or to more than one (ie, to at least one) of the grammatical object of the article. As an example, "an element" refers to one element or more than one element.

[0024] The term "retrovirus" refers to viruses of the Retroviridae family. The term "lentivirus" refers to viruses of the genus Lentivirus within the Retroviridae family. Retroviruses are unique in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, making them one of the most effective gene delivery vectors. HIV, SIV, and FIV are all examples of retroviruses.

[0025] The term "retroviral vector" refers to a vector derived from at least a portion of a retroviral genome, and particularly includes the self-inactivating lentiviral vectors provided in Milone et al., Mol. Ther. 17(8): 1453–1464 (2009). Other examples of clinically useful lentiviral vectors include, but are not limited to, gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Non-clinical lentiviral vectors are also available and are known to those skilled in the art.

[0026] Lentiviral vectors and lentiviral vector backbone genomes are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey, et al., (1998) J. Virol. 72:9873-9880; Dull, et al., (1998) J. Virol. 72:8463-8471, U.S. Pat. No. 6,013,516, and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety.

[0027] The term "stable integration" is also called "stable transfection", which refers to the integration of exogenous polynucleotides into the host cell genome after introduction into the host cell and their long-term stable expression in the host cell (Stable Gene Expression); in contrast, transient transfection and transient expression (Transient Expression) are involved.

[0028] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0029] Portions of the antibodies or antibody fragments thereof of the present invention can exist in a variety of forms, wherein the antigen binding domain is expressed as part of a continuous polypeptide chain comprising, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of the present invention comprises an antibody fragment. In another aspect, the antibody fragment of the present invention is an scFv. The exact amino acid sequence boundaries of a given CDR can be determined using any one or a combination of a number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme).

[0030] The term "anti-PD-1 antibody" refers to antibodies that specifically bind to PD-1. PD-1 (programmed death receptor-1) is a surface protein whose primary function is to inhibit T cell activity by binding to its ligand, PD-L1, expressed on some tumor cells, thereby helping tumor cells evade immune system attack. By using anti-PD-1 antibodies to specifically bind to PD-1, thereby blocking the immune escape signaling pathway mediated by the binding of PD-1 and PD-L1, tumor cells can effectively prevent them from evading immune surveillance and attack.

[0031] The term "identity" refers to the subunit sequence identity between two polymer molecules, such as two nucleic acid molecules (such as two DNA molecules or two RNA molecules), or between two polypeptide molecules. When a subunit position in both molecules is occupied by the same monomeric subunit, for example, if a position in two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of paired or homologous positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer of 10 subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, then the two sequences are 90% homologous.

[0032] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise expressly limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly shown. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0033] The term "MOI" or "Multiplicity of Infection (MOI)" refers to the number of virus particles added to each cell during infection. For example, when one million virus particles are added to one million cells, MOI = 1.

[0034] The terms "pharmaceutically acceptable carrier" and "excipient" are found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), in particular Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMSM102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol.

[0035] The term "VSV-G" refers to the envelope glycoprotein of the Indiana strain of Vesicular Stomatitis Virus. VSV-G can bind to the low-density lipoprotein receptor (LDL-R) that is widely present on the surface of various cells, including various cancer cells, and has a wide range of infectivity.

[0036] The term "biologically active portion" refers to a biologically active portion of a protein, generally referring to a region or structure of the protein that is directly involved in its biological function.

[0037] The term "pharmaceutically acceptable excipient or carrier" herein includes, but is not limited to, diluents, solubilizers, emulsifiers, preservatives, preservatives and / or adjuvants.

[0038] Herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, or elements, or groups of steps or elements, but not the exclusion of any other steps, or elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including," "having," "containing," and "comprising" are used synonymously.

[0039] As used herein, the terms "transfection," "transformation," and "transduction" are used synonymously to refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell, packaging cell, or the like. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0040] "Embodiments": Reference throughout this specification to "some embodiments" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, various appearances of the foregoing phrase throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0041] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0042] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0043] Example 1 of the present invention provides an oncolytic virus, wherein the oncolytic virus is a lentiviral vector, and the lentiviral vector comprises a polynucleotide encoding the p16 protein or a biologically active portion thereof comprising the CDKNA gene.

[0044] In a further embodiment, the amino acid sequence of the p16 protein is as shown in SEQ ID NO: 1; the amino acid sequence of the p16 protein is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1.

[0045] In a further implementation, the nucleic acid sequence of the polynucleotide comprising the CDKN2A gene is shown in SEQ ID NO: 2; the nucleic acid sequence of the polynucleotide is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2.

[0046] In a further embodiment, the method further comprises a polynucleotide encoding an anti-PD-1 antibody.

[0047] In a further implementation process, the anti-PD-1 antibody encodes a recombinant anti-PD-1 antibody, and the polynucleotide encoding the recombinant anti-PD-1 antibody sequentially encodes from the 5' end to the 3' end: a secretory protein signal peptide, an anti-PD-1 antibody heavy chain, a 2A peptide, a secretory protein signal peptide, and an anti-PD-1 antibody light chain.

[0048] In a further implementation, the amino acid sequence of the HCDR1 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 3; the amino acid sequence of the HCDR2 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 4; the amino acid sequence of the HCDR3 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 5;

[0049] The amino acid sequence of the LCDR1 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 6; the amino acid sequence of the LCDR2 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 7; and the amino acid sequence of the LCDR3 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 8.

[0050] In a further implementation, the amino acid sequence of the anti-PD-1 antibody heavy chain is as shown in SEQ ID NO:9; the amino acid sequence of the anti-PD-1 antibody heavy chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9; the amino acid sequence of the anti-PD-1 antibody light chain is as shown in SEQ ID NO:10; the amino acid sequence of the anti-PD-1 antibody light chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10.

[0051] In a further embodiment, the secretory protein signal peptide is an IL-10 signal peptide; and the 2A peptide is an FT2A peptide.

[0052] In a further implementation, the amino acid sequence of the IL-10 signal peptide is as shown in SEQ ID NO:11; the amino acid sequence of the IL-10 signal peptide is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11; the amino acid sequence of the FT2A peptide is as shown in SEQ ID NO:12; the amino acid sequence of the FT2A peptide is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:12.

[0053] In a further implementation, the viral glycoprotein of the lentiviral vector is VSV-G; the amino acid sequence of VSV-G is as shown in SEQ ID NO: 13; the amino acid sequence of VSV-G is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13.

[0054] An embodiment of the present invention also provides a pharmaceutical composition comprising the above-mentioned oncolytic virus and a pharmaceutically acceptable excipient or carrier.

[0055] The embodiments of the present invention also provide the use of the above-mentioned oncolytic virus or pharmaceutical composition in the preparation of tumor suppressor drugs.

[0056] In a further embodiment, the tumor is an LDL-R + Tumor; LDL-R with CDKN2A gene mutation + Tumors include liver cancer, glioblastoma, pancreatic ductal adenocarcinoma, pancreatic cancer, colorectal cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, non-small cell lung cancer, and melanoma.

[0057] The following are specific embodiments

[0058] Example 1

[0059] The lentiviral vector containing the CDKN2A gene, LVV1, was packaged.

[0060] 1. Packaging Lentiviral Vectors

[0061] S1. Prepare the lentiviral vector packaging system:

[0062] (1) According to the lentiviral vector packaging system shown in Tables 1 and 2 below, a lentiviral vector containing the CDKN2A gene d was packaged:

[0063] Prepare solution A, the ingredients are shown in the following table 1.

[0064] Table 1

[0065]

[0066]

[0067] The transfer plasmid 1 comprises a lentiviral backbone genome, a CDKN2A gene, and a polynucleotide encoding GFP ("Green Flourescent Protein"). The p16 protein and GFP are connected via an FT2A peptide (SEQ ID NO: 12).

[0068] The amino acid sequence of the p16 protein is shown in SEQ ID NO: 1;

[0069] p16 protein (UniProtKB number: Q8N726-1):

[0070] MVRRFLVTLRIRRACGPPRVRVFVVHIPRLTGEWAAPGAPAAVALVLML LRSQRLGQQPLPRRPGHDDGQRPSGGAAAAPRRGAQLRRPRHSHPTRARRC PGGLPGHAGGAAPGRGAAGRARCLGPSARGPG (SEQ ID NO: 1);

[0071] CDKN2A gene (NCBI Reference Sequence: NM_058195.3):

[0072] atggtgcgcaggttcttggtgaccctccggattcggcgcgcgtgcggcccgccgcgagtgagggttttcgtggttcacatcccgcggctcacgggggagt gggcagcgccaggggcgcccgccgctgtggccctcgtgctgatgctactgaggagccagcgtctagggcagcagccgcttcctagaagaccaggtcatgat gatgggcagcgcccgagtggcggagctgctgctgctccacggcgcggagcccaactgcgccgaccccgccactctcacccgacccgtgcacgacgctgccc gggagggcttcctggacacgctggtggtgctgcaccgggccggggcgcggctggacgtgcgcgatgcctggggccgtctgcccgtggacctggctga(SEQ ID NO:2);

[0073] The amino acid sequence of the FT2A peptide is shown in SEQ ID NO: 12;

[0074] FT2A peptide:

[0075] RRKRGSGERGSLLTCGDVEENPGP(SEQ ID NO:12);

[0076] The pMD2.G plasmid contains a polynucleotide encoding wild-type VSV-G (including its signal peptide);

[0077] The amino acid sequence of the full-length protein of wild-type VSV-G (its signal peptide is underlined) is shown in SEQ ID NO: 13;

[0078] Wild-type VSV-G full-length protein:

[0079] MKCLLYLAFLFIGVNC KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNNNHNDLIGTALQVKMPKSHKAIQADGMNCHASKWVCCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAV IVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHNGVRLPSGVWEEMADKDLFAAAR FPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPCSPVDLSYLAPKNPGTGPAETIINGTLKYEETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGV LRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVEEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSZASFFFLIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLG(SEQ ID NO:13).

[0080] (2) Prepare Solution B, the ingredients of which are shown in Table 2 below.

[0081] Table 2

[0082]

[0083] Transfer solution B into solution A with a pipette, blow down, shake vigorously for 2 minutes, and let it stand for 10 minutes.

[0084] S2. Introducing the solution A and solution B (packaging system) into HEK-293T cells:

[0085] (1) On Day 0, remove HEK-293T cells from the incubator and remove the original culture medium using an electric aspirator. Add 9.5-10 mL of the culture medium containing the mixture of A and B solutions to the plates, then return the plates to the 5% CO2, 37°C incubator for incubation. Record the start time of the incubation.

[0086] (2) After 6 hours, the HEK-293T cell culture medium was replaced with fresh 10% FBS and the cells were returned to the 37°C incubator with 5% CO2 for further culture.

[0087] (3) 48 hours after transfection, the virus solution was collected for the first time and completely fresh HEK-293T cell culture medium FBS was added. The cells were returned to the 37°C incubator with 5% CO2 and continued to be cultured. The collected lentiviral vectors were labeled and stored in a 4°C refrigerator.

[0088] (4) The virus solution was collected for the second time 72 hours after transfection.

[0089] (5) Take out the virus liquid collected for the first time, together with the virus liquid collected for the second time, centrifuge at 500×g for 3 minutes, take the supernatant, filter the virus liquid using a 0.45μm filter membrane and a 50mL syringe, and filter it into a sterilized ultracentrifuge tube.

[0090] (6) After the virus solution is strictly balanced, centrifuge it at 50,000 × g at 4°C for 150 min. After the centrifugation, a small amount of white precipitate will be visible at the bottom of the centrifuge tube. Mark it and discard the supernatant. Be careful not to aspirate the white precipitate when discarding the supernatant with an aspirator. Resuspend each tube of virus by repeatedly pipetting at the marked position with 200 μl of F12, and try not to create too many bubbles.

[0091] (7) Collect all resuspended LVV1 viruses into one tube, shake well, centrifuge briefly to remove water droplets on the inner wall of the tube cap, divide into smaller packages as needed, and store in a -80℃ refrigerator.

[0092] 2. Inject the LVV1 into PANC-1 tumor cell model mice

[0093] 2×10 6 PANC-1 tumor cells (adult pancreatic cancer cells) were added to each mouse and divided into 3 groups, with 5 tumor-bearing mice in each group.

[0094] On Day 0, the LVV1 and gemcitabine were orthotopically injected into two groups of tumor-bearing mice according to the administration method shown in Table 3 below.

[0095] Table 3

[0096] 1 group 5 control group 2 groups 5 Orthotopic injection of 1E7 TU as described in LVV1 3 groups 5 Gemcitabine drug control, 120 mg / Kg body weight

[0097] Starting from the third day, the tumor growth was observed every day, the tumor size was recorded, and the tumor volume was calculated according to the following formula: V = ab 2 / 2 (V-volume, a-long diameter of tumor, b-short diameter of tumor). Tumor volume of each group (mm 3 ) changes are shown in Table 4 below (mean ± standard deviation).

[0098] Table 4

[0099]

[0100] As shown in Table 4, in situ injection of the LVV1 containing the CDKN2A gene can effectively inhibit the CDKN2A gene mutation, LDL-R + The invention can inhibit the growth of tumor cells and PANC-1 tumor cells, and its effect of inhibiting tumor growth is significantly better than the anti-tumor drug gemcitabine commonly used in the prior art.

[0101] Example 2

[0102] 1. Packaging LVV2 containing CDKN2A gene and recombinant anti-PD-1 antibody gene

[0103] Referring to the method for packaging the LVV1 in Example 1, a lentiviral vector comprising the CDKN2A gene and a polynucleotide encoding a recombinant anti-PD-1 antibody was packaged, LVV2; wherein the transfer plasmid 1 was replaced with a transfer plasmid 2 comprising a lentiviral backbone genome and a tandem expression cassette, wherein the tandem expression cassette comprises a strong promoter CASI, the CDKN2A gene and a polynucleotide encoding the recombinant anti-PD-1 antibody; the structure of the tandem expression cassette is as follows: Figure 1 shown.

[0104] The polynucleotide encoding the recombinant anti-PD-1 antibody sequentially encodes from the 5' end to the 3' end: IL-10 signal peptide, anti-PD-1 antibody heavy chain, the FT2A peptide, IL-10 signal peptide, and anti-PD-1 antibody light chain;

[0105] The nucleic acid sequence of the strong promoter CASI is shown in SEQ ID NO: 16:

[0106]

[0107] The amino acid sequence of the IL-10 signal peptide is shown in SEQ ID NO: 11:

[0108] MHSSALLCCLVLLTGVRA(SEQ ID NO:11)

[0109] The amino acid sequence of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 9:

[0110] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:9);

[0111] The amino acid sequence of the VH region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 14:

[0112] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLE WMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCA RRDYRFDMGFDYWGQGTTVTVSS(SEQ ID NO:14)

[0113] The amino acid sequences of the HCDR1-3 regions of the heavy chain of the anti-PD-1 antibody are shown in SEQ ID NOs: 3-5, respectively;

[0114] HCDR1 region of the anti-PD-1 antibody heavy chain:

[0115] GYTFTNYY (SEQ ID NO: 3)

[0116] HCDR2 region of the anti-PD-1 antibody heavy chain:

[0117] INPSNGGT (SEQ ID NO: 4)

[0118] The HCDR3 region of the anti-PD-1 antibody heavy chain:

[0119] ARRDYRFDMGFDY (SEQ ID NO: 5)

[0120] The amino acid sequence of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 10;

[0121] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:10)

[0122] The amino acid sequence of the VL region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 15:

[0123] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRL LIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGG GTKVEIK(SEQ ID NO:15)

[0124] The amino acid sequences of the LCDR1-3 regions of the anti-PD-1 antibody light chain are shown in SEQ ID NOs: 6-8, respectively;

[0125] LCDR1 region of the anti-PD-1 antibody light chain:

[0126] KGVSTSGYSY (SEQ ID NO: 6)

[0127] LCDR2 region of the anti-PD-1 antibody light chain:

[0128] LAS (SEQ ID NO: 7)

[0129] LCDR3 region of the anti-PD-1 antibody light chain:

[0130] QHSRDLPLT (SEQ ID NO: 8).

[0131] 2. Detecting the efficiency of LVV2 in killing tumor cells

[0132] Day 0: thaw frozen PBMCs from healthy individuals (thaw methods are well known to those skilled in the art); prepare two groups of mixed cells as follows: 1×10 5 The PBMCs were mixed with 5 × 10 5 PANC-1 cells carrying a luciferase reporter gene were mixed, and the two groups of mixed cells were resuspended separately in a mixed cell culture medium, wherein the mixed cell culture medium includes: 1640 culture medium + 10% FBS;

[0133] At an MOI of 1, LVV2 was added to one of the mixed cell culture systems; the two mixed cell groups were inoculated into three wells of an ELISA plate, respectively. After culturing for 2 hours, substrate was added, and chemiluminescence detection was performed using an ELISA plate to calculate the killing efficiency. The specific calculation method was as follows: the well containing only target PANC-1 cells was used as a reference well, and its fluorescence value was the total fluorescence value of the original tumor cell count; the fluorescence value of the tumor remaining after killing in each well with or without the addition of LVV2 was the residual fluorescence value, and the killing efficiency was calculated as follows: Killing efficiency (%) = (total fluorescence value - residual fluorescence value) / total fluorescence value × 100%. The killing results are shown in FIG. Figure 2 As shown;

[0134] Depend on Figure 2 It can be seen that when LVV2 is added, the efficiency of PBMCs in killing Huh7 tumor cells can be significantly improved.

[0135] In summary, the oncolytic virus of the present invention is a lentiviral vector, and the lentiviral vector contains the CDKN2A gene and the anti-PD-1 antibody gene, which can effectively inhibit the growth of cancers associated with CDKN2A gene mutations, thereby effectively solving the problem of biosafety risks in the delivery of tumor suppressor genes by existing oncolytic viruses.

[0136] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. An oncolytic virus, characterized in that The oncolytic virus is a lentiviral vector, which contains a polynucleotide encoding the p16 protein or a biologically active portion thereof including the CDKN2A gene.

2. The oncolytic virus according to claim 1, characterized in that The amino acid sequence of the p16 protein is shown in SEQ ID NO: 1; the amino acid sequence of the p16 protein is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

1.

3. The oncolytic virus according to claim 2, characterized in that The nucleic acid sequence of the polynucleotide is shown in SEQ ID NO: 2; the nucleic acid sequence of the polynucleotide comprising the CDKN2A gene is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

2.

4. The oncolytic virus according to any one of claims 1 to 3, characterized in that Also included are polynucleotides encoding anti-PD-1 antibodies.

5. The oncolytic virus according to claim 4, characterized in that The anti-PD-1 antibody encodes a recombinant anti-PD-1 antibody, and the polynucleotide encoding the recombinant anti-PD-1 antibody sequentially encodes from the 5' end to the 3' end: a secretory protein signal peptide, an anti-PD-1 antibody heavy chain, a 2A peptide, a secretory protein signal peptide, and an anti-PD-1 antibody light chain.

6. The oncolytic virus according to claim 5, characterized in that The amino acid sequence of the HCDR1 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 3; the amino acid sequence of the HCDR2 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 4; and the amino acid sequence of the HCDR3 region of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO:

5. The amino acid sequence of the LCDR1 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 6; the amino acid sequence of the LCDR2 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 7; and the amino acid sequence of the LCDR3 region of the anti-PD-1 antibody light chain is shown in SEQ ID NO:

8.

7. The oncolytic virus according to claim 6, characterized in that The amino acid sequence of the anti-PD-1 antibody heavy chain is shown in SEQ ID NO: 9; the amino acid sequence of the anti-PD-1 antibody heavy chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9; the amino acid sequence of the anti-PD-1 antibody light chain is shown in SEQ ID NO: 10; the amino acid sequence of the anti-PD-1 antibody light chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

10.

8. The oncolytic virus according to claim 5, characterized in that The secretory protein signal peptide is IL-10 signal peptide; the 2A peptide is FT2A peptide.

9. The oncolytic virus according to claim 8, characterized in that The amino acid sequence of the IL-10 signal peptide is shown in SEQ ID NO: 11; the amino acid sequence of the IL-10 signal peptide is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11; the amino acid sequence of the FT2A peptide is shown in SEQ ID NO: 12; the amino acid sequence of the FT2A peptide is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

12.

10. The oncolytic virus according to claim 4, characterized in that The viral glycoprotein of the lentiviral vector is VSV-G; the amino acid sequence of VSV-G is shown in SEQ ID NO: 13; the amino acid sequence of VSV-G is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

13.

11. A pharmaceutical composition, characterized in that Comprising the oncolytic virus according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient or carrier.

12. Use of the oncolytic virus according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 in the preparation of tumor suppressor drugs.

13. The use according to claim 12, characterized in that The tumor is an LDL-R with a CDKN2A gene mutation + Tumor; LDL-R with CDKN2A gene mutation + Tumors include liver cancer, glioblastoma, pancreatic ductal adenocarcinoma, pancreatic cancer, colorectal cancer, head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, non-small cell lung cancer, and melanoma.

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