Retroviral vector and application thereof in preparation of tumor inhibition drugs

Abstract

The invention relates to the field of biological medicine, in particular to a retroviral vector and application thereof in preparation of tumor inhibition drugs, the retroviral vector comprises polynucleotide encoding p53 protein or a bioactive part thereof, the surface of the retroviral vector comprises a targeting molecule binding to PD-L1, and the targeting molecule comprises a targeting molecule binding to PD-L1. The retrovirus vector can effectively inhibit tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to a retroviral vector and its application in the preparation of anti-tumor drugs. Background Art

[0002] TP53 gene mutation is the most common genetic variation in human malignant tumors. According to statistics, about 50% of human tumors are related to TP53 gene mutation, and TP53 gene mutation exists in 30%-40% of breast cancers, 50% of lung cancers and 70% of colon cancers; almost 100% of small cell lung cancers have TP53 gene mutation. In addition, animal experiments show that 100% of mice expressing abnormal TP53 gene protein develop tumors.

[0003] A large number of existing studies have shown that wild-type p53 protein plays a biological function of "gene guard" in cells, monitoring the integrity and stability of the cell genome. More importantly, wild-type p53 protein can initiate the process of programmed cell death, induce cell suicide, prevent cells with malignant tendencies from further dividing and proliferating, and thus prevent the occurrence of cancer.

[0004] PD-L1 (programmed death ligand 1) is a surface protein, and its main function is to inhibit the activity of T cells by binding to its receptor PD-1, thereby helping tumor cells evade the attack of the immune system. The mutation of TP53 gene may affect the cell cycle, apoptosis and other anti-tumor defense mechanisms, and thus indirectly affect the immune environment of tumor cells.

[0005] Some studies have shown that there is a correlation between the mutation of TP53 gene and the expression of PD-L1 in some cancers. For example, in non-small cell lung cancer (NSCLC), there is a certain correlation between the mutation of TP53 gene and the high expression of PD-L1. This may be because the cell stress and changes in the immune microenvironment caused by TP53 gene mutation promote the increase of PD-L1 expression.

[0006] Therefore, for the treatment of tumors related to TP53 gene mutation, PD-L1 has the potential to be used as a target.

[0007] In the prior art, the TP53 gene is mainly carried by adenovirus; although adenovirus as a vector has the advantages of wide host range, high infection efficiency, large packaging capacity, high expression level and no integration risk, etc., adenovirus as a vector also has the following problems: it cannot integrate into the host genome, so it can only be transiently expressed, and its ability to inhibit tumor growth is poor.

[0008] There is a need for a drug that can effectively inhibit PD-L1 + related to TP53 gene mutation and relatively long-term and stably inhibit tumor growth, which has not been met yet. Summary of the Invention

[0009] In order to improve the above technical problems, on the one hand, the present invention provides a retroviral vector, characterized in that,

[0010] (1) The retroviral vector contains a polynucleotide encoding p53 protein or a biologically active part thereof; and

[0011] (2) The surface of the retroviral vector contains a targeting molecule that binds to PD-L1.

[0012] In one embodiment, the p53 protein contains an amino acid sequence as shown in SEQ ID NO:1 or having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:1;

[0013] Preferably, the amino acid sequence of the p53 protein is as shown in SEQ ID NO:1.

[0014] In one embodiment, the targeting molecule contains an antibody specific for binding to PD-L1 and fragments thereof;

[0015] Preferably, the heavy chain CDR1, CDR2 and CDR3 of the antibody and its fragments are respectively as shown in SEQ ID NO:10-12, and the light chain CDR1, CDR2 and CDR3 are respectively as shown in SEQ ID NO:13-15.

[0016] In one embodiment, the targeting molecule contains a transmembrane peptide segment;

[0017] Preferably, the transmembrane peptide segment is the transmembrane region of CD3ζ, CD4, CD8α or CD28;

[0018] More preferably, the transmembrane peptide segment is the transmembrane region of CD8α.

[0019] In one embodiment, the targeting molecule further contains a hinge region;

[0020] Preferably, the hinge region is the hinge region of CD8α or CD28.

[0021] On the other hand, the present invention also provides a pharmaceutical composition, which contains a pharmaceutically acceptable excipient or carrier and the aforementioned retroviral vector provided by the present invention.

[0022] On the other hand, the present invention provides the use of the aforementioned retroviral vector and pharmaceutical composition in the preparation of a tumor-inhibiting drug.

[0023] In one embodiment, the tumor is PD-L1 +A tumor, the PD-L1 + The tumors include non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), melanoma, renal cell carcinoma, head and neck squamous cell carcinoma (HNSCC), bladder cancer, gastric cancer, esophageal cancer, liver cancer, triple-negative breast cancer (TNBC), Hodgkin lymphoma, colorectal cancer, pancreatic cancer, ovarian cancer, and prostate cancer;

[0024] Preferably, the tumor is liver cancer.

[0025] Beneficial effects

[0026] The retroviral vector provided by the present invention can stably integrate the TP53 gene into the genome of PD-L1 + tumor cells related to TP53 gene mutation, so that the p53 protein is stably expressed in PD-L1 + tumor cells for a long time, effectively inhibiting tumor growth. Description of the drawings

[0027] Figure 1 Shows the detection results of the infection efficiency of the targeted lentiviral vector (VSVG-anti-PD-L1) (i.e., Figure 1 A) and the non-targeted lentiviral vector (VSVG) (i.e., Figure 1 B) in mixed Huh-7 cells and overexpressed Huh-7 cells. Detailed implementation manners

[0028] Definitions

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] The terms "a" and "an" refer to one or more than one (i.e., at least one) grammatical object of the article. As an example, "an element" refers to one element or more than one element.

[0031] The term "retrovirus" refers to a virus of the family Retroviridae. The term "lentivirus" refers to a virus of the genus Lentivirus in the family Retroviridae. The uniqueness of retroviruses lies in their ability to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, so they are one of the most effective methods of gene delivery vectors. HIV, SIV, and FIV are all examples of retroviruses.

[0032] The term "retroviral vector" refers to a vector derived from at least a portion of a retroviral genome, and particularly includes self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453–1464 (2009). Other examples of clinically available lentiviral vectors include, but are not limited to, for example, gene delivery technologies from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0033] The term "stable integration", also known as "stable transfection", refers to the integration of an exogenous polynucleotide into the genome of a host cell after introduction into the host cell, with stable expression in the host cell over the long term (Stable Gene Expression); in contrast thereto is transient transfection and transient expression (Transient Expression).

[0034] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen. The antibody can be polyclonal or monoclonal, multichain or single-chain, or a complete immunoglobulin, and can be derived from a natural source or from a recombinant source. The antibody can be a tetramer of immunoglobulin molecules.

[0035] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain and heavy chain variable regions are continuously linked, for example, by a synthetic linker (such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv used herein can have the VL and VH variable regions in either order, for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0036] Portions of the antibodies or antibody fragments 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 that includes, for example, single-domain antibody fragments (sdAb), single-chain antibodies (scFv), humanized antibodies, or bispecific antibodies (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 scFv in the present invention. The exact amino acid sequence boundaries of a given CDR can be determined using any one or a combination of many 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).

[0037] The term "identity" refers to subunit sequence identity between two polymer molecules, such as between two nucleic acid molecules (e.g., two DNA molecules or two RNA molecules), or between two polypeptide molecules. The subunits at corresponding positions in the two molecules are occupied by the same monomeric subunit; for example, if the positions in two DNA molecules are both occupied by adenine, they are homologous or identical at that position. Homology between two sequences is a direct function of the number of paired or homologous positions; for example, if half of the positions in two sequences (e.g., 5 positions in a polymer of 10 subunits in length) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, the two sequences are 90% homologous.

[0038] The terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, and polymers thereof. Unless otherwise expressly restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly shown sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixture of bases 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)).

[0039] The term "MOI", i.e., "Multiplicity of Infection (MOI)", refers to the number of viral particles added to each cell during the infection process. For example, when one million viral particles are added to one million cells, MOI = 1.

[0040] The terms "pharmaceutically acceptable carrier" and "excipient" can be found in pharmacopoeias known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th Edition, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMSM102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous absorbents, water-in-oil and oil-in-water emulsions, emulsion polyethyleneglycols (polyethyleneglycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethyleneglycol.

[0041] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, rectal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, for example, both terms encompass solid and fluid (e.g., diffuse or circulating) tumors. The term "cancer" or "tumor" as used herein includes pre-malignant as well as malignant cancers and tumors.

[0042] The term "treatment" as used herein refers to a process. A therapeutic effect is obtained by reducing, inhibiting, alleviating, or eliminating a disease state.

[0043] The term "prevention" as used herein refers to the prevention or prophylactic treatment of a disease or disease state.

[0044] The term "tumor suppression" refers to a biological effect that can manifest in various ways, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in cancer cell proliferation, a decrease in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition.

[0045] In this document, the terms "transfection", "transformation", and "transduction" are used synonymously and refer to the process of introducing exogenous nucleic acid into a host cell or packaging cell. A "transfected", "transformed", or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary test cell and its progeny.

[0046] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0047] Unless otherwise specified, the raw materials and reagents used in the following embodiments are commercially available products or can be prepared by known methods.

[0048] Example 1: Packaging a lentiviral vector (targeted lentiviral vector) containing the TP53 gene and targeting PD-L1 + cells

[0049] 1. Constructing a membrane-bound anti-PD-L1 antibody

[0050] The structure of the membrane-bound anti-PD-L1 antibody from the N-terminus to the C-terminus is in turn: CD8α signal peptide, scFv specifically binding to PD-L1 (scFv-PD-L1), CD8α hinge region, CD8α transmembrane region;

[0051] The heavy chain variable region (VH) of the scFv-PD-L1 is linked to the light chain variable region (VL) of the scFv-PD-L1 through a GS linker peptide;

[0052] (1) The CD8α signal peptide contains the amino acid sequence shown in SEQ ID NO:3;

[0053] (2) The VH region of the scFv-PD-L1 contains the amino acid sequence shown in SEQ ID NO:4;

[0054] (3) The linker peptide contains the amino acid sequence shown in SEQ ID NO:5;

[0055] (4) The VL region of the scFv-PD-L1 contains the amino acid sequence shown in SEQ ID NO:6;

[0056] (5) The CD8α hinge region contains the amino acid sequence shown in SEQ ID NO:7;

[0057] (6) The CD8α transmembrane region contains the amino acid sequence shown in SEQ ID NO:8;

[0058] CD8α signal peptide:

[0059] MALPVTALLLPLALLLHAARP (SEQ ID NO:3);

[0060] VH region of scFv-PD-L1:

[0061] QIQLVQSGPELKNPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKW MGWINTHTGEPTYADDFKGRFAFSSETSASSAYLQINNLKNDDMATYFCAK GTHREEIPAWFAYWGQGTLVTVSA (SEQ ID NO:4);

[0062] The CDRs of the VH region of scFv-PD-L1 are respectively:

[0063] HCDR1: NYGMN (SEQ ID NO:10)

[0064] HCDR2: WINTHTGEPTYADDFKG (SEQ ID NO:11)

[0065] HCDR3: GTHREEIPAWFAY (SEQ ID NO:12);

[0066] GS linker peptide:

[0067] GGGGSGGGGSGGGGS (SEQ ID NO:5);

[0068] VL region of scFv-PD-L1:

[0069] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPELLIYKVSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPPTFGAGTKLELKR (SEQ ID NO:6);

[0070] CDRs of the VL region of scFv-PD-L1 are respectively:

[0071] LCDR1: RSSQSIVHSNGNTYLE (SEQ ID NO:13)

[0072] LCDR2: KVSNLFS (SEQ ID NO:14)

[0073] LCDR3: FQGSHVPPT (SEQ ID NO:15);

[0074] Hinge region of CD8α:

[0075] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:7);

[0076] Transmembrane region of CD8α:

[0077] IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:8).

[0078] 2. Packaging the targeted lentiviral vector

[0079] S1. Prepare the lentiviral vector packaging system:

[0080] (1) According to the lentiviral vector packaging system shown in Table 1 and Table 2 below, package the targeted lentiviral vector containing the TP53 gene and targeting PD-L1:

[0081] Prepare Solution A, and its components are shown in Table 1 below.

[0082] Table 1

[0083]

[0084] Among them, the transfer plasmid contains a lentiviral backbone genome, a polynucleotide encoding the p53 protein, and a polynucleotide encoding GFP ("Green Flourescent Protein", green fluorescent protein); the p53 protein and GFP are linked by a P2A peptide (SEQ ID NO: 16);

[0085] The p53 protein contains the amino acid sequence shown in SEQ ID NO: 1;

[0086] p53 protein:

[0087] MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID NO: 1);

[0088] The polynucleotide (TP53 gene) encoding the p53 protein contains the nucleotide sequence shown in SEQ ID NO: 2;

[0089] The polynucleotide (TP53 gene) encoding the p53 protein:

[0090]

[0091] RRKRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO:16);

[0092] The envelope plasmid contains a polynucleotide encoding wild-type VSV-G and a polynucleotide encoding the membrane-type expressed anti-PD-L1 antibody; the method for constructing the envelope plasmid by conventional molecular cloning methods is well known to those skilled in the art;

[0093] The full-length protein of wild-type VSV-G contains the amino acid sequence shown in SEQ ID NO:9;

[0094] Wild-type VSV-G full-length protein:

[0095] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNNHNDLIGTALQVKMPKSHKAIQADGMNCHASKWVCCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHNGVRLPSGVWEEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPCSPVDLSYLAPKNPGTGPAETIINGTLKYEETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVEEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSZASFFFLIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLG (SEQ ID NO:9).

[0096] (2) Prepare Solution B, and the components are shown in Table 2 below.

[0097] Table 2

[0098]

[0099] Transfer Solution B to Solution A with a pipette, pipette up and down and shake vigorously for 2 min, and let stand for 10 min.

[0100] S2. Introduce the A liquid and B liquid (packaging system) into HEK-293T cells:

[0101] (1) On Day 0, take out HEK-293T cells from the incubator, remove the original culture with an electric suction device, 4 - 5 plates at a time, add 9.5 - 10 mL of the culture medium mixed with A + B liquid, and put it back into the 5% CO₂ 37°C incubator for culture. Record the start time of culture.

[0102] (2) After 6 h, replace it with fresh HEK-293T cell culture medium containing 10% FBS, and put it back into the 5% CO₂ 37°C incubator for continuous culture.

[0103] (3) After 48 h of transfection, collect the first virus solution for the first time, and add completely fresh HEK-293T cell culture medium with FBS, then put it back into the 5% CO₂ 37°C incubator for continuous culture. Mark the collected lentiviral vector and store it in a 4°C refrigerator.

[0104] (4) Collect the second virus solution 72 h after transfection.

[0105] (5) Take out the virus solution collected for the first time, together with the virus solution collected for the second time, centrifuge at 500×g for 3 min, take the supernatant, filter the virus solution using a 0.45 μm filter membrane and a 50 mL syringe, and filter it into a sterilized ultra-high speed centrifuge special centrifuge tube.

[0106] (6) After strictly balancing the virus solution, centrifuge at 4°C at 50,000×g for 150 min. After centrifugation, a small amount of white precipitate can be seen at the bottom of the centrifuge tube. Make a mark, discard the supernatant, and note that when aspirating the supernatant with a suction device, do not aspirate the white precipitate. Resuspend each tube of virus by repeatedly pipetting with 200 μl of F12 at the marked place, and try not to cause too many bubbles.

[0107] (7) Concentrate all the resuspended viruses into one tube, shake well, briefly centrifuge to remove the water droplets on the inner wall of the tube cap, aliquot according to requirements, and store in an -80°C refrigerator.

[0108] 3. Package non-targeted lentiviral vector

[0109] Refer to the packaging method of the above-mentioned targeted lentiviral vector to package a non-targeted lentiviral vector whose surface does not contain the membrane-type expressed anti-PD-L1 antibody; the specific method is to replace the envelope plasmid contained in the A liquid in S1 with the wild-type VSV-G envelope plasmid pMD2.G.

[0110] Example 2: Influence of the presence or absence of membrane-type expressed anti-PD-L1 antibody on the infection efficiency of lentiviral vector infecting PD-L1 + cells

[0111] On Day 0, 1×10 5 Huh-7 cells and 1×10 5 Huh-7 cells overexpressing PD-L1 were taken, mixed, and according to MOI = 1, the targeted lentiviral vector was added to the mixed cells. On Day 2, flow cytometry was used to detect the expression efficiency of GFP in the mixed cells, and the results are as Figure 1 shown in B;

[0112] The targeted lentiviral vector was replaced with a non-targeted lentiviral vector, and the above operation was repeated. On Day 2, flow cytometry was used to detect the expression efficiency of GFP in the mixed cells, and the results are as Figure 1 shown in A.

[0113] As can be seen from Figure 1 A, in the mixed cells, the infection efficiency of the non-targeted lentiviral vector on Huh-7 cells and Huh-7 cells overexpressing PD-L1 is quite equivalent;

[0114] As can be seen from Figure 1 B, in the mixed cells, the targeted lentiviral vector expresses anti-PD-L1 antibody on its surface, and specifically binds to PD-L1 through the membrane-bound anti-PD-L1 antibody contained on its surface. The infection efficiency of the targeted lentiviral vector on Huh-7 cells overexpressing PD-L1 is significantly better than its infection efficiency on Huh-7 cells not expressing PD-L1, and is significantly better than the infection efficiency of the non-targeted lentiviral vector on Huh-7 cells overexpressing PD-L1.

[0115] Example 3: Comparing the effects of non-targeted lentiviral vector and adenovirus on inhibiting tumor growth

[0116] 2×10 6 Huh-7 tumor cells overexpressing PD-L1 per mouse were subcutaneously injected into 25 NKG mice (purchased from Cyagen Biosciences), and they were divided into 5 groups, with 5 tumor-bearing mice in each group.

[0117] The construction method of the Huh-7 cell line (adult hepatocellular carcinoma cell line) overexpressing PD-L1 is well known to those skilled in the art.

[0118] On Day 0, using the non-targeted lentiviral vector, adenovirus containing the TP53 gene (TP53 adenovirus, purchased from Shenzhen Sunway Biotech Co., Ltd.), and gemcitabine, in situ injection was administered to 3 groups of tumor-bearing mice according to the dosing methods shown in Table 3 below.

[0119] Table 3

[0120] Group 1 5 animals Control group Group 2 5 animals In-situ injection of 1E7 TU non-targeted lentiviral preparation Group 3 5 animals In-situ injection of 1E9 IFU TP53 adenoviral preparation Group 4 5 animals Gemcitabine drug control, 120 mg / Kg body weight

[0121] From day 3, the tumor growth was observed daily, the tumor size was recorded, and the tumor volume was calculated according to the following formula: V = ab 2 / 2 (V - volume, a - major axis of the tumor, b - minor axis of the tumor). The changes in the tumor volume (mm 3 ) are shown in Table 4 below (mean ± standard deviation).

[0122] Table 4

[0123]

[0124] As can be seen from Table 4, in-situ injection of the non-targeted lentiviral vector can effectively inhibit the growth of PD-L1 + tumors, and its effect on inhibiting tumor growth is significantly better than that of the TP53 adenovirus and gemcitabine.

[0125] Example 4: Comparison of the effects of non-targeted and targeted lentiviral vectors on inhibiting tumor growth

[0126] 2×10 6 per mouse of Huh-7 tumor cells overexpressing PD-L1 + were subcutaneously injected into 15 NKG mice and divided into 3 groups, with 5 tumor-bearing mice in each group.

[0127] On Day 0, using the targeted lentiviral vector and the non-targeted lentiviral vector, in-situ injection was administered to two groups of tumor-bearing mice respectively according to the dosing method shown in Table 5 below.

[0128] Table 5

[0129] Group 1 5 animals Control group Group 2 5 animals In-situ injection of 1E7 TU non-targeted lentiviral preparation Group 3 5 animals In-situ injection of 1E7 TU targeted lentiviral preparation

[0130] From day 3, the tumor growth was observed daily, the tumor size was recorded, and the tumor volume was calculated according to the following formula: V = ab 2 / 2 (V - volume, a - major axis of the tumor, b - minor axis of the tumor). The changes in the tumor volume (mm 3 ) are shown in Table 6 below (mean ± standard deviation).

[0131] Table 6

[0132]

[0133] As can be seen from Table 6, in-situ injection of the targeted lentiviral vector can effectively inhibit the growth of PD-L1 + tumors, and its effect on inhibiting the growth of PD-L1 + tumors is significantly better than that of the non-targeted lentiviral vector.

[0134] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present application (including any definitions herein) shall prevail. However, any reference, article, publication, patent, patent publication, and patent application cited herein does not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country of the world.

[0135] The subsection headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0136] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A retroviral vector, characterized in that, (a) the retroviral vector comprises a polynucleotide encoding p53 protein or a bioactive portion thereof; and (b) the surface of the retroviral vector comprises a targeting molecule that binds to PD-L1.

2. The retroviral vector according to claim 1, wherein the p53 protein comprises an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO:1; Preferably, the amino acid sequence of the p53 protein is as shown in SEQ ID NO:

1.

3. The retroviral vector according to claim 1 or 2, wherein the targeting molecule comprises an antibody specific for binding to PD-L1 and fragments thereof; Preferably, the heavy chain CDR1, CDR2 and CDR3 of the antibody and its fragments are respectively as shown in SEQ ID NO:10-12, and the light chain CDR1, CDR2 and CDR3 are respectively as shown in SEQ ID NO:13-15.

4. The retroviral vector according to any one of claims 1-3, wherein the targeting molecule comprises a transmembrane peptide segment; Preferably, the transmembrane peptide segment is the transmembrane region of CD3ζ, CD4, CD8α or CD28; More preferably, the transmembrane peptide segment is the transmembrane region of CD8α.

5. The retroviral vector according to claim 4, wherein the targeting molecule further comprises a hinge region; Preferably, the hinge region is the hinge region of CD8α or CD28.

6. A pharmaceutical composition, which comprises the retroviral vector according to any one of claims 1-5 and a pharmaceutically acceptable excipient or carrier.

7. Use of the retroviral vector according to any one of claims 1-6 and the pharmaceutical composition according to claim 7 in the preparation of a tumor-inhibiting drug; Preferably, the tumor is PD-L1 + tumor, which includes non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), melanoma, renal cell carcinoma, head and neck squamous cell carcinoma (HNSCC), bladder cancer, gastric cancer, esophageal cancer, liver cancer, triple-negative breast cancer (TNBC), Hodgkin lymphoma, colorectal cancer, pancreatic cancer, ovarian cancer, and prostate cancer; More preferably, the tumor is liver cancer.

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