Recombinant adenovirus vector tumor vaccine, preparation method therefor, and use thereof

Abstract

A recombinant adenovirus vector tumor vaccine, a preparation method therefor, and use thereof. An antigen encoded by the recombinant adenovirus vector tumor vaccine comprises any one or a combination of two or more of antigenic peptide epitopes of a KRAS protein. The recombinant adenovirus vector tumor vaccine encodes multiple KRAS tumor-specific antigenic peptides, and exhibits a good killing effect on tumors driven by KRAS mutations, especially on lung tumors. The vaccine can be administered via nebulized inhalation, intramuscular injection followed by nebulized inhalation, nebulized inhalation followed by intramuscular injection, intramuscular injection, or simultaneous intramuscular injection and nebulized administration. After nebulization, the vaccine can reach the lungs via nasal or oral inhalation, generating anti-tumor protective immune responses in the respiratory tract and lungs and throughout the body, thereby enhancing the utilization rate and therapeutic efficacy of the vaccine.

Description

A recombinant adenovirus vector tumor vaccine, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant adenovirus vector tumor vaccine, its preparation method, and its application. Background Technology

[0002] According to the latest global cancer burden data released by the International Agency for Research on Cancer (IARC) of the World Health Organization in 2020, lung cancer was the leading cause of cancer incidence and mortality in China in 2022, with over 1.06 million new cases, accounting for approximately 42% of the global new lung cancer diagnoses (2.5 million); and over 730,000 deaths from lung cancer, accounting for approximately 40% of the global lung cancer deaths (1.8 million). Research on cancer treatment drugs is currently a hot topic internationally. Besides surgery, chemotherapy, and radiotherapy, biological immunotherapy has developed rapidly in recent years, including cell therapy, antibody therapy, and vaccine therapy, and is currently the most promising cancer treatment method. Cancer vaccination is one type of immunotherapy. Its principle is to use tumor cells or tumor antigens to induce specific cellular and humoral immune responses in the body, that is, to activate the patient's own immune system to enhance the body's anti-cancer ability, thereby preventing tumor growth, spread, and recurrence, and ultimately achieving the goal of controlling or even eliminating the tumor. The basic principle of tumor vaccine development is to enhance the immune system’s ability to recognize and kill tumor cells containing specific antigens by targeting tumor-associated antigens (TAAs) or tumor-specific antigens (TSA).

[0003] TAAs are human autoantibodies expressed in normal cells but abnormally highly expressed in tumor cells. These include: tumor / reproductive antigens, typically expressed only on immune-granted germ cells, such as MAGE-A and NY-ESO-1; cell-directed differentiation antigens, usually not expressed in adult tissues, such as GP100, PSA, PAP, MART-1, and tyrosinase; and antigens abnormally highly expressed on tumor cells, such as HER2 and MUC-1. TAAs exhibit a degree of central tolerance and lack complete tumor specificity. When these proteins reach the T cell recognition threshold, they may elicit an anti-tumor immune response, but may also induce autoimmunity in normal tissues. Furthermore, because these antigens are also expressed in healthy tissues, they often result in low affinity for T cell recognition.

[0004] Tumor neoantigens (TAAs) are newly formed antigens produced by tumor cells due to various tumor-specific alterations, such as genomic mutations, RNA splicing dysregulation, post-translational modification disorders, and integrated viral open reading frames. TSAs are considered non-self and elicit immune responses unaffected by central and peripheral tolerance; they are also known as tumor neoantigens. TSAs possess unique tumor specificity and the significant advantage of being absent in normal tissues, providing ideal targets for effective personalized cancer treatment. Vaccines based on neoantigens rather than traditional TAAs offer the following advantages: First, neoantigens are expressed only by tumor cells, thus triggering a truly tumor-specific T-cell response and preventing off-target damage to non-tumor tissues. Second, neoantigens are novel epitopes derived from somatic mutations; T cells targeting neoantigens can bypass the negative selection effect in the thymus, enhancing the tumor-specific immune response. Furthermore, the neoantigen-specific T-cell response enhanced by immunotherapy has the ability to be durable and generate post-treatment immune memory, offering hope for long-term prevention of disease recurrence.

[0005] Studies have shown that high-frequency tumor-specific mutations in lung cancer are commonly found in genes such as KRAS, EGFR, ALK, ROS1, BRAF, and RET. Among these, KRAS protein mutations G12V, G12D, G12C, G12A, G12R, G12S, G13D, G13C, G13E, Q61H, Q61R, Q61K, and Q61L cover common mutation types. Targeting common specific mutant antigens in lung cancer can induce a highly efficient immune response against lung cancer. It also shows therapeutic effects against other Kras-driven cancers such as pancreatic cancer and colorectal cancer.

[0006] Therefore, developing vaccines based on specific mutant antigens is of great significance for the prevention and treatment of Kras-driven cancers such as lung cancer. Summary of the Invention

[0007] In order to better prevent and treat lung cancer, reduce the side effects of conventional radiotherapy and chemotherapy, and increase the treatment methods for lung cancer, this invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a recombinant adenovirus vector, wherein the antigen encoded by the recombinant adenovirus vector comprises any one or more combinations of antigenic peptide epitopes of the KRAS protein.

[0009] Preferably, the antigenic peptide epitope is selected from any one or a combination of two or more of the human KRAS protein mutation sites G12C, G12V, G12D, G12A, G12R, G12S, G13D, G13C, G13E, Q61H, Q61R, Q61K and Q61L.

[0010] Preferably, the recombinant adenovirus vector encoding antigen comprises any one or more combinations of KRAS antigenic peptide epitopes shown in SEQ ID NO:1-13, or one or more combinations of sequences having more than 70% identity with any sequence or sequence combination shown in SEQ ID NO:1-13.

[0011] Furthermore, the recombinant adenovirus vector encoding antigen comprises any one or more combinations of KRAS antigenic peptide epitopes shown in SEQ ID NO:14-31, or one or more combinations of sequences having more than 70% identity with the sequences shown in SEQ ID NO:14-31.

[0012] More preferably, the recombinant adenovirus vector is a recombinant human adenovirus or a chimpanzee adenovirus.

[0013] Preferably, the human adenovirus is selected from: AdHu2, AdHu4, AdHu5, AdHu7, AdHu11, AdHu26, AdHu35, AdHu55, and more preferably AdHu5.

[0014] Furthermore, the antigenic peptide epitope encoded by the recombinant adenovirus vector is selected from any one or more of the following human KRAS protein mutations: G12C / D / V / A / R / S, G13D / C / E, and Q61H / R / K / L.

[0015] Furthermore, the KRAS epitopes are selected from any one or more combinations of SEQ ID NO:1-13.

[0016] Furthermore, the KRAS epitopes are selected from any combination of two of SEQ ID NO:1-13. For example, a combination of SEQ ID NO:1 and 3.

[0017] Furthermore, the KRAS epitopes are selected from any combination of three of SEQ ID NO:1-13. For example, a combination of SEQ ID NO:1, 2, and 3.

[0018] Furthermore, the KRAS epitopes are selected from any combination of four of SEQ ID NO:1-13. For example, a combination of SEQ ID NO:1-4.

[0019] Furthermore, the KRAS epitopes are selected from any combination of five of SEQ ID NO:1-13. For example, combinations of SEQ ID NO:1-5, combinations of SEQ ID NO:1-4 and 6, combinations of SEQ ID NO:1-4 and 7, and combinations of SEQ ID NO:1-4 and 10.

[0020] Furthermore, the KRAS epitopes are selected from any combination of six of SEQ ID NO:1-13. For example, combinations of SEQ ID NO:1-4, 7, and 10, and combinations of SEQ ID NO:1-6.

[0021] Furthermore, the KRAS epitopes are selected from any combination of seven of SEQ ID NO:1-13. For example, combinations of SEQ ID NO:1-4, 6, 7, 10, combinations of SEQ ID NO:1-6, 7, and combinations of SEQ ID NO:1-6, 10.

[0022] Furthermore, the KRAS epitopes are selected from any combination of eight of SEQ ID NO:1-13. For example, combinations of SEQ ID NO:1-6, 7, and 10.

[0023] Furthermore, the KRAS epitopes are selected from any combination of nine of SEQ ID NO:1-13. For example, a combination of SEQ ID NO:1-9.

[0024] Furthermore, the KRAS epitopes are selected from any combination of ten of SEQ ID NO:1-13. For example, combinations of SEQ ID NO:1-10, and combinations of SEQ ID NO:1-6 and 10-13.

[0025] Furthermore, the KRAS epitopes are selected from any combination of eleven of SEQ ID NO:1-13. For example, combinations of SEQ ID NO:1-6, 7, and 10-13.

[0026] Furthermore, the KRAS epitopes are selected from any combination of twelve of SEQ ID NO:1-13.

[0027] Furthermore, the KRAS epitopes are selected from any combination of thirteen of SEQ ID NO:1-13. For example, combinations of SEQ ID NO:1-13.

[0028] Furthermore, the antigenic peptide epitope encoded by the recombinant adenovirus vector has more than 70% identity with the above-mentioned antigenic peptide epitope sequence.

[0029] Preferably, the antigenic peptides encoded by the recombinant adenovirus vector are spaced by 0-30 bases, and more preferably by a single base spacer or a GS linker spacer.

[0030] Preferably, the GS connector sequence is selected from any one of GGSGGGGSGG, GSGSGSGSGSGS, GGSGSGGSGG, GGSLGGGGSG, or GGGGSGGGGS.

[0031] Furthermore, the genome of the recombinant adenovirus vector also encodes a fusion protein that promotes the presentation of cellular immune antigens and enhances cellular immunity.

[0032] Furthermore, the fusion protein includes LC3B, IFN-γ, IL12, GM-CSF, IL21, CCL3, or CCL5.

[0033] Furthermore, the fusion protein is LC3B, IL12, or CCL5.

[0034] Furthermore, the fusion proteins can be linked by self-cleaving peptides, which include IRES, Furin, P2A, or T2A, more preferably P2A or T2A.

[0035] In a second aspect, the present invention provides a recombinant adenovirus vector tumor vaccine, wherein the recombinant adenovirus vector tumor vaccine comprises the recombinant adenovirus vector described in the first aspect.

[0036] Preferably, the unit dose of the vaccine is 0.05-6 mL, for example: 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL. More preferably, it is 0.05-1 mL.

[0037] Preferably, the recombinant adenovirus content in the vaccine is 1×10⁻⁶. 8 ~9×10 12 VP / mL, for example: 1×10 8 VP / mL, 5×10 8 VP / mL, 9×10 8 VP / mL, 1×10 9 VP / mL, 5×10 9 VP / mL, 9×10 9 VP / mL, 1×10 10 VP / mL, 5×10 10 VP / mL, 9×10 10 VP / mL, 1×10 11 VP / mL, 5×10 11 VP / mL, 9×10 11 VP / mL, 1×10 12 VP / mL, 5×10 12 VP / mL, 9×10 12 VP / mL.

[0038] Preferably, the recombinant adenovirus vector tumor vaccine further includes pharmaceutically acceptable excipients.

[0039] Furthermore, the pharmaceutically acceptable excipients include, but are not limited to, one or more of the following: buffers, protectants, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, inactivators, or human albumin.

[0040] Preferably, the buffer includes, but is not limited to, one or more of HEPES, HIS, TRIS, PB, succinic acid, and citric acid.

[0041] Preferably, the protective agent includes, but is not limited to, one or more of gelatin, ethanol, diethylaminetetraacetic acid (EDTA), disodium diethylaminetetraacetic acid (EDTA-2Na), and magnesium chloride.

[0042] Preferably, the stabilizer includes, but is not limited to, one or more of sucrose, mannitol, fucose, and maltose.

[0043] Preferably, the surfactant includes, but is not limited to, one or more of Tween, Span, and glycerol.

[0044] Preferably, the osmotic pressure regulator includes, but is not limited to, sodium chloride, or may be omitted.

[0045] In one specific embodiment of the present invention, the excipient components include mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, and glycerin.

[0046] Preferably, the mannitol concentration in the vaccine is 5-220 mg / mL, for example: 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL.

[0047] More preferably, the mannitol concentration in the vaccine is 5-100 mg / mL.

[0048] Preferably, the sodium chloride concentration in the vaccine is 0.5-30 mg / mL, for example: 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL.

[0049] More preferably, the concentration of sodium chloride in the vaccine is 1-15 mg / mL.

[0050] Preferably, the HEPES concentration in the vaccine is 0.05-10 mg / mL, for example: 0.05 mg / mL, 0.07 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL.

[0051] More preferably, the HEPES concentration in the vaccine is 0.1-5 mg / mL.

[0052] Preferably, the concentration of polysorbate 80 in the vaccine is 0.01-13 mg / mL, for example: 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL.

[0053] More preferably, the concentration of polysorbate 80 in the vaccine is 0.05-7 mg / mL.

[0054] Preferably, the glycerol concentration in the vaccine is 0.1-5 mg / mL, for example: 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL.

[0055] More preferably, the glycerol concentration in the vaccine is 1-5 mg / mL.

[0056] Preferably, the concentration of magnesium chloride in the vaccine is 0.1-5 mg / mL, for example: 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL.

[0057] More preferably, the concentration of magnesium chloride in the vaccine is 0.1-1 mg / mL.

[0058] Preferably, the sucrose concentration in the vaccine is 0.5-100 mg / mL, for example: 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL.

[0059] More preferably, the sucrose concentration in the vaccine is 2-60 mg / mL.

[0060] Preferably, the recombinant adenovirus vector tumor vaccine is a mucosal immunizing agent.

[0061] Furthermore, the mucosal immunomodulatory agent is a nasal drop, aerosol, spray, powder, liquid preparation, lyophilized preparation, gel, microsphere, liposome, film, or suspension.

[0062] Preferably, the mucosal immunomodulator is an inhaled formulation, more preferably nasal or oral, and even more preferably a nebulized inhaler.

[0063] Furthermore, the recombinant adenovirus is atomized by the drug delivery device to form particles smaller than 15 μm.

[0064] Preferably, the inhaled drug delivery formulation is a liquid formulation or a dry powder formulation.

[0065] Preferably, the mucosa includes nasal mucosa, oral mucosa, or lung mucosa.

[0066] Preferably, the dosage form of the recombinant adenovirus vector tumor vaccine can also be an injectable formulation.

[0067] Furthermore, the administration methods of the recombinant adenovirus vector tumor vaccine include, but are not limited to, nebulized inhalation, intramuscular injection followed by nebulized inhalation, nebulization followed by intramuscular injection, intramuscular injection, or simultaneous intramuscular injection and nebulized inhalation.

[0068] Thirdly, the present invention provides an antigenic peptide comprising any one or more combinations of antigenic peptide epitopes as shown in SEQ ID NO:1-13 or any one or more combinations of antigenic peptide epitopes having more than 70% identity with the sequences shown in SEQ ID NO:1-13.

[0069] Furthermore, the amino acid sequences of SEQ ID NO:1-13 are as follows:

[0070] Furthermore, when the antigenic peptide contains two or more different antigenic peptide epitopes, the different antigenic peptide epitopes are connected by a GS Linker. More preferably, the GS Linker is selected from any one of GGSGGGGSGG, GSGSGSGSGSGS, GGSGSGGSGG, GGSLGGGGSG, or GGGGSGGGGS.

[0071] Furthermore, the sequence of the antigenic peptide is as shown in any one of SEQ ID NO:1-13 or has more than 70% identity with it.

[0072] Furthermore, the aforementioned 70% identity can be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0073] Furthermore, the antigenic peptide epitope sequences of SEQ ID NO:1-13 can be arbitrarily combined to form antigenic peptides covering different Kras mutations;

[0074] Furthermore, the antigenic peptide epitope sequences of SEQ ID NO:1 and SEQ ID NO:3 combine to form the diantigenic peptide P2 (SEQ ID NO:14).

[0075] Furthermore, the antigenic peptide epitope sequences of SEQ ID NO:1-3 combine to form the triantigenic peptide P3 (SEQ ID NO:15).

[0076] Furthermore, the antigenic peptide epitope sequences of SEQ ID NO:1-4 combine to form the tetraantigenic peptide P4 (SEQ ID NO:16).

[0077] Furthermore, the tetrapeptide P4 is combined with SEQ ID NO:5, 6, 7, and 10 to form pentaantigen peptides P5.1, P5.2, P5.3, and P5.4 (SEQ ID NO:17-20).

[0078] Furthermore, the tetrapeptide P4 combines with SEQ ID NO:7 and 10 to form the hexaantigen peptide P6.1 (SEQ ID NO:21).

[0079] Furthermore, the antigenic peptide epitope sequences of SEQ ID NO:1-6 combine to form the hexaantigenic peptide P6.2 (SEQ ID NO:22).

[0080] Furthermore, the pentagonal peptide P5.2 combines with SEQ ID NO:7 and 10 to form the heptagonal peptide P7.1 (SEQ ID NO:23).

[0081] Furthermore, the hexaantigen peptide P6.2 is combined with SEQ ID NO:7 and 10 to form heptaantigen peptides P7.2 (SEQ ID NO:24) and P7.3 (SEQ ID NO:25), respectively.

[0082] Furthermore, the hexaantigen peptide P6.2 combines with SEQ ID NO:7 and 10 to form the octaantigen peptide P8 (SEQ ID NO:26).

[0083] Furthermore, the hexaantigen peptide P6.2 combines with the epitope sequences of SEQ ID NO:7-9 to form the nonaantigen peptide P9 (SEQ ID NO:27).

[0084] Furthermore, the hexaantigen peptide P6.2 combines with the antigen peptide epitope sequences of SEQ ID NO:7-10 to form the decaantigen peptide P10.1 (SEQ ID NO:28).

[0085] Furthermore, the hexaantigen peptide P6.2 combines with the epitope sequence of SEQ ID NO:10-13 to form the decaantigen peptide P10.2 (SEQ ID NO:29).

[0086] Furthermore, the hexaantigen peptide P6.2 combines with the epitope sequences of SEQ ID NO:7 and SEQ ID NO:10-13 to form the undecapeptide peptide P11 (SEQ ID NO:30).

[0087] Furthermore, the hexaantigen peptide P6.2 combines with the antigen peptide epitope sequences of SEQ ID NO:7-13 to form the thirteen-antigen peptide P13 (SEQ ID NO:31).

[0088] Specifically, the combinations of antigenic peptide epitope sequences at each Kras mutation site are as follows:

[0089] Table 1. Combinations of antigenic peptide epitope sequences at various Kras mutation sites.

[0090] The specific amino acid sequences of SEQ ID NO:14-31 are as follows:

[0091] Furthermore, the sequence of the antigenic peptide is as shown in any one of SEQ ID NO:1-31 or has more than 70% identity with it.

[0092] Fourthly, the present invention provides a nucleic acid molecule that encodes the antigenic peptide described in the third aspect.

[0093] Preferably, the nucleic acid molecule is DNA or mRNA.

[0094] Furthermore, the nucleic acid molecule contains a gene encoding a fusion protein that promotes the presentation of cellular immune antigens and / or enhances cellular immunity.

[0095] Furthermore, the fusion protein is selected from one or more of LC3B, IFN-γ, IL12, GM-CSF, IL21, CCL3, or CCL5.

[0096] Furthermore, the fusion proteins can be directly linked or linked via self-cleaving peptides, such as IRES, Furin, P2A, or T2A.

[0097] Fifthly, the present invention provides a host cell comprising the recombinant adenovirus vector described in the first aspect or the nucleic acid molecule described in the fourth aspect.

[0098] Preferably, the host cell is selected from any one of the following: Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, or NK cells.

[0099] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the vaccine described in the second aspect and pharmaceutically permissible excipients.

[0100] In a seventh aspect, the present invention provides an aerosol comprising the vaccine described in the second aspect or the pharmaceutical composition described in the sixth aspect.

[0101] Preferably, the aerosol is formed by atomizing the vaccine described in the second aspect or the pharmaceutical composition described in the sixth aspect.

[0102] Furthermore, the particle size of the aerosol is less than 15 μm, for example: 0.01 μm, 0.05 μm, 0.1 μm, 0.25 μm, 0.5 μm, 0.75 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm.

[0103] Preferably, the particle size of the aerosol is 0.25-15 μm, more preferably 5-10 μm.

[0104] Eighthly, the present invention provides the use of the recombinant adenovirus vector of the first aspect, the vaccine of the second aspect, the nucleic acid molecule of the fourth aspect, or the pharmaceutical composition of the sixth aspect in the preparation of products for the prevention and / or treatment of tumors.

[0105] Preferably, the tumor is a Kras mutation-driven cancer; more preferably, the cancer includes lung cancer, pancreatic cancer, or colorectal cancer.

[0106] Preferably, the tumor is lung cancer, which includes non-small cell lung cancer, lung metastases from pancreatic cancer, or lung metastases from colorectal cancer, and more preferably non-small cell lung cancer.

[0107] Preferably, the application includes preventing tumor occurrence, treating tumors, or preventing tumor recurrence after surgery.

[0108] In a ninth aspect, the present invention provides the use of the recombinant adenovirus vector described in the first aspect in the preparation of a mucosal delivery immunomodulatory composition.

[0109] In a tenth aspect, the present invention provides a method for preparing the recombinant adenovirus vector tumor vaccine described in the second aspect, the method comprising the following steps:

[0110] (1) Construct the recombinant adenovirus vector described in the first aspect;

[0111] (2) The recombinant adenovirus vector and the backbone plasmid carrying most of the adenovirus genome were co-transfected into packaging cells, and the recombinant packaging replication was obtained to obtain a defective adenovirus.

[0112] (3)Optionally, pharmaceutically acceptable excipients are added to obtain the recombinant adenovirus vector tumor vaccine.

[0113] Preferably, the backbone plasmid is an AdEasy system or an Admax system, more preferably an Admax system.

[0114] Preferably, the packaging cells are Hala cells, HEK293 cells, or their derived cell lines.

[0115] In one aspect, the present invention provides a treatment method for lung cancer, comprising administering to a lung cancer patient the vaccine described in the sixth aspect or the pharmaceutical composition described in the eighth aspect.

[0116] Preferably, the vaccine or pharmaceutical composition can also be combined with chemotherapy, radiotherapy, small molecule inhibitors (such as PD-1), immune checkpoint inhibitors (such as PD-L1 antibodies) or cell immunotherapy to treat lung cancer.

[0117] The beneficial effects of this invention are:

[0118] I. The recombinant adenovirus vector tumor vaccine of the present invention encodes multiple Kras tumor-specific antigens, thereby improving the killing effect on Kras-driven tumor cells (such as lung cancer, pancreatic cancer, colorectal cancer, pancreatic cancer lung metastases, colorectal cancer lung metastases, etc.).

[0119] Second, the recombinant adenovirus vector tumor vaccine of the present invention can be administered via multiple routes. In particular, when administered via nebulized inhalation, compared with intramuscular injection of the same formulation, it can generate not only a systemic immune response but also mucosal immunity, which is more beneficial for the treatment and prevention of lung cancer.

[0120] Third, the recombinant adenovirus vector tumor vaccine of the present invention produces particles of 0.25-15μm after atomization. These particles can reach the lungs when inhaled through the nasal cavity or mouth, generating an anti-tumor protective immune response against the respiratory tract, lungs, and the whole body, thereby enhancing the utilization rate and therapeutic effect of the vaccine.

[0121] IV. The recombinant adenovirus vector tumor vaccine of the present invention can be used in people who have undergone surgical resection of tumors (such as lung cancer) to prevent postoperative tumor recurrence; it can also be used in tumor patients who have failed recommended therapies to prolong their survival. Attached Figure Description

[0122] Figure 1 shows the morphology of virus particles before and after atomization of the recombinant adenovirus vector tumor vaccine.

[0123] Figure 2 shows the results of ELIPSOT detection of IFN-γ secretion in mouse spleen and lung cells after immunization with Ad5-G12V via different immunization routes. A represents spleen cells and B represents lung cells.

[0124] Figure 3 shows the results of ELIPSOT detection of IFN-γ secretion in mouse spleen cells after immunization with recombinant adenovirus vector vaccines expressing single mutant antigenic peptides G12 and G13 of KRAS.

[0125] Figure 4 shows the results of ELIPSOT detection of IFN-γ secretion in mouse spleen cells after immunization with recombinant adenovirus vector vaccine expressing Q61 single mutant antigen peptides of KRAS.

[0126] Figure 5 shows the results of IFN-γ secretion in mouse spleen cells as detected by ELIPSOT after immunization with each recombinant adenovirus vector vaccine;

[0127] Figure 6 shows the effects of different cytokines on the immunogenicity of the recombinant adenovirus vector tumor vaccine;

[0128] Figure 7 shows the therapeutic effect of the recombinant adenovirus vector tumor vaccine on a lung cancer model;

[0129] Figure 8 shows the preventive and therapeutic effects of the recombinant adenovirus vector tumor vaccine on a pancreatic cancer model.

[0130] Figure 9 shows the therapeutic effect of the recombinant adenovirus vector tumor vaccine on a colorectal cancer model. Detailed Implementation

[0131] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0132] Example 1: Preparation of Recombinant Adenovirus Vector Tumor Vaccine

[0133] 1.1 The target antigen peptides were synthesized and constructed into the shuttle vector pDC316 by enzyme digestion. The shuttle vectors that were correctly identified by sequencing were labeled.

[0134] 1.2 The shuttle plasmid and the backbone plasmid of the AdMax adenovirus system were co-transfected to package the virus strain, and the packaged virus was named. The specific procedure is as follows:

[0135] Before transfection, HEK293 cells were seeded in six-well plates. The backbone plasmid and shuttle plasmid were transfected using transfection reagents, with three replicate wells transfected. After transfection, confluent cells were passaged into cell culture flasks and cultured in MEM medium. Cell cytotoxicity was observed daily. Cytotoxicity was characterized by enlarged, rounded cells resembling grapes, and the appearance of obvious plaques. The virus was harvested when most cells exhibited pathotropic effects and detached from the base. The virus was further seeded into continuously cultured HEK293 cells to a suitable scale, cultured for 48 hours, and then harvested. After lysis, crude purification, and further purification using chromatography media, the stock solution was aseptically filtered to obtain the final product.

[0136] 1.3 Take the recombinant adenovirus vector tumor vaccine stock solution prepared in 1.2 (containing 1×10⁻⁶ virus particles). 9 Add 50 mg mannitol, 5 mg sodium chloride, 1 mg HEPES, 0.2 mg polysorbate 80, 4 mg glycerol, 0.2 mg magnesium chloride and 30 mg sucrose to VP, and mix to obtain 1 mL of recombinant adenovirus vector tumor vaccine preparation.

[0137] Example 2: Effect of atomization on the morphology of recombinant adenovirus vector tumor vaccine virus particles

[0138] The recombinant adenovirus vector tumor vaccine preparation was nebulized and inhaled using a nebulizer. The aerosols were collected, and the virus morphology of the nebulized samples was observed by transmission electron microscopy to study the effect of the nebulization process on the morphology of virus particles.

[0139] As shown in Figure 1, the adenovirus in the recombinant adenovirus vector tumor vaccine inhaled before and after nebulization exhibits a typical hexagonal morphology, and the nebulization process has no effect on the virus morphology.

[0140] Example 3: Effect of nebulization on vaccine virus activity

[0141] Three batches of inhaled vaccine were nebulized using a nebulizer. The nebulized samples were collected into centrifuge tubes, and the viral infection titer (IFU) was measured. The IFU recovery rate was calculated according to Formula 1, and the results are shown in Table 2. IFU recovery rate (%) = (IFU after nebulization / IFU before nebulization) × 100% (Formula 1)

[0142] Table 2. Effects of nebulization on the viral activity of recombinant adenovirus vector tumor vaccine.

[0143] As shown in Table 2, the IFU recovery rate of the recombinant adenovirus vector tumor vaccine after nebulization reached over 96.6%, and nebulization had no significant effect on the viral activity of the recombinant adenovirus vector tumor vaccine, indicating that the recombinant adenovirus vector tumor vaccine has stable performance.

[0144] Example 4: Effect of nebulization on the number of vaccine virus particles

[0145] Three batches of inhaled vaccine were nebulized using a nebulizer. The nebulized samples were collected in centrifuge tubes, and the viral particle count (VP) was determined using ultraviolet spectrophotometry. The recovery rate was calculated according to Formula 2, and the results are shown in Table 3. VP recovery rate (%) = (VP after nebulization / VP before nebulization) × 100% (Formula 2)

[0146] Table 3. Effects of nebulization on vaccine virus particle number

[0147] As shown in Table 3, the VP recovery rate of the recombinant adenovirus vector tumor vaccine after nebulization is close to 100%, and nebulization has no significant effect on the number of viral particles in the recombinant adenovirus vector tumor vaccine, indicating that the performance of the recombinant adenovirus vector tumor vaccine is stable.

[0148] Example 5: Immunological evaluation of Ad5-G12V via different immunization pathways

[0149] According to the method in Example 1, the candidate adenovirus vector vaccine Ad5-G12V containing only sequence 3 was prepared, and the empty vector Ad-Null was also prepared. Six- to eight-week-old humanized HLA-A*11:01 female mice were randomly divided into six groups of three. Ad5-G12V was administered via five routes: intramuscular injection, inhalation, inhalation followed by intramuscular injection, intramuscular injection followed by inhalation, simultaneous inhalation, and intramuscular injection. The empty vector Ad-Null was administered only via simultaneous inhalation and intramuscular injection. Immunization schedule: All groups received two injections on days 0 and 7. The intramuscular injection dose was 1E10VP / mouse, 100μL; the inhalation dose was 5E9VP / mouse, 50μL.

[0150] On day 14, mice were dissected, and spleen and lung tissue were harvested from each experimental group. Spleen cells were collected after grinding and seeded at a rate of 1E5 cells / well in 96-well plates pre-coated with IFN-γ antibody. Splenocytes from each experimental group were replicated, and the corresponding peptide stimulant G12V was added to each well to a final concentration of 10 μg / mL. Each experimental group also included a no-stimulation control well and a PMA-stimulated positive control well. The 96-well plates were incubated overnight at 37°C and 5% humidity in a CO2 incubator for 16–24 h before Elispot staining. Specific staining procedure: Discard cells, add 200 μL PBS / well, wash 5 times, and pat dry; prepare biotinylated detection antibody at a 1:100 ratio, 100 μL / well, and incubate at 37°C in the dark for 1 hour; add 200 μL PBS / well, wash 5 times; add 100 μL Streptavidin-HRP working solution to each well, and incubate at 37°C in the dark for 1 hour; add 200 μL PBS / well, wash 5 times; add 100 μL of freshly prepared AEC chromogenic substrate, and develop at room temperature in the dark for 5-30 minutes, stopping the development time according to the spot formation. Wash both sides of each well and the base with deionized water / tap water 3-5 times, place the plate in a cool, shaded place at room temperature to air dry, then close the base and count the spots. After grinding the mouse lung tissue, take 4E5 cells / well and seed them into a 96-well plate pre-coated with IFN-γ antibody. Lung cells from each experimental group were replicated, and the corresponding polypeptide stimulant G12V was added to each well to a final concentration of 10 μg / mL. Each experimental group also included a non-stimulated control well and a PMA-stimulated positive control well. The 96-well plates were incubated overnight at 37°C and 5% humidity in a CO2 incubator for 16–24 h before Elispot staining.

[0151] As shown in Figure 2, each mutated antigen group can effectively activate the immune system to produce a specific immune response. Among them, the group that is simultaneously inhaled and injected intramuscularly has the highest number of IFN-γ cells produced by overall stimulation, inducing the strongest response.

[0152] Example 6: Immunogenicity evaluation of individual mutant antigenic peptides G12 and G13 of KRAS

[0153] Following the method in Example 1, candidate adenovirus vector vaccines containing only sequences 1-9 were prepared. Six- to eight-week-old humanized HLA-A*11:1 female mice were randomly divided into 10 groups of 3 mice each. The groups were designated as Ad5-G12V, Ad5-G12D, Ad5-G12C, Ad5-G12R, Ad5-G12A, Ad5-G12S, Ad5-G13D, Ad5-G13C, and Ad5-G13E, respectively. An empty vector was used as a control. Immunization was performed on days 0 and 7, with two injections in total. Immunization was administered via simultaneous inhalation and intramuscular injection; 50 μL was administered via inhalation, and 100 μL via intramuscular injection.

[0154] On day 14, mice were dissected, and spleen tissue was collected from each experimental group. Spleen cells were then extracted and seeded at a ratio of 1E5 cells / well in 96-well plates pre-coated with IFN-γ antibody. Splenocytes from each experimental group were replicated, and the corresponding polypeptide stimulants G12V, G12D, G12C, G12R, G12A, G12S, G13D, G13C, and G13E were added to each well, with a final concentration of 10 μg / mL. Each experimental group also included a non-stimulated control well and a PMA-stimulated positive control well. The 96-well plates were incubated overnight at 37°C and 5% humidity in a CO2 incubator for 16–24 h before Elispot staining.

[0155] As shown in Figure 3, all mutated antigen groups effectively activated the immune system to produce specific immune responses. Among them, the G12 mutation site produced the highest number of IFN-γ cells overall, which was more effective than the individual antigen peptides from the G13 mutation site. This indicates that G12V has the highest immunogenicity and induced the strongest response.

[0156] Example 7 Immunogenicity evaluation of individual mutant antigenic peptides of KRAS Q61

[0157] A candidate adenovirus vector vaccine containing only sequences 10-13 was prepared according to the method in Example 1. Six- to eight-week-old humanized HLA-A*01:01 female mice were randomly divided into five groups of three (Ad5-Q61H, Q61K, Q61R, and Q61L), with an empty vector serving as a control. Immunization was performed on days 0 and 7 (days 7), for a total of two injections. Immunization was administered via simultaneous inhalation and intramuscular injection; 50 μL was administered via inhalation, and 100 μL via intramuscular injection.

[0158] On day 14, mice were dissected, and spleen tissue was collected from each experimental group. Spleen cells were then extracted and seeded at a ratio of 1E5 cells / well in 96-well plates pre-coated with IFN-γ antibody. Each experimental group had duplicate wells containing the corresponding peptide stimulants Q61H, Q61K, Q61R, and Q61L, respectively, at a final concentration of 10 μg / mL. Each experimental group also included a no-stimulation control well and a PMA-stimulated positive control well. The 96-well plates were incubated overnight at 37°C and 5% humidity in a CO2 incubator for 16–24 h before Elispot staining.

[0159] As shown in Figure 4, the Q61 mutations Q61H, Q61K, Q61R, and Q67L share neoantigens and can effectively activate the immune system to produce a specific immune response.

[0160] Example 8: Cellular Immunological Activity of Recombinant Adenovirus Vector Tumor Vaccine

[0161] Following the method described in Example 1, recombinant adenovirus vector tumor vaccines were designed and packaged according to sequences 1-4, 7, 10, 16, 22, 26, and 31, respectively, and labeled as Ad-Kn-LC3B, with n being 1, 2, 3, 4, 7, 10, 16, 22, 26, and 31. The encoded antigen peptide sequence was fused with LC3B. Control vaccines without the Kras antigen peptide and empty Ad were also packaged and labeled as Ad-LC3B and Ad-null.

[0162] HLA-A*11:01 humanized mice were randomly assigned to groups of three. On days 0 and 7, mice were immunized with the corresponding recombinant adenovirus vector tumor vaccine via simultaneous intramuscular injection and inhalation, respectively. The intramuscular dose was 1E10Vp / mouse, and the inhalation dose was 5E9VP / mouse. Mice were sacrificed on day 14, and spleen cells were harvested. The cellular immune response levels of different antigenic peptides and immunization routes were assessed using the IFN-γELISpot method. K1-K4, K7, and K10 were stimulating peptides, while K16, K22, K26, and K31 were stimulating peptides prepared as mixtures of antigens from various combinations. The final concentration of each peptide was 10 μg / mL.

[0163] As shown in Figure 5, compared with the control group, all experimental groups produced a higher level of antigen peptide-specific cellular immune response, with G12V exhibiting the highest immunogenicity.

[0164] Example 9: Effects of different cytokines on cellular immunity of recombinant adenovirus vector tumor vaccine

[0165] Following the method described in Example 1, recombinant adenovirus vector tumor vaccines were designed and packaged from sequences 26 and 31, respectively. The encoded antigenic peptide sequences were fused with IL12, GMCSF, CCL5, and IFN-γ for expression to enhance the immune response. These were labeled Ad-K26-LC3B-IL12, Ad-K26-LC3B-GMCSF, Ad-K26-LC3B-CCL5, Ad-K26-LC3B-IFN-γ, Ad-K31-LC3B-IL12, Ad-K31-LC3B-GMCSF, Ad-K31-LC3B-CCL5, and Ad-K31-LC3B-IFN-γ, respectively. An empty Ad vector was used as a negative control vaccine, labeled Ad-null.

[0166] HLA-A*11:01 humanized mice were randomly assigned to groups of three. On days 0 and 7, mice were immunized with the corresponding recombinant adenovirus vector tumor vaccine via inhalation at a dose of 5E9VP per mouse. Mice were sacrificed on day 14, and spleen cells were harvested. The effects of cytokines on the cellular immune response to the fusion antigen peptide were assessed using the IFN-γ ELISpot assay. The stimulating peptide was a mixture of all antigen peptides, with each peptide having a final concentration of 10 μg / mL.

[0167] As shown in Figure 6, compared with the control group, all cytokines enhanced the level of antigen peptide-specific cellular immune responses. Among these cytokines, IL-12 elicited the highest level of cellular immune response in the inhalation group.

[0168] Example 10: Therapeutic effect of recombinant adenovirus vector tumor vaccine on lung cancer model

[0169] Immune system reconstruction was performed using HSCs, with HLA matching at HLA-A*11:01 for evaluation. Six- to eight-week-old female mice were subcutaneously inoculated with 2.5E6 NCIH-441 lung cancer tumor cells, resulting in tumors with a volume of 150-250 mm². 3 Mice were then divided into two treatment groups: the Ad-K31-LC3B-IL12 group and the buffer group, with 5 mice in each group. The dosing schedule was four administrations on days 0, 7, 14, and 21. The first two administrations were a combination of intramuscular and inhalation, while the last two were inhalation administrations. The intramuscular dose was 1E10VP / mouse, and the inhalation dose was 5E9VP / mouse. One week after the last administration, the tumor volume (TV) was calculated using the formula: 1 / 2 × a × b. 2 , where a and b are the length and width of the tumor, respectively. The mouse tumor volume reached 2500 mm². 3 The trial was terminated, and the tumor inhibition rate (TGI) was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100%

[0170] In the formula: Ti is the mean tumor volume of the treatment group on day i of drug administration, T0 is the mean tumor volume of the treatment group on day 0 of drug administration; Vi is the mean tumor volume of the solvent control group on day i of drug administration; V0 is the mean tumor volume of the solvent control group on day 0 of drug administration.

[0171] As shown in Figure 7, the TGI was 73.2% compared to the buffer group control, indicating that the recombinant adenovirus vector tumor vaccine can effectively inhibit tumor growth.

[0172] Example 11: Preventive and therapeutic effects of recombinant adenovirus vector tumor vaccine on a pancreatic cancer model.

[0173] Using HSC immune system reconstitution and HLA typing (HLA-A*11:01) for evaluation, 6-8 week old female mice were subcutaneously inoculated with 5E6 PANC1 lung cancer cells on day D-1 to model tumors. A prophylactic dosing strategy was adopted, with mice divided into two groups (Ad-K31-LC3B-IL12 group and buffer group) starting one day after tumor implantation, with 5 mice in each group. The dosing schedule was four administrations on days 0, 7, 21, and 28. The first two administrations were simultaneous intramuscular and inhalation, and the last two were inhalation administrations. The intramuscular dose was 1E10VP / mouse, and the inhalation dose was 5E9VP / mouse. The experiment was terminated after 2 weeks following the last administration, and the tumor inhibition rate was calculated.

[0174] As shown in Figure 8, compared with the buffer group, the TGI of the Ad-K31-LC3B-IL12 group was 83.3%, indicating a significant tumor-suppressing effect.

[0175] Example 12: Therapeutic effect of recombinant adenovirus vector tumor vaccine on a colorectal cancer model

[0176] Mouse colon cancer MC38 cells were genetically modified to express human HLA-A11.1 and KRAS G12V proteins. The genetically modified cells were named HLA-A11.1 / hKRAS*G12V MC38, and were grown at a rate of 1×10⁻⁶. 6 A tumor animal model was established by inoculating 6-8 week old HLA-A*11:1 humanized C57BL / 6 mice with 0.1 mL / mcg cells. The tumor volume reached 100-200 mm². 3Treatment began in groups. The dosing schedule consisted of four administrations on days 0, 7, 14, and 21. The first two administrations were a combination of intramuscular and inhalation, while the latter two were inhalation-only. The test drug was Ad-G12V. Three groups were established: a high-dose group, a low-dose group, and a buffer control group, with eight animals in each group. The high-dose group received an inhalation dose of 1.5E10VP / animal, while the low-dose group received an inhalation dose of 5E9VP / animal. The first two intramuscular injections for both the high- and low-dose groups were 1E10VP / animal. Animals were observed for one week after the last administration, and the tumor inhibition rate (TGI) was calculated.

[0177] As shown in Figure 9, compared with the Ad-null group, the high-dose and low-dose Ad-G12V groups had significant tumor suppression effects, with a TGI of 72.6% in the high-dose group and 66.4% in the low-dose group.

[0178] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A recombinant adenovirus vector, characterized in that: The antigen encoded by the recombinant adenovirus vector contains any one or more combinations of antigenic peptide epitopes of the KRAS protein.

2. The recombinant adenovirus vector according to claim 1, characterized in that: The antigenic peptide epitope is selected from any one or a combination of two or more of the human KRAS protein mutation sites G12C, G12V, G12D, G12A, G12R, G12S, G13D, G13C, G13E, Q61H, Q61R, Q61K and Q61L.

3. The recombinant adenovirus vector according to claim 1, characterized in that: The antigenic peptide epitope is selected from any one or more combinations of SEQ ID NO:1-13; or One or more combinations of sequences that have more than 70% identity with the sequences shown in SEQ ID NO:1-13.

4. The recombinant adenovirus vector according to claim 1, characterized in that: The antigenic peptide epitopes are as shown in any one of SEQ ID NO:14-31; or One or more of the sequences that have more than 70% identity with the sequence shown in SEQ ID NO:14-31.

5. The recombinant adenovirus vector according to any one of claims 1-4, characterized in that: The genome of the recombinant adenovirus vector also encodes a fusion protein that promotes the presentation of cellular immune antigens and / or enhances cellular immunity.

6. The recombinant adenovirus vector according to claim 5, characterized in that: The fusion protein includes LC3B, IFN-γ, IL12, GM-CSF, IL21, CCL3, or CCL5.

7. A recombinant adenovirus vector tumor vaccine, characterized in that: The recombinant adenovirus vector tumor vaccine comprises the recombinant adenovirus vector as described in any one of claims 1-6.

8. The recombinant adenovirus vector tumor vaccine according to claim 7, characterized in that: The recombinant adenovirus vector tumor vaccine is in the form of an inhaler or an injectable; preferably, the recombinant adenovirus is atomized by a drug delivery device to form particles smaller than 15 μm.

9. An antigenic peptide, characterized in that: The antigenic peptide comprises any one or more combinations of antigenic peptide epitopes as shown in SEQ ID NO:1-13, or any one or more combinations of antigenic peptide epitopes having more than 70% identity with the sequence shown in SEQ ID NO:1-13.

10. The antigenic peptide according to claim 9, characterized in that: The sequence of the antigenic peptide is as shown in any of SEQ ID NO:14-31 or has more than 70% identity with it.

11. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antigenic peptide according to any one of claims 9-10.

12. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a recombinant adenovirus vector as described in any one of claims 1-6 or a recombinant adenovirus vector tumor vaccine as described in any one of claims 7-8; preferably, the pharmaceutical composition further comprises pharmaceutically permissible excipients.

13. An aerosol, characterized in that: The aerosol comprises the recombinant adenovirus vector of any one of claims 1-6, the recombinant adenovirus vector tumor vaccine of any one of claims 7-8, or the pharmaceutical composition of claim 12.

14. The aerosol according to claim 13, characterized in that: The particle size of the aerosol is ≤15μm.

15. The use of the recombinant adenovirus vector of any one of claims 1-6, the recombinant adenovirus vector tumor vaccine of any one of claims 7-8, the nucleic acid molecule of claim 11, or the pharmaceutical composition of claim 12 in the preparation of products for the prevention or treatment of tumors, or for the prevention of postoperative recurrence of tumors; preferably, the use is carried out by means of administration including nebulized inhalation and / or intramuscular injection.

16. The application according to claim 15, characterized in that: The tumor is a Kras mutation-driven cancer; preferably, the cancer includes lung cancer, pancreatic cancer, or colorectal cancer.

17. The application according to claim 16, characterized in that: The lung cancers mentioned include non-small cell lung cancer, lung metastases from pancreatic cancer, or lung metastases from colorectal cancer.

18. The use of the recombinant adenovirus vector according to any one of claims 1-6 in the preparation of a mucosal delivery immunomodulatory composition; preferably, the mucosa includes oral mucosa, respiratory mucosa or lung mucosa.

19. A method for preparing the recombinant adenovirus vector tumor vaccine according to any one of claims 7-8, characterized in that: The preparation method includes the following steps: (1) Construct the recombinant adenovirus vector according to any one of claims 1-6; (2) The recombinant adenovirus vector and the backbone plasmid carrying most of the adenovirus genome were co-transfected into packaging cells, and the recombinant packaging replication was obtained to obtain a defective adenovirus. (3)Optionally, pharmaceutically acceptable excipients are added to obtain the recombinant adenovirus vector tumor vaccine.

20. The preparation method according to claim 19, characterized in that: The packaging cells are Hala cells, HEK293 cells, or their derived cell lines.

Images