Lung cancer AAV vector therapeutic vaccine as well as preparation method and application thereof

Abstract

The invention provides a lung cancer therapeutic vaccine based on an AAV vector as well as a preparation method and application of the lung cancer therapeutic vaccine. The screened inhalation type AAV vaccine for coding the KRAS antigen and / or the EGFR antigen and / or the ALK antigen and / or the ROS1 antigen and suitable for inhalation administration is used for treating lung cancer, compared with an intramuscular injection immunization mode of the same preparation, triple immunization effects of humoral immunization, cellular immunization and mucosal immunization can be generated, and the killing effect on lung tumor cells is improved.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to a lung cancer AAV vector therapeutic vaccine and a preparation method and use thereof. Background Art

[0002] The research on cancer treatment drugs is a hot topic in the world today. In addition to 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 method for treating tumors. Tumor vaccination is a type of immunotherapy. Its principle is to use tumor cells or tumor antigens to induce the body to produce specific cellular immunity and humoral immune responses, that is, to activate the patient's own immune system to enhance the body's anti-cancer ability, thereby preventing the growth, spread, and recurrence of tumors, and ultimately achieving the goal of controlling or even eliminating tumors. 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 (TAA) or tumor-specific antigens (TSA).

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

[0004] TSAs are newly formed antigens produced by tumor cells due to various tumor-specific changes, such as genomic mutations, dysregulated RNA splicing, disordered post-translational modifications, and integrated viral open reading frames. TSAs are considered non-self and trigger immune responses that are not affected by central and peripheral tolerance. They are also called tumor neoantigens. TSAs have the distinct advantages of unique tumor specificity and absence in normal tissues, providing ideal targets for effective personalized treatment of tumors. Vaccines based on neoantigens rather than traditional TAAs have the following advantages: First, neoantigens are only expressed by tumor cells and can therefore trigger true tumor-specific T cell responses, thereby preventing "off-target" damage to non-tumor tissues. Second, neoantigens are new epitopes derived from somatic mutations. T cells specific for neoantigens can bypass the negative selection effect in the thymus, enhancing tumor-specific immune responses. In addition, the neoantigen-specific T cell responses enhanced by immunotherapy have the ability to persist and generate post-treatment immune memory, which offers 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 them, KRAS protein G12V, G12D, G12C, G12A, G12R, G12S, G13D, G13C, Q61H, Q61R, Q61K, and Q61L mutations, EGFR protein T790M, L858R, G719C / A / S, S768I, and G 796S, ALK protein L1196M, L1152R, G1202R, G1269A, S1206Y, C1156Y, F1174C, D1203N, L1198F, ROS1 protein L2026M, S1986Y / F, G2032R, D2033N, L1951R mutations are the most common mutation types. Targeting specific mutant antigens commonly found in lung cancer can induce an efficient immune response against lung cancer.

[0006] Currently, the possible methods of tumor vaccine administration include: (1) subcutaneous injection (2) intravenous injection (3) intradermal injection (4) intramuscular injection (5) inhalation. Among them, inhalation vaccines can induce a triple immune effect of humoral immunity, cellular immunity, and mucosal immunity. For lung tumor cells, they can induce an in situ immune response and enhance the immune efficacy of lung cancer. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an inhaled AAV vector vaccine for treating lung cancer.

[0008] The term "vaccine" refers to a composition suitable for use in animals (including humans) that, upon administration, induces an immune response strong enough to minimally help prevent, ameliorate or treat a tumor.

[0009] The term "delivery system" refers to a preparation or composition that regulates the spatial, temporal, and dosage distribution of a biologically active ingredient in an organism.

[0010] The term "AAV vector vaccine" refers to a vaccine that uses AAV to carry an encoded antigen sequence, infects human cells after immunization, and then translates into the target antigen. The present invention provides an AAV vector tumor vaccine, characterized in that the antigen encoded by the AAV vector vaccine comprises one or more antigenic peptide epitopes selected from EGFR and / or KRAS and / or ALK and / or ROS1, preferably, prepared from one or more separated: epitope antigen gene sequences of EGFR, epitope antigen gene sequences of KRAS, epitope antigen gene sequences of ALK, and epitope antigen gene sequences of ROS1; preferably, the vaccine is a preparation for intramuscular injection or inhalation administration, more preferably, a nebulized inhalation administration preparation.

[0011] Specifically, it is characterized in that each antigenic peptide epitope encoded by the AAV vector is independently selected from human KRAS protein G12C / D / V / A / R / S, G13D / C, Q61H / R / K / L mutations, and / or EGFR protein L718Q, T790M, L858R, G719C / A / S, S768I, G796S, and / or ALK protein L1196M, L1152R, G12 02R, G1269A, S1206Y, C1156Y, F1174C / L / V, D1203N, L1198F, I1171T, and / or ROS1 protein L2026M, S1986Y / F, G2032R, D2033N, L1951R mutations; preferably, the antigenic peptide epitope combination encoded by the AAV vector is human KRAS protein G12C / D / V / A, G13D / C, Q61H / R / K / L mutations; and / or EGFR protein L718Q, T790M, L858R, G719C / A / S, S768I, G796S, and ALK protein L1196M, L1152R, G1202R, G1269A, S1206Y, C1156Y, F1174C / L / V, D1203N, L1198F, I1171T; ROS1 Protein L2026M, S1986Y / F, G2032R, D2033N, L1951R mutations; More preferably, the antigenic peptide epitope combination encoded by the AAV vector has a sequence combination with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more or 100% identity with the above combination; More preferably, the KRAS epitope encoded by the AAV vector is selected from SEQ ID NO: 1-12, EGFR epitope is selected from 1 or more of SEQ ID NO: 13-20, ALK epitope is selected from 1 or more of SEQ ID NO: 21-32, ROS1 epitope is selected from 1 or more of SEQ ID NO: 33-38; More preferably, the KRAS epitope encoded by the AAV vector is selected from sequences 1 to 5, 7 of SEQ ID NO: 1-12, the EGFR epitope is selected from sequences 13 to 16, 19, 20 of SEQ ID NO: 13-20, the ALK epitope is selected from sequences 21 to 27, 30 to 32 of SEQ ID NO: 21-32, and ROS1 epitope is selected from sequences 33, 34, 36 to 38 of SEQ ID NO: 33-38; More preferably, the KRAS epitope encoded by the AAV vector is selected from sequences 1 to 12, the EGFR epitope is selected from sequences 13 to 20, the ALK epitope is selected from sequences 21 to 27, 30 to 32 of SEQ ID NO: 21-32, and ROS1 epitope is selected from sequences 33, 34, 36 to 38 of SEQ ID NO: 33-38. NO: 21-32 sequences, ROS1 epitopes are selected from SEQ ID NO: 33-38 sequences;Preferably, the AAV vector encodes an antigenic peptide epitope that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, or 100% identical to the sequence of SEQ ID NO: 1-38. ;

[0012] Specifically, it is characterized in that each antigenic peptide epitope encoded by the AAV vector is 10-30 amino acids in length, preferably, the antigenic peptide epitope is 20-30 amino acids in length, and more preferably, the antigenic peptide epitope is 22-28 amino acids in length.

[0013] Specifically, it is characterized in that the number of antigenic peptides encoded by the AAV vector is 12-38, preferably, the number of antigenic peptides is 15-38, and more preferably, the number of antigenic peptides is 25-38.

[0014] Specifically, it is characterized in that there are 0-30 base intervals between the antigenic peptides encoded by the AAV vector, preferably, a single base interval or a GS linker interval. Preferably, the GS linker sequence is selected from GGSGGGGSGG (SEQ ID NO:41), GSGSGSGSGS (SEQ ID NO:42), GSGGSGGSGG (SEQ ID NO:43), GGSLGGGGSG (SEQ ID NO:44), GGGGSGGGGS (SEQ ID NO:45). Specifically, it is characterized in that the AAV vector vaccine is characterized in that the genome contained in the AAV vector vaccine further includes a nucleic acid sequence selected from the ITR region, the promoter region, and / or the poly(A) tail.

[0015] Specifically, it is characterized in that the AAV vector is a wild-type or recombinant AAV vector, and the preferred AAV vectors are type 1, type 3, type 4, type 5, type 6, type 9, and type 10, and the more preferred AAV vectors are type 5, type 6, and type 9.

[0016] Specifically, it is characterized in that the AAV vector vaccine is a mucosal immune preparation; the mucosal immune preparation is nasal drops, aerosols, sprays, powder sprays, powders, liquid preparations, freeze-dried preparations, gels, microspheres, liposomes, films, and suspensions.

[0017] Specifically, it is characterized in that the mucosal administration preparation is an inhalation administration preparation, preferably, a nasal inhalation preparation or an oral inhalation preparation; more preferably, the inhalation administration preparation is a liquid inhalation preparation or a dry powder inhalation preparation.

[0018] Specifically, it is characterized in that the AAV vector vaccine contains pharmaceutically acceptable excipients.

[0019] Specifically, it is characterized in that the AAV vector vaccine dosage form is an aerosol inhaler, and the vaccine forms particles of less than 10 μm after being aerosolized by an aerosol delivery device.

[0020] Specifically, it is characterized in that the AAV vector vaccine prescription also contains other excipients, and the excipients are one or more combinations of sodium chloride, magnesium chloride, poloxamer 188, and sucrose.

[0021] The present invention provides use of an AAV vector vaccine in the preparation of a vaccine for treating mammalian tumors.

[0022] Specifically, the invention is characterized by: use in preparing a composition for treating lung cancer or preventing recurrence of lung cancer after surgery; preferably, use in preparing a composition for treating non-small cell lung cancer; or use in preparing a composition for preventing recurrence of lung cancer after surgery.

[0023] Specifically, it is characterized in that the AAV vector vaccine is immunized through the mucosa.

[0024] Specifically, the mucosa includes oral mucosa or respiratory mucosa. Preferably, the respiratory mucosa is lung mucosa.

[0025] Specifically, it is characterized in that the AAV vector vaccine is a single drug and / or combined with chemotherapy, and / or combined with radiotherapy, and / or combined with small molecule inhibitors, and / or combined with immune checkpoint inhibitors, and / or combined with cellular immunotherapy; preferably, combined with PD-1 or PD-L1 antibodies, or combined with TCR-T.

[0026] The beneficial effects of the present invention include:

[0027] 1. One aspect of the present invention provides an inhaled AAV vector vaccine for lung cancer, as well as its preparation method and use. This invention utilizes an inhaled AAV vector vaccine encoding KRAS antigen, EGFR antigen, ALK antigen, and / or ROS1 antigen to treat lung cancer. Compared to intramuscular injection of the same formulation, this vaccine can produce a triple immune effect of humoral, cellular, and mucosal immunity, thereby increasing the killing of lung tumor cells.

[0028] 2. In one aspect of the present invention, primary immunization through mRNA vaccine or adenovirus intramuscular injection and enhanced inhalation administration of AAV viral vector vaccine can not only induce high humoral and cellular immune responses, but also produce mucosal immune responses, achieving a better triple protection effect.

[0029] 3. In one aspect of the present invention, the vaccine provided by the present invention can be used for lung cancer patients who have failed recommended treatments to prolong the patients' survival.

[0030] 4. In one aspect of the present invention, the vaccine provided by the present invention can be used for people who have undergone lung cancer surgical resection to prevent the recurrence of lung cancer after surgery.

[0031] 5. In one aspect of the present invention, the AAV vector vaccine of the present invention can produce particles with a size between 3-10 μm and good uniformity after being atomized by suitable equipment. The particles can reach the lungs by inhalation through the nasal cavity or oral cavity, thereby generating a protective immune response to the entire respiratory tract and lungs, enhancing the effective utilization rate of the vaccine and improving the effect of the vaccine. DETAILED DESCRIPTION

[0032] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0033] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0034] Example 1: Preparation of AAV vector vaccine

[0035] 1. Artificially synthesize the designed target antigen gene sequence and recombinantly form the pAAV-ITR-GOI plasmid;

[0036] 2. Transform the successfully identified pAAV-ITR-GOI plasmid, packaging plasmid pRepCapX, and auxiliary plasmid pHelper into competent cells respectively. After small-scale plasmid shaking, further large-scale plasmid amplification is performed. The plasmid is extracted using a plasmid extraction kit and used for double enzyme digestion and identification.

[0037] 3. Expand suspended HEK239 cells to a density of 1.6×10 6 When the concentration of 1:1 was 1:1 / ml, three plasmid transfections were performed and the virus was harvested after 72 h of culture.

[0038] 4. Use Tween 80 to lyse the virus harvest solution, lyse at 37°C for 4 hours, and then centrifuge at 4500 rpm for 20 minutes to harvest the supernatant;

[0039] 5. After filtering the centrifugal supernatant with a 0.04-0.8 μm depth filter, use an ultrafiltration membrane package to concentrate the liquid to a suitable volume for later use;

[0040] 6. Load the concentrated liquid into the filler containing AAVX, elute the target virus with AAVX elution buffer containing sodium citrate and magnesium chloride, and then collect the liquid;

[0041] 7. Prepare 15%, 25%, 40%, and 60% iodixanol solutions; add the virus feed solution, 60%, 40%, 25%, and 15% iodixanol solutions to the ultracentrifuge tube in sequence; centrifuge at 69,000 rpm for 1.5 hours and collect the target virus bands;

[0042] 8. Ultrafilter the target virus 30 times and exchange the solution into DS buffer. Detect Vg, solid rate and other indicators.

[0043] Example 2: Application of inhaled lung cancer vaccine

[0044] To test the potential of inhaled AAV-based vaccines to enhance immune responses against lung cancer, we designed and packaged a recombinant AAV vector encoding the Kras G12C antigen. Immunization was performed by intramuscular injection or inhalation. Female Kras G12C mice were randomly divided into groups of 10 and immunized with AAV at different doses (1E13 Vg / mouse in the high-dose group and 5E12 Vg / mouse in the low-dose group) and routes of immunization on days 0 and 28. Blood samples were collected on days 28 (before the second immunization) and 42, and the frequencies of antigen-specific CD4+ and CD8+ cytokine-secreting T cells were assessed using ICS assays (IFNγ, TNF, and IL-2). On day 42, bronchoalveolar lavage fluid (BALF) was collected, mice were sacrificed, and splenocytes were harvested and stimulated with overlapping peptide libraries of the antigen protein to assess cellular immune responses. Serum IgG and BALF IgA were measured by ELISA.

[0045] Immunization groups:

[0046] Group dose Route of administration 1 5E12 Vg / piece intramuscular injection 2 1E13Vg / piece intramuscular injection 3 5E12 Vg / piece Inhalation 4 1E13Vg / piece Inhalation

[0047] The table below shows the effect of an inhaled AAV lung cancer vaccine on the frequency of Kras G12C-specific CD4+ and CD8+ cytokine-secreting T cells (IFNγ, TNF, and IL2), as assessed by the ICS assay. As shown in the table, inhaled AAV vaccine significantly increased the frequency of CD4+ and CD8+ cytokine-secreting T cells.

[0048]

[0049] The following table shows the IFNγ ELISpot assay to assess the cellular immune response levels across different immune pathways. As shown in the table, inhaled formulations significantly increased the cellular immune response levels.

[0050]

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0052] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience, and preference of those skilled in the art.

[0053] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

[0054] Sequence Listing

[0055] SEQ ID NO: 1

[0056] KRAS G12C:MTEYKLVVVGACGVGKSALTIQLI

[0057] SEQ ID NO: 2

[0058] KRAS G12D:MTEYKLVVVGADGVGKSALTIQLI

[0059] SEQ ID NO: 3

[0060] KRAS G12V:MTEYKLVVVGAVGVGKSALTIQLI

[0061] SEQ ID NO: 4

[0062] KRAS G12A:MTEYKLVVVGAAGVGKSALTIQLI

[0063] SEQ ID NO: 5

[0064] KRAS G12R:MTEYKLVVVGARGVGKSALTIQLI

[0065] SEQ ID NO: 6

[0066] KRAS G12S:MTEYKLVVVGASGVGKSALTIQLI

[0067] SEQ ID NO: 7

[0068] KRAS G13D:MTEYKLVVVGAGDVGKSALTIQLI

[0069] SEQ ID NO: 8

[0070] <h2 style=";text-align:left;direction:ltr">KRAS G13C:MTEYKLVVVGAGCVGKSALTIQLI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0071] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0072] <h2 style=";text-align:left;direction:ltr"> KRAS Q61H:ETCLLLDILDTAGHEEYSAMRDQYMR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0073] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0074] <h2 style=";text-align:left;direction:ltr"> KRAS Q61R:ETCLLDILDTAGREEYSAMRDQYMR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0075] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0076] <h2 style=";text-align:left;direction:ltr"> KRAS Q61K:ETCLLDILDTAGKEEYSAMRDQYMR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0077] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0078] <h2 style=";text-align:left;direction:ltr"> KRAS Q61L:ETCLLDILDTAGLEYSAMRDQYMR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0079] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0080] <h2 style=";text-align:left;direction:ltr"> EGFR L718Q:KETEFKKIKVQGSGAFGTVYKGLW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0081] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:14<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0082] <h2 style=";text-align:left;direction:ltr"> EGFR T790M:GICLTSTVQLIMQLMPFGCLLDYV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0083] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0084] <h2 style=";text-align:left;direction:ltr"> EGFR L858R:TPQHVKITDFGRAKLLGAEEKEYH<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0085] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:16<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0086] <h2 style=";text-align:left;direction:ltr"> EGFR G719C:KETEFKKIKVLCSGAFGTVYKGLW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0087] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:17<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0088] <h2 style=";text-align:left;direction:ltr"> EGFR G719A:KETEFKKIKVLASGAFGTVYKGLW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:18<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr">EGFR G719S:KETEFKKIKVLSSGAFGTVYKGLW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:19<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0092] <h2 style=";text-align:left;direction:ltr"> EGFR S768I:KEILDEAYVMAIVDNPHVCRLLGI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 20<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> EGFR G796S:STVQLITQLMPFSCLLDYVREHKD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO: 21<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] <h2 style=";text-align:left;direction:ltr"> ALK L1196M:GVSLQSLPRFILMELMAGGDLKSFSEQ ID NO:22<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0097] <h2 style=";text-align:left;direction:ltr"> ALK L1152R:DPSPLQVAVKTRPEVCSEQDELDFSEQ ID NO:23<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0098] <h2 style=";text-align:left;direction:ltr"> ALK G1202R:PRFILLELMAGRDLKSFLRETRPRSEQ ID NO:24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0099] <h2 style=";text-align:left;direction:ltr"> ALK G1269A:TCPGPGRVAKIADFGMARDIYRASSEQ ID NO:25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0100] <h2 style=";text-align:left;direction:ltr"> ALK S1206Y:LLELMAGGDLKYFLRETRPRPSQPSEQ ID NO:26<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0101] <h2 style=";text-align:left;direction:ltr"> ALK C1156Y:LQVAVKTLPEVYSEQDELDFLMEASEQ ID NO:27<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0102] <h2 style=";text-align:left;direction:ltr"> ALK F1174C:DFLMEALIISKCNHQNIVRCIGVSSEQ ID NO:28<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0103] <h2 style=";text-align:left;direction:ltr"> ALK F1174C:DFLMEALIISKLNHQNIVRCIGVSSEQ ID NO:29<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0104] <h2 style=";text-align:left;direction:ltr"> ALK F1174C:DFLMEALIISKNNHQNIVRCIGVSSEQ ID NO:30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0105] <h2 style=";text-align:left;direction:ltr"> ALK D1203N:RFILLELMAGGNLKSFLRETRPRPSEQ ID NO:31

[0106] ALK L1198F:LQSLPRFILLEFMAGGDLKSFLRESEQ ID NO:32

[0107] ALK I1171T:DFLMEALITSKNNHQNIVRCIGVSSEQ ID NO:33

[0108] ROS1 L2026M:VCLLNEPQYIIMELMEGGDLLTYLSEQ ID NO:34

[0109] ROS1 S1986Y:IKVAVKTLKKGYTDQEKIEFLKEASEQ ID NO:35

[0110] ROS1 S1986F:IKVAVKTLKKGFTDQEKIEFLKEA

[0111] SEQ ID NO:36

[0112] ROS1 G2032R:PQYIILELMEGRDLLTYLRKARMA

[0113] SEQ ID NO:37

[0114] ROS1 D2033N:QYIILELMEGGNLLTYLRKARMAT

[0115] SEQ ID NO:38

[0116] ROS1 L1951R:AFPREKLTLRLLRGSGAFGEVYEG

[0117] SEQ ID NO:39

[0118] MTEYKLVVGACGVGKSALTIQLIGSGGSGGSGGMTEYKLVVVGADGVGKSALTIQLIGSGGSGGSGGMTEYKLVVVGAGDVGKSALTIQLIGSGGSGGSGGGETCLLDILDTAGHEEYSAMRDQYMRGSGGSGGSGGGETCLLDILDTAGREEYSAMRDQYMRGSGGSGGSGGKETEFKKIKVQGSGAFGTVYKGLWGSGGSGGSGGGICLTSTVQLIMQLMPFGCLLDYVGSGGSGGSGGTPQHVKITDFGRAKLLGAEEKEYHGSGGSGGSGGKETEFKKIKVLCSGAFGTVYKGLWGSGGSGGSGGKEILDEAYVMAIVDNPHVCRLLGIGSGGSGGSGGSTVQLITQLMPFSCLLDYVREHKDGSGGSGGSGGVSLQSLPRFILMELMAGGDLKSFGSGGSGGSGGDPSPLQVAVKTRPEVCSEQDELDFGSGG SGGSGGPRFILLELMAGRDLKSFLRETRPRGSGGSGGSCGPGRVAKIADFGMARDIYRASGSGGSGGLLELMAGGDLKYFLRETRPRPPSQPGSGGSGGSGGLQVAVKTLPEVYSEQDELDFLMEAGSGGSGGSGGDFLMEAILIISKCNHQNIVRCIGVSGSGGSGGRFILLELMAGGNLKSFLRETRPRPGSGGSGGSGGGLQSLPRFILLEFMAGGDLKSFLREGSGGSGGDFLMEALITSKNNHQNIVRCIGVSGSGGSGGVCLLNEPQYIIMELMEGGDLLTYLGSGGSGGSGGGIKVAVKTLKKGYTDQEKIEFLKEAGSGGSGGSGGPQYIILELMEGRDLLTYLRKARMAGSGGSGGSGGQYIILELMEGGNLLTYLRKARMATGSGGSGGAFPREKLTLRLLRGSGAFGEVYEG

[0119] SEQ ID NO: 40

[0120] MTEYKLVVGACGVGKSALTIQLIGSGGSGGMTEYKLVVVGADGVGKSALTIQLIGSGGSGGMTEYKLVVVGAVGVGSALTIQLIGSGGSGGMTEYKLVVVGAAGVGKSALTIQLIGSGGSGGMTEYKLVVVGAGARGVGKSALTIQLIGSGGSGGMTEYKLVVVGAGDVGKSALTIQLIGSGGSGGSGGMTEYKLVVVGAGCVGKSALTIQLIGSGGSGGSGGETCLLDILDTAGHEEYSAMRDQYMRGSGGSGGSGGETCLLDILDTAGREEYSAMRDQYMRGSGGSGGSGGETCLLDILDTAGKEEYSAMRDQYMRGSGGSGGSGGETCLLDILDTA GLEEYSAMRDQYMRGSGGSGGSGGKETEFKKIKVQGSGAFGTVYKGLWGSGGSGGGICLTSTVQLIMQLMPFGCLLDYVGSGGSGGSGGTPQHVKITDFGRAKLLGAEEKEYHGSGGSGGKETEFKKIKVLCSGAFGTVYKGLWGSGGSGGSGGKETEFKKIKVLASGAFGTVYKGLWGSGGSGGSGGKETEFKKIKVLSSGAFGTVYKGLWGSGGSGGSGGKEILDEAYVMAIVDNPHVCRLLGIGSGGSGGSGGSTVQLITQLMPFSCLLDYVREHKDGSGGSGGSGGVSLQSLPRFILMELMAGDLKSFGSGGSGGSGGDPSPLQVAVKTRPEVCSEQDELDFGSGGSGGSGGGPRFILLELMAGRDLKSFLRETRPR GSGGSGGTCPGPGRVAKIADFGMARDIYRASGSGGSGGLLELMAGGDLKYFLRETRPRPSQPGSGGSGGSGGLQVAVKTLPEVYSEQDELDFLMEAGSGGSGGSGGDFLMEALIISKCNHQNIVRCIGVSGSGGSGGDFLMEALIISKLNHQNIVRCIGVSGSGGSGGDFLMEALIISKNNHQNIVRCIGVSGSGGSGGRFILLELMAGGNLKSFLRETRPRPGSGGSGGSGGLQSLPRFILLEFMAGGDLKSFLREGSGGSGGSGGDFLMEALITSKNNHQNIVRCIGVSGSGGSGGVCLLNEPQYIIMELMEGGDLLTYLGSGGSGGSGGIGKVAVKTLKKGYTDQEKIEFLKEAGSGGSGGSGGIKVAVKTLKKGFTDQEKIEFLKEAGSGGSGGSGGPQYIILELMEGRDLLTYLRKARMAGSGGSGGSGGGQYIILELMEGGNLLTYLRKARMATGSGGSGGAFPREKLTLRLLRGSGAFGEVYEG

[0121] SEQ ID NO:41

[0122] GGSGGGGSGG

[0123] SEQ ID NO:42

[0124] GSGSGSGSGSGS

[0125] SEQ ID NO:43

[0126] GSGGSGGSGG

[0127] SEQ ID NO:44GGSLGGGGSGSEQ ID NO:45GGGGSGGGGS。

Claims

1. An AAV vector vaccine, characterized in that The AAV vector-encoded antigen comprises one or more antigenic peptide epitopes selected from EGFR and / or KRAS and / or ALK and / or ROS1, preferably, it is prepared from one or more separated: epitope antigen gene sequence of EGFR, epitope antigen gene sequence of KRAS, epitope antigen gene sequence of ALK and epitope antigen gene sequence of ROS1; more preferably, the vaccine is a preparation for intramuscular injection or inhalation administration, more preferably, it is a preparation for nebulized inhalation administration.

2. The AAV vector vaccine according to claim 1, characterized in that Each antigenic peptide epitope encoded by the AAV vector is independently selected from the group consisting of human KRAS protein G12C / D / V / A / R / S, G13D / C, Q61H / R / K / L mutations, and / or EGFR protein L718Q, T790M, L858R, G719C / A / S, S768I, G796S, and / or ALK protein L1196M, L1152R, G1202R, G126 9A, S1206Y, C1156Y, F1174C / L / V, D1203N, L1198F, I1171T, and / or ROS1 protein L2026M, S1986Y / F, G2032R, D2033N, L1951R mutations; preferably, the antigen peptide epitope combination encoded by the AAV vector is human KRAS protein G12C / D / V / A, G13D / C, Q6 1H / R / K / L mutations; and / or EGFR protein L718Q, T790M, L858R, G719C / A / S, S768I, G796S, and ALK protein L1196M, L1152R, G1202R, G1269A, S1206Y, C1156Y, F1174C / L / V, D1203N, L1198F, I1171T; ROS1 protein L 2026M, S1986Y / F, G2032R, D2033N, L1951R mutations; More preferably, the antigenic peptide epitope combination encoded by the AAV vector has a sequence combination with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more or 100% identity with the above combination; More preferably, the KRAS epitope encoded by the AAV vector is selected from SEQ ID NO: 1-12, EGFR epitope is selected from 1 or more of SEQ ID NO: 13-20, ALK epitope is selected from 1 or more of SEQ ID NO: 21-32, ROS1 epitope is selected from 1 or more of SEQ ID NO: 33-38; More preferably, the KRAS epitope encoded by the AAV vector is selected from sequences 1 to 5, 7 of SEQ ID NO: 1-12, the EGFR epitope is selected from sequences 13 to 16, 19, 20 of SEQ ID NO: 13-20, the ALK epitope is selected from sequences 21 to 27, 30 to 32 of SEQ ID NO: 21-32, and ROS1 epitope is selected from sequences 33, 34, 36 to 38 of SEQ ID NO: 33-38; More preferably, the KRAS epitope encoded by the AAV vector is selected from sequences 1 to 12, the EGFR epitope is selected from sequences 13 to 20, the ALK epitope is selected from sequences 21 to 27, 30 to 32 of SEQ ID NO: 21-32, and ROS1 epitope is selected from sequences 33, 34, 36 to 38 of SEQ ID NO: 33-38. NO:21-32 sequence, ROS1 epitope is selected from SEQ ID NO:33-38 sequence.

3. The AAV vector vaccine according to claim 1-2, characterized in that The AAV vector encodes an antigenic peptide epitope selected from one or more combinations of SEQ ID NO: 1-38 sequences and sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with the sequences; preferably, the AAV vector encodes an antigenic peptide epitope for a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 39-40.

4. The AAV vector vaccine according to any one of claims 1 to 3, characterized in that Each antigenic peptide epitope encoded by the AAV vector is 10-30 amino acids in length, preferably, the antigenic peptide epitope is 20-30 amino acids in length, and more preferably, the antigenic peptide epitope is 22-28 amino acids in length.

5. The AAV vector vaccine according to any one of claims 1 to 4, characterized in that The number of antigenic peptides encoded by the AAV vector is 12-38, preferably, the number of antigenic peptides is 15-38, and more preferably, the number of antigenic peptides is 25-38.

6. The AAV vector vaccine according to any one of claims 1 to 5, characterized in that There are 0-30 base intervals between the antigenic peptides encoded by the AAV vector, preferably, a single base interval or a GS linker interval, preferably, the GS linker sequence is selected from GGSGGGGSGG (SEQ ID NO: 41), GSGSGSGSGS (SEQ ID NO: 42), GSGGSGGSGG (SEQ ID NO: 43), GGSLGGGGSG (SEQ ID NO: 44), GGGGSGGGGS (SEQ ID NO: 45).

7. The AAV vector vaccine according to any one of claims 1 to 6, characterized in that The genome contained in the AAV vector vaccine further includes a nucleic acid sequence selected from the ITR region, the promoter region, and / or the poly(A) tail.

8. The AAV vector vaccine according to any one of claims 1 to 7, characterized in that The AAV vector is a wild-type or recombinant AAV vector. The preferred AAV vectors are type 1, type 3, type 4, type 5, type 6, type 9, and type 10, and the more preferred AAV vectors are type 5, type 6, and type 9.

9. The AAV vector vaccine according to any one of claims 1 to 8, characterized in that The AAV vector vaccine is a mucosal immune preparation; the mucosal immune preparation is nasal drops, aerosols, sprays, powder sprays, powders, liquid preparations, freeze-dried preparations, gels, microspheres, liposomes, films, and suspensions.

10. The AAV vector vaccine according to any one of claims 1 to 9, characterized in that The mucosal administration preparation is an inhalation administration preparation, preferably, a nasal inhalation preparation or an oral inhalation preparation; more preferably, the inhalation administration preparation is a liquid inhalation preparation or a dry powder inhalation preparation.

11. The AAV vector vaccine according to any one of claims 1 to 10, characterized in that The AAV vector is a wild-type or recombinant AAV vector. The preferred AAV vectors are type 1, type 3, type 4, type 5, type 6, type 9, and type 10, and the more preferred AAV vectors are type 5, type 6, and type 9.

12. The AAV vector vaccine according to any one of claims 1 to 11, characterized in that The AAV vector vaccine contains pharmaceutically acceptable excipients.

13. The AAV vector vaccine according to any one of claims 1 to 12, characterized in that The AAV vector vaccine dosage form is an aerosol inhaler, and the vaccine forms particles of less than 10 μm after being aerosolized by an aerosol delivery device.

14. The AAV vector vaccine according to any one of claims 1 to 13, characterized in that The AAV vector vaccine prescription also contains other excipients, which are one or more combinations of sodium chloride, magnesium chloride, poloxamer 188, and sucrose.

15. Use of the AAV vector vaccine according to any one of claims 1-14 in the preparation of a vaccine for treating mammalian tumors; preferably, for the treatment of lung cancer.

16. The use according to claim 15, characterized in that Use in preparing a preparation for treating lung cancer or preventing recurrence of lung cancer after surgery; preferably, use in preparing a preparation for treating non-small cell lung cancer; or use in preparing a preparation for preventing recurrence of lung cancer after surgery.

17. The use according to any one of claims 15-16, characterized in that AAV vector vaccines are administered through mucosal immunization.

18. The use according to any one of claims 15 to 17, wherein the mucosa comprises oral mucosa or respiratory mucosa, preferably, the respiratory mucosa is lung mucosa.

19. The use according to any one of claims 15 to 18, characterized in that AAV vector vaccines are monotherapy and / or combined with chemotherapy, and / or combined with radiotherapy, and / or combined with small molecule inhibitors, and / or combined with immune checkpoint inhibitors, and / or combined with cellular immunotherapy; preferably, combined with PD-1 or PD-L1 antibodies, or combined with TCR-T.

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