An EBV immunogenic polypeptide, its combination and application

Abstract

This invention provides an EBV immunogenic peptide, combinations thereof, and applications. The EBV immunogenic peptide comprises any of the amino acid sequences shown in SEQ ID NO:7-12 and SEQ ID NO:21. This invention also discloses a kit containing the EBV immunogenic peptide or combinations thereof, as well as other drugs for the prevention and / or treatment of EBV-related diseases, such as NK cells. This invention can achieve durable inhibition of EBV-positive nasopharyngeal carcinoma in humanized mouse models and enhance the infiltration and effector function of T cells and NK cells in the tumor microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an EBV immunogenic polypeptide, its combination and application. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is the leading malignant tumor of the nasopharynx, with a significantly geographically variable incidence. Although its incidence is low in most parts of the world, NPC, particularly the undifferentiated nonkeratinizing subtype, is prevalent in North Africa and Southeast Asia. Currently, the standard treatment for NPC is limited to concurrent chemoradiotherapy, which is effective for patients with locally acquired disease. However, highly aggressive and potentially metastatic undifferentiated NPC often leads to failure of first-line treatment. Furthermore, over 75% of patients are diagnosed at an advanced stage due to delayed presentation, resulting in a poor prognosis. Therefore, there is an urgent need for more effective treatment options for advanced, incurable nasopharyngeal carcinoma.

[0003] Most undifferentiated nasopharyngeal carcinomas (NPCs) are etiologically associated with Epstein-Barr virus (EBV) infection. EBV can be detected in precancerous lesions and is crucial to the oncogenic process of NPCs by inducing genomic instability and imposing immunosuppression. These properties make EBV an attractive candidate for targeted therapy of NPCs. Therefore, therapeutic tumor vaccination has emerged as a promising strategy to combat EBV-infected cancer cells by stimulating an individual's T-cell immunity against viral antigens. To date, various forms of vaccines have been developed, primarily focusing on dendritic cells pulsed with EBV antigens and recombinant viruses expressing EBV antigens. Other approaches based on cancer stem cell lysates, synthetic peptides, plasmid DNA, and mRNA have also been evaluated. Early clinical trials have demonstrated the safety and immunogenicity of therapeutic EBV vaccines, but only a subset of patients exhibited strong T-cell responses. Wild-type EBNA1, LMP1, and LMP2 oncoproteins of the prototypical EBV strain (e.g., B95-8) are the most common targeted antigens. It is worth noting that recent large-scale whole-genome sequencing has identified abundant genomic variations in cancer-associated EBV strains. Similar to how somatic mutations in cancer cells can generate new antigens, unidentified nonsynonymous mutations in the EBV genome may encode novel immunogenic epitopes for vaccine design, a possibility that has not yet been experimentally verified.

[0004] In addition to antigen selection, cancer vaccines often require combination therapy to overcome immune evasion and maximize the therapeutic efficacy against established solid tumors. Currently, combination therapeutic vaccination with other therapies is also an area of ​​active research in NPC treatment. With increasing recognition of the crucial role of innate immune responses in anti-cancer immunity, activation of innate immune cells holds promise for providing multi-layered and durable tumor control. NK cells represent a specialized population of effector cells, naturally targeting newly formed, abnormally transformed cells and virus-infected cells. Importantly, NK cell-mediated cytotoxicity is independent of human leukocyte antigen (HLA), enabling it to eliminate cancer cells that evade T cell-mediated immune surveillance due to HLA loss. Studies have shown that a higher proportion of NK cells in the tumor microenvironment (TME) is associated with EBV. + Improved prognosis is associated with NPC. A case report documented a gradual reduction in brain metastases in a patient with advanced nasopharyngeal carcinoma following NK cell metastasis. Safety and partial clinical response were also observed in a phase I study of NK cells in combination with cetuximab for recurrent nasopharyngeal carcinoma, demonstrating the translational potential of adoptive NK cell therapy. To date, NK cell therapy in combination with therapeutic vaccines for EBV... + The potential synergistic effects of NCP have not yet been explored.

[0005] The standard treatment for nasopharyngeal carcinoma (NPC) is generally concurrent chemoradiotherapy. For early-stage patients (stage I or II) without cervical lymph node metastasis, radiotherapy alone is the primary treatment. For advanced patients with high-risk factors such as significantly elevated plasma EBV copy numbers, concurrent chemoradiotherapy is necessary. However, highly aggressive and metastatic undifferentiated NPC often leads to first-line treatment failure. In recent years, immune checkpoint inhibitors have been used as second-line and later-line treatments for NPC, but the overall response rate (ORR) has consistently been below 30%, failing to meet clinical needs. Similarly, early clinical trials of EBV-related tumor mRNA therapeutic vaccines have demonstrated safety and immunogenicity, but only a subset of patients showed strong T-cell responses. Cell therapy is now an actively researched area in NPC treatment. Clinical trials have investigated EBV-targeted chimeric antigen receptor T-cell (TCR-T) and CAR-T therapies for advanced EBV-positive NPC patients, showing good tolerability and preliminary efficacy. However, to date, no studies have explored the combined use of NK cell therapy and therapeutic vaccination for EBV. + Potential synergistic effects of NPCs.

[0006] Therefore, there is an urgent need to develop a therapeutic vaccine that can cover a wider population and to explore suitable combination therapy strategies to enhance efficacy. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the limitations of existing therapeutic tumor vaccines that target wild-type oncoproteins of the prototype EBV strain. These vaccines are unable to cope with the large number of nonsynonymous mutations in the EBV genome, resulting in limited efficacy in certain populations. Furthermore, their anti-tumor effects are not ideal when used alone. The present invention provides an EBV immunogenic polypeptide and its combination and application.

[0008] In this invention, the inventors performed genomic mutation sequencing analysis on numerous cancer-associated EBV strains and predicted common epitopes with high-frequency nonsynonymous mutations covering a wide range of human leukocyte antigens (HLA). The inventors demonstrated that a multi-epitope mRNA vaccine constructed from the predicted epitopes could elicit an antigen-specific T-cell response, but in PBMC humanized mouse EBV... + In NPC models, the control of tumor growth was ineffective. To further investigate effective multimodal therapy, the inventors expanded highly pure and cytotoxic human NK cells in vitro using an optimized NK cell culture system as a combination therapy. Combined administration of mRNA vaccine and NK cells significantly improved therapeutic efficacy by persistently inhibiting or eradicating NPC tumors in humanized mice. The inventors further demonstrated that the combination therapy improved the infiltration of human T cells and NK cells into the tumor microenvironment and enhanced their effector function. Therefore, the inventors' findings suggest that simultaneous therapeutic vaccination and NK cell therapy could be a potential combination therapy strategy for EBV+NPC.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0010] A first aspect of the present invention provides an EBV immunogenic peptide comprising an amino acid sequence as shown in any of SEQ ID NO:7-12 and SEQ ID NO:21.

[0011] In some embodiments of the present invention, the EBV immunogenic peptide comprises an amino acid sequence as shown in any of SEQ ID NO:1-6 and SEQ ID NO:15.

[0012] A second aspect of the present invention provides a combination of EBV immunogenic peptides, the combination comprising two or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-12 and SEQ ID NO:19-24.

[0013] In some embodiments of the present invention, the combination comprises two or more selected from the group consisting of EBV immunogenic peptides comprising amino acid sequences as shown in SEQ ID NO:1-6 and SEQ ID NO:13-18.

[0014] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:7, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:8-12 and SEQ ID NO:19-24.

[0015] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:8, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:9-12 and SEQ ID NO:19-24.

[0016] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:9, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-8, SEQ ID NO:10-12 and SEQ ID NO:19-24.

[0017] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:10, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-9, SEQ ID NO:11-12 and SEQ ID NO:19-24.

[0018] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:11, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-10, SEQ ID NO:12 and SEQ ID NO:19-24.

[0019] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:12, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-11 and SEQ ID NO:19-24.

[0020] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:21, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-12, SEQ ID NO:19-20 and SEQ ID NO:22-24.

[0021] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:1, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:2-6 and SEQ ID NO:13-18.

[0022] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:2, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:3-6 and SEQ ID NO:13-18.

[0023] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:3, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-2, SEQ ID NO:4-6 and SEQ ID NO:13-18.

[0024] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:4, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-3, SEQ ID NO:5-6 and SEQ ID NO:13-18.

[0025] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:5, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-4, SEQ ID NO:6 and SEQ ID NO:13-18.

[0026] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:6, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-5 and SEQ ID NO:13-18.

[0027] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:15, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-6, SEQ ID NO:13-14 and SEQ ID NO:16-18.

[0028] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:7 or 19, an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:8 or 20, an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:9 or 21, an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:10 or 22, an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:11 or 23, and an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:12 or 24.

[0029] In some embodiments of the present invention, the combination comprises: an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:1 or 13, an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:2 or 14, an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:3 or 15, an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:4 or 16, an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:5 or 17, and an EBV immunogenic peptide comprising an amino acid sequence as shown in SEQ ID NO:6 or 18.

[0030] In some embodiments of the present invention, the combination comprises two or more, preferably six, EBV immunogenic peptides selected from the amino acid sequences shown in SEQ ID NO: 7-12.

[0031] In some embodiments of the present invention, the combination comprises two or more, preferably six, EBV immunogenic peptides selected from the amino acid sequences shown in SEQ ID NO:1 to 6.

[0032] In some embodiments of the present invention, the EBV immunogenic peptides in the combination are connected by a linker, such as the (GS)5 linker; and / or, the combination comprises, from the N-terminus to the C-terminus, six EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-12, preferably comprising, from the N-terminus to the C-terminus, six EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-6.

[0033] A third aspect of the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a combination of EBV immunogenic peptides as described in the first aspect of the present invention or as described in any of the second aspects of the present invention.

[0034] In some embodiments of the present invention, the EBV immunogenic peptide or combination of the EBV immunogenic peptide comprises one or more nucleotide sequences selected from SEQ ID NO:31-36 and SEQ ID NO:43-48, preferably comprising one or more nucleotide sequences selected from SEQ ID NO:25-30 and SEQ ID NO:37-42.

[0035] In some embodiments of the present invention, the nucleic acid further comprises a nucleotide sequence encoding one or more selected from the 5'UTR, signal peptide, MHCI transport domain, 3'UTR, and polyA tail.

[0036] In some embodiments of the present invention, the nucleic acid satisfies one or more of the following conditions:

[0037] (1) The 5'UTR is the Xenopus β-globin 5'UTR, and the preferred nucleotide sequence is shown in SEQ ID NO:52;

[0038] (2) The signal peptide is an IgK signal peptide, preferably with a nucleotide sequence as shown in SEQ ID NO:50;

[0039] (3) The nucleotide sequence of the MHCI transport domain is shown in SEQ ID NO:54, preferably a nucleotide sequence encoding a 6×His tag;

[0040] (4) The 3'UTR is the VEEF 3'UTR, preferably with the nucleotide sequence shown in SEQ ID NO:53, and more preferably with two copies of the 3'UTR;

[0041] (5) The polyA tail contains 120 to 140 adenine nucleotides, preferably 130;

[0042] (6) The nucleic acid comprises, from the 5' end to the 3' end, the following in sequence: 5'UTR, signal peptide, the EBV immunogenic peptide or a combination of the EBV immunogenic peptides, MHCI transport domain, 3'UTR and polyA tail;

[0043] (7) One or both ends of the EBV immunogenic peptide or a combination of the EBV immunogenic peptides further comprise a nucleotide sequence encoding a linker, such as the (GS)5 linker; and,

[0044] (8) The combination of EBV immunogenic peptides contains six nucleotide sequences as shown in SEQ ID NO:31-36 from the 5' end to the 3' end, preferably six nucleotide sequences as shown in SEQ ID NO:25-30.

[0045] In some embodiments of the present invention, the nucleic acid is DNA, and preferably also includes a promoter.

[0046] In some embodiments of the present invention, the promoter is the T7 promoter, with a preferred sequence as shown in SEQ ID NO:49.

[0047] In some embodiments of the present invention, the nucleic acid is RNA, preferably mRNA.

[0048] In some embodiments of the present invention, the nucleic acid further comprises a 5' cap structure; and / or, the nucleic acid further comprises modifications, pseudouridine (Ψ) and / or N1-methylpseudouridine (m1Ψ) modifications.

[0049] In some embodiments of the present invention, the nucleic acid comprises a sequence as shown in SEQ ID NO:55.

[0050] A fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the third aspect of the present invention.

[0051] In some embodiments of the present invention, the backbone of the recombinant expression vector is pUC57 or pRV1.

[0052] A fifth aspect of the present invention provides a transformant comprising a nucleic acid as described in the third aspect of the present invention or a recombinant expression vector as described in the fourth aspect of the present invention; the transformant is a non-animal or non-plant variety.

[0053] In some embodiments of the present invention, the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0054] In some embodiments of the present invention, the prokaryotic cells are bacteria, such as Escherichia coli.

[0055] A sixth aspect of the present invention provides a lipid nanoparticle comprising a nucleic acid as described in the third aspect of the present invention.

[0056] A seventh aspect of the present invention provides a pharmaceutical composition comprising one or more selected from the following: EBV immunogenic peptides as described in the first aspect of the present invention, combinations as described in the second aspect of the present invention, nucleic acids as described in the third aspect of the present invention, and lipid nanoparticles as described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0057] In some embodiments of the present invention, one or more selected from the EBV immunogenic peptides as described in the first aspect of the present invention, the combinations as described in the second aspect of the present invention, the nucleic acids as described in the third aspect of the present invention, the recombinant expression vectors as described in the fourth aspect of the present invention, the transformants as described in the fifth aspect of the present invention, the lipid nanoparticles as described in the sixth aspect of the present invention, and the pharmaceutical compositions as described in the seventh aspect of the present invention are used in the preparation of medicaments for the prevention and / or treatment of EBV-related diseases.

[0058] In some embodiments of the present invention, the drug is a vaccine, such as an mRNA vaccine.

[0059] In some embodiments of the present invention, the disease is a tumor, such as nasopharyngeal carcinoma.

[0060] An eighth aspect of the present invention provides a kit comprising a kit A and a kit B, wherein kit A comprises one or more selected from the EBV immunogenic peptides as described in the first aspect of the present invention, combinations as described in the second aspect of the present invention, nucleic acids as described in the third aspect of the present invention, lipid nanoparticles as described in the sixth aspect of the present invention, and pharmaceutical compositions as described in the seventh aspect of the present invention; and kit B comprises other drugs for the prevention and / or treatment of EBV-related diseases, such as cell therapy preparations.

[0061] In addition to the combined therapy of the EBV mRNA vaccine and adoptive NK cell therapy described in this application, other cell therapies, such as TCR-T and CAR-T, can also be used in combination; other EBV-related tumors can also be treated with the EBV mRNA vaccine designed and constructed by the inventors.

[0062] In some embodiments of the present invention, the cell therapy preparation comprises one or more selected from TCR-T cells, CAR-T cells and NK cells, preferably comprising NK cells.

[0063] In some embodiments of the present invention, the method for preparing NK cells includes culturing them using an activation medium comprising a first cytokine, a glycogen synthase kinase inhibitor, and an NK basal medium.

[0064] In some embodiments of the present invention, the activation culture medium satisfies one or more of the following conditions:

[0065] The first cytokine includes one or more of IL-2, IL-15, IL-21, or IL-18;

[0066] The glycogen synthase kinase inhibitors include GSK3βi;

[0067] The NK basal culture medium includes NK serum-free culture medium; and,

[0068] The activated culture medium also includes a plasma substitute.

[0069] In some embodiments of the present invention, the activation culture medium satisfies one or more of the following conditions:

[0070] The concentration of IL-2 is 500–2000 IU / mL; and / or the concentration of IL-15 is 5–15 ng / mL;

[0071] The GSK3βi includes one or more of TWS119, LY2090314, or CHIR-98014; and / or, the concentration of the glycogen synthase kinase inhibitor is 0.1–0.5 μmol / mL; and,

[0072] The volume of the plasma substitute accounts for 0.09% to 9% of the total volume of the activation culture medium.

[0073] In some embodiments of the present invention, the method for preparing the NK cells includes:

[0074] (1) The culture flask is coated with a coating solution to obtain a coated culture flask; the coating solution includes antibody, recombinant human fibronectin and buffer solution;

[0075] (2) Peripheral blood mononuclear cells were activated and cultured in the coated culture flask using the activated culture medium to obtain activated cells;

[0076] (3) The cell suspension in the coated culture flask is transferred to a culture bag, and the activated cells are cultured in the culture bag using the activation medium and the proliferation medium to obtain the NK cells; the proliferation medium includes a second cytokine and the NK basal medium.

[0077] In some embodiments of the present invention, the method for preparing NK cells satisfies one or more of the following conditions:

[0078] The antibody includes one or more of anti-CD16 antibody, anti-CD337 antibody, or anti-CD314; preferably, the concentration of the anti-CD16 antibody is 1–10 μg / mL.

[0079] The recombinant human fibronectin includes one or more of RetroNectin, FibroNectin, or NbroNectin; preferably, the concentration of RetroNectin is 25–50 μg / mL.

[0080] The buffer solution includes one or more of DPBS, PBS, or HBSS;

[0081] The second cytokine includes one or more of IL-2, IL-21, or IL-18, wherein the concentration of IL-2 is preferably 500–2000 IU / mL; and / or, the NK basal medium includes NK serum-free medium; and,

[0082] The proliferation medium also includes a plasma substitute.

[0083] In some embodiments of the present invention, the activation culture includes any of the following methods: 1) cell separation and first activation replenishment; or 2) cell resuscitation and second activation replenishment.

[0084] In some embodiments of the present invention, the activation culture satisfies one or more of the following conditions: In step 1), the cell separation includes: separating lymphocytes using a lymphocyte separation solution to obtain peripheral blood mononuclear cells, washing and resuspending them, and then inoculating them into the coated culture flask for culture; preferably, the inoculation density of the peripheral blood mononuclear cells in the coated culture flask is 1×10⁻⁶. 6 ~2×10 6 per mL.

[0085] In some embodiments of the present invention, the first activation replenishment includes: after the peripheral blood mononuclear cells are seeded into the coated culture flask, the activation culture medium is added to the coated culture flask every 2 to 4 days.

[0086] In some embodiments of the present invention, in step 2), the cell resuscitation includes: washing and resuspending frozen peripheral blood mononuclear cells, and then inoculating them into the coated culture flask for culture.

[0087] In some embodiments of the present invention, the seeding density of the frozen peripheral blood mononuclear cells inoculated into the coated culture flask is 1 × 10⁻⁶. 6 ~2×10 6 per mL.

[0088] In some embodiments of the present invention, the second activation replenishment includes: after seeding the frozen peripheral blood mononuclear cells into the coated culture flask, adding the activation culture medium to the coated culture flask every 2-4 days; and,

[0089] In 1) or 2), the culture conditions for activation culture are 36.5–37.5°C and 4.5–5.5% (v / v) CO2.

[0090] In some embodiments of the present invention, the kit is used in the preparation of medicaments for the prevention and / or treatment of EBV-related diseases.

[0091] In some embodiments of the present invention, the disease is a tumor.

[0092] In some embodiments of the present invention, the tumor is nasopharyngeal carcinoma.

[0093] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0094] The reagents and raw materials used in this invention are all commercially available.

[0095] The positive and progressive effects of this invention are as follows:

[0096] The EBV immunogenic peptides of the present invention contain shared epitopes predicted from high-frequency nonsynonymous mutations to have strong HLA binding affinity and broad HLA coverage. mRNA vaccines designed based on the EBV immunogenic peptides of the present invention or combinations thereof, which maximize population coverage, can elicit a strong antigen-specific T-cell response.

[0097] The combined therapy of EBV mRNA vaccine and adoptive NK cell therapy based on the kit of this invention can persistently inhibit or eliminate NPC tumors in humanized mice, synergistically improving the therapeutic effect. Simultaneously, it can enhance the infiltration of human T cells and NK cells in the tumor microenvironment and improve their effector function, and can be used as a treatment for EBV. + A novel and effective strategy for NPCs. Attached Figure Description

[0098] Figure 1 This section describes the prediction of immunogenic EBV epitopes with broad HLA coverage from genomic nsSNVs of cancer-associated EBV strains. (A) Schematic diagram of the prediction process. (B) Heatmap showing the distribution of EBV genomic variations in different tumor types. The right side shows the occurrence frequency, HLA coverage, and exact amino acid substitution information for each mutation in 283 sequencing strains. Purple mutations were selected for vaccine construction due to their predicted strong HLA binding affinity and broad HLA coverage. Meta-information, including tumor type and sex, is plotted at the bottom.

[0099] Figure 2 The human immune system was established in NOG mice to evaluate the immunogenicity and therapeutic potential of mRNA vaccines. Flow cytometry characterization of human CD45 expression in humanized mouse PMBCs at different time points.

[0100] Figure 3 This section describes the expression of the designed mRNA in vitro and in vivo. (A) Template plasmid map used for the production of nEL and EGFP mRNA. The plasmid map was created by SnapGene. (B) Immunoblotting of HEK293 cells transfected with 500 ng nEL mRNA in culture medium and cell lysis buffer. Transfection was performed using Lipofectamine MessengerMAX (ThermoFisher, LMRNA001). Untransfected cell samples were used as controls. (C) 5 μg EGFP mRNA was combined with 4 μL of InstantFECT liposomes and injected intramuscularly. Liposomes were injected alone as a control. In vivo imaging was performed on anesthetized mice 24 hours after injection. (D) Photographs of tumor tissue harvested from the control and vaccine groups.

[0101] Figure 4 The nEL mRNA vaccine induced EBV in humanized mice. + Antigen-specific T-cell immune response in NPCs. (A) Animal experimental protocol. CNE2-EBV cells were subcutaneously implanted into NOG mice transplanted with human PBMCs. One week after tumor implantation, mice were treated with nEL mRNA vaccine (vaccine) or liposome (control) every 3 to 4 days (n = 5-6 per group). Spleen and tumor samples were collected at the end of the experiment for subsequent analysis. (B) Schematic diagram of multi-epitope nEL mRNA molecules. (C) Tumor growth curves of vaccinated and control mice (ordinary two-dimensional ANOVA, ns, no significant difference, P>0.05). (D) Mean tumor weight of vaccinated and control mice (unpaired two-tailed t-test, ****P<0.05). (E and F) Post-vaccination response of vaccinated and control mice to the corresponding epitopes. Representative image of spleen IFN-γ ELISpot detection (E) and quantification of IFN-γ positive spleen cells (F, unpaired two-tailed t-test, ****P<0.0001). (G) IHC staining of human CD4 and CD8 in tumor sections; scale bar 100 μm. All data are expressed as mean ± SEM.

[0102] Figure 5Phenotypic characteristics of expanded NK cells from peripheral blood in vitro. (A) Flow cytometry characteristics of NKG2D, NKp30, PD-1, granzyme B, and perforin expression in expanded NK cells. (B) Mean viability and purity of NK cells, and percentage of expanded NK cells expressing NKG2D, NKp30, PD-1, granzyme B, and perforin in nine separate experiments. (C) Cytotoxicity assays of expanded NK cells against leukemia cell lines K562 and CNE2-EBV (n=3 per group).

[0103] Figure 6 The aim was to establish human immunity in NOG mice to evaluate combination therapy. (A) Quantification of human CD45 expression in humanized mouse PMBCs at different time points. (B) Body weight changes in each group. Body weight changes in a group of non-humanized NOG mice were recorded as a control. Data are expressed as mean ± standard deviation.

[0104] Figure 7 It is the synergistic effect of nEL mRNA vaccine and NK cells that promotes EBV in humanized mice. + Eradication of NPC. (A) Animal experimental protocol. CNE2-EBV cells were subcutaneously implanted into NOG mice transplanted with human PBMCs. One week after tumor inoculation, mice were treated every 3 to 4 days with mRNA vaccine and NK cells (Vac+NK), NK cells alone (NK), or saline and liposomes (control) (n=6 per group). Blood and tumor samples were collected for analysis at the end of the experiment. (B) Tumor growth curves for each group (conventional two-way ANOVA and Tukey multiple comparison test, ***P<0.001, ****P<0.0001). (C) Photographs of representative subcutaneous tumors and harvested tumor tissue from each group. Cross symbols indicate tumors eradicated by combined treatment. (D) Mean tumor weight for each group (conventional one-way ANOVA and Tukey multiple comparison test, *P<0.05, ****P<0.0001). (E and F) HE staining (E) and IHC staining (F) of tumor sections from each group. Scale bar: 500 μm (top), 100 μm (bottom, magnified area). All data are expressed as mean ± SEM.

[0105] Figure 8 The combination therapy mobilized EBV-targeting agents in humanized mice. +T-cell and NK-cell immunity in NPCs. (A) Quantification of human IFN-γ (left) and human TNF-α (right) concentrations in the serum of humanized mice in each group (ordinary one-way ANOVA and Tukey multiple comparison test, ns, no significant difference, P>0.05, *P<0.05, ****P<0.0001; data are expressed as mean ± SEM). (B) IHC staining of human IFN-γ and granzyme B in tumor sections of each group. (C) IHC staining of human CD56, CD4, and CD8 in tumor sections of each group. Scale bar, 200 μm. Detailed Implementation

[0106] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0107] Example 1: mRNA vaccine against EBV in humanized mice + The function of NPCs

[0108] In this invention, to design an mRNA vaccine with maximized population coverage, the inventors conducted a large-scale analysis of genomic mutations in cancer-associated EBV strains and predicted shared epitopes that may have strong HLA binding affinity and broad HLA coverage for constructing the mRNA vaccine. To evaluate the immunogenicity and therapeutic potential of the mRNA vaccine, the inventors established a humanized mouse EBV vaccine... + NPC models are used to test the efficacy of mRNA vaccines.

[0109] The specific experimental steps are as follows:

[0110] A. Predicting EBV epitopes

[0111] EBV genome sequencing data were collected from BioProjects (PRJNA522388 and PRJEB24495), and sequence reads were mapped to the EBV reference genome (NC_007605) using BWA-mem. Genomic variants were invoked using HaplotypeCaller. After variant quality filtering, mutant peptides were submitted to NetMHCpan-4.1 and NetMHCIIpan-4.0 for binding affinity prediction of different HLA allele classes. Population coverage was calculated based on HLA alleles with strong binding affinity for each predicted EBV epitope.

[0112] B. Construction and management of mRNA vaccines

[0113] Based on the previously reported DNA backbone (cited in: Kuwentrai C, Yu J, Rong L, Zhang BZ, Hu YF, Gong HR, et al. Intradermal delivery of receptor-binding domain of SARS-CoV-2 spike protein with dissolvable microneedles to induce humoral and cellular responses in mice. Bioeng Transl Med 2021; 6:e10202.), the inventors developed a plasmid template called pRV1 for mRNA synthesis. In short, the Xenopus β-globin 5'UTR and VEEF 3'UTR were inserted into the pUC57 plasmid (Addgene: Vector Database-pUC57), and a 130 bp polyadenine (A) tail was cloned after the 3'UTR. nEL represents the coding sequence of ENBA1, LMP1, LMP2A, and LMP2B epitopes linked by (GS)5, or EGFP, as subcloned into pRV1. Each epitope was encoded as a 30-amino acid peptide with a mutation at amino acid position 15. The two coding sequences were fused at the 5' end to an Igk signal peptide and at the 3' end to an MHCI transport domain (MITD) with a 6×His tag, yielding pRV1-nEL and pRV1-EGFP. The plasmids were linearized by SapI digestion and transcribed in vitro using the HiScribe T7 mRNA kit (NEB, E2040), introducing pseudouridine (Ψ) and / or N1-methylpseudouridine (m1Ψ) modifications. The mRNA products were purified by lithium chloride precipitation and 5' capped using a vaccinia virus capping system (NEB, M2080). The capped mRNA products were stored at -80°C until use.

[0114] C. Cell lines and cell culture

[0115] K562 and 293T cell lines were purchased from ATCC. Human EBV + The NPC cell line CNE2-EBV was generously provided by Professor Kwan Sin-yuen of the University of Hong Kong. All cell lines were certified under the material transfer protocol and tested for mycoplasma contamination. All cells were cultured in RPMI 1640 medium (Gibco, 11875093) containing 10% FBS (Gibco, 10099141), 100 U / ml penicillin, and 100 mg / ml streptomycin (Gibco, 15140122) at 37°C in a humidified incubator with 5% CO2.

[0116] D. Constructing humanized mouse EBV + NPC Model

[0117] NOD.Cg-Prkdc scid Il2rg tm1Sug JicCrl (NOG) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were conducted in a specific pathogen-free (SPF) laboratory environment at Shenzhen Lingfu Top Biotechnology Co., Ltd., under the guidance of the Institutional Animal Care and Use Committee, approval number TOP-IACUC-2023-0267. To establish humanized mice, human peripheral blood mononuclear cells (PBMCs) were purified from leukocyte layers of healthy donors using Ficoll–Hypaque (Biochrom, L6115) density gradient centrifugation. The isolated PBMCs were centrifuged at 6 × 10⁶ cells per mouse. 6 The dose was administered intravenously. Informed consent was obtained from the donors, and the study was approved by the BGI Institutional Review Board, approval number BGI-IRB 22046-T1. This was to establish humanized mouse EBV. + NPC model, 5×10 6 CNE2-EBV cells were subcutaneously injected into the right abdomen of mice. The tumors grew to an average size of 50-100 mm before treatment. 3 Tumor volume is calculated by measuring the length and width of each tumor using calipers, using the formula V = length × width. 2 ×0.5. On days 0, 14, 21 and 28 of mouse modeling, 100 μL of anticoagulated blood was collected from the orbital cavity and stored at 4℃. The human CD45 (BD, 555483) clustering was detected by flow cytometry to verify the reconstruction of the humanized mouse immune system.

[0118] E. humanized mouse EBV + NPC infusion of mRNA vaccine

[0119] The mRNA vaccine is prepared by mixing nEL mRNA with InstantFECT liposomes donated by PGR-Solutions. It is administered every 3-4 days for a total of three treatments. For each therapeutic vaccination, 10 μg of nEL mRNA is diluted with serum-free medium and mixed with 20 μL of 0.9% saline and 4 μL of InstantFECT, for a total volume of 100 μL. The mixture is then incubated with EBV. +Multiple subcutaneous injections were administered near the tumor site in NPC model mice, with an average injection rate at each site. Mice were sacrificed and dissected at the experimental endpoint, and labeled according to group. Tumor tissue (if available) was collected to measure tumor size and weight; fresh spleen was harvested, ground into single cells, and subjected to an ELISApot assay for IFN-γ; peripheral blood samples were collected from mice for quantitative detection of human IFN-γ and TNF-α; embedded paraffin sections were used for immunohistochemical staining to detect human CD4. + T cells, CD8 + T cell tissue infiltration.

[0120] The specific experimental results are as follows:

[0121] Statistical analysis was performed on the experimental data. Statistical significance was determined using GraphPad Prism 8.0 with standard two-way ANOVA, unpaired two-tailed t-test, standard two-way ANOVA, or as otherwise described in the legend. P-values ​​are represented in the graph as follows: ns: not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0122] 1. Predicting EBV epitopes with broad HLA coverage from genomic mutations in cancer-associated EBV strains

[0123] Genomic mutations present in tumors not only reflect the characteristics of tumor evolution but also serve as valuable targets for immunotherapy. Neoantigens generated by tumor-specific nonsynonymous single nucleotide variants (nsSNVs) or small-scale genomic insertions and deletions (indels) have been shown to be immunogenic for the development of effective cancer vaccines. Since viruses can generate a large number of genomic mutations during their evolution, the inventors proposed that specific nsSNVs or indels occurring in the genome of cancer-associated EBV may encode immunogenic epitopes suitable for constructing vaccines against EBV-associated malignancies. Therefore, using the non-cancer-derived B95-8 EBV strain as a reference, the inventors analyzed the genomic variations of 283 cancer-associated EBV strains and predicted the potential immunogenic epitopes encoded by these mutations. Figure 1 (A).

[0124] Therefore, the inventors discovered high-frequency nsSNVs in EBV oncogenes, including EBNA1, LMP1, LMP2A, and LMP2B. Immune responses to specific epitopes are typically limited to individuals possessing HLA molecules capable of binding to and expressing that specific epitope. To design a T-cell epitope-based EBV vaccine effective against diverse populations, the inventors further predicted immunogenic EBV epitopes with broad HLA coverage among these mutations. To this end, the inventors predicted binding affinity across different HLA allele classes for variant peptides encoded by nsSNVs. The calculated HLA coverage for each mutation is shown in the results. Figure 1 In the right two columns of B. Finally, six HLAI and HLAII allele epitopes predicted to have broad population coverage were selected for subsequent mRNA vaccine construction (Table 1). Of the six selected mutations, five were located in previously identified T-cell epitopes of EBNA1 and LMP2. One mutation in LMP1, Cys84Gly, corresponds to a novel epitope that has not yet been reported.

[0125] Table 1. List of EBV epitopes predicted by cancer-associated EBV strains

[0126]

[0127]

[0128] Table 2. Sequence list of predicted EBV amino acid epitope mutation sites in cancer-associated EBV strains.

[0129]

[0130] Table 3. List of normal EBV amino acid epitope sequences predicted from cancer-associated EBV strains.

[0131]

[0132] Table 4. Sequence list of predicted EBV nucleotide epitope mutation sites in cancer-associated EBV strains.

[0133]

[0134]

[0135] Table 5. List of normal EBV nucleotide epitope sequences predicted from cancer-associated EBV strains.

[0136]

[0137]

[0138] Table 6. Sequence List of mRNA Vaccine-Related Elements

[0139]

[0140] Full-length sequence of the multi-epitope EBV mRNA vaccine (SEQ ID NO:55):

[0141]

[0142] 2. mRNA vaccine induces antigen-specific T cell responses against EBV+NPC in humanized mice.

[0143] The human immune system was reconstructed in a mouse model transplanted with human PBMCs, thus providing a useful tool for evaluating cancer vaccines and immunotherapies. To assess the immunogenicity and therapeutic potential of the predicted human HLA-restricted epitopes, the inventors synthesized a multi-epitope EBV mRNA vaccine and tested it in humanized mouse EBV... + Its efficacy was tested in an NPC model. This model was established by inoculating undifferentiated CNE2-EBV NPC cells into PBMC humanized mice. Figure 4 A and Figure 2 Synthetic mRNA vaccines, called nEL vaccines, consist of RNA molecules encoding all six epitopes, which are linked by non-immunogenic glycine / serine linkers. Figure 3 A and Figure 4 (B). When transfected into HEK-293T cells, the mRNA expression product nEL was successfully detected in the culture medium and cell lysis buffer. Figure 3 (B). In vivo imaging of mice via intramuscular injection of EGFP-encoded mRNA in a liposome complex also demonstrated the in vivo transfection and expression capabilities of the designed mRNA. Figure 3 (C).

[0144] EBV in repeated nEL vaccinations + In NPC mice, significant tumor growth inhibition was observed only on days 24 and 27. Figure 4 C and Figure 3 Consistently, at the end of the experiment, the weight of tumor tissue collected from the vaccine group was significantly lower than that from the control group (D). Figure 4 To further investigate whether the vaccine could elicit an antigen-specific T-cell response, the inventors tested the responsiveness of two groups of collected spleen cells to six epitopes using IFN-γELISpot. The results showed that mice vaccinated with the nEL vaccine produced T-cell responses to nearly 100% of the predicted epitopes, while control mice showed almost no such response. Figure 4 (E and F). Furthermore, human CD4 in tumor lesions of mice vaccinated with nEL. + and CD8 + T cell infiltration was significantly higher than in the control group. Figure 4 (G). In summary, the above data indicate that the nEL vaccine elicited a strong T-cell response to each predicted epitope and remodeled the tumor microenvironment, although in controlling humanized mouse EBV + The model showed limited effectiveness in tumor growth within the NPC model.

[0145] Example 2: mRNA vaccine combined with NK cell adoptive therapy for EBV in humanized mice + The function of NPCs

[0146] In this invention, the multi-epitope mRNA vaccine constructed by the inventors can elicit a strong antigen-specific T-cell response, but in PBMC humanized mice with EBV... + In the NPC model, controlling tumor growth showed moderate efficacy. To further investigate effective multimodal therapy, the inventors developed an optimized NK cell culture process, using in vitro expanded human NK cells with high purity and cytotoxicity as a combination therapy, namely, mRNA vaccine combined with NK cell adoptive therapy on humanized mouse EBV. + The function of NPCs.

[0147] The specific experimental steps are as follows:

[0148] A. Constructing humanized mouse EBV + NPC Model

[0149] The same steps as in Example 1, D.

[0150] B. Expansion and detection of NK cells in vitro

[0151] (1) Expansion of NK cells in vitro

[0152] NK cells were prepared from peripheral blood mononuclear cells (PBMCs) of healthy donors and expanded using the inventor's optimized NK cell culture protocol. Culture flasks were pre-coated with 5 μg / mL CD16 antibody (T&LBiotechnology, TL-201) and 25 μg / mL RetroNectin (Takara, T110A). Then, they were cultured at a rate of 1 × 10⁻⁶ cells / mL. 6PBMCs at a concentration of [cells / mL] were seeded into coated culture flasks, 40 mL of serum-free medium was added, and the flasks were incubated at 37°C and 5% CO2. The activation medium consisted of X-VIVO 10 serum-free medium (Lonza, 04-380Q) supplemented with 1000 IU / mL human IL-2 (Beijing Sihuan, S20040008), 10 ng / mL human IL-15 (Med Chem Express, HY-P7371), 0.1 μmol / mL GSK3βi-TWS119 (Med Chem Express, HY-10590), and 10% plasma substitute (Jia Ke, X990206). The activation medium was replaced on day 3. To initiate NK cell expansion, NK cells were transferred to aerated culture bags (Nipro, NCB-22) on days 7-9 and cultured in expansion medium for up to 21 days. The amplification medium consisted of X-VIVO 10 serum-free medium supplemented with 1000 IU / mL human IL-2 and 1% plasma substitute. Total cell number and cell viability were calculated using AO / PI (Countstar, 1250T) staining. NK cell purity was calculated on day 21 by quantifying CD56 / CD16+CD3-NK cells.

[0153] (2) Flow cytometry detection of NK cells

[0154] NK cells were identified by surface staining with antibodies against human CD3 (BD, 552852), CD56 (BD, 555518), and CD16 (BD, 561304). For phenotypic analysis of NK cell activation, expanded NK cells were stained with antibodies against human NKG2D (BD, 561815) and NKp30 (BD, 558407). For analysis of NK cell effector function, NK cells were pre-incubated with Brefeldin A (BD, 555029) for 5 hours, followed by permeabilization and fixation using BD Cytofix / Cytoperm (BD, 554722) according to the manufacturer's instructions. After fixation and permeabilization, intracellular cytokine staining was performed using antibodies against human granzyme B (BD, 561142) and perforin (BD, 567722). All samples were collected using a NovoCyte Advanteon flow cytometry system.

[0155] (3) NK cell toxicity detection

[0156] NK cells were respectively compared with 1×10 4 K562 and 1×10 4CNE2-EBV cells were co-cultured as target cells at different effector / target ratios (5:1, 10:1, 20:1, and 50:1). The effector / target cell mixtures were centrifuged at 100 g for 2 minutes in 96-well plates and then incubated at 37°C for 4–6 hours. The culture medium was subsequently analyzed using an LDH cytotoxicity assay kit (Beyotime, C0017). NK-specific killing percentage (%) = 100 × (OD value of apoptotic cells in co-culture - OD value of target cells - OD value of effector cells) / (OD value of apoptotic cells - OD value of target cells).

[0157] C. Humanized mouse EBV + NPC infusion of mRNA vaccine and NK cells

[0158] (1) Humanized mouse EBV + NPC drug administration

[0159] EBV in humanized mice + On days 7, 10, and 14 after the establishment of the NPC model, participants were administered mRNA vaccine intramuscularly or NK cell preparation intravenously according to their experimental groups: a control group, an NK cell group, and a combination therapy group. The control group received an infusion of physiological saline containing 1% human serum albumin and an intramuscular injection of liposomes; the NK cell group received an infusion of NK cell preparation; and the combination therapy group received an intramuscular injection of mRNA vaccine and an infusion of NK cells. For mRNA vaccination, 10 μg of nEL mRNA was diluted with serum-free medium and mixed with 20 μL of 0.9% physiological saline and 4 μL of InstantFECT. The mixture was then incubated with EBV. + Multiple subcutaneous injections were administered near the tumor site in NPC model mice, with a total volume of 100 μL. For NK cell therapy, NK cells were counted and resuspended in physiological saline containing 1% human serum albumin (Hualan Biological, S10950009), followed by 1.5 × 10⁻⁶ cells. 7 The NK cell preparation was administered to mice via intravenous injection at a volume of 100 μL.

[0160] (2) Enzyme-linked immunosorbent assay

[0161] Human IFN-γ and TNF-α in mouse peripheral blood were quantitatively detected using an enzyme-linked immunosorbent assay (ELISA) kit (MEIMIAN, 0033H1 and 0122H1) according to the manufacturer's instructions. Briefly, serially diluted mouse serum was added to the wells of a plate and incubated at 37°C for 1 hour. The plate was washed six times with PBS containing 0.05% Tween-20 and incubated at 37°C for 1 hour with HRP-bound goat anti-mouse antibody. Colorimetric development was performed using TMB solution, and absorbance was measured at 450 nm using an ELISA reader. Pre-immunization mouse serum samples were used as a negative control.

[0162] (3) Human IFN-γ ELISpot detection

[0163] According to the manufacturer's instructions, cytokine-specific enzyme-linked immunospot (ELISpot) kits (Mabtech, 3420-2HPW) were used to analyze IFN-γ-producing human spleen cells from vaccinated or control mice. Briefly, spleen cells were analyzed at 1 × 10⁻⁶. 5 Cells were seeded at a concentration of [number] cells / well and stimulated for 20 hours at 37°C with a different synthetic peptide (GL Biochem Shanghai Ltd.) at 0.2 μg / ml. Spleen cells not stimulated with synthetic peptides served as a negative control. Cells were washed and incubated at 37°C for 1 hour with biotinylated anti-IFN-γ monoclonal antibody, followed by incubation at 37°C for another 1 hour with streptavidin-HRP conjugate. The plates were then washed and developed with BCIP / NBT substrate for 20 minutes. Spots were recorded and counted using an immunospot analyzer.

[0164] (4) Immunostaining and histology

[0165] Tumor tissue was fixed in 4% PFA solution (Servicebio, G1101) for 24 hours, then dehydrated with graded ethanol at 4°C until the paraffin embedding procedure (Leica, EG1160). Paraffin sections (10 mm) were dewaxed in xylene and rehydrated with graded ethanol and ddH2O. For H&E staining, sections were stained with Harris hematoxylin and 2% eosin (Servicebio, G1005). For immunostaining, antigen retrieval was performed, followed by blocking with 10% goat serum (Solarbio, SL038) for 1 hour, then incubating overnight at 4°C. Major antibodies against CD56 (1:200, Affinity, DF7832), granzyme B (1:200, Affinity, AF0175), perforin (1:200, Affinity, DF6004), CD4 (1:100, Abcam, DF16080), CD8 (1:100, Abcam, ab17147), IFN-γ (1:100, Affinity, DF6045), and EBNA1 (1:100, Santa Cruz, 81581) were used. Finally, sections were incubated at 37°C for 30 minutes, visualized with the corresponding HRP-labeled secondary antibody using a DAB staining kit (Servicebio, G1212), and subsequently counterstained with hematoxylin (Servicebio, G1004). The images were taken using a Nikon Eclipse-E100 optical microscope.

[0166] The specific experimental results are as follows:

[0167] Statistical analysis was performed on the experimental data. Statistical significance was determined using GraphPad Prism 8.0 with standard two-way ANOVA, unpaired two-tailed t-test, standard two-way ANOVA, or as otherwise described in the legend. P-values ​​are represented in the graph as follows: ns: not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0168] 1. Producing high-quality human NK cells using an optimized amplification system.

[0169] To ensure a continuous production of qualified human NK cells for combination therapy with mRNA vaccines, the inventors developed an optimized NK cell expansion protocol, including the initial population of PBMCs, activation, and proliferation time windows. The inventors further examined the effector phenotypes of the expanded NK cells in more detail using flow cytometry, such as... Figure 5 As shown in Figure A, expression of NK cell activation receptors NKG2D and NKp30 was observed in over 90% of the expanded cells, while expression of the immune checkpoint PD1 was absent. Simultaneously, most NK cells (over 90%) expressed granzyme B and perforin, indicating a cytotoxic phenotype (see Figure A). Figure 5 (B). The optimized NK amplification method was replicated in nine independent experiments, yielding an average of 3.3 × 10^9 CD3. - CD56 + NK cells, with a survival rate exceeding 90% and a purity exceeding 90%. Figure 5 (B). Notably, the expanded NK cells exhibited strong cytotoxicity (>60%) against K562 and CNE2-EBV cells at E / T ratios of 20:1 and 50:1, indicating that the expanded cells were functionally normal. Figure 5 (C). In summary, our optimized NK cell expansion system can stably produce cytotoxic NK cells with high survival rates and purity.

[0170] 2. Combining mRNA vaccines and NK cell therapy to promote EBV levels in humanized mice + NPC eradication

[0171] The inventors evaluated the combination therapy in humanized mice with EBV by administering nEL vaccine and intravenous NK cells multiple times. + Tumor control capabilities in NPC models ( Figure 7 (A). By detecting persistent human CD45 in mouse peripheral blood. + Cells confirmed the successful implantation of the human immune system in a tumor model. Figure 6 (A). Compared with the control group, both combination therapy and NK cell therapy alone significantly inhibited tumor growth (A). Figure 7(B). Importantly, the combination therapy demonstrated a stronger antitumor effect and effectively eradicated established NPCs in humanized mice. Complete tumor regression was observed in 50% (3 out of 6) of the mice treated with the combination therapy. Figure 7 (C). Furthermore, the mean weight of residual tumors in the combination therapy group was significantly lower than in the other groups (C). Figure 7 (D). During the 24-day monitoring period following initial treatment, the combination therapy was well tolerated and demonstrated a strong safety profile, with no abnormal symptoms or weight loss observed in any of the treated mice. Figure 6 (B).

[0172] To further characterize the tumor-reducing effect of the combination therapy, the inventors performed H&E staining on tumor sections obtained from all groups. Histological image analysis showed that mice receiving the combination therapy had a lower tumor burden than mice in the control group or the NK cell therapy group. Figure 7 (E, above image). A magnified view shows loose cancerous tissue, resembling liquefactive necrosis, with extensive infiltration of immune cells in tumor sections from mice treated with NK cells and combination therapy. Figure 7 (See figure below). Simultaneously, the inventors examined changes in EBV oncogene expression in NPCs after treatment. Histological analysis of tumor sections immunostained with a specific anti-EBNA1 antibody showed widespread expression of this oncogene in control mice. In contrast, large areas of tumors treated with NK cells or combination therapy showed significantly reduced or undetectable EBNA1 levels, indicating reduced EBNA1 replication or elimination of tumor cells after treatment. Figure 7 (F).

[0173] 3. The underlying immune response induced by the combined mRNA vaccine and NK cell therapy

[0174] To further understand the underlying immune response driving the observed therapeutic effect, the inventors collected serum samples from all mice and detected higher levels of systemic human tumor necrosis factor-α (hTNF-α) and interferon-γ (hIFN-γ) in mice treated with the combination therapy compared to the control group and the NK cell therapy group. Figure 8 (A) This indicates that the combination therapy enhances the antitumor response through synergistic effects. Considering the extensive immune cell infiltration observed in H&E-stained tumor sections of treated mice, the inventors further investigated the immune response in the tumor microenvironment using IHC staining. Figure 8 As shown in Figure B, the levels of effector cytokines human granzyme B and IFN-γ were significantly elevated in tumors treated with NK cells and combination therapy, indicating the induction of strong cytotoxicity in the tumor microenvironment. The inventors also observed substantial infiltration of human NK cells in the aforementioned treated tumors by staining human CD56. Figure 8 (C, above figure). Further analysis of T cell-mediated responses using immunostaining of human CD4 and CD8 showed that, compared with tumors treated with NK cells alone or the control group, tumors treated with the combination therapy had significantly higher T cell levels, particularly infiltrating CD4 cells. + T cells ( Figure 8 (C, middle and bottom figures). In summary, the above results indicate that combined mRNA vaccine and NK cell therapy can simultaneously induce EBV-targeting responses in humanized mice. + The NPC's potent T-cell and NK-cell mediated immune response.

Claims

1. An EBV immunogenic peptide fragment, characterized in that, The EBV immunogenic peptide comprises any of the amino acid sequences shown in SEQ ID NO:7-12 and SEQ ID NO:21; Preferably, the EBV immunogenic peptide comprises an amino acid sequence as shown in any of SEQ ID NO:1-6 and SEQ ID NO:

15.

2. A combination of EBV immunogenic peptides, characterized in that, The combination comprises two or more selected from the group consisting of EBV immunogenic peptides containing amino acid sequences as shown in SEQ ID NO:7-12 and SEQ ID NO:19-24; Preferably, the combination comprises two or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-6 and SEQ ID NO:13-18.

3. The combination of claim 2, wherein, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:7, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:8–12 and SEQ ID NO:19–24; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:8, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:9–12, and SEQ ID NO:19–24; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:9, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7–8, SEQ ID NO:10–12, and SEQ ID NO:19–24; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:10, and one or more EBV immunogenic peptides selected from the group consisting of SEQ ID NO:7–9, SEQ ID NO:11–12, and SEQ ID NO:19–24; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:11, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7–10, SEQ ID NO:12, and SEQ ID NO:19–24; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:12, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7–11 and SEQ ID NO:19–24; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:21, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-12, SEQ ID NO:19-20 and SEQ ID NO:22-24; Preferably, the combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:1, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:2–6 and SEQ ID NO:13–18; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:2, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:3-6, and SEQ ID NO:13-18; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:3, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1–2, SEQ ID NO:4–6, and SEQ ID NO:13–18; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:4, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1–3, SEQ ID NO:5–6, and SEQ ID NO:13–18; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:5, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1–4, SEQ ID NO:6, and SEQ ID NO:13–18; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:6, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1–5 and SEQ ID NO:13–18; or, The combination comprises: an EBV immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:15, and one or more selected from the group consisting of EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-6, SEQ ID NO:13-14 and SEQ ID NO:16-18.

4. The combination of claim 2 or 3, wherein, The combination comprises: an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:7 or 19; an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:8 or 20; an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:9 or 21; an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:10 or 22; an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:11 or 23; and an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:12 or 24. Preferably, the combination comprises: an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:1 or 13, an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:2 or 14, an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:3 or 15, an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:4 or 16, an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:5 or 17, and an EBV immunogenic peptide containing the amino acid sequence shown in SEQ ID NO:6 or 18.

5. The combination according to any one of claims 2 to 4, wherein The combination comprises two or more, preferably six, EBV immunogenic peptides selected from the amino acid sequences shown in SEQ ID NO: 7-12. Preferably, the combination comprises two or more, preferably six, EBV immunogenic peptides selected from the amino acid sequences shown in SEQ ID NO:1 to 6. More preferably, the EBV immunogenic peptides in the combination are connected by a linker, such as the (GS)5 linker; and / or, the combination comprises, from the N-terminus to the C-terminus, six EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:7-12, preferably comprising, from the N-terminus to the C-terminus, six EBV immunogenic peptides comprising the amino acid sequences shown in SEQ ID NO:1-6.

6. An isolated nucleic acid, comprising, The nucleic acid comprises a nucleotide sequence encoding a combination of EBV immunogenic peptides as described in claim 1 or as described in any one of claims 2 to 5.

7. The nucleic acid of claim 6, wherein The EBV immunogenic peptide or combination of the EBV immunogenic peptides comprises one or more nucleotide sequences selected from SEQ ID NO:31-36 and SEQ ID NO:43-48, preferably comprising one or more nucleotide sequences selected from SEQ ID NO:25-30 and SEQ ID NO:37-42; and / or, the nucleic acid further comprises a nucleotide sequence encoding one or more of the 5'UTR, signal peptide, MHCI transport domain, 3'UTR, and polyA tail; Preferably, the nucleic acid satisfies one or more of the following conditions: (1) The 5'UTR is the Xenopus β-globin 5'UTR, and the preferred nucleotide sequence is shown in SEQ ID NO:52; (2) The signal peptide is an IgK signal peptide, preferably with a nucleotide sequence as shown in SEQ ID NO:50; (3) The nucleotide sequence of the MHCI transport domain is shown in SEQ ID NO:54, preferably a nucleotide sequence encoding a 6×His tag; (4) The 3'UTR is the VEEF 3'UTR, preferably with the nucleotide sequence shown in SEQ ID NO:53, and more preferably with two copies of the 3'UTR; (5) The polyA tail contains 120 to 140 adenine nucleotides, preferably 130; (6) The nucleic acid comprises, from the 5' end to the 3' end, the following in sequence: 5'UTR, signal peptide, the EBV immunogenic peptide or a combination of the EBV immunogenic peptides, MHCI transport domain, 3'UTR and polyA tail; (7) One or both ends of the EBV immunogenic peptide or a combination of the EBV immunogenic peptides further comprise a nucleotide sequence encoding a linker, such as the (GS)5 linker; and, (8) The combination of EBV immunogenic peptides contains six nucleotide sequences as shown in SEQ ID NO:31-36 from the 5' end to the 3' end, preferably six nucleotide sequences as shown in SEQ ID NO:25-30.

8. The nucleic acid of claim 6 or 7, wherein The nucleic acid is DNA, and preferably also contains a promoter; Preferably, the promoter is a T7 promoter, with a preferred sequence as shown in SEQ ID NO:

49.

9. The nucleic acid of claim 6 or 7, wherein The nucleic acid is RNA, preferably mRNA; Preferably, the nucleic acid further comprises a 5' cap structure; and / or, the nucleic acid further comprises modifications, such as pseudouridine (Ψ) and / or N1-methylpseudouridine (m1Ψ) modifications; More preferably, the nucleic acid comprises a sequence as shown in SEQ ID NO:

55.

10. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 8; Preferably, the backbone of the recombinant expression vector is pUC57 or pRV1.

11. A transformant characterized in that, The transformant comprises the nucleic acid as described in any one of claims 6 to 9 or the recombinant expression vector as described in claim 10; the transformant is a non-animal or non-plant variety; Preferably, the host cell of the transformant is a eukaryotic cell or a prokaryotic cell; More preferably, the prokaryotic cell is a bacterium, such as Escherichia coli.

12. A lipid nanoparticle characterized in that, The lipid nanoparticles comprise the nucleic acid as described in claim 9.

13. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises one or more selected from the EBV immunogenic peptides of claim 1, combinations of any one of claims 2 to 5, nucleic acids of any one of claims 6 to 9, and lipid nanoparticles of claim 12, as well as a pharmaceutically acceptable carrier.

14. Use of one or more of the following in the preparation of a medicament for the prevention and / or treatment of EBV-related diseases: the EBV immunogenic peptide as described in claim 1, the combination of any one of claims 2 to 5, the nucleic acid as described in any one of claims 6 to 9, the recombinant expression vector as described in claim 10, the transformant as described in claim 11, the lipid nanoparticle as described in claim 12, and the pharmaceutical composition as described in claim 13; Preferably, the drug is a vaccine, such as an mRNA vaccine; and / or, the disease is a tumor, such as nasopharyngeal carcinoma.

15. A kit comprising, The kit includes kit A and kit B. Kit A contains one or more selected from the EBV immunogenic peptides as described in claim 1, combinations as described in any one of claims 2 to 5, nucleic acids as described in any one of claims 6 to 9, lipid nanoparticles as described in claim 12, and pharmaceutical compositions as described in claim 13. Kit B contains other drugs for the prevention and / or treatment of EBV-related diseases, such as cell therapy preparations. Preferably, the cell therapy formulation comprises one or more selected from TCR-T cells, CAR-T cells and NK cells, and preferably comprises NK cells.

16. The kit of claim 15, wherein The method for preparing NK cells includes culturing them in an activation medium, which includes a first cytokine, a glycogen synthase kinase inhibitor, and NK basal medium. Preferably, the activation medium meets one or more of the following conditions: The first cytokine includes one or more of IL-2, IL-15, IL-21, or IL-18; The glycogen synthase kinase inhibitors include GSK3βi; The NK basal culture medium includes NK serum-free culture medium; and, The activated culture medium also includes a plasma substitute; More preferably, the activation medium meets one or more of the following conditions: The concentration of IL-2 is 500–2000 IU / mL; and / or the concentration of IL-15 is 5–15 ng / mL; The GSK3βi includes one or more of TWS119, LY2090314, or CHIR-98014; and / or, the concentration of the glycogen synthase kinase inhibitor is 0.1–0.5 μmol / mL; and, The volume of the plasma substitute accounts for 0.09% to 9% of the total volume of the activation culture medium.

17. The kit of claim 16, wherein The method for preparing the NK cells includes: (1) The culture flask is coated with a coating solution to obtain a coated culture flask; the coating solution includes antibody, recombinant human fibronectin and buffer solution; (2) Peripheral blood mononuclear cells were activated and cultured in the coated culture flask using the activated culture medium to obtain activated cells; (3) The cell suspension in the coated culture flask is transferred to a culture bag, and the activated cells are proliferated and cultured in the culture bag using the activation medium and the proliferation medium to obtain the NK cells; the proliferation medium includes a second cytokine and the NK basal medium. Preferably, the method for preparing NK cells satisfies one or more of the following conditions: The antibody includes one or more of anti-CD16 antibody, anti-CD337 antibody, or anti-CD314; preferably, the concentration of the anti-CD16 antibody is 1–10 μg / mL. The recombinant human fibronectin includes one or more of RetroNectin, FibroNectin, or NbroNectin; preferably, the concentration of RetroNectin is 25–50 μg / mL. The buffer solution includes one or more of DPBS, PBS, or HBSS; The second cytokine includes one or more of IL-2, IL-21, or IL-18, wherein the concentration of IL-2 is preferably 500–2000 IU / mL; and / or, the NK basal medium includes NK serum-free medium; and, The proliferation medium also includes a plasma substitute.

18. The kit of claim 17, wherein The activation culture includes any of the following methods: 1) cell isolation and first activation replenishment; or 2) cell resuscitation and second activation replenishment; Preferably, the activation culture satisfies one or more of the following conditions: In step 1), the cell separation includes: separating lymphocytes using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing and resuspending them, and then inoculating them into the coated culture flask for culture; preferably, the inoculation density of the peripheral blood mononuclear cells in the coated culture flask is 1×10⁻⁶. 6 ~2×10 6 More preferably, the first activation replenishment solution includes: after seeding the peripheral blood mononuclear cells into the coated culture flask, adding the activation culture medium to the coated culture flask every 2 to 4 days; In step 2), the cell resuscitation includes: washing and resuspending frozen peripheral blood mononuclear cells, and then inoculating them into the coated culture flask for culture; preferably, the inoculation density of the frozen peripheral blood mononuclear cells in the coated culture flask is 1×10⁻⁶. 6 ~2×10 6 Cells / mL; more preferably, the second activation replenishment solution comprises: after seeding the frozen peripheral blood mononuclear cells into the coated culture flask, adding the activation culture medium to the coated culture flask every 2-4 days; and, In 1) or 2), the culture conditions for activation culture are 36.5–37.5°C and 4.5–5.5% (v / v) CO2.

19. The use of the kit as described in any one of claims 15 to 18 in the preparation of medicaments for the prevention and / or treatment of EBV-related diseases; Preferably, the disease is a tumor; More preferably, the tumor is nasopharyngeal carcinoma.

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