FomA-targeted mRNA vaccine and application thereof in immunotherapy of esophageal squamous carcinoma

Abstract

The invention relates to an mRNA (messenger Ribonucleic Acid) vaccine targeting FomA and application of the mRNA vaccine in immunotherapy of esophageal squamous carcinoma in the technical field of tumor immunotherapy. The problems that in the prior art, a specific targeting vaccine for the fusobacterium nucleatum membrane protein FomA lacks, traditional antibiotics can only kill extracellular bacteria and cannot remove intracellular colonized fusobacterium nucleatum, and cellular immunity cannot be effectively activated to remove fusobacterium nucleatum colonized in tumor tissue; the immunotherapy drug resistance of esophageal squamous carcinoma caused by fusobacterium nucleatum infection is difficult to reverse. According to the technical scheme, an mRNA molecule with a fusobacterium nucleatum FomA extracellular domain tandem repeat sequence and a lipid nanoparticle delivery system are coded, an extramembrane segment tandem repeat immune enhancement technology is adopted for design, two extracellular ring sequences are repeated for 2-5 times and connected through a connector sequence, lipid nanoparticles are composed of optimized quaternary lipids, and the lipid nanoparticles are prepared from the quaternary lipids. And a PD-1 monoclonal antibody is combined to activate FomA specific CD8 + T cell immunoreaction, so that comprehensive removal of intracellular and extracellular bacteria and bacteria targeted immune remodeling are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor immunotherapy, and in particular to an mRNA vaccine targeting FomA and its application in immunotherapy of esophageal squamous cell carcinoma. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is a common digestive tract malignancy. Recent studies have shown that the microbial composition of the tumor microenvironment is closely related to tumor development and progression. Fusobacterium nucleatum (Fn), an anaerobic Gram-negative bacterium, has been found to be abnormally enriched in various cancers and is particularly associated with poor prognosis in ESCC.

[0003] Fusobacterium nucleatum can colonize tumor cells, promoting tumor progression and inducing immunotherapy resistance through multiple mechanisms. FomA (Fusobacterium outer membrane protein A) is the major outer membrane protein of Fusobacterium nucleatum. It has a β-barrel transmembrane structure, with its extracellular domain exposed on the bacterial surface, making it an ideal immunotherapy target.

[0004] After searching and analyzing, several related technical solutions have been discovered in the field of targeted therapy of bacteria in the tumor microenvironment. CN117137884A discloses a nanomedicine composition based on bacterial cell membranes. This technology uses a double-layer structure design of bacterial cell membrane-encapsulated liposomes. The drug delivery system contains a drug that kills Fusobacterium nucleatum, which is enriched at the tumor site through the EPR effect. However, this technology mainly relies on passive targeting, which has limited specificity. The bacterial membrane source poses safety risks and does not involve active immune activation mechanisms.

[0005] EP3536706A1 proposes a fusion protein technology for delivering heterologous peptides within outer membrane vesicles. This technology utilizes FhuD2 as a carrier protein, fused to and expressed with heterologous peptide sequences, and utilizes bacterial outer membrane vesicles as a natural adjuvant. However, this technology is limited to the FhuD2 carrier protein and does not target specific membrane proteins of Fusobacterium nucleatum. Furthermore, the outer membrane vesicle preparation process is complex, making batch consistency difficult to control.

[0006] Traditional vaccine technologies, such as protein and peptide vaccines, still face numerous challenges when targeting bacterial membrane proteins. First, the expression and purification processes for these proteins are complex, leading to high costs. Second, the protein components of these vaccines are less stable and require strict storage environments. Furthermore, the immune responses they induce are often weak, typically requiring adjuvants to enhance their effects. Finally, precise regulation of in vivo expression levels and duration is difficult.

[0007] While traditional antibiotics can effectively kill extracellular Fusobacterium nucleatum, they have limited effectiveness against bacteria that have already colonized tumor cells. While anti-anaerobic drugs such as metronidazole have good bactericidal activity against Fusobacterium nucleatum, they primarily target extracellular bacteria and have difficulty penetrating cell membranes to eliminate intracellular colonized bacteria. Furthermore, long-term antibiotic use can easily lead to drug resistance and may disrupt the normal balance of the bacterial flora.

[0008] In contrast, mRNA vaccine technology offers a range of advantages, such as high production efficiency, rapid sequence optimization, and good safety, and has achieved significant success in COVID-19 vaccine development. However, there are currently no reports on the application of mRNA vaccine technology to the key membrane protein FomA of Fusobacterium nucleatum, and this field remains elusive.

[0009] In summary, the existing technologies for the targeted treatment of Fusobacterium nucleatum have the following deficiencies: 1. Lack of specific targeting technology for FomA, a key membrane protein of Fusobacterium nucleatum; 2. Existing drug delivery systems rely primarily on passive sterilization, making it difficult to eliminate bacteria colonized in tumor tissues; 3. Traditional antibiotic treatment can only kill extracellular bacteria and has limited effect on intracellular colonization of Fusobacterium nucleatum; 4. There is a lack of therapeutic approaches that can activate cellular immunity and directly eliminate host cells colonized by bacteria; 5. Relevant technologies have not yet achieved ideal results in overcoming immunotherapy resistance caused by Fusobacterium nucleatum. Summary of the Invention

[0010] Technical issues

[0011] The technical problem to be solved by the present invention is that the existing technology lacks a specific targeted vaccine against the membrane protein FomA of Fusobacterium nucleatum. Traditional antibiotic treatment can only kill extracellular bacteria but cannot eliminate Fusobacterium nucleatum colonized in cells. It cannot effectively activate cellular immunity to eliminate Fusobacterium nucleatum colonized in tumor tissues, and it is difficult to reverse the immunotherapy resistance of esophageal squamous cell carcinoma caused by Fusobacterium nucleatum infection.

[0012] Technical Solution To solve the above technical problems, the present invention provides an mRNA vaccine targeting FomA, comprising an mRNA molecule encoding a tandem repeat sequence of the extracellular domain of Fusobacterium nucleatum FomA and a lipid nanoparticle delivery system.

[0013] The mRNA molecule is prepared by using T7 promoter-driven in vitro transcription technology, and comprises a 5' cap structure, a 5' untranslated region, an open reading frame encoding a tandem repeat sequence of the FomA extracellular domain, a 3' untranslated region and a polyadenylic acid tail.

[0014] The open reading frame encoding the tandem repeat sequence of the FomA extracellular domain is designed using the extracellular segment multimerization immune enhancement technology. Based on the β-barrel structural characteristics of the FomA protein, the extracellular loop region exposed to the bacterial surface is selected for repeated expression to enhance the efficiency of antigen presentation and T cell recognition ability. It contains two types of FomA extracellular domain repeat sequences, the first FomA extracellular domain repeat sequence is 2 to 5 repeats of the amino acid sequence SEQ ID NO: 1, and the second FomA extracellular domain repeat sequence is 2 to 5 repeats of the amino acid sequence SEQ ID NO: 2, and the amino acid sequence is derived from the standard strain of Fusobacterium nucleatum ATCC 25586. The two repeat sequences are sequentially connected by a linker sequence, and the linker sequence is a glycine-serine linker.

[0015] The lipid nanoparticle delivery system is prepared using microfluidics technology, using a bilayer structure to completely encapsulate mRNA molecules in the core. The average particle size measured by dynamic light scattering is 70 to 100 nm, and the encapsulation efficiency measured by fluorescence quantification is 90 to 98%. The lipid nanoparticles are prepared using a microfluidics system, with a controlled aqueous phase to organic phase flow rate ratio of 2:1 to 6:1 and a mixing temperature of 20 to 30°C. The resulting lipid nanoparticles are uniformly spherical with a polydispersity index of less than 0.3.

[0016] The lipid nanoparticles contain an ionizable lipid, phosphatidylcholine, cholesterol, and a PEGylated lipid. The ionizable lipid is DLin-MC3-DMA or ALC-0315, the phosphatidylcholine is DSPC or DOPC, and the PEGylated lipid is DMG-PEG2000 or ALC-0159. The molar ratio of the components is 40-50:10-20:32-40:1.5-5.

[0017] The mRNA sequence purity as determined by capillary electrophoresis is no less than 95%, and the 5' capping efficiency is no less than 90%. After storage at -80°C for 12 months, the lipid nanoparticles maintain an mRNA integrity of no less than 85% and an encapsulation efficiency of no less than 90%.

[0018] The present invention also provides a method for preparing the mRNA vaccine, comprising the following steps: Step 1: designing an mRNA sequence encoding the tandem repeat sequence of the FomA extracellular domain based on the amino acid sequence of the Fusobacterium nucleatum FomA protein, and synthesizing the mRNA sequence by in vitro transcription at 30-40°C for 3-8 hours using T7 RNA polymerase; Step 2: preparing a lipid mixture solution comprising ionizable lipids, phosphatidylcholine, cholesterol and PEGylated lipids, and dissolving them in an organic solvent; Step 3: Mixing the mRNA aqueous solution with the lipid organic solution through a microfluidic system to form lipid nanoparticles; Step 4: Remove the organic solvent and replace the buffer through tangential flow filtration to finally obtain the mRNA vaccine.

[0019] The present invention also provides the use of the mRNA vaccine in the preparation of an immunotherapy drug for Fusobacterium nucleatum-positive esophageal squamous cell carcinoma. The treatment is directed to the determination of the Fusobacterium nucleatum load by qPCR method to be not less than The mRNA vaccine, combined with a PD-1 monoclonal antibody, activated FomA-specific CD8+ T cell immune responses to eliminate Fusobacterium nucleatum-colonized tumor cells.

[0020] The mRNA vaccine is administered at a dose of 1 to 10 mg / kg body weight by intramuscular injection, 1 to 2 times per week for 3 to 8 consecutive doses. The PD-1 monoclonal antibody is administered at a dose of 1 to 10 mg / kg body weight by intravenous injection, 1 to 3 times per week. Compared to the control group using the PD-1 monoclonal antibody alone, the tumor growth inhibition rate after combined treatment increased by 50 to 90%.

[0021] The FomA-specific IgG antibody titer induced by the mRNA vaccine reached 1:1000 to 1:50000 on days 7 to 21 after administration. The FomA-specific CD8+ T cell response peaked on days 7 to 21 after administration, and the immune memory was maintained for at least 4 weeks.

[0022] Beneficial effects The present invention has the following beneficial effects compared to the prior art: 1. The tandem repeats of the extracellular segment have a significant immune-enhancing effect: The design uses 2 to 5 repeats of the FomA extracellular domain. Based on the principles of protein structural biology, the surface-exposed extracellular loop region is selected for repeated expression. Compared with single expression, the titer of FomA-specific IgG antibodies is increased by 5 to 15 times, achieving broad-spectrum targeting.

[0023] 2. Outstanding ability to eliminate intracellular bacteria: By activating the FomA-specific CD8+ T cell immune response through mRNA vaccines, it can directly eliminate host cells colonized by Fusobacterium nucleatum, solving the technical problem that traditional antibiotics can only kill extracellular bacteria but cannot eliminate intracellular colonized bacteria. The bacterial load of tumor tissue is reduced by more than 90%.

[0024] 3. Immunotherapy has an outstanding effect in reversing drug resistance: mRNA vaccine combined with PD-1 monoclonal antibody treatment can increase the tumor growth inhibition rate by 50-90% compared with the use of PD-1 monoclonal antibody alone, effectively reversing the immunotherapy resistance caused by Fusobacterium nucleatum infection.

[0025] 4. Lipid nanoparticles have high delivery efficiency: using optimized lipid composition and preparation process, the encapsulation efficiency reaches 90-98%, and the cell transfection efficiency is 2-5 times higher than that of traditional liposomes. At the same time, it has good stability and safety.

[0026] 5. Advantages over traditional antibiotic treatment: Although traditional antibiotics such as metronidazole have a good bactericidal effect on extracellular Fusobacterium nucleatum, they cannot penetrate the cell membrane to eliminate intracellular bacteria. The mRNA vaccine of the present invention can identify and eliminate host cells colonized by bacteria by activating cellular immune responses, thereby achieving comprehensive elimination of intracellular and extracellular bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic structural diagram of the FomA-mRNA vaccine of the present invention; Figure 2 Schematic diagram of the protein structure of the tandem repeat sequence of the FomA extracellular domain of the present invention; Figure 3 It is a process flow chart of the preparation method of the present invention; Figure 4 This is a comparison chart of the experimental results of the FomA-mRNA vaccine efficacy of the present invention. DETAILED DESCRIPTION

[0028] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] Example 1 The purpose of this example is to construct an immunotherapy-resistant model of esophageal squamous cell carcinoma in mice infected with Fusobacterium nucleatum and colonized with Fusobacterium nucleatum, providing a reliable animal model for subsequent evaluation of vaccine treatment efficacy.

[0030] Mouse esophageal organoid culture and gene editing: Esophageal epithelial cells were isolated from C57BL / 6 mice (6-8 weeks old) and an esophageal organoid culture system was established. The culture medium consisted of Advanced DMEM / F12 basal medium supplemented with B27 supplement, N2 supplement, EGF (50 ng / mL), Noggin (100 ng / mL), R-spondin 1 (500 ng / mL), Wnt3a (100 ng / mL), and Y-27632 (10 μM).

[0031] A mouse model of esophageal cancer with a defined genetic background was constructed using a lentiviral vector system to introduce oncogenic mutations. Target genes included: Trp53 knockout, Pten knockout, Smad4 knockout, KrasG12D overexpression, and Myc overexpression (TPSKM model). Lentiviral infection efficiency, measured by fluorescent labeling, exceeded 85%.

[0032] TPSKM organoid characterization validation: H&E staining revealed that wild-type esophageal organoids possessed a similar structural composition to esophageal tissue, with an outer layer of cells displaying proliferative activity and an inner stratum corneum. Compared to wild-type organoids, TPSKM organoids exhibited an increased proportion of nuclei and darker hematoxylin staining, lacking the normal keratinized structure. Ki67 immunofluorescence staining revealed significantly enhanced proliferative activity in TPSKM organoids.

[0033] Establishment of subcutaneous transplant tumor model: TPSKM organoids ( The cells were subcutaneously transplanted into the right axilla of C57BL / 6 mice. Approximately 14 days later, strong fluorescence signals were detected through in vivo imaging. Pathological and immunohistochemical results revealed that the tumor tissue was esophageal squamous cell carcinoma (ESCC), expressing squamous cell carcinoma markers such as CK5 and CK14.

[0034] Fn infection model construction: Fusobacterium nucleatum ATCC 25586 ( CFU) and TPSKM organoids ( The Fn-infected mouse ESCC model was established. A control group was implanted with only organoid cells.

[0035] Immunotherapy resistance verification: Seven days after tumor establishment, mice were treated with mouse PD-1 monoclonal antibody (Bio X Cell, 100 μg / mouse, twice a week for 3 consecutive weeks). The results showed: Control group (not infected with Fn): Tumor growth was significantly inhibited after PD-1 monoclonal antibody treatment, and the tumor volume on day 21 was reduced by 45% compared with that before treatment; Fn infection group: The tumor continued to progress after PD-1 monoclonal antibody treatment, and on the 21st day, the tumor volume increased by 180% compared with before treatment, showing resistance to immunotherapy.

[0036] Fn colonization verification: Immunofluorescence co-localization of Fn-16S rRNA (red fluorescence) and CK5 (green fluorescence), a marker for esophageal squamous cell carcinoma (ESCC), revealed that Fn colonized in the cytoplasm of CK5-positive cells in the resistant group, suggesting that Fn promotes ESCC progression and immunotherapy resistance through intracellular colonization.

[0037] Through this example, a Fn-infected and colonized mouse ESCC immunotherapy resistance model was successfully constructed. This model can stably reproduce the immunotherapy resistance phenomenon caused by Fn infection, providing a reliable experimental platform for subsequent vaccine treatment effect evaluation.

[0038] Example 2 The purpose of this example is to prepare and characterize an mRNA vaccine with three repeats of the FomA extracellular domain and verify its basic performance parameters.

[0039] Materials and reagents: T7 RNA polymerase (New England Biolabs); 5' cap analog (Trilink); nucleoside triphosphates ATP, CTP, GTP, and UTP (Sigma-Aldrich); ionizable lipid DLin-MC3-DMA; phosphatidylcholine DSPC; cholesterol; PEGylated lipid DMG-PEG2000; and anhydrous ethanol.

[0040] FomA-mRNA sequence design: Based on the amino acid sequence of the FomA protein from the standard strain of Fusobacterium nucleatum ATCC 25586, an mRNA encoding tandem repeats of the FomA extracellular domain was designed. The amino acid sequence of the protein encoded by this mRNA is shown in SEQ ID NO: 4. The first FomA extracellular domain sequence, SEQ ID NO: 1, was repeated three times, and the second FomA extracellular domain sequence, SEQ ID NO: 2, was repeated three times. The two repeats were connected by a glycine-serine linker sequence, GSGGSGGGGSGG.

[0041] In vitro transcription and synthesis of mRNA: like Figure 1 As shown, the overall structure of the mRNA vaccine includes an mRNA molecule and a lipid nanoparticle delivery system. The mRNA molecule is prepared using T7 promoter-driven in vitro transcription technology. T7 RNA polymerase is used to synthesize mRNA by in vitro transcription at 37°C. The reaction system contains: 10U of T7 RNA polymerase, 7.5mM ATP, 7.5mM CTP, 1.5mM GTP, 7.5mM UTP, 6mM 5' cap analog, 1μg DNA template, and reaction buffer to a total volume of 20μL. After reacting at 37°C for 5 hours, DNase I was added to digest the DNA template, and the mRNA was purified by LiCl precipitation.

[0042] Lipid nanoparticle preparation: like Figure 2As shown, the protein structure and linkage of the tandem repeats of the FomA extracellular domain contain two FomA extracellular domain repeats. A lipid mixture containing DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 at a molar ratio of 45:15:37:3, with a total lipid concentration of 15 mM, was prepared and dissolved in anhydrous ethanol. mRNA was dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.5 mg / mL. The mRNA aqueous solution and the lipid ethanol solution were mixed using a microfluidic system with a controlled aqueous to organic phase flow rate ratio of 4:1 and a mixing temperature of 23°C to form lipid nanoparticles. The ethanol was removed by tangential flow filtration and replaced with PBS buffer.

[0043] like Figure 3 As shown, the preparation process includes the following steps: Step 1: designing an mRNA sequence encoding the tandem repeat sequence of the FomA extracellular domain based on the amino acid sequence of the Fusobacterium nucleatum FomA protein, and synthesizing the mRNA sequence by in vitro transcription at 30-40°C for 3-8 hours using T7 RNA polymerase; Step 2: preparing a lipid mixture solution comprising ionizable lipids, phosphatidylcholine, cholesterol and PEGylated lipids, and dissolving them in an organic solvent; Step 3: Mixing the mRNA aqueous solution with the lipid organic solution through a microfluidic system to form lipid nanoparticles; Step 4: Remove the organic solvent and replace the buffer through tangential flow filtration to finally obtain the mRNA vaccine.

[0044] Quality Control Testing: Capillary electrophoresis assayed mRNA purity of 96.8%, and the 5' capping efficiency was 92.3%. Transmission electron microscopy revealed the lipid nanoparticles to be uniformly spherical. Dynamic light scattering analysis revealed an average particle size of 82.18 nm, with a polydispersity index of 0.21. RiboGreen fluorescence quantification demonstrated an encapsulation efficiency of 95.2%.

[0045] Cell transfection experiments: Transfection experiments were conducted using HEK293T cells. The vaccine was diluted to a final concentration of 1 μg / mL, and FomA protein expression was assessed by Western blot 24 hours after transfection. The results showed that the transfection efficiency of the vaccine presented in this invention was 2.3-fold higher than that of traditional liposome transfection.

[0046] This example verifies the feasibility of the preparation process of the FomA extracellular domain 3-repeat mRNA vaccine. The prepared vaccine meets the design requirements and has high purity, high encapsulation efficiency and good transfection efficiency.

[0047] Example 3 The purpose of this example is to verify the therapeutic effect of FomA-mRNA vaccine combined with PD-1 monoclonal antibody in the esophageal squamous cell carcinoma mouse model infected with Fusobacterium nucleatum.

[0048] Animal model construction: The Fn-infected and colonized mouse ESCC immunotherapy resistance model constructed in Example 1 was used. On day 0, Fn-colonized human organoids were subcutaneously inoculated ( ) + Fn bacteria ( CFU).

[0049] Grouping and dosing: Mice were randomly divided into 3 groups, 10 in each group; different vaccines were injected intramuscularly from day 7 to day 21 (twice a week): 1. PD-1 monoclonal antibody group: anti-PD-1 antibody 100 μg / mouse; 2. Combination treatment group: anti-PD-1 antibody 100 μg / animal + FomA-mRNA vaccine 10 μg / animal; 3. FomA antibody control group: anti-PD-1 antibody 100 μg / mouse + anti-FomA antibody 10 mg / kg.

[0050] On day 28, tumor volume, bacterial load, immune response and other indicators were evaluated.

[0051] Comparison of in vivo efficacy: like Figure 4 As shown in A, the experimental results of the treatment effects of each group show the comparison results of the tumor volumes of each group on the 28th day, among which the tumor volumes of the PD-1 monoclonal antibody group, combined treatment group and FomA antibody control group were 1397±156mm³, 101±23mm³ and 653±76mm³, respectively.

[0052] The tumor volume of the combined treatment group was significantly lower than that of the other groups, demonstrating the superiority of the combined treatment.

[0053] Bacterial load test: The number of 16S rRNA gene copies of Fusobacterium nucleatum in tumor tissue was detected by qPCR. The gene copy number / g was significantly lower than that of the other groups, proving that the vaccine can effectively eliminate Fusobacterium nucleatum in tumor tissues.

[0054] Immune response testing: ELISA assays were used to detect FomA-specific IgG antibodies in serum. The antibody titer in the vaccine group was 1:2000 on day 7, reaching 1:8000 on day 14, and maintaining at 1:15000 on day 21. Flow cytometry assays were used to measure the proportion of FomA-specific CD8+ T cells in the spleen, which was 3.2% in the vaccine group, significantly higher than in the other groups.

[0055] Immune microenvironment analysis: Flow cytometry analysis of tumor-infiltrating lymphocytes showed that the proportion of CD8+ T cells in the combined treatment group was 18.7%, and the proportion of regulatory T cells was 6.8%, indicating that the combined treatment can improve the tumor immune microenvironment.

[0056] This example verifies that the FomA-mRNA vaccine combined with PD-1 monoclonal antibody has a significant anti-tumor effect, can effectively eliminate Fusobacterium nucleatum in tumor tissues, activate specific immune responses, and improve the tumor immune microenvironment.

[0057] Example 4 The purpose of this example is to verify the effects of different repetition times (2 times, 3 times, 4 times, 5 times) on the immune enhancement effect of the FomA extracellular domain.

[0058] Repetition gradient design: Four FomA extracellular domain sequences with different repeat numbers were designed: 2 repeats (FomA-2×), 3 repeats (FomA-3×), 4 repeats (FomA-4×), and 5 repeats (FomA-5×). Each sequence used the same linker sequence and preparation process.

[0059] Cell transfection experiments: HEK293T cells were transfected, and FomA protein expression was detected by Western blot. The results showed that the relative expression levels of FomA in the FomA-2× group were 1.5, 2.6 in the FomA-3× group, 2.4 in the FomA-4× group, and 2.1 in the FomA-5× group. The highest protein expression level was observed in the 3-replicate group.

[0060] Immunogenicity assessment: Immunogenicity was assessed in C57BL / 6 mice. Each group of eight mice received intramuscular injections of 10 μg of the vaccine at varying doses. Blood was collected on day 14 to measure FomA-specific IgG antibody titers.

[0061] ELISA test results showed that the antibody titer of the FomA-2× group was 1:5000, the FomA-3× group was 1:15000, the FomA-4× group was 1:12000, and the FomA-5× group was 1:9000. The antibody titer of the 3-repeat group was the highest, verifying the superiority of the 3-repeat design.

[0062] CD8+ T cell activation assay: Flow cytometry analysis of the proportion of FomA-specific CD8+ T cells in the spleen revealed that the percentages were 2.1% in the FomA-2× group, 3.2% in the FomA-3× group, 2.8% in the FomA-4× group, and 2.3% in the FomA-5× group. The three-repetition group exhibited the best CD8+ T cell activation effect.

[0063] This example demonstrates that three repeats are the optimal choice for the tandem repeat design of the FomA extracellular domain, and performs best in terms of protein expression, antibody production, and cellular immune activation.

[0064] Example 5 The purpose of this example is to verify the effect of different lipid compositions on mRNA delivery efficiency and optimize the lipid nanoparticle formulation.

[0065] Lipid formulation design: Design 5 different lipid formulations: Formula A: DLin-MC3-DMA: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5; Formulation B: DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 = 45:15:37:3; Formula C: DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 = 40:20:37:3; Formula D: DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 = 45:10:40:5; Formula E: DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 = 45:20:32:3; Performance parameter detection: The particle size and polydispersity index were determined by dynamic light scattering, the encapsulation efficiency was determined by RiboGreen assay, and the delivery efficiency was determined by cell transfection assay.

[0066] The results show: Particle size: Formulation A is 95.2nm, Formulation B is 81.2nm, Formulation C is 78.5nm, Formulation D is 88.5nm, and Formulation E is 85.3nm; Encapsulation efficiency: 89.2% for formula A, 95.2% for formula B, 94.1% for formula C, 93.1% for formula D, and 91.8% for formula E; Transfection efficiency: Formula A is 1.8 times, Formula B is 2.3 times, Formula C is 2.0 times, Formula D is 1.9 times, and Formula E is 1.7 times; Formula B performed best in all indicators, verifying the superiority of the 45:15:37:3 molar ratio.

[0067] Cytotoxicity assessment: The cytotoxicity of the different formulations was assessed using the MTT assay. After 24 hours, cell viability was 78% for Formulation A, 89% for Formulation B, 85% for Formulation C, 82% for Formulation D, and 80% for Formulation E. Formulation B exhibited the lowest cytotoxicity.

[0068] This example verifies the important influence of lipid composition on delivery efficiency and determines the optimal lipid formulation.

[0069] Example 6 The purpose of this example is to optimize the dosage and frequency of FomA-mRNA vaccine administration and determine the optimal treatment plan.

[0070] Dose gradient design: Four different doses were designed: 1 mg / kg, 3 mg / kg, 5 mg / kg, and 10 mg / kg, with each dose set at two dosing frequencies: once a week and twice a week.

[0071] Efficacy evaluation: The therapeutic effects of different doses and frequencies were evaluated in the esophageal squamous cell carcinoma model infected with Fusobacterium nucleatum. The results showed: 1 mg / kg once a week: tumor inhibition rate 45%; 3 mg / kg once a week: tumor inhibition rate 78%; 5 mg / kg once a week: tumor inhibition rate 85%; 10 mg / kg once a week: tumor inhibition rate 87%; 3 mg / kg twice a week: tumor inhibition rate 82%; 5 mg / kg twice a week: tumor inhibition rate 88%; Safety Assessment: Serum liver function indicators ALT and AST, and renal function indicators BUN and Cr were measured. Mild liver function abnormalities were observed in the 10 mg / kg group, while doses of 5 mg / kg and below showed good safety.

[0072] Antibody response: The FomA-specific IgG antibody titers in different dose groups were: 1:2000 in the 1 mg / kg group, 1:8000 in the 3 mg / kg group, 1:15000 in the 5 mg / kg group, and 1:18000 in the 10 mg / kg group.

[0073] Taking into account the efficacy and safety, 3-5 mg / kg once a week was determined to be the optimal dosing regimen.

[0074] Example 7 The purpose of this example is to evaluate the immune memory and long-term protective effects induced by FomA-mRNA vaccine.

[0075] Immune memory testing: Mice that completed treatment were observed over a long period of time, with regular testing for FomA-specific antibody and T cell responses. Results showed that eight weeks after treatment, FomA-specific IgG antibody titers remained above 1:5000, and the proportion of FomA-specific CD8+ T cells was 1.8%, demonstrating that the vaccine was able to establish long-lasting immune memory.

[0076] Secondary challenge experiment: Sixty days after the end of treatment, surviving mice were challenged with a second tumor inoculation. 85% (17 / 20) of the mice in the combination treatment group developed protective immunity to the second challenge, while no protection was seen in the control group, demonstrating that the vaccine can establish long-term immune protection.

[0077] Memory T cell analysis: Flow cytometry was used to detect central memory T cells (TCM) and effector memory T cells (TEM). The proportion of TCM in the vaccine group was 2.3%, and the proportion of TEM was 4.1%, both significantly higher than those in the control group, demonstrating that the vaccine can induce the formation of memory T cells.

[0078] This example verifies that the FomA-mRNA vaccine can induce persistent immune memory and long-term protective effects.

[0079] Example 8 The purpose of this example is to verify the effect of different linker sequences on vaccine efficacy.

[0080] Connector design: Design 3 different linker sequences: Linker A: GSGGSGGGGSGG (present invention) Connector B: GGGGSGGGGS; Connector C: GGGGS; Immunogenicity comparison: The same animal model and dosing regimen were used to compare the immune effects of different linkers.

[0081] Antibody titers: 1:15,000 for connector A, 1:12,000 for connector B, and 1:8,000 for connector C. Connector A showed the best effect.

[0082] CD8+ T cell activation: 3.2% in the Connexon A group, 2.8% in the Connexon B group, and 2.1% in the Connexon C group. Connexon A had the strongest activation effect.

[0083] Protein stability: The stability of the protein secondary structure was detected by circular dichroism spectroscopy. The protein structure of linker group A was the most stable.

[0084] This example verifies the superiority of the GSGGSGGGGSGG linker sequence.

[0085] Example 9 The purpose of this example is to verify the differential clearance effect of FomA-mRNA vaccine on intracellular and extracellular Fusobacterium nucleatum and compare it with traditional antibiotic treatment.

[0086] Construction of in vitro bacterial infection model: The human esophageal squamous cell carcinoma cell line KYSE-150 was used to establish an in vitro Fn infection model. The cells were seeded in 24-well plates at a density of After 24 hours of culture, Fusobacterium nucleatum ATCC 25586 was added (MOI = 100:1) and co-cultured for 2 hours to establish the infection model. Extracellular bacteria were killed by gentamicin treatment (100 μg / mL, 2 hours), while intracellular colonized bacteria were retained.

[0087] Quantitative detection of intracellular and extracellular bacteria: Using a differentiated bacterial quantification method: 1. Total bacterial load: Direct cell lysis and detection of Fn 16S rRNA gene copy number by qPCR; 2. Extracellular bacterial load: Collect culture supernatant and detect by qPCR; 3. Intracellular bacterial load: total bacterial load minus extracellular bacterial load.

[0088] Treatment plan design: Infected cells were divided into 4 groups: 1. Control group: no treatment; 2. Metronidazole group: metronidazole 50 μg / mL treatment for 24 hours; 3. FomA-mRNA vaccine group: Transfected with FomA-mRNA vaccine 1 μg / mL, and 24 hours later, FomA-specific CD8+ T cells were added (effector-target ratio 10:1); 4. Combined treatment group: metronidazole + FomA-mRNA vaccine combined treatment.

[0089] Extracellular bacteria removal effect: Control group: extracellular bacterial load Gene copies / mL, intracellular bacterial load Gene copies / mL; Metronidazole group: extracellular bacterial load Gene copies / mL (clearance rate 99.9%), intracellular bacterial load gene copies / mL (clearance rate 12.5%); FomA-mRNA vaccine group: extracellular bacterial load gene copies / mL (clearance rate 8.2%), intracellular bacterial load gene copies / mL (clearance rate 93.4%); Combination treatment group: extracellular bacterial load Gene copies / mL (clearance rate 99.9%), intracellular bacterial load gene copies / mL (clearance rate 94.4%).

[0090] Cellular immune activation assay: Flow cytometry was used to detect the expression of CD69 and CD107a, activation markers of CD8+ T cells. The proportion of CD69+CD8+ T cells in the FomA-mRNA vaccine group was 45.2%, and the proportion of CD107a+CD8+ T cells was 38.7%, significantly higher than in the other groups, demonstrating that the vaccine can effectively activate cytotoxic T cell responses.

[0091] Cytotoxicity assay: The LDH release assay was used to test the T cell cytotoxicity against infected cells. The FomA-mRNA vaccine group showed a cytotoxicity of 78.5%, while the metronidazole group showed only 15.2%, demonstrating that the vaccine can directly kill bacterially infected host cells by activating CD8+ T cells.

[0092] Mechanism analysis: Immunofluorescence co-localization assays revealed that extracellular Fn (red fluorescence) was significantly reduced after metronidazole treatment, but intracellular Fn (red fluorescence co-localized with nuclear DAPI) remained largely unchanged. Furthermore, after FomA-mRNA vaccine treatment, the number of Fn-infected cells (CK5+Fn+ double-positive cells) was significantly reduced, indicating that the vaccine indirectly eliminates intracellular bacteria by clearing infected host cells.

[0093] This example verifies the unique advantages of FomA-mRNA vaccine in clearing intracellular bacteria, solves the technical problem that traditional antibiotics cannot clear intracellular colonized bacteria, and provides a new strategy for the treatment of tumors related to Fusobacterium nucleatum infection.

[0094] Comparative Example 1 The purpose of this comparative example is to verify the advantages of the tandem repeat design over the full-length FomA protein.

[0095] Comparative solution preparation: An mRNA vaccine encoding the complete FomA protein sequence (FomA-full-length, amino acid sequence SEQ ID NO: 3) was prepared, including the complete transmembrane and intracellular domains, without a tandem repeat design. The same preparation conditions and lipid nanoparticle delivery system were used.

[0096] Animal model construction: The Fn-infected and colonized mouse ESCC immunotherapy resistance model constructed in Example 1 was used. On day 0, Fn-colonized human organoids were subcutaneously inoculated ( ) + Fn bacteria ( CFU).

[0097] Grouping and dosing: Mice were randomly divided into two groups, 10 in each group; different vaccines were injected intramuscularly from day 7 to day 21 (twice a week): 1. Combination treatment group: anti-PD-1 antibody 100 μg / animal + FomA-mRNA vaccine 10 μg / animal; 2. Combined treatment control group: anti-PD-1 antibody 100 μg / mouse + FomA full-length mRNA vaccine 10 μg / mouse

[0098] Comparison of in vivo efficacy: like Figure 4 As shown in B, the tumor volume on day 28 was 398±45 mm³ in the combined treatment control group and 101±23 mm³ in the combined treatment group.

[0099] Antibody titer detection: On the 28th day, the antibody titer of the FomA-full-length vaccine group was 1:3000, and that of the FomA-3× vaccine group was 1:15000, a 5-fold increase.

[0100] Bacterial load: The bacterial load of tumor tissue in the FomA-full-length vaccine group was gene copies / g, FomA-3× vaccine group gene copies / g, and the clearance efficiency was increased by 4.8 times.

[0101] The comparison verified the significant advantages of tandem repeat design over full-length protein.

[0102] Comparative Example 2 The purpose of this comparative example is to verify the advantages of mRNA vaccine treatment over traditional antibiotic treatment.

[0103] Animal model construction: The Fn-infected and colonized mouse ESCC immunotherapy resistance model constructed in Example 1 was used. On day 0, Fn-colonized human organoids were subcutaneously inoculated ( ) + Fn bacteria ( CFU).

[0104] Grouping and dosing: Mice were randomly divided into 4 groups, with 10 mice in each group. Different treatment regimens were given from day 7 to day 21: 1. Combination treatment group: anti-PD-1 antibody 100 μg / animal + FomA-mRNA vaccine 10 μg / animal, intravenous injection, twice a week; 2. Metronidazole control group: anti-PD-1 antibody 100 μg / animal + metronidazole 200 mg / kg, orally twice a day; 3. Metronidazole group: metronidazole 200 mg / kg, orally twice a day; 4. Blank control group: treated with normal saline.

[0105] On day 28, tumor volume, bacterial load, immune response and other indicators were evaluated.

[0106] Comparison of in vivo efficacy: like Figure 4 As shown in C, the tumor volume on the 28th day was: 1856±198 mm³ in the blank control group, 1623±145 mm³ in the metronidazole alone group, 752±89 mm³ in the metronidazole control group, and 101±23 mm³ in the combined treatment group.

[0107] Bacterial load test: Detection of 16S rRNA gene copy number of Fusobacterium nucleatum in tumor tissue by qPCR: Blank control group: Gene copy number / g; Metronidazole alone group: Gene copy number / g (mainly to eliminate extracellular bacteria) Metronidazole control group: Gene copy number / g; Combined treatment group: Gene copy number / g; Analysis of intracellular and extracellular bacteria: Detection of intracellular Fn by immunofluorescence in tissue sections: Metronidazole control group: the proportion of intracellular Fn-positive cells was 23.5%, indicating that metronidazole had limited effect on clearing intracellular bacteria; In the combined treatment group, the proportion of intracellular Fn-positive cells was 2.1%, which was significantly lower than that in the metronidazole group.

[0108] Drug resistance analysis: After 4 weeks of continuous treatment, the sensitivity of Fn to metronidazole was tested: Metronidazole group: MIC increased from the initial 2 μg / mL to 16 μg / mL, indicating obvious drug resistance; mRNA vaccine group: There is no drug resistance problem because the bacteria are eliminated through immune mechanisms.

[0109] Side effect assessment: The metronidazole group experienced obvious gastrointestinal reactions and a weight loss of 15%; the mRNA vaccine group had no obvious side effects and the weight change was <5%.

[0110] The comparison verified the significant advantages of mRNA vaccine therapy over traditional antibiotic treatment, especially in eliminating intracellular bacteria, avoiding the development of drug resistance and reducing side effects.

[0111] Based on the above embodiments and comparative examples, the core innovation of the present invention lies in the development of an mRNA vaccine targeting the membrane protein FomA of Fusobacterium nucleatum for the first time. It adopts an extracellular domain design with 2 to 5 tandem repeats to enhance immunogenicity, realizes efficient intracellular delivery through an optimized lipid nanoparticle delivery system, activates FomA-specific cellular immune response, can directly eliminate host cells colonized by Fusobacterium nucleatum, and effectively reverses the immunotherapy resistance of esophageal squamous cell carcinoma caused by Fusobacterium nucleatum infection.

[0112] Compared with traditional antibiotic treatment, the present invention has the following outstanding advantages: 1. Ability to eliminate intracellular bacteria: Traditional antibiotics such as metronidazole mainly kill extracellular bacteria and have limited effectiveness against intracellular Fusobacterium nucleatum (elimination rate is only 12.5%). However, the mRNA vaccine of this invention activates the CD8+ T cell immune response, which can identify and eliminate bacterially infected host cells, thereby indirectly eliminating intracellular bacteria (elimination rate reaches 93.4%). 2. Avoid drug resistance: Long-term use of antibiotics can easily lead to drug resistance, while immunotherapy works through a multi-target mechanism and is less likely to cause drug resistance; 3. Better safety: It avoids the gastrointestinal side effects of antibiotics and the damage to normal flora.

[0113] Compared with the existing technology, the present invention has significant advantages in targeting specificity, immune activation ability, therapeutic effect and safety, and provides a new technical solution for personalized immunotherapy of esophageal squamous cell carcinoma.

Claims

1. An mRNA vaccine targeting FomA, characterized in that The invention comprises an mRNA sequence encoding a tandem repeat sequence of the extracellular domain of FomA of Fusobacterium nucleatum and lipid nanoparticles; the tandem repeat sequence of the extracellular domain of FomA is an extramembrane segment multimerization immune enhancement sequence; the mRNA sequence comprises a 5' cap structure, a 5' untranslated region, an open reading frame encoding the tandem repeat sequence of the extracellular domain of FomA, a 3' untranslated region and a polyadenylation tail; the tandem repeat sequence encoding the extracellular domain of FomA comprises a first FomA extracellular domain repeat sequence as shown in SEQ ID NO:1 and a second FomA extracellular domain repeat sequence as shown in SEQ ID NO:2, and the two FomA extracellular domain repeat sequences are sequentially connected by a linker sequence; the lipid nanoparticle completely encapsulates the mRNA sequence in the inner core through a bilayer structure, with an average particle size of 70 to 100 nm and an encapsulation efficiency of 90 to 98%.

2. The mRNA vaccine according to claim 1, wherein The first FomA extracellular domain repeat sequence is 2 to 5 repeats of the amino acid sequence SEQ ID NO: 1, and the second FomA extracellular domain repeat sequence is 2 to 5 repeats of the amino acid sequence SEQ ID NO: 2; the amino acid sequence is derived from the standard strain of Fusobacterium nucleatum ATCC 25586.

3. The mRNA vaccine according to claim 1, characterized in that The linker sequence is a glycine-serine linker.

4. The mRNA vaccine according to claim 1, characterized in that The lipid nanoparticles are prepared by a microfluidic system, the flow rate ratio of the aqueous phase to the organic phase is controlled to be 2:1 to 6:1, and the mixing temperature is controlled at 20-30°C. The formed lipid nanoparticles are uniformly spherical and have a polydispersity index of less than 0.

3.

5. The mRNA vaccine according to claim 1, characterized in that The purity of the mRNA sequence detected by capillary electrophoresis is not less than 95%, and the capping efficiency of the 5' cap structure is not less than 90%; after the lipid nanoparticles are stored at -80°C for 12 months, the mRNA integrity retention rate is not less than 85%, and the encapsulation efficiency retention rate is not less than 90%.

6. The mRNA vaccine according to claim 1, characterized in that The lipid nanoparticles contain ionizable lipids, phosphatidylcholine, cholesterol and PEGylated lipids; the ionizable lipids are DLin-MC3-DMA or ALC-0315, the phosphatidylcholine is DSPC or DOPC, and the PEGylated lipids are DMG-PEG2000 or ALC-0159; and the molar ratio of the components is 40-50:10-20:32-40:1.5-5.

7. Use of the mRNA vaccine according to claim 1 in the preparation of an immunotherapy drug for Fusobacterium nucleatum-positive esophageal squamous cell carcinoma, characterized in that: The treatment is aimed at determining that the load of Fusobacterium nucleatum is not less than The mRNA vaccine was used in combination with PD-1 monoclonal antibody to eliminate tumor cells colonized with Fusobacterium nucleatum by activating FomA-specific CD8+ T cell immune response.

8. The use according to claim 7, characterized in that The dosage of the mRNA vaccine is 1 to 10 mg / kg body weight, administered by intramuscular injection, with a frequency of 1 to 2 times per week, and continuously administered 3 to 8 times; the dosage of the PD-1 monoclonal antibody is 1 to 10 mg / kg body weight, administered by intravenous injection, with a frequency of 1 to 3 times per week; compared with the control group using PD-1 monoclonal antibody alone, the tumor growth inhibition rate after combined treatment is increased by 50 to 90%.

9. The use according to claim 7, characterized in that The FomA-specific IgG antibody titer induced by the mRNA vaccine reaches 1:1000-1:50000 on the 7th to 21st day after administration; the FomA-specific CD8+ T cell response reaches a peak on the 7th to 21st day after administration, and the immune memory is maintained for no less than 4 weeks.

10. A method for preparing the mRNA vaccine according to claim 1, characterized in that: The following steps are involved: Step 1: designing an mRNA sequence encoding the tandem repeat sequence of the FomA extracellular domain based on the amino acid sequence of the Fusobacterium nucleatum FomA protein, and synthesizing the mRNA sequence by in vitro transcription at 30-40°C for 3-8 hours using T7 RNA polymerase; Step 2: preparing a lipid mixture solution comprising ionizable lipids, phosphatidylcholine, cholesterol and PEGylated lipids, and dissolving them in an organic solvent; Step 3: Mixing the mRNA aqueous solution with the lipid organic solution through a microfluidic system to form lipid nanoparticles; Step 4: Remove the organic solvent and replace the buffer through tangential flow filtration to finally obtain the mRNA vaccine.

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