Antigen MNA and application thereof as well as vaccine and medicine prepared from antigen MNA

Abstract

The invention discloses an antigen MNA and application thereof, and a vaccine and a medicine prepared from the antigen MNA, and belongs to the technical field of biological medicines. The novel tumor neoantigen (MNA) found and designed by the invention has strong immunogenicity and can effectively activate T cell immune response; and the polypeptide is only expressed in tumor cells, is not influenced by central immune tolerance, and can be used for accurately targeting the tumor cells and reducing the damage to normal tissues. The circRNA is used as an expression vector of the new antigen MNA, so that the stability is high, the expression is efficient, the strong new antigen specific T cell reaction is induced, and an adjuvant is not needed. The invention provides a new thought for the development and personalized immunotherapy of tumor new antigen vaccines, is helpful to be popularized to more high mutation load tumor types, and has wide clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an antigen MNA and its application and the vaccine and medicine prepared therefrom. Background Art

[0002] In recent years, tumor immunotherapy has become increasingly mature. Cancer immunotherapy represented by immune checkpoint blockade and adoptive cell therapy has made good progress in clinical trials, but it still faces challenges such as some patients not responding to expensive immune checkpoint drugs, and adoptive cell therapy has poor persistence in the body, difficulty entering solid tumors, and side effects. In order to expand tumor treatment options, it is crucial to develop more suitable new immunotherapies. In addition to immunosuppressive factors in the tumor microenvironment, the lack of immunogenicity of tumor cells is another reason for their escape from immune surveillance. Therefore, finding new antigens with stronger immunogenicity has become the next key issue in immunotherapy. Summary of the invention

[0003] In this invention, a new tumor neoantigen named MNA was discovered and designed using B16F10 mouse melanoma as a model. The designed neoantigen MNA was cloned into circRNA to construct a melanoma neoantigen vaccine circRNA based on circRNA. MNA , which can stably express the above tumor neoantigens. After effective delivery into the body by lipid nanoparticles, its immune response and anti-tumor efficacy were evaluated. The results showed that circRNA MNA The vaccine effectively inhibited tumor growth and triggered robust antigen-specific T cell responses in both preventive and therapeutic mouse tumor models.

[0004] therefore,

[0005] The primary purpose of the present invention is to provide an antigen MNA, whose amino acid sequence is shown in SEQ ID NO.1; and whose nucleotide sequence is shown in SEQ ID NO.2.

[0006] The second aspect of the present invention aims to provide the use of the antigen MNA for preparing a melanoma vaccine or a drug for treating melanoma.

[0007] Further,

[0008] The nucleotide sequence of the antigen MNA is cloned into circRNA to construct a circRNA based on circRNA that stably expresses MNA MNA As a melanoma vaccine or drug for the treatment of melanoma.

[0009] Furthermore,

[0010] The vaccine or drug is delivered via lipid nanoparticle encapsulation of circRNA MNA .

[0011] The third aspect of the present invention is to provide a melanoma vaccine.

[0012] Specifically, the nucleotide sequence of the antigen MNA is cloned into circRNA to construct a circRNA based on circRNA to stably express MNA MNA as a melanoma vaccine.

[0013] Furthermore, the vaccine is delivered via lipid nanoparticle encapsulation of circRNA MNA .

[0014] The fourth aspect of the present invention is to provide a drug for treating melanoma.

[0015] Specifically, the nucleotide sequence of the antigen MNA is cloned into circRNA to construct a circRNA based on circRNA to stably express MNA MNA As a drug for the treatment of melanoma.

[0016] Further,

[0017] The drug is delivered via lipid nanoparticle encapsulation of circRNA MNA .

[0018] The preparation method of the new antigen MNA of the present invention and the verification of its application effect in anti-tumor immunotherapy are as follows:

[0019] 1. Synthesize circRNA to express new antigen MNA

[0020] The present invention adopts the group I intron autocatalytic strategy to clone the designed new antigen epitope MNA (MNA: Melanomaneoantigen) into the circRNA vector, and then generates a circRNA encoding the new antigen MNA through in vitro transcription and cyclization, which is named circRNA MNA The final product was purified by high performance liquid chromatography. Figure 1 )

[0021] Denaturing agarose gel RNA electrophoresis confirmed circRNA MNA The relative sizes of their linear precursors. RNase R digestion experiments revealed that most purified circRNAs MNA The circRNA was resistant to RNase R, while the linear precursor was almost completely degraded. Reverse transcription-qPCR, Sanger sequencing, and RNase H-mediated specific cleavage further verified the circRNAMNA Cyclization. Figure 2 )

[0022] The present invention detects circRNA by ELISA experiment MNA The secretory expression of the new antigen in the supernatant of HEK293 and B16F10 cells after transfection. The above shows that the circRNA vector for expressing the new antigen constructed by the present invention is stable and can achieve MNA antigen expression. In addition, we used the new antigen epitope M30 in the literature as a reference (Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Sebastian Kreiter. Nature volume 520, pages 692–696 (2015), the new antigen M30 was reported to have good immunogenicity), and circRNA encoding M30 was generated. M30 The secretory expression of M30 was also detected in the supernatant. MNA and circRNA M30 The antigen expression efficiency is close to that of Figure 3 )

[0023] Next, LNPs were used to encapsulate and deliver circRNA MNA For in vivo experiments. MNA The encapsulation efficiency is greater than 97%, and the average diameter is about 82 nm. MNA After the complex was co-incubated with cells in vitro, circRNA was confirmed by ELISA MNA Successfully delivered into cells and expressing new antigens, circRNA M30 The CCK-8 assay also confirmed the circRNA MNA The biocompatibility of the drug was good. With the increase of dose and time, there was no significant change in cell viability and proliferation. Figure 4 )

[0024] 2. circRNA MNA Vaccination significantly inhibits tumor growth in mice

[0025] The present invention first detected circRNA MNA The ability of the vaccine to trigger innate and adaptive immune responses. MNA Vaccines and LNP-circRNA M30The vaccine was injected intramuscularly into C57BL / 6J mice, and peripheral blood was extracted 24 h after injection for ELISA assay to detect the secretion of pro-inflammatory cytokines. The results showed that compared with the control group, circRNA MNA Innate immune responses, such as TNF-α and IL-6 secretion, were observed in the vaccine group. To detect the novel antigen-specific T cell response, spleen cells from immunized mice were collected and exposed to the same antigens for IFN-γ ELISpot assay, and the results showed that circRNA vaccination MNA Splenocytes from mice that received the vaccine produced a strong response to the new antigens. MNA and circRNA M30 The antigen expression efficiency of the vaccine is similar to that of the vaccine, but the circRNA expressing the new antigen MNA of the present invention is MNA Mice vaccinated with circRNA M30 The immune response of mice to the vaccine was more obvious. The systemic toxicity of the vaccine was evaluated by hematoxylin and eosin (H&E) staining of major organs, and no obvious toxic side effects were observed. Figure 5 )

[0026] To evaluate circRNA MNA Whether the immune response triggered by the vaccine can be converted into anti-tumor effects in mice was evaluated in the invasive B16F10 melanoma model. First, in the preventive model, C57BL / 6J mice were first vaccinated with two doses of vaccine and then transplanted with tumor cells. The results showed that compared with the control group, circRNA MNA Vaccine group and circRNA M30 The vaccine groups all significantly inhibited tumor growth. MNA Mice vaccinated with circRNA M30 The tumors in the vaccinated mice grew more slowly and the tumor suppression effect was more significant. Vaccination did not change the weight of the mice. Figure 6 )

[0027] IFN-γ ELISpot test results showed that circRNA vaccination MNA Splenocytes and peripheral blood mononuclear cells (PBMCs) of mice vaccinated with the vaccine responded strongly to the new antigens. Flow cytometry showed that, in contrast, the circRNA MNA In mice immunized with the vaccine, CD4 + and CD8 + T cells showed enhanced function, with the most significant increase in IFN-γ and TNF-α expression. MNA In the tumors of immunized mice, CD4 + and CD8+ T cell infiltration was significantly more abundant. Figure 7 )

[0028] Similarly, in a therapeutic model, C57BL / 6J mice were transplanted with B16F10 cells and then vaccinated with two doses of vaccine, and the above results were consistent ( Figure 8-9 ). These results prove that circRNA MNA The vaccine can effectively inhibit tumor growth and trigger robust antigen-specific T cell responses.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The novel tumor neoantigen (MNA) discovered and designed by the present invention showed significant tumor suppression effect and strong immune response in melanoma models, without obvious toxic side effects. The present invention aims to explore more applicable new immunotherapy, focusing on neoantigens. Neoantigens have strong immunogenicity and can effectively activate T cell immune response; they are only expressed in tumor cells, are not affected by central immune tolerance, can accurately target tumor cells, and reduce damage to normal tissues.

[0031] 2. The present invention uses circRNA as the expression vector of the new antigen MNA. The advantages of the circRNA vaccine format are: high stability; efficient expression, inducing strong antigen-specific T cell response; no adjuvant is required, circRNA itself has an immunostimulatory effect, and can be used as a self-adjuvant to enhance the vaccine effect, simplifying the vaccine preparation process.

[0032] 3. The new antigen MNA showed dual effects in the preventive and therapeutic models of mouse B16F10 melanoma, with a significant reduction in tumor volume. Compared with the reported new antigen M30, the tumor inhibition effect of the new antigen MNA was more significant.

[0033] 4. This invention provides new ideas for the development of tumor neoantigen vaccines and personalized immunotherapy, which will help to extend to more tumor types with high mutation loads and has broad prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : The synthesis process of circRNA expressing the tumor neoantigen of the present invention and the product detection results;

[0035] A. circRNA MNA Schematic diagram of the cyclization by group I intron autocatalysis; B, the result of liquid chromatography purification of the final product.

[0036] Figure 2 :Characterization of the circRNA of the present invention MNAThe result of the ring structure;

[0037] A. Detection of linear precursor RNA and circRNA by denaturing agarose gel RNA electrophoresis MNA Size results; B, linear precursor RNA and circRNA MNA Denaturing agarose gel RNA electrophoresis results after RNase R treatment; C, DNA gel electrophoresis showing PCR analysis results; D, Sanger sequencing results showing circRNA MNA Splicing site; E, schematic diagram of RNase H detection.

[0038] Figure 3 :ELISA assay to detect circRNA MNA Secretory expression results of neoantigens in the supernatant after transfection of HEK293 and B16F10 cells by Hiperfect.

[0039] Figure 4 :LNP-circRNA MNA characterization and expression of the complex and results of safety assessment;

[0040] A. LNP-circRNA obtained by dynamic light scattering MNA Representative intensity-size distribution of LNP-circRNA by ELISA. MNA The expression of new antigens was detected in the supernatant after the complex was co-incubated with HEK293 and B16F10 cells; C, the expression of new antigens was detected by CCK-8 assay along with LNP-circRNA MNA The changes in cell viability with the increase of the complex dosage; D. CCK-8 test to determine the changes in cell viability with the increase of LNP-circRNA MNA The extension of the action time of the complex will lead to changes in cell proliferation.

[0041] Figure 5 :circRNA MNA Vaccine-induced innate and adaptive immune responses outcomes;

[0042] A. Evaluation of circRNA MNA Experimental timeline of vaccine (10 µg circRNA per mouse) induced immune response; B, ELISA assay for circRNA MNA The results of the secretion of cytokine TNF-α in peripheral blood after vaccination, , ; C. ELISA determination of circRNA MNA The results of the secretion of cytokine IL-6 in peripheral blood after vaccination, , ; D, representative images and statistical results of IFN-γ spot formation after restimulation of splenocytes by IFN-γ ELISpot, , ; E, H&E staining results of the main organs of each group, scale bar, 200 μm.

[0043] Figure 6 :circRNA MNA The vaccine significantly inhibited tumor growth in a preventive mouse tumor model;

[0044] A, Schematic diagram of the immune strategy of the preventive mouse tumor model and the monitoring of tumor volume after inoculation of B16F10 cells; B, Representative images of subcutaneous melanoma tissues dissected after 22 days in the preventive mouse model (n=5 per group); C, Tumor volume growth curve of mice with transplanted tumors after vaccine immunization in the preventive mouse model, , , ; D. Statistical results of tumor tissue weight of mice transplanted with tumors after vaccine immunization in preventive mouse model, , ; E. Body weight change curve of tumor-transplanted mice after vaccine immunization in the preventive mouse model.

[0045] Figure 7 : In a preventive mouse tumor model, circRNA MNA The vaccine induces robust antigen-specific T cell responses;

[0046] A. Images and statistical results of IFN-γ spot formation in spleen cells measured by IFN-γ ELISpot in the preventive mouse model. , , ; B. Images and statistical results of IFN-γ spot formation in PBMCs measured by IFN-γ ELISpot in a preventive mouse model, , ; C, IFN-γ in restimulated splenocytes measured by flow cytometry in a preventive mouse model + and TNF-α + CD4 + and CD8 + T cell percentage results, , , , ; D. Detection of control and circRNAs by flow cytometry in a preventive mouse model MNA Results of the proportion of infiltrating immune cells in subcutaneous B16F10 tumors immunized with vaccines, , .

[0047] Figure 8 :circRNA MNA The vaccine significantly inhibited tumor growth results in a therapeutic mouse tumor model;

[0048] A, Schematic diagram of the immune strategy of the therapeutic mouse tumor model and the monitoring of tumor volume after inoculation of B16F10 cells; B, Representative images of subcutaneous melanoma tissues dissected after 20 days in the therapeutic mouse model (n=5 per group); C, Tumor volume growth curve of mice with transplanted tumors after vaccine immunization in the therapeutic mouse model, , , ; D. Statistical results of tumor tissue weight of mice transplanted with tumors after vaccine immunization in therapeutic mouse model, , , ; E. Body weight change curve of tumor-transplanted mice after vaccine immunization in the therapeutic mouse model.

[0049] Fig. 9 : In therapeutic mouse tumor models, circRNA MNA The vaccine induces robust antigen-specific T cell responses;

[0050] A. Images and statistical results of IFN-γ spot formation in restimulated splenocytes measured by IFN-γ ELISpot in a therapeutic mouse model. ; B. Images and statistical results of IFN-γ spot formation in PBMCs measured by IFN-γ ELISpot in a therapeutic mouse model, , ; C, IFN-γ in restimulated splenocytes measured by flow cytometry in a therapeutic mouse model + and TNF-α + CD4 + and CD8 + T cell percentage results, , , , ; D. Detection of control and circRNAs by flow cytometry in therapeutic mouse models MNA Results of the proportion of infiltrating immune cells in subcutaneous B16F10 tumors immunized with vaccines, , , . DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0052] The experimental mice used in the present invention are 6-8 week old female C57BL / 6J mice, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., and fostered in the Department of Experimental Animals of Central South University (Changsha, Hunan), in an SPF barrier environment. All experimental protocols involving animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the United States and approved by the Animal Ethics and Welfare Committee of Central South University. The cells B16F10 and HEK293 used in the present invention were ordered from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured in RPMI-1640 complete medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution) at 37 °C and 5% CO2.

[0053] The melanoma neoantigen MNA polypeptide, neoantigen nucleotide sequence and circRNA skeleton (SEQ ID NO.3) of the present invention were commissioned to Shanghai Sangon Biotechnology Co., Ltd. for synthesis.

[0054] The experimental results of the present invention were statistically analyzed: t-test was used to evaluate the difference between the two groups. p<0.05 was used to indicate statistical significance, and all p values ​​were tested using a two-sided test. All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0 software.

[0055] Example 1: circRNA MNA Production and purification of MNA antigens

[0056] The self-splicing circularization strategy based on type I intron ribozymes (also known as the replacement intron exon (PIE) system) is a common technology for in vitro synthesis of circRNA. The principle is to add homology arms at both ends of the sequence, insert exons E1 and E2, spacer, IRES and coding sequence between the 3'intron and 5'intron sequences, synthesize PrecursorRNA through in vitro transcription reaction, and then 2+The circRNA was formed by two ester exchange reactions assisted by GTP. The T7 promoter, homology arms, elements from the replacement intron-exon construct, spacer, CVB3 IRES, MNA neoantigen sequence and Flag tag sequence were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. and cloned into a plasmid (SEQ ID NO.3) for the generation of circRNA backbone and subsequent in vitro transcription reaction. Then, the plasmid was linearized by PCR amplification or restriction digestion, and the product was purified by agarose electrophoresis and gel extraction. After sequencing verification, it was used as a template for in vitro transcription and circRNA was synthesized using HiScribe™ T7 HighYield RNA Synthesis Kit according to the manufacturer's protocol. MNA Precursor. For the in vitro transcription reaction, we optimized and adjusted the appropriate T7 RNA polymerase concentration (7.5 U / uL), rNTP concentration (7.5 mM), and in vitro transcription reaction time (16 h) to maximize transcription efficiency. The transcribed RNA product was treated with DNase I for 30 minutes to digest the DNA template. After DNase I digestion, additional GTP was added to a final concentration of 2 mM, and the reaction was then incubated at 55°C for 15 minutes to catalyze the circularization of circRNA. The column-purified RNA was then heated at 65°C for 3 min and cooled on ice for 3 min. The reaction was treated with RNase R at 37°C for 30 minutes to further enrich circRNA. The RNase R-treated RNA was column-purified. For high-performance liquid chromatography, a 7.8×300 mm size exclusion column with a particle size of 5µm and a pore size of 2000Å was used and run at a flow rate of 0.8 mL / minute in RNase-free TE buffer. Collect circRNA-enriched MNA The fractions were then column purified and treated with Quick CIP phosphatase. Finally, we produced and purified circRNA MNA (See Figure 1 B, The second half of the main peak was collected to produce highly pure circRNA MNA ), used to express MNA antigens.

[0057] Example 2: Denaturing agarose gel RNA electrophoresis

[0058] Prepare 1% denaturing agarose gel to separate and identify the size of RNA fragments. Take 1 g of agarose and add it to 72 mL of enzyme-free water, heat it to melt, and then add 10x MOPS Buffer. When the agarose cools to 50-60°C, add 18 mL of 37% formaldehyde and an appropriate amount of fluorescent dye, mix well, pour it into the electrophoresis tank and cool it for later use. Mix the RNA sample to be tested and the ssRNA ladder with RNA loading Dye according to the manufacturer's instructions (RNA sample: RNA loading Dye=1:1, ssRNAladder: RNA loading Dye=1:4), heat it at 70°C for 10 min to deform the sample, and then immediately place it on ice. After electrophoresis, use a gel imaging system to take pictures and observe. circRNA was confirmed using denaturing agarose gel RNA electrophoresis MNA and the relative sizes of its linear precursors, as expected (see Figure 2 A, Linear precursor RNA was used as a control).

[0059] Example 3: RNase R digestion experiment, reverse transcription-qPCR, Sanger sequencing, and RNase H cleavage experiment prove circRNA MNA The ring form

[0060] For RNase R digestion, 20 U / μL RNase R was incubated with linear precursor RNA or purified circRNA at 37 °C for 30 min, and then the enzyme was inactivated by heating at 70 °C for 10 min before direct RNA electrophoresis identification. RNase R is a 3'→5' exonuclease that can degrade linear RNA but has no degradation effect on circular RNA. The results showed that linear precursor RNA was almost completely degraded after RNase R treatment, and the electrophoretic band disappeared. circRNA MNA It is resistant to RNase R, and the electrophoresis band is clearly visible, indicating that it has a ring structure (see Figure 2 B, Linear precursor RNA was used as a negative control).

[0061] For reverse transcription-qPCR, circRNA MNA Reverse transcription was performed on the linear precursor RNA to generate cDNA. CircRNA was amplified by qPCR using specific primers. MNA The results showed that the linear precursor RNA could amplify the F2 / R2 fragment, but not the F1 / R1 fragment; while the circRNA MNA Both the circularization site region (F1 / R1) and the linear region (F2 / R2) can be amplified (see Figure 2 C).

[0062] The sequence of primer F1 is: 5'-GAGATCATAGTCAGCCTCCTCT-3'

[0063] The sequence of primer R1 is: 5'-TGGAGGGACTTGAACCCACA-3'

[0064] The sequence of primer F2 is: 5'-GTTCAAGTCCCTCCACCCC-3'

[0065] The sequence of primer R2 is: 5'-AAACAGGCGCACAAAGGTAC-3'

[0066] For Sanger sequencing, circRNA MNA The qPCR products of the cyclization site region were recovered and sent to Qingke Company for Sanger sequencing. The sequences returned by the company were compared with DNASTAR software, and the peak graph was viewed with chromas software to judge the quality of sequencing. The comparison results showed that circRNA MNA The sequence of the cyclization site is exactly the same as the designed sequence, circRNA MNA Forming a covalently closed ring structure (see Figure 2 D).

[0067] For RNase H cleavage, 500 ng of purified circRNA was added MNA and its linear precursor, 50 pmol of sense or antisense ssDNA probe (antisense ssDNA probe and circRNA MNA After incubating a total of 18 μL of the system (complementary to a specific region of the linear precursor RNA, with a sense probe as a control) and RNase H buffer at 50°C for 10 minutes, 2 μL of RNase H was added to the reaction, gently mixed, and incubated at 37°C for 1 hour. RNA was resolved by agarose gel electrophoresis. RNase H is an endoribonuclease that can specifically degrade the RNA phosphodiester bond hybridized to the DNA chain, so it can cut the RNA chain in the RNA / DNA hybrid chain. The results showed that after the linear precursor RNA hybridized with the antisense probe, it was treated with RNase H, and the RNA was cut into two short fragments, resulting in multiple electrophoretic bands. circRNA MNA After hybridization with the antisense probe and treatment with RNase H, circRNA MNA The cleavage is linear, the size remains unchanged, and the electrophoresis bands remain consistent (see Figure 2 E).

[0068] The sequence of the antisense ssDNA probe is: 5'-GAGTTCAGCTCTGGA-3'

[0069] The sequence of the sense ssDNA probe is: 5'-TCCAGAGCTGAACTC-3'.

[0070] Example 4: ELISA assay proves circRNA MNA Can effectively express MNA antigens

[0071] 1) HEK293 cells and B16F10 melanoma cells were seeded into 6-well plates, respectively, and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 until the cell density reached 70%-80%.

[0072] 2) Use liposome transfection reagents (such as Hiperfect) to transfect circRNA MNA Transfected into HEK293 cells and B16F10 cells, or circRNA was expressed via LNP MNA In the delivered cells, an untreated group and a linear precursor group were set as controls. After treatment, the cells were cultured for 48 hours and the cell supernatant was collected for ELISA detection.

[0073] 3) Use Flag tag ELISA kit (synthetic circRNA MNA The Flag sequence was added to express the MNA fusion protein with a Flag tag for easy detection) and the operation was performed according to the instructions.

[0074] 4) Dilute the cell supernatant and standard in appropriate proportions and add them to the 96-well plate pre-coated with Flag monoclonal antibody, add 50 μL sample to each well, and incubate at 37°C for 1 hour.

[0075] 5) Discard the liquid in the wells and wash five times with washing buffer, soaking for 1 minute each time.

[0076] 6) Add 50 μL of enzyme labeling reagent (HRP-labeled secondary antibody) to each well and incubate at 37°C for 1 hour.

[0077] 7) Discard the liquid in the wells and wash 5 times.

[0078] 8) Add 100 μL TMB colorimetric solution to each well and incubate at 37°C in the dark for 15 minutes.

[0079] 9) Add 50 μL of stop solution, mix gently, and immediately measure the absorbance (OD value) at 450 nm using a microplate reader.

[0080] 10) Data analysis: Set the blank well to zero, draw a standard curve based on the OD value of the standard, and calculate the concentration of MNA antigen in the cell supernatant. MNA MNA antigen expression levels in the treated group versus the untreated group and the linear precursor group, and evaluation of circRNA MNA expression efficiency.

[0081] 11) Experimental results: circRNA MNA In the treated groups, significant MNA antigen expression was detected in the supernatants of HEK293 cells and B16F10 cells; no MNA antigen expression was detected in the untreated group and linear precursor group, and the OD values ​​were close to the background values.

[0082] 12) Experimental conclusion: The results of ELISA assay showed that circRNA MNA The MNA antigen can be effectively expressed and secreted in HEK293 cells and B16F10 cells, verifying the circRNA MNA Functionality and reliability as a vaccine vector (see Figure 3 ). The new antigen epitope M30 in the literature was used as a reference, and circRNA encoding M30 was generated. M30 The secretory expression of M30 was also detected in the supernatant. MNA and circRNA M30 The antigen expression efficiency is close to that of

[0083] Example 5: LNP encapsulation of circRNA MNA For in vivo experiments

[0084] Experimental purpose: To verify lipid nanoparticle (LNP) encapsulation of circRNA MNA The efficiency and stability of the vaccine were evaluated, and its feasibility as a vaccine delivery system was assessed to facilitate subsequent in vivo experiments.

[0085] LNP-circRNA MNA Preparation:

[0086] 1) Prepare lipid phase: ALC-0315, DSPC, cholesterol and ALC-0159 were mixed in a molar ratio of 50:10:38.5:1.5 and dissolved in anhydrous ethanol to form a lipid phase. The role of each component in the lipid phase is: ALC-0315: ionizable lipid, used to encapsulate RNA; DSPC: auxiliary lipid, enhance the stability of LNP; cholesterol: regulate the fluidity and stability of LNP; ALC-0159: PEGylated lipid, prevent LNP aggregation and prolong its blood circulation time.

[0087] 2) Prepare the aqueous phase:MNA Dissolved in 100 mM citrate buffer (pH 4.0) at an N / P ratio of 6.

[0088] 3) Microfluidic mixing: Using a microfluidic mixing device, the lipid phase and the aqueous phase were mixed at a volume ratio of 1:3 to prepare LNP-circRNA MNA The chaotic mixing characteristics induce fluid folding in the laminar state to obtain uniform and high encapsulation efficiency of LNPs in a controllable and reproducible manner.

[0089] 4) Dialysis purification: LNP-circRNA MNA The complex was dialyzed against PBS at 4°C overnight to remove unencapsulated circRNA. MNA After dialysis, LNP-circRNA MNA The complex was stored in 10% sucrose-PBS and kept at 4°C until use.

[0090] LNP-circRNA MNA Characterization:

[0091] 1) Particle size determination: LNP-circRNA was measured using a dynamic light scattering instrument MNA The results showed that LNP-circRNA MNA The average particle size was 82 nm, indicating that it had a good particle size distribution (see Figure 4 A).

[0092] 2) Encapsulation efficiency determination: Use Quant-iT RiboGreen RNA detection kit to determine the total circRNA MNA Concentration and unencapsulated circRNA MNA The results showed that LNP-circRNA MNA The encapsulation efficiency is greater than 97%, indicating that it has a high encapsulation efficiency (see Figure 4 A).

[0093] 3) MNA antigen expression was then detected by ELISA (see Figure 4 B). The results showed that circRNA MNA It was successfully delivered into cells and expressed new antigens. LNP-encapsulated circRNA M30 With consistent effects, LNP-circRNA MNA and LNP-circRNA M30 The antigen expression efficiency is close to that of

[0094] Example 6: CCK8 cell viability assay proves LNP-circRNA MNA Security

[0095] 1) Experimental purpose: Evaluation of LNP-circRNA by CCK-8 assay MNA The effects of the complex on cell viability / cell proliferation were investigated to verify its biocompatibility and safety.

[0096] 2) Cell culture: B16F10 melanoma cells and HEK293 cells were seeded in 96-well plates, respectively, and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 for 12 hours to allow the cells to adhere and enter the logarithmic growth phase.

[0097] 3) Dose-dependent experiment: Add different concentrations of LNP-circRNA to the 96-well plate MNA The complexes (0, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL) were set up with 5 replicate wells for each concentration. MNA The complexes were incubated at 37°C, 5% CO2 for 24 h.

[0098] 4) Time-dependent experiment: 100 ng LNP-circRNA MNA The complex was added to the cells and incubated for 0, 12, 24, 36, 48, and 60 hours, respectively. Five replicate wells were set at each time point, and an untreated group was set as a control.

[0099] 5) CCK-8 assay: After incubation, add 10 μL of CCK-8 reagent to each well. Incubate the 96-well plate at 37°C and 5% CO2 for 1 hour. Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0100] 6) The results showed that LNP-circRNA in B16F10 and HEK293 cells MNA There was no significant difference in cell viability among the treatments (0, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL). MNA There was no significant difference in cell proliferation between the treated group and the untreated group (see Figure 4 C). Conclusion: With the LNP-circRNA MNA The increase of concentration and the extension of treatment time did not significantly change the cell viability. MNAThe complex had no effect on the viability of B16F10 cells and HEK293T cells within the experimental dose range (0-250 ng / mL) and time range (0-60 hours), indicating that it has good biocompatibility and safety.

[0101] Example 7: Animal Experiment

[0102] Objective: To evaluate LNP-circRNA in animal experiments MNA The vaccine's immunogenicity, preventive and therapeutic anti-tumor effects verified the safety and efficacy of the new antigen MNA in a mouse melanoma model.

[0103] 1) Immunogenicity study:

[0104] Female C57BL / 6J mice aged 6-8 weeks were randomly divided into LNP-circRNA MNA Group, LNP-circRNA M30 The mice were inoculated with LNP-circRNA by intramuscular injection. MNA Vaccine / LNP-circRNA M30 The vaccine was given to mice in the control group (10 μg / mouse, 100 μL), and the control group mice were inoculated with an equal volume of LNP-PBS. 24 hours after immunization, peripheral blood was collected from mice, and the concentrations of IL-6 and TNF-α in the serum were detected using ELISA kits to evaluate the innate immune response induced by the vaccine. 7 days after immunization, the mice were killed and the spleens were isolated. The specific response of splenocytes to the new antigens was detected by IFN-γ ELISpot. The main organs of the mice, such as the heart, liver, spleen, lung, and kidney, were collected for hematoxylin-eosin (H&E) staining to evaluate the systemic toxicity of the vaccine (see Figure 5 A).

[0105] The results showed that LNP-circRNA MNA The levels of IL-6 and TNF-α in the peripheral blood of the vaccinated mice were significantly increased, indicating that the vaccine can induce a strong innate immune response (see Figure 5 BC). IFN-γ ELISpot assay showed that LNP-circRNA MNA The spleen cells of the vaccinated mice produced a strong specific response to the new antigen, and the number of spots was significantly higher than that of the control group (see Figure 5 D). LNP-circRNA M30 Vaccination can also induce immune response in mice, but vaccination with circRNA MNA Mice vaccinated with circRNA M30 The immune response of mice to the vaccine was more obvious. MNANo obvious pathological changes were observed in the main organs of the mice in the vaccination group, indicating that the vaccine has good safety (see Figure 5 E).

[0106] 2) Preventive tumor experiments:

[0107] Female C57BL / 6J mice aged 6-8 weeks were randomly divided into LNP-circRNA MNA Group, LNP-circRNA M30 The mice were inoculated with LNP-circRNA 2 weeks and 1 week before tumor inoculation. MNA Vaccine / LNP-circRNA M30 The vaccine (10 μg / mouse, 100 μL) was administered to mice in the control group, and the same volume of LNP-PBS was administered to mice in the control group. 5 B16F10 melanoma cells were subcutaneously inoculated into the flank of mice. Starting from 10 days after tumor cell inoculation, the tumor volume was measured every 3 days using a vernier caliper. The calculation formula was: V = length × width × width / 2 (mm 3 After the experiment, the mice were killed and the tumors, spleens and peripheral blood were collected for immunological tests (see Figure 6 A).

[0108] The results showed that LNP-circRNA MNA The tumor volume of mice in the vaccinated group was significantly smaller than that in the control group, indicating that the vaccine can effectively inhibit tumor growth in the preventive mouse tumor model, and the vaccination of circRNA MNA Vaccines vs circRNAs M30 The tumor suppression effect of vaccines is more significant (see Figure 6 BD). However, it had no effect on the body weight of mice (see Figure 6 E).

[0109] Immune response analysis: IFN-γ ELISpot was used to detect the specific response of splenocytes and peripheral blood mononuclear cells (PBMCs) to new antigens. The results showed that in the preventive tumor model, LNP-circRNA MNA The spleen cells and PBMCs of the vaccinated mice responded strongly to the neoantigens, as shown by a significant increase in IFN-γ spots, indicating that MNA induced neoantigen-specific T cell responses (see Figure 7 AB). Analysis of CD4 infiltrating lymphocytes (TILs) and splenocytes by flow cytometry. + and CD8 + The proportion and function of T cells. The results showed that in the preventive tumor model, LNP-circRNA MNACD4 + and CD8 + T cells showed enhanced function, with significantly increased expression of IFN-γ and TNF-α. MNA Tumor infiltrating CD4 + and CD8 + T cells are more abundant (see Figure 7 CD). And inoculation of circRNA MNA Mice vaccinated with circRNA M30 The anti-tumor immune response of mice given the vaccine was more pronounced.

[0110] 3) Therapeutic tumor experiments:

[0111] Female C57BL / 6J mice aged 6-8 weeks were randomly divided into LNP-circRNA MNA Group, LNP-circRNA M30 There were 5 mice in each group. 1×10 5 B16F10 melanoma cells were subcutaneously inoculated into the flank of mice. Five days after tumor inoculation, mice were inoculated with LNP-circRNA by intramuscular injection. MNA Vaccine / LNP-circRNA M30 The vaccine (10 μg / mouse, 100 μL) was administered once a week to strengthen the immune response. The control group mice were inoculated with an equal volume of LNP-PBS. Tumor monitoring: The tumor volume was measured every 3 days using a vernier caliper starting from the 8th day after tumor cell inoculation. The calculation formula was V = length × width × width / 2 (mm 3 After 20 days of the experiment, the mice were killed, and the tumors, spleens, and peripheral blood were collected for antigen-specific T cell analysis by flow cytometry and ELISPOT (see Figure 8 A).

[0112] The results showed that LNP-circRNA MNA The tumor volume of mice in the vaccinated group was significantly smaller than that in the control group, indicating that the vaccine can effectively inhibit tumor growth in the therapeutic mouse tumor model, and the vaccination of circRNA MNA Vaccines vs circRNAs M30 The tumor suppression effect of vaccines is more significant (see Figure 8 BD). However, it had no effect on the body weight of mice (see Figure 8 E).

[0113] Immune response analysis: IFN-γ ELISpot was used to detect the specific response of splenocytes and peripheral blood mononuclear cells (PBMCs) to new antigens. The results showed that in the therapeutic tumor model, LNP-circRNA MNA Splenocytes and PBMCs of vaccinated mice responded strongly to the new antigens (see Fig. 9 AB). Analysis of CD4 infiltrating lymphocytes (TILs) and splenocytes by flow cytometry. + and CD8 + The proportion and function of T cells. The results showed that in the therapeutic tumor model, LNP-circRNA MNA CD4 + and CD8 + T cells showed enhanced function, with significantly increased expression of IFN-γ and TNF-α. MNA Tumor infiltrating CD4 + and CD8 + T cells are more abundant (see Fig. 9 CD). And inoculation of circRNA MNA Mice vaccinated with circRNA M30 The anti-tumor immune response of mice given the vaccine was more pronounced.

[0114] IFN-γ ELISpot assay workflow:

[0115] a. Sample preparation:

[0116] Isolation of spleen cells: Immunized mice were sacrificed and the spleen was removed under sterile conditions. The spleen was ground and filtered through a 70 μm cell sieve to obtain a single cell suspension. Red blood cells were removed using red blood cell lysis buffer, washed twice with PBS, and resuspended in RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin). Splenocytes were counted and the cell concentration was adjusted to 5×10 6 Pieces / mL.

[0117] Peripheral blood mononuclear cells (PBMCs) isolation: Collect peripheral blood from mice and separate PBMCs by density gradient centrifugation using lymphocyte separation fluid (such as Ficoll). Wash twice with PBS and resuspend in RPMI-1640 complete medium. Count PBMCs and adjust the cell concentration to 1×10 6 Pieces / mL.

[0118] b. Use the commercially available Mouse IFN-γ ELISpot detection kit (Dakewei) according to the instructions.

[0119] c. Add 100 μL PBS to each well of the Anti-IFN-γ pre-coated PVDF plate and incubate at room temperature for 10 minutes to activate the membrane surface.

[0120] d. Cell stimulation and incubation: Add 100 μL of spleen cell suspension (5×10 5 cells) or PBMCs suspension (1×10 5 cells). Add MNA antigen peptides at a final concentration of 2 μg / mL to stimulate the cells. Set up a positive control: add PMA (50 ng / mL) and ionomycin (1 μg / mL). Negative control: only add RPMI-1640 complete medium.

[0121] e. Incubate the PVDF plate in a 37°C, 5% CO2 incubator overnight (16-24 hours).

[0122] f. Washing and antibody incubation: Discard the liquid in the wells and wash three times with washing buffer, soaking for 1 minute each time.

[0123] g. Add 100 μL of biotinylated detection antibody (1:1000 dilution) to each well and incubate at room temperature for 1 hour. Discard the detection antibody and wash 3 times.

[0124] h Enzyme labeling and color development: Add 100 μL of streptavidin-HRP solution (1:1000 dilution) to each well and incubate at room temperature for 1 hour. Discard the streptavidin-HRP solution and wash 3 times.

[0125] i Add 100 μL of AEC chromogenic substrate to each well and incubate at room temperature in the dark for 15-30 minutes until the spots are clearly visible.

[0126] j Discard the color development substrate and wash the PVDF plate with distilled water to terminate the reaction.

[0127] k Spot counting: After the PVDF plate is dried, the number of spots in each well is counted using an ELISpot plate reader. Technical support was provided by Beijing Dakewei Biotechnology Co., Ltd to ensure the accuracy and reproducibility of spot counting.

[0128] H&E staining experimental process:

[0129] a. The main organ tissues dissected were fixed with 4% paraformaldehyde and dehydrated with alcohol before being embedded in paraffin and sectioned.

[0130] b. Bake the tissue sections in a 65°C oven for 2 hours to allow the sections to adhere firmly.

[0131] c. Dewaxing and hydration: xylene 2 times, 10 minutes each time (dewaxing); 100% ethanol, 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, distilled water, 5 minutes each time (hydration)

[0132] d. Soak the slices in hematoxylin staining solution for 2 minutes. Then soak them in warm water for 5 minutes to turn blue, and rinse the slices in running water for 30 minutes to make the cell nucleus stain clearer.

[0133] e. Soak the sections in eosin staining solution and stain for 30 seconds.

[0134] f. Dehydration and sealing: Dehydrate the slices in 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol solutions in turn for 5 minutes each time; after drying at room temperature for 10 minutes, add neutral resin to the slices, cover with coverslips, and dry at room temperature. Observe under a microscope and take photos.

[0135] Flow cytometry experimental process:

[0136] a. Collect splenocytes and tumor infiltrating lymphocytes. Splenocytes are used to detect CD4 + and CD8 + The production of IFN-γ and TNF-α by T cells; tumor infiltrating lymphocytes were used to detect CD45 + 、CD3 + 、CD4 + and CD8 + The proportion of T cells.

[0137] b. For spleen cells, BFA (25 ng / mL) was added to block the efflux of cytokines (IFN-γ and TNF-α) before flow cytometry experiments. Splenocytes were plated in 24-well plates (2×10 6 / well) for 5-6 hours and stimulated with 2 μg / mL antigen peptide. Splenocytes treated with PMA and ionomycin were used as positive controls.

[0138] c. Surface marker staining: TILs and splenocytes were incubated with anti-mouse Fc receptor blocker (BD 553141) at room temperature for 5 minutes to block nonspecific binding. The following surface marker antibodies were added and incubated at 4°C in the dark for 30 minutes: anti-CD8 (Alexa Fluor 700, Thermo Fisher 56-0081-82), anti-CD4 (BV605, BD 563151), anti-CD3 (BV786, BD 564010), anti-CD45 (APC-CY7, BD557659). Unbound antibodies were removed by washing with PBS twice.

[0139] d. Intracellular cytokine staining: Permeabilize and fix the cells using the BD Cytofix / Cytoperm kit. Add intracellular cytokine antibodies anti-IFN-γ (FITC, BD 554411) and anti-TNF-α (PerCP-Cy5.5, BD 566510) and incubate at 4°C in the dark for 30 minutes. Wash twice with PBS to remove unbound antibodies.

[0140] e. Data were acquired using a BD FACSCanto II flow cytometer and analyzed using FlowJo software.

Claims

1. An antigen MNA, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1; its nucleotide sequence is shown in SEQ ID NO.

2.

2. The use of the antigen MNA according to claim 1, characterized in that: Used for preparing melanoma vaccine or drug for treating melanoma.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the antigen MNA was cloned into circRNA to construct a circRNA based on circRNA to stably express MNA MNA As a melanoma vaccine or drug for the treatment of melanoma.

4. The use according to claim 3, characterized in that: The vaccine or drug is delivered via lipid nanoparticle encapsulation of circRNA MNA .

5. A melanoma vaccine, characterized in that: The nucleotide sequence of the antigen MNA was cloned into circRNA to construct a circRNA based on circRNA to stably express MNA MNA As a melanoma vaccine, the nucleotide sequence of the antigen MNA is shown in SEQ ID NO.

2.

6. The melanoma vaccine according to claim 5, characterized in that The vaccine delivers circRNA via lipid nanoparticle encapsulation MNA .

7. A drug for treating melanoma, characterized in that: The nucleotide sequence of the antigen MNA was cloned into circRNA to construct a circRNA based on circRNA to stably express MNA MNA As a drug for treating melanoma, the nucleotide sequence of the antigen MNA is shown in SEQ ID NO.

2.

8. The melanoma therapeutic drug according to claim 7, characterized in that: The drug is delivered via lipid nanoparticle encapsulation of circRNA MNA .

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