An antigen MNA and its application, as well as the vaccines and drugs prepared therefrom

Abstract

The present invention discloses an antigen MNA, as well as a vaccine and a drug prepared therefrom, belonging to the technical field of biomedicine. The novel tumor neoantigen (MNA) discovered and designed by the present invention has strong immunogenicity, can effectively activate T cell immune responses, is only expressed in tumor cells, is not affected by central immune tolerance, can accurately target tumor cells, and reduces damage to normal tissues. Moreover, by using circRNA as an expression vector for the neoantigen MNA, it has high stability, high-efficiency expression, induces strong neoantigen-specific T cell responses, and does not require adjuvants. The present invention provides new ideas for the development of tumor neoantigen vaccines and personalized immunotherapy, helps to be extended to more tumor types with high mutation burdens, and has broad clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an antigen MNA, its application, and vaccines and drugs prepared therefrom. Background Art

[0002] In recent years, cancer immunotherapy has become increasingly mature. Cancer immunotherapies represented by immune checkpoint blockade and adoptive cell therapy have shown good progress in clinical trials. However, some patients still do not respond to expensive immune checkpoint drugs, and adoptive cell therapy faces challenges such as poor persistence in the body, difficulty in entering solid tumors, and side effects. To expand treatment options for tumors, it is crucial to develop more applicable new immunotherapies. In addition to immunosuppressive factors in the tumor microenvironment, insufficient 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 the present invention, using B16F10 murine melanoma as a model, a novel tumor neoantigen was discovered and designed, named MNA. The designed neoantigen MNA was cloned into circRNA to construct a melanoma neoantigen vaccine circRNA MNA that can stably express the above-mentioned tumor neoantigen. After being effectively delivered into the body through lipid nanoparticles, its immune response and anti-tumor efficacy were evaluated. The results showed that the circRNA MNA vaccine could effectively inhibit tumor growth and trigger a robust antigen-specific T cell response in both prophylactic and therapeutic murine tumor models.

[0004] Therefore,

[0005] The primary object 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 object of the present invention is to provide the application of the antigen MNA for preparing a melanoma vaccine or a drug for treating melanoma.

[0007] Furthermore,

[0008] it is to clone the nucleotide sequence of the antigen MNA into circRNA to construct a circRNA MNA that stably expresses MNA as a melanoma vaccine or a drug for treating melanoma.

[0009] Even further,

[0010] The vaccine or drug is delivered by lipid nanoparticles encapsulating circRNA MNA .

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

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

[0013] Furthermore, the vaccine is delivered by lipid nanoparticles encapsulating circRNA MNA .

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

[0015] Specifically, the nucleotide sequence of antigen MNA is cloned into circRNA to construct a circRNA that stably expresses MNA based on circRNA MNA as a drug for treating melanoma.

[0016] Furthermore,

[0017] the drug is delivered by lipid nanoparticles encapsulating circRNA MNA .

[0018] The preparation method of the neoantigen MNA of the present invention is provided, and its application effect in anti-tumor immunotherapy is verified. Specifically as follows:

[0019] 1. Synthesize circRNA for expressing neoantigen MNA

[0020] The present invention adopts the group I intron self-catalytic strategy. After cloning the designed neoantigen epitope MNA (MNA: Melanoma neoantigen) into the circRNA vector, the circRNA encoding the neoantigen MNA is generated through in vitro transcription reaction and cyclization, named circRNA MNA . The final product is purified by high performance liquid chromatography. ( Figure 1 )

[0021] The relative sizes of circRNA MNA and its linear precursor are confirmed by denaturing agarose gel RNA electrophoresis. Through the RNase R digestion experiment, it is found that most of the purified circRNA MNA is resistant to RNase R, while the linear precursor is almost completely degraded. Reverse transcription-qPCR, Sanger sequencing, and RNase H-mediated specific cleavage further verify circRNAMNA Circularization. ( Figure 2 )

[0022] The present invention detected the secreted expression of neoantigens in the supernatants of HEK293 and B16F10 cells after transfection of circRNA MNA by ELISA experiment. The above indicates that the circRNA vector constructed by the present invention for expressing neoantigens is stable and can achieve the expression of MNA antigens. In addition, we used the neoantigen epitope M30 in the literature as a reference

[0023] (Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Sebastian Kreiter. Nature volume 520, pages 692–696 (2015), and the neoantigen M30 was reported to have good immunogenicity), and at the same time generated circRNA encoding M30, circRNA M30 , and the secreted expression of M30 in the supernatant was also detected. The antigen expression efficiency of circRNA MNA and circRNA M30 is close. ( Figure 3 )

[0024] Next, LNP was used to encapsulate and deliver circRNA MNA for in vivo experiments. The encapsulation efficiency of circRNA MNA is greater than 97%, and the average diameter is about 82 nm. After co-incubation of the LNP-circRNA MNA complex with cells in vitro, ELISA detection confirmed that circRNA MNA was successfully delivered into cells and expressed neoantigens, and circRNA M30 had the same effect. The biocompatibility of circRNA MNA was also verified by the CCK-8 assay. With the increase of dose and time, the cell viability and proliferation did not change significantly. ( Figure 4 )

[0025] 2. Vaccination with circRNA MNA significantly inhibited tumor growth in mice

[0026] The present invention first detected the stimulating ability of circRNA MNA vaccine to trigger innate and adaptive immune responses. LNP-PBS control, LNP-circRNA MNA vaccine and LNP-circRNA M30Vaccines were intramuscularly injected 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, innate immune responses such as TNF-α and IL-6 secretion were observed in the circRNA MNA vaccine group. To detect the neoantigen-specific T cell response, splenocytes from immunized mice were collected and exposed to the same antigen for IFN-γ ELISpot assay. The results showed that splenocytes from mice vaccinated with circRNA MNA vaccine had a strong response to the neoantigen. In addition, in contrast, although the antigen expression efficiencies of circRNA MNA and circRNA M30 were similar, mice vaccinated with circRNA MNA expressing the novel antigen MNA of the present invention had a more obvious immune response than those vaccinated with circRNA M30 vaccine. The systemic toxicity of the vaccine was evaluated by hematoxylin and eosin (H&E) staining of major organs, and no obvious toxic or side effects were observed. ( Figure 5 )

[0027] To evaluate whether the immune response induced by circRNA MNA vaccine could be translated into anti-tumor effects in mice, the present invention was evaluated using an 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, both the circRNA MNA vaccine group and the circRNA M30 vaccine group significantly inhibited tumor growth. However, the tumors in mice vaccinated with circRNA MNA vaccine grew more slowly than those in mice vaccinated with circRNA M30 vaccine, and the tumor suppression effect was more significant. Vaccination did not change the body weight of the mice. ( Figure 6 )

[0028] The results of IFN-γ ELISpot detection showed that splenocytes and peripheral blood mononuclear cells (PBMCs) from mice vaccinated with circRNA MNA vaccine had a strong response to the neoantigen. By flow cytometry, it was found that, in contrast, in mice immunized with circRNA MNA vaccine, CD4 + and CD8 + T cells showed enhanced function, and the expression of IFN-γ and TNF-α increased most significantly. Compared with control mice, in tumors from mice immunized with circRNA MNA , CD4 + as well as CD8+ T cell infiltration was significantly more abundant. ( Figure 7 )

[0029] Similarly, in the therapeutic model, that is, after transplanting B16F10 cells into C57BL / 6J mice and then inoculating two doses of the vaccine, the same results as above were obtained ( Figures 8 - 9 ). These results demonstrated that the circRNA MNA vaccine could effectively inhibit tumor growth and trigger a robust antigen-specific T cell response.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. The novel tumor neoantigen (MNA) discovered and designed by the present invention showed significant tumor inhibitory effects and strong immune responses in melanoma models, with no obvious toxic side effects. The present invention aims to explore more applicable new immunotherapies and focuses on neoantigens. Neoantigens have strong immunogenicity and can effectively activate T cell immune responses; and are only expressed in tumor cells, not affected by central immune tolerance, can precisely target tumor cells, and reduce damage to normal tissues.

[0032] 2. The present invention uses circRNA as an expression vector for the neoantigen MNA. The advantages of the circRNA vaccine form are: high stability; efficient expression, inducing strong antigen-specific T cell responses; no need for adjuvants, as circRNA itself has immunostimulatory effects and can be used as a self-adjuvant to enhance the vaccine effect, simplifying the vaccine preparation process.

[0033] 3. The neoantigen MNA showed dual effects in prophylactic and therapeutic models of murine B16F10 melanoma, with a significant reduction in tumor volume. And compared with the reported neoantigen M30, the tumor inhibitory effect of the neoantigen MNA is more significant.

[0034] 4. The present invention provides new ideas for the development of tumor neoantigen vaccines and personalized immunotherapy, helps to be extended to more tumor types with high mutation burdens, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : The synthesis process of circRNA expressing the tumor neoantigen of the present invention and the detection results of the products;

[0036] A, Schematic diagram of the circularization of circRNA MNA through the self-catalysis of group I introns; B, Results of the purification of the final product by liquid chromatography.

[0037] Figure 2 : Characterizing the circRNA of the present invention MNAResults of the circular structure;

[0038] A. Detection of linear precursor RNA and circRNA by denaturing agarose gel RNA electrophoresis MNA Results of the size; B. Linear precursor RNA and circRNA MNA Results of denaturing agarose gel RNA electrophoresis after treatment with RNase R; C. DNA gel electrophoresis showing the results of PCR analysis; D. Splicing sites of circRNA shown by Sanger sequencing results; E. Schematic diagram of RNase H detection. MNA

[0039] Figure 3 : Detection of circRNA MNA Results of the secreted expression of neoantigens in the supernatant after transfection of HEK293 and B16F10 cells with Hiperfect.

[0040] Figure 4 : LNP-circRNA MNA Results of the characterization, expression and safety evaluation of the complex;

[0041] A. Representative intensity-size distribution plot of LNP-circRNA MNA obtained by dynamic light scattering; B. Expression results of neoantigens detected in the supernatant after co-incubation of LNP-circRNA MNA complex with HEK293 and B16F10 cells measured by ELISA; C. Changes in cell viability determined by CCK-8 assay with increasing dose of LNP-circRNA MNA complex; D. Changes in cell proliferation determined by CCK-8 assay with the prolongation of the action time of LNP-circRNA MNA complex.

[0042] Figure 5 : circRNA MNA Results of the induction of innate and adaptive immune responses by the vaccine;

[0043] A. Experimental timeline for evaluating the immune response induced by circRNA MNA vaccine (10 µg circRNA per mouse); B. Secretion results of cytokine TNF-α in peripheral blood after vaccination with circRNA MNA vaccine measured by ELISA, , ; C. Secretion results of cytokine IL-6 in peripheral blood after vaccination with circRNA MNA vaccine measured by ELISA, ,​ ; D, Representative pictures and statistical results of IFN-γ spot formation in splenocytes after restimulation measured by IFN-γ ELISpot, , ; E, H&E staining results of major organs in each group, scale bar, 200 μm.

[0044] Figure 6 : circRNA MNA vaccine significantly inhibits tumor growth in prophylactic mouse tumor models;

[0045] A, Schematic diagram of the immunization strategy of the prophylactic mouse tumor model and tumor volume monitoring after inoculation with B16F10 cells; B, Representative pictures of subcutaneous melanoma tissues dissected 22 days after vaccination in the prophylactic mouse model (n = 5 per group); C, Tumor volume growth curves of transplanted tumor mice after vaccination with the vaccine in the prophylactic mouse model, , , ; D, Statistical results of the weights of tumor tissues of transplanted tumor mice after vaccination with the vaccine in the prophylactic mouse model, , ; E, Body weight change curves of transplanted tumor mice after vaccination with the vaccine in the prophylactic mouse model.

[0046] Figure 7 : In the prophylactic mouse tumor model, circRNA MNA vaccine induces robust antigen-specific T cell responses;

[0047] A, Pictures and statistical results of IFN-γ spot formation in splenocytes measured by IFN-γ ELISpot in the prophylactic mouse model, , , ; B, Pictures and statistical results of IFN-γ spot formation in PBMCs measured by IFN-γ ELISpot in the prophylactic mouse model, , ; C, IFN-γ + and TNF-α + CD4 + and CD8 + T cell percentage results in restimulated splenocytes measured by flow cytometry in the prophylactic mouse model, , , , ; D, Proportion results of infiltrating immune cells in subcutaneous B16F10 tumors in control and circRNA MNA vaccine-immunized mice detected by flow cytometry in the prophylactic mouse model, , 。

[0048] Figure 8 : circRNA MNA vaccine significantly inhibits tumor growth in a therapeutic mouse tumor model;

[0049] A, Schematic diagram of the immunization strategy of the therapeutic mouse tumor model and tumor volume monitoring after inoculation with B16F10 cells; B, Representative pictures of subcutaneous melanoma tissues obtained by dissection after 20 d in the therapeutic mouse model (n = 5 per group); C, Tumor volume growth curve of tumor-bearing mice after vaccination in the therapeutic mouse model, , , ; D, Statistical results of the weights of tumor tissues of tumor-bearing mice after vaccination in the therapeutic mouse model, , , ; E, Body weight change curve of tumor-bearing mice after vaccination in the therapeutic mouse model.

[0050] Figure 9 : In the therapeutic mouse tumor model, circRNA MNA vaccine induces robust antigen-specific T cell responses;

[0051] A, IFN-γ spot formation pictures and statistical results in restimulated splenocytes measured by IFN-γ ELISpot in the therapeutic mouse model, ; B, IFN-γ spot formation pictures and statistical results in PBMC measured by IFN-γ ELISpot in the therapeutic mouse model, , ; C, IFN-γ + and TNF-α + in restimulated splenocytes measured by flow cytometry in the therapeutic mouse model + and CD8 + T cell percentage results, , , , ; D, Proportion results of infiltrating immune cells in subcutaneous B16F10 tumors detected by flow cytometry in control and circRNA MNA vaccine-immunized therapeutic mouse models, , , 。 Detailed implementation manners

[0052] The technical solution of the present invention will be further described below through specific embodiments. The specific embodiments do not represent a limitation on 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.

[0053] The experimental mice used in the present invention were 6-8-week-old female C57BL / 6J mice, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., and fostered in the Experimental Animal Department of Central South University (Changsha, Hunan), in a SPF-level 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 were 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 Institute of Life 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.

[0054] The melanoma neoantigen MNA polypeptide, the neoantigen nucleotide sequence, and the circRNA backbone (SEQ ID NO. 3) of the present invention were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0055] The test results of the present invention were all analyzed statistically: the t-test was used to evaluate the differences between two groups. p < 0.05 was used to indicate statistical significance, and all p-values were tested two-sided. All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0 software.

[0056] Example 1: circRNA MNA Production and purification for expressing the MNA antigen

[0057] The self-splicing and circularization strategy based on group I intron ribozyme (also known as the permuted intron exon (PIE) system) is a commonly used technique for in vitro synthesis of circRNA. Its principle is to add homologous arms at both ends of the sequence, insert exon E1 and E2, spacer, IRES, and the coding sequence between the 3'intron and 5'intron sequences, synthesize PrecursorRNA through in vitro transcription reaction, and then in Mg 2+Two transesterification reactions occur with the assistance of GTP to form circRNA. We synthesized and cloned the T7 promoter, homology arms, elements from the replacement intron-exon construct, spacer, CVB3 IRES, MNA neoantigen sequence, and Flag tag sequence from Sangon Biotech (Shanghai) Co., Ltd. into a plasmid (SEQ ID NO.3) for generating the circRNA backbone and subsequent in vitro transcription reactions. Then, the plasmid was linearized by PCR amplification or digestion with enzymes, and the product was purified by agarose gel electrophoresis and gel extraction. After sequencing verification, it was used as a template for in vitro transcription, and circRNA was synthesized using the HiScribe™ T7 HighYield RNA Synthesis Kit according to the manufacturer's protocol. MNA Precursor. For the in vitro transcription reaction, we optimized and adjusted to determine the appropriate T7 RNA polymerase concentration (7.5 U / μL), rNTP concentration (7.5 mM), and in vitro transcription reaction time (16 h) to maximize the 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 then the reaction mixture was incubated at 55 °C for 15 minutes to catalyze the cyclization of circRNA. Then, the column-purified RNA was heated at 65 °C for 3 min and cooled on ice for 3 min. The reaction mixture 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 5 µm and a pore size of 2000 Å was used and run at a flow rate of 0.8 mL / minute in RNase-free TE buffer. The fractions rich in circRNA MNA were collected, 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 high-purity circRNA MNA ), for expressing the MNA antigen.

[0058] Example 2: Denaturing agarose gel RNA electrophoresis

[0059] 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).

[0060] Example 3: RNase R digestion experiment, reverse transcription-qPCR, Sanger sequencing, and RNase H cleavage experiment prove circRNA MNA The ring form

[0061] 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).

[0062] 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 (seeFigure 2 C).

[0063] The sequence of primer F1 is: 5’-GAGATCATAGTCAGCCTCCTCT-3’

[0064] The sequence of primer R1 is: 5’-TGGAGGGACTTGAACCCACA-3’

[0065] The sequence of primer F2 is: 5’-GTTCAAGTCCCTCCACCCC-3’

[0066] The sequence of primer R2 is: 5’-AAACAGGCGCACAAAGGTAC-3’

[0067] For Sanger sequencing, the qPCR products of the circularization site region of circRNA MNA were recovered and sent to Qingke Company for Sanger sequencing. The sequences returned by the company were aligned using DNASTAR software, and the peak maps were viewed with chromas software to judge the quality of the sequencing. The alignment results showed that the sequence of the circularization site of circRNA MNA was completely consistent with the designed sequence, and circRNA MNA formed a covalently closed circular structure (see Figure 2 D).

[0068] For RNase H cleavage, 500 ng of purified circRNA MNA and its linear precursor, 50 pmol of sense or antisense ssDNA probe (the antisense ssDNA probe is complementary to specific regions of circRNA MNA and linear precursor RNA, and the sense probe is used as a control), and RNase H buffer were incubated in a total volume of 18 μL at 50 °C for 10 minutes. Then, 2 μL of RNase H was added to the reaction, gently mixed, and incubated at 37 °C for 1 hour. The RNA was analyzed by agarose gel electrophoresis. RNase H is an endoribonuclease that can specifically degrade the RNA phosphodiester bond hybridized to the DNA strand, so it can cleave the RNA strand in the RNA / DNA hybrid strand. The results showed that after the linear precursor RNA was hybridized with the antisense probe and treated with RNase H, the RNA was cleaved into two short fragments, and multiple electrophoretic bands appeared. After circRNA MNA was hybridized with the antisense probe and treated with RNase H, circRNA MNA was cleaved into a linear form with the same size, and the electrophoretic bands remained consistent (see Figure 2 E).

[0069] The sequence of the antisense ssDNA probe is: 5’-GAGTTCAGCTCTGGA-3’

[0070] The sequence of the sense ssDNA probe is: 5'-TCCAGAGCTGAACTC-3'.

[0071] Example 4: ELISA assay proves that circRNA MNA can effectively express the MNA antigen

[0072] 1) HEK293 cells and B16F10 melanoma cells were respectively seeded in 6-well plates 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%.

[0073] 2) The circRNA was transfected into HEK293 cells and B16F10 cells using a liposome transfection reagent (such as Hiperfect), or the circRNA was delivered into the cells through LNP. An untreated group and a linear precursor group were set as controls. After treatment, the cells were cultured for another 48 hours, and the cell supernatants were collected for ELISA detection. MNA MNA

[0074] 3) A Flag-tag ELISA detection kit (the Flag sequence was added during the synthesis of circRNA MNA to express the Flag-tagged MNA fusion protein for easy detection) was used and operated according to the instructions.

[0075] 4) The cell supernatants and standards were diluted at appropriate ratios and then added to a 96-well plate pre-coated with anti-Flag monoclonal antibody, 50 μL of sample was added to each well, and incubated at 37°C for 1 hour.

[0076] 5) The liquid in the wells was discarded and washed 5 times with washing buffer, each time soaked for 1 minute.

[0077] 6) 50 μL of enzyme-labeled reagent (HRP-labeled secondary antibody) was added to each well and incubated at 37°C for 1 hour.

[0078] 7) The liquid in the wells was discarded and washed 5 times.

[0079] 8) 100 μL of TMB chromogenic solution was added to each well and incubated at 37°C in the dark for 15 minutes.

[0080] 9) 50 μL of stop solution was added, gently mixed, and immediately the absorbance (OD value) was measured at 450 nm wavelength using an enzyme-linked immunosorbent assay reader.

[0081] 10) Data analysis: Zero the blank wells, plot the standard curve based on the OD values of the standards, and calculate the concentration of MNA antigen in the cell supernatant. Compare the expression levels of MNA antigen in the circRNA MNA treatment group with those in the untreated group and the linear precursor group to evaluate the expression efficiency of circRNA MNA .

[0082] 11) Experimental results: In the circRNA MNA treatment group, 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 the linear precursor group, and the OD values were close to the background values.

[0083] 12) Experimental conclusion: The ELISA assay results showed that circRNA MNA could be effectively expressed and secrete MNA antigen in HEK293 cells and B16F10 cells, verifying the functionality and reliability of circRNA MNA as a vaccine vector (see Figure 3 ). Using the novel antigen epitope M30 in the literature as a reference, circRNA encoding M30 was simultaneously generated, and circRNA M30 also detected the secreted expression of M30 in the supernatant. The antigen expression efficiencies of circRNA MNA and circRNA M30 were close.

[0084] Example 5: Encapsulation of circRNA MNA with LNP for in vivo experiments

[0085] Experimental purpose: To verify the efficiency and stability of encapsulating circRNA MNA with lipid nanoparticles (LNP) and evaluate its feasibility as a vaccine delivery system for subsequent in vivo experiments.

[0086] Preparation of LNP-circRNA MNA :

[0087] 1) Preparation of the lipid phase: Mix ALC-0315, DSPC, cholesterol, and ALC-0159 in a molar ratio of 50:10:38.5:1.5 and dissolve in anhydrous ethanol to form the lipid phase. The functions of the components in the lipid phase: ALC-0315: ionizable lipid for encapsulating RNA; DSPC: helper lipid to enhance the stability of LNP; cholesterol: regulate the fluidity and stability of LNP; ALC-0159: PEGylated lipid to prevent LNP aggregation and prolong its blood circulation time.

[0088] 2) Preparation of the aqueous phase: Dissolve circRNAMNA Dissolve it in 100 mM citrate buffer (pH 4.0) to prepare a solution with an N / P ratio of 6.

[0089] 3) Microfluidic mixing: Use a microfluidic mixing device to mix the lipid phase and the aqueous phase at a volume ratio of 1:3 to prepare LNP-circRNA MNA complexes. Induce fluid folding in the laminar flow state through chaotic mixing characteristics to obtain uniformly and highly encapsulated LNPs in a controllable and reproducible manner.

[0090] 4) Dialysis purification: Dialyze the LNP-circRNA MNA complexes overnight at 4 °C with PBS to remove unencapsulated circRNA MNA and ethanol. After dialysis, store the LNP-circRNA MNA complexes in 10% sucrose-PBS and store them at 4 °C for later use.

[0091] Characterization of LNP-circRNA MNA :

[0092] 1) Particle size measurement: Use a dynamic light scattering instrument to measure the particle size distribution of LNP-circRNA MNA . The results show that the average particle size of LNP-circRNA MNA is 82 nm, indicating that it has a good particle size distribution (see Figure 4 A).

[0093] 2) Encapsulation efficiency measurement: Use the Quant-iT RiboGreen RNA Assay Kit to measure the total circRNA MNA concentration and the concentration of unencapsulated circRNA MNA respectively. The results show that the encapsulation efficiency of LNP-circRNA MNA is greater than 97%, indicating that it has a high encapsulation efficiency (see Figure 4 A).

[0094] 3) Subsequently, detect the expression of MNA antigen by ELISA (see Figure 4 B). The results show that circRNA MNA is successfully delivered into cells and expresses neoantigens. The circRNA encapsulated by LNP M30 has a consistent effect, and the antigen expression efficiencies of LNP-circRNA MNA and LNP-circRNA M30 are close.

[0095] Example 6: CCK8 cell viability assay proves the safety of LNP-circRNA MNA ​

[0096] 1) Experimental purpose: To evaluate the effect of the LNP-circRNA MNA complex on cell viability / cell proliferation by CCK-8 assay, and to verify its biocompatibility and safety.

[0097] 2) Cell culture: B16F10 melanoma cells and HEK293 cells were respectively seeded into 96-well plates 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.

[0098] 3) Dose-dependent experiment: Different concentrations of the LNP-circRNA MNA complex (0, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL) were added to the 96-well plates, with 5 replicates for each concentration. The cells were co-incubated with the LNP-circRNA MNA complex at 37 °C and 5% CO2 for 24 hours.

[0099] 4) Time-dependent experiment: 100 ng of the LNP-circRNA MNA complex was added to the cells and incubated for 0, 12 hours, 24 hours, 36 hours, 48 hours, and 60 hours respectively. 5 replicates were set for each time point, and an untreated group was set as the control.

[0100] 5) CCK-8 assay: After the incubation, 10 μL of CCK-8 reagent was added to each well. The 96-well plates were further incubated at 37 °C and 5% CO2 for 1 hour. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader.

[0101] 6) Results showed that there were no significant differences in cell viability among the cells treated with LNP-circRNA MNA at concentrations of 0, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, and 250 ng / mL in B16F10 and HEK293 cells. There were also no significant differences in cell proliferation between the LNP-circRNA MNA treatment group and the untreated group at different incubation times (0, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours) (see Figure 4 C). Conclusion: With the increase in the concentration of LNP-circRNA MNA and the prolongation of the treatment time, there was no obvious change in cell viability. LNP-circRNA MNAThe complex had no effect on the viability of B16F10 cells and HEK293T cells within the experimental dose range (0 - 250 ng / mL) and experimental time range (0 - 60 hours), indicating its good biocompatibility and safety.

[0102] Example 7: Animal Experiment

[0103] Experimental purpose: To evaluate the immunogenicity, prophylactic and therapeutic anti-tumor effects of LNP-circRNA MNA vaccine, and to verify the safety and effectiveness of the neoantigen MNA in a murine melanoma model.

[0104] 1) Immunogenicity study:

[0105] Female C57BL / 6J mice aged 6 - 8 weeks were selected and randomly divided into the LNP-circRNA MNA group, the LNP-circRNA M30 group and the control group LNP-PBS, with 5 mice in each group. The mice were inoculated with LNP-circRNA MNA vaccine / LNP-circRNA M30 vaccine (10 μg / mouse, 100 μL) by intramuscular injection. The mice in the control group were inoculated with an equal volume of LNP-PBS. At 24 hours after immunization, peripheral blood of the mice was collected, and ELISA kits were used to detect the concentrations of IL-6 and TNF-α in the serum to evaluate the innate immune response induced by the vaccine. At 7 days after immunization, the mice were sacrificed and the spleens were isolated. The specific response of splenocytes to the neoantigen was detected by IFN-γ ELISpot. The main organs of the mice such as the heart, liver, spleen, lung, and kidney were collected and stained with hematoxylin and eosin (H&E) to evaluate the systemic toxicity of the vaccine (see Figure 5 A).

[0106] The results showed that the levels of IL-6 and TNF-α in the peripheral blood of the mice in the LNP-circRNA MNA vaccine inoculation group were significantly increased, indicating that the vaccine could induce a strong innate immune response (see Figure 5 B - C). The IFN-γ ELISpot detection showed that the splenocytes of the mice in the LNP-circRNA MNA vaccine inoculation group had a strong specific response to the neoantigen, and the number of spots was significantly higher than that of the control group (see Figure 5 D). The inoculation of LNP-circRNA M30 vaccine could also cause an immune response in mice, but the immune response of the mice inoculated with circRNA MNA vaccine was more obvious than that of the mice inoculated with circRNA M30 vaccine. The inoculation of LNP-circRNA MNANo obvious pathological changes were observed in the major organs of the vaccinated mice, indicating that the vaccine has good safety (see Figure 5 E).

[0107] 2) Prophylactic tumor experiment:

[0108] Female C57BL / 6J mice at 6 - 8 weeks of age were randomly divided into the LNP-circRNA MNA group, the LNP-circRNA M30 group, and the control group LNP-PBS, with 5 mice in each group. Two weeks and one week before tumor inoculation, the mice were inoculated with LNP-circRNA MNA vaccine / LNP-circRNA M30 vaccine (10 μg / mouse, 100 μL), and the mice in the control group were inoculated with an equal volume of LNP-PBS. After immunization, 1×10 5 B16F10 melanoma cells were subcutaneously inoculated into the flanks of the mice. Starting from 10 days after tumor cell inoculation, the tumor volume was measured every 3 days using a vernier caliper, and the calculation formula was: V = length × width × width / 2 (mm 3 ). After the experiment, the mice were sacrificed, and the tumors, spleens, and peripheral blood were collected for immunological tests (see Figure 6 A).

[0109] The results showed that the tumor volume of the mice in the LNP-circRNA MNA vaccine group was significantly smaller than that of the control group, indicating that the vaccine could effectively inhibit tumor growth in the prophylactic mouse tumor model, and the anti-tumor effect of inoculating the circRNA MNA vaccine was more significant than that of inoculating the circRNA M30 vaccine (see Figure 6 B - D). However, it had no effect on the body weight of the mice (see Figure 6 E).

[0110] Analysis of immune response: The specific response of splenocytes and peripheral blood mononuclear cells (PBMCs) to neoantigens was detected by IFN-γ ELISpot. The results showed that in the prophylactic tumor model, the splenocytes and PBMCs of the mice in the LNP-circRNA MNA vaccine group had a strong response to neoantigens, manifested as a significant increase in IFN-γ spots, indicating that MNA induced a neoantigen-specific T cell response (see Figure 7 A - B). The proportion and function of CD4 + and CD8 + T cells in tumor-infiltrating lymphocytes (TILs) and splenocytes were analyzed by flow cytometry. The results showed that in the prophylactic tumor model, the LNP-circRNA MNAIn the vaccinated group, mouse CD4 + and CD8 + T cells showed enhanced function and significantly increased IFN-γ and TNF-α expression. In the LNP-circRNA MNA vaccinated group, the proportion of tumor-infiltrating CD4 + and CD8 + T cells in mice was more abundant (see Figure 7 C-D). Moreover, the anti-tumor immune response of mice vaccinated with circRNA MNA vaccine was more obvious than that of mice vaccinated with circRNA M30 vaccine.

[0111] 3) Therapeutic tumor experiment:

[0112] Female C57BL / 6J mice aged 6-8 weeks were selected and randomly divided into the LNP-circRNA MNA group, the LNP-circRNA M30 group and the control group LNP-PBS, with 5 mice in each group. 1×10 5 B16F10 melanoma cells were subcutaneously inoculated into the flanks of the mice. Five days after tumor inoculation, the mice were inoculated with LNP-circRNA MNA vaccine / LNP-circRNA M30 vaccine (10 μg / mouse, 100 μL) by intramuscular injection, and a second dose was given 1 week later to boost the immune response. The control group mice were inoculated with an equal volume of LNP-PBS. Tumor monitoring: From the 8th day after tumor cell inoculation, the tumor volume was measured every 3 days using a vernier caliper, and the calculation formula was V = length × width × width / 2 (mm 3 ). After 20 days of the experiment, the mice were sacrificed, and tumors, spleens and peripheral blood were collected for antigen-specific T cell analysis by flow cytometry and ELISPOT (see Figure 8 A).

[0113] The results showed that the tumor volume of mice in the LNP-circRNA MNA vaccinated group was significantly smaller than that of the control group, indicating that the vaccine could effectively inhibit tumor growth in the therapeutic mouse tumor model. Moreover, the tumor inhibitory effect of mice vaccinated with circRNA MNA vaccine was more significant than that of mice vaccinated with circRNA M30 vaccine (see Figure 8 B-D). However, it had no effect on the body weight of the mice (see Figure 8 E).

[0114] Immune response analysis: The specific responses of splenocytes and peripheral blood mononuclear cells (PBMCs) to neoantigens were detected by IFN-γ ELISpot. The results showed that in the therapeutic tumor model, LNP-circRNA MNA vaccination group, the splenocytes of mice and PBMCs showed strong responses to neoantigens (see Figure 9 A - B). The proportions and functions of CD4 + and CD8 + T cells in tumor-infiltrating lymphocytes (TILs) and splenocytes were analyzed by flow cytometry. The results showed that in the therapeutic tumor model, LNP-circRNA MNA vaccination group, the CD4 + and CD8 + T cells showed enhanced functions, and the expressions of IFN-γ and TNF-α were significantly increased. In the LNP-circRNA MNA vaccination group, the proportions of tumor-infiltrating CD4 + and CD8 + T cells were more abundant (see Figure 9 C - D). Moreover, the anti-tumor immune response of mice vaccinated with circRNA MNA vaccine was more obvious than that of mice vaccinated with circRNA M30 vaccine.

[0115] IFN-γ ELISpot experimental procedure:

[0116] a. Sample preparation:

[0117] Isolation of splenocytes: The immunized mice were sacrificed, and the spleens were removed under sterile conditions. The spleens were ground and filtered through a 70 μm cell strainer to obtain a single-cell suspension. Red blood cells were removed using red blood cell lysis buffer and washed twice with PBS, then resuspended in RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin). The splenocytes were counted and the cell concentration was adjusted to 5×10 6 cells / mL.

[0118] Isolation of peripheral blood mononuclear cells (PBMCs): The peripheral blood of mice was collected, and PBMCs were isolated by density gradient centrifugation using lymphocyte separation medium (such as Ficoll). They were washed twice with PBS and resuspended in RPMI-1640 complete medium. The PBMCs were counted and the cell concentration was adjusted to 1×10 6 cells / mL.

[0119] b. Use a commercially available Mouse IFN-γ ELISpot detection kit (Dakewei) and operate according to the instructions.

[0120] c. Add 100 μL of 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.

[0121] d. Cell stimulation and incubation: Add 100 μL of splenocyte suspension (5×10 5 cells) or PBMCs suspension (1×10 5 cells) to each well. Stimulate the cells with MNA antigen peptide at a final concentration of 2 μg / mL. Set up a positive control: add PMA (50 ng / mL) and ionomycin (1 μg / mL). Negative control: add only RPMI-1640 complete medium.

[0122] e. Incubate the PVDF plate in a 37°C, 5% CO2 incubator overnight (16 - 24 hours).

[0123] f. Washing and antibody incubation: Discard the liquid in the wells and wash 3 times with washing buffer, soaking for 1 minute each time.

[0124] g. Add 100 μL of biotinylated detection antibody (diluted 1:1000) to each well and incubate at room temperature for 1 hour. Discard the detection antibody and wash 3 times.

[0125] h. Enzyme-labeled reaction and color development: Add 100 μL of streptavidin-HRP solution (diluted 1:1000) to each well and incubate at room temperature for 1 hour. Discard the streptavidin-HRP solution and wash 3 times.

[0126] i. Add 100 μL of AEC color development substrate to each well and incubate at room temperature in the dark for 15 - 30 minutes until the spots are clearly visible.

[0127] j. Discard the color development substrate and wash the PVDF plate with distilled water to terminate the reaction.

[0128] k. Spot counting: After drying the PVDF plate, use an ELISpot reader to count the number of spots in each well. Technical support is provided by Beijing Dakewei Biotechnology Co., Ltd to ensure the accuracy and reproducibility of spot counting.

[0129] H&E staining experimental procedure:

[0130] a. After fixing the dissected main organ tissues with 4% paraformaldehyde and dehydrating with alcohol, perform paraffin embedding and sectioning.

[0131] b. Bake the tissue sections in an oven at 65°C for 2 hours to firmly attach the sections.

[0132] c. Dewaxing and hydration: twice in xylene for 10 minutes each (dewaxing); in 100% ethanol, 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, and distilled water for 5 minutes each (hydration).

[0133] d. Immerse the sections in hematoxylin staining solution for 2 minutes. Then immerse in warm water for 5 minutes to blue the sections, and rinse the sections with running water for 30 minutes to make the nuclear staining clearer.

[0134] e. Immerse the sections in eosin staining solution for 30 seconds.

[0135] f. Dehydration and mounting: Immerse the sections successively in 50% ethanol, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol solutions for dehydration for 5 minutes each; after drying at room temperature for 10 minutes, drop neutral resin on the sections, cover with a coverslip, and air dry at room temperature. Observe and photograph under a microscope.

[0136] Flow cytometry experimental procedure:

[0137] a. Collect splenocytes and tumor-infiltrating lymphocytes. Splenocytes are used to detect the production of IFN-γ and TNF-α by CD4 + and CD8 + T cells; tumor-infiltrating lymphocytes are used to detect the proportions of CD45 + 、CD3 + 、CD4 + and CD8 + T cells infiltrating in each group of tumors.

[0138] b. For splenocytes, add BFA (25 ng / mL) before flow cytometry experiment to block the exocytosis of cytokines (IFN-γ and TNF-α). Culture the splenocytes in a 24-well plate (2×10 6 / well) for 5 - 6 hours and stimulate with 2 μg / mL antigen peptide. Use splenocytes treated with PMA and ionomycin as positive controls.

[0139] c. Surface marker staining: Incubate TILs and splenocytes with anti-mouse Fc receptor blocker (BD 553141) at room temperature for 5 minutes to block non-specific binding. Add the following surface marker antibodies and incubate in the dark at 4°C 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). Wash twice with PBS to remove unbound antibodies.

[0140] d. Intracellular cytokine staining: Permeabilize and fix the cells using the BD Cytofix / Cytoperm kit. Add the intracellular cytokine antibodies anti-IFN-γ (FITC, BD 554411) and anti-TNF-α (PerCP-Cy5.5, BD 566510) and incubate in the dark at 4°C for 30 minutes. Wash twice with PBS to remove unbound antibodies.

[0141] e. Collect data using a BD FACSCanto II flow cytometer and analyze the data 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; the melanoma vaccine or drug for treating melanoma is to clone the nucleotide sequence of antigen MNA into circRNA, construct circRNAMNA based on circRNA for stably expressing MNA; and then deliver circRNAMNA by encapsulating lipid nanoparticles.

3. A melanoma vaccine, characterized in that: The nucleotide sequence of the antigen MNA is cloned into circRNA, and a circRNAMNA based on circRNA that stably expresses MNA is constructed as a melanoma vaccine. The nucleotide sequence of the antigen MNA is shown in SEQ ID NO.2; the vaccine is delivered by encapsulating the circRNAMNA in lipid nanoparticles.

4. A drug for treating melanoma, characterized in that: The nucleotide sequence of the antigen MNA is cloned into circRNA, and a circRNAMNA based on circRNA that stably expresses MNA is constructed as a drug for treating melanoma. The nucleotide sequence of the antigen MNA is shown in SEQ ID NO.2; the drug is delivered by lipid nanoparticle encapsulation of circRNAMNA.

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