Broad-spectrum anti-tumor percutaneous immune mRNA vaccine and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a broad-spectrum anti-tumor percutaneous immune mRNA vaccine and a preparation method thereof. The broad-spectrum anti-tumor percutaneous immune mRNA vaccine comprises a gene sequence of an antigen shared by induced pluripotent stem cells and tumors, a medicinal carrier of the broad-spectrum anti-tumor percutaneous immune mRNA vaccine is dendritic cell targeting lipid nanoparticles, and mRNA molecules are encapsulated in the lipid nanoparticles. The shared antigen comprises an antigen with an amino acid sequence of SEQ NO: 1-14. The mRNA percutaneous immune vaccine with broad-spectrum tumor prevention is constructed based on the tumor cell-iPSC shared new antigen, a more ideal application effect is expected to be achieved, and the mRNA percutaneous immune vaccine is convenient and safe to use, simple in process and beneficial to popularization.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and more specifically, to a broad-spectrum anti-tumor transdermal immunization mRNA vaccine based on antigens shared by induced pluripotent stem cells (iPSCs) and tumors and a preparation method thereof. Background Art

[0002] The research and development of human tumor vaccines has continued for more than 120 years. The basic principle of tumor vaccines is to utilize specific antigens to activate the immune system's ability to recognize and kill tumor cells, including prophylactic tumor vaccines and therapeutic tumor vaccines. Therapeutic tumor vaccines induce the production of specific antibodies, effector cells, and specific immune memory cells that can eliminate tumors in tumor patients.

[0003] The stimulation of tumor antigens can increase the diversity and breadth of T cell responses. The selection of specific antigens is the key to constructing an effective vaccine. Currently, the research and development of tumor vaccines, especially broad-spectrum tumor vaccines, mainly face obstacles such as insufficient specific targeting ability, low immunogenicity, and lack of antigen specificity among different patients and different tumor tissues. Given the extremely complexity of tumorigenesis factors, developing a broad-spectrum prophylactic tumor vaccine starting from the commonalities of tumor cells may be a more effective strategy. Existing research teams have shown that certain antigen components are shared among different cancer cells. By using whole cell component vaccines of tumor tissues to induce a broad-spectrum immune response against cancer cells, it can play a preventive and therapeutic role in melanoma, lung cancer, and liver cancer.

[0004] The principle of mRNA vaccines is to deliver mRNA molecules encoding antigens (usually pathogens / cancer cells) to antigen-presenting cells through a carrier. The antigen-presenting cells (APCs) translate the mRNA into proteins, thereby stimulating the body to produce a specific immune response against the antigen, recognizing and eliminating pathogens or tumor cells. mRNA vaccines have great advantages in terms of safety and production efficiency. Compared with DNA vaccines and virus vector-based vaccines, mRNA vaccines do not need to enter the nucleus to exert their effects and do not pose a risk of genomic integration and insertional mutagenesis. Compared with live attenuated vaccines and inactivated vaccines, mRNA vaccines eliminate any concerns related to endotoxins and infections. Compared with peptide vaccines, mRNA vaccines can encode full-length tumor antigens, enabling APCs to present multiple epitopes of class I and II specific HLAs simultaneously or cross-present. The natural property of the short-lived mRNA can avoid the continuous expression of the corresponding protein, thereby better controlling their transient activity and not generating anti-vector immune resistance, and can be repeatedly inoculated. Since mRNA vaccines only provide the necessary antigens, they promote the development of more antigen-specific adaptive immunity compared with whole cell vaccines, greatly reducing the probability of adverse reactions.

[0005] Current vaccination is mainly administered through routes such as intramuscular, subcutaneous, intradermal intrasegmental, and intravenous injection. Although needle vaccination can effectively achieve the desired immune response, there are still certain risks in terms of safety and compliance. Compared with needle vaccination, the new vaccine delivery method provided by the new type of vaccine can provide new technologies and methods. Compared with traditional vaccines that utilize the skin, transdermal immunization has the following significant advantages: 1. Painless. It improves the recipient's compliance, which is particularly important for the success of the childhood immunization program. 2. Convenient. The recipient can operate by themselves, thus reducing the dependence on clinical professionals and saving costs. During the epidemic, this has important practical significance. 3. Safe. It can avoid the infection risk of needle injection, and is particularly suitable for people such as diabetic patients who need to avoid wound infection as much as possible. In addition, transdermal immunization can bypass the first-pass effect of the liver, avoid the irritation or destruction of the drug to the gastrointestinal tract, can reduce the fluctuation of blood drug concentration, and reduce toxic and side reactions. Therefore, transdermal immunization is considered a new generation of immunization means to replace injection and oral vaccination, which is conducive to improving the coverage rate and safety of vaccination.

[0006] Based on the above, the development of a broad-spectrum anti-tumor transdermal immunization mRNA vaccine is of great significance for tumor prevention. Summary of the Invention

[0007] The object of the present invention is to provide a broad-spectrum anti-tumor transdermal immunization mRNA vaccine, which administers immune antigens in the form of mRNA drugs to stimulate specific immunity in the body and achieve the purpose of tumor prevention.

[0008] Due to the high similarity between tumor cells and stem cells (such as the potential for unlimited proliferation), the present invention innovatively selects induced pluripotent stem cells (iPSCs) as the antigen source. iPSCs are obtained through reprogramming technology, have the characteristics of embryonic stem cells and have no ethical controversy. Key discovery: iPSCs share specific tumor-associated antigens (TAAs) with a variety of tumors, and these antigens are lowly expressed in normal tissues, which can effectively avoid the risk of immune rejection.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In the first aspect, the present invention discloses a broad-spectrum anti-tumor transdermal immunization mRNA vaccine, which contains the gene sequence of the antigen shared by induced pluripotent stem cells and tumors.

[0011] Further, the shared antigen includes antigens with the amino acid sequence of SEQ NO: 1-14.

[0012] Further, the nucleic acid sequence of the shared antigen has the structure from the N-terminus to the C-terminus: T7 promoter - 5'UTR - Kozak sequence - ORF - stop codon - 3'UTR - polyA - restriction enzyme site;

[0013] The ORF includes multivalent antigen and adjuvant encoding sequences, which are connected to each other by P2A peptide and T2A peptide encoding sequences.

[0014] Furthermore, the pharmaceutical carrier is dendritic cell-targeting lipid nanoparticles, and the mRNA molecule is encapsulated in the lipid nanoparticles. Furthermore, the anti-tumor mRNA vaccine provided by the present invention comprises:

[0015] (1) Antigen encoding module: Multivalent tumor antigen gene sequences are connected by P2A / T2A self-cleaving peptides.

[0016] (2) Translation regulation module: Comprising a T7 promoter, 5'UTR, Kozak sequence and 3'UTR.

[0017] (3) Delivery system: Dendritic cell-targeting ethosomes (Eths).

[0018] (4) Transdermal drug delivery system: Using electrospray technology to prepare a drug-loaded Eths / silk fibroin (SF) fiber membrane patch.

[0019] The tumors include but are not limited to melanoma, breast cancer, and pancreatic cancer.

[0020] In a second aspect, the present invention discloses a method for preparing the broad-spectrum anti-tumor transdermal immune mRNA vaccine in the first aspect, comprising the following steps:

[0021] (1) Screening suitable iPSCs-tumor shared neoantigens through literature research and bioinformatics;

[0022] (2) Synthesizing mRNA encoding multivalent tumor antigens using in vitro transcription technology and modifying it;

[0023] (3) Loading the obtained IVT mRNA and adjuvant into DCs-targeting lipid nanocarriers to prepare a tumor vaccine, and using the electrospray method to spray the vaccine onto a silk fibroin membrane to prepare a vaccine patch.

[0024] The immunological activity study of the vaccine shows that the mRNA-LNP vaccine loaded with tumor antigens can activate the maturation of DCs, significantly upregulate the surface CD80, CD86 and other maturation markers of DCs. And it can activate the effector differentiation of lymph node T cells. It indicates that the mRNA vaccine can effectively activate in vitro immunity.

[0025] Anti-tumor experiments have shown that mRNA vaccines designed based on neoantigens shared by iPSCs and tumors can inhibit the growth of various tumors, including melanoma, breast cancer, pancreatic cancer, etc., and have no significant cross-toxicity. These data together indicate that mRNA vaccines provide an effective strategy for tumor prevention and treatment by efficiently activating antigen-specific T cell responses, remodeling the immune microenvironment, and inducing immune memory. Description of the Drawings

[0026] Figure 1 The figure shows the map of the in vitro transcription template plasmid PUC57.

[0027] Figure 2 The figure shows the experimental data of the in vitro induction of DCs maturation by mRNA vaccines. The results show that mRNA vaccines can effectively stimulate the expression of the DCs cell maturation marker molecules CD80 and CD86.

[0028] Figure 3 The figure shows the data of the proportion of CD80 and CD86 positive cells in the lymph nodes of mice immunized transdermally with mRNA vaccines. The results show that mRNA vaccines can effectively stimulate the maturation of DCs cells in vivo.

[0029] Figure 4 The figure shows the experimental data of T cell proliferation. The results show that mRNA vaccines can effectively stimulate T cell proliferation.

[0030] Figure 5 The figure shows the experimental data of T cell killing of tumor cells. The results show that the vaccine can activate the ability of T cells to kill various tumor cells.

[0031] Figure 6 The figure shows the proportion of CD80 and CD86 positive cells in the spleen, lymph nodes and tumor tissues of mice after transdermal immunization with mRNA vaccines. The data indicate that transdermal immunization can stimulate the maturation of DCs and promote their infiltration into tumor tissues.

[0032] Figure 7 The figure shows the proportion of CD4 and CD8 positive T cells in the spleen, lymph nodes and tumor tissues of mice after transdermal immunization with mRNA vaccines. The data show that transdermal immunization can activate CD4 and CD8 positive T cells and promote their infiltration into tumor tissues.

[0033] Figure 8 The figure shows the proportion of regulatory T cells (Tregs) in the spleen, lymph nodes and tumor tissues of mice after transdermal immunization with mRNA vaccines. The data indicate that transdermal immunization can significantly reduce the number of Treg cells, which is beneficial for tumor clearance.

[0034] Figure 9The figure shows the secretion levels of relevant cytokines in the sera of mice after transdermal immunization with mRNA vaccines. The data show that transdermal immunization can effectively stimulate mice to secrete immune-related cytokines.

[0035] Figure 10 The figure shows the tumor growth curves of mice and photos of tumor masses. The data show that transdermal immunization with mRNA vaccines can effectively inhibit the growth of various tumors, and some immunized mice did not even develop tumors.

[0036] Figure 11 The figure shows the blood routine data of mice transdermally immunized with mRNA vaccines. The results show that there are no obvious abnormalities in the blood cells of mice transdermally immunized with mRNA vaccines.

[0037] Figure 12 The figure shows the blood biochemical data of mice transdermally immunized with mRNA vaccines. The results show that the functions of the liver, kidneys, heart, etc. of mice transdermally immunized with mRNA vaccines are normal. Figure 11 and 12 It shows that the transdermal immunization mRNA vaccine prepared by the present invention has good biosafety. Detailed Embodiments

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used for the present invention and do not limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0039] Example 1: In vitro transcription and synthesis of mRNA

[0040] (1) Screening of target antigen sequences: Collect MHC class I antigens shared by iPSCs and cancer cells from the literature and the NCBI database, and test the antigenicity of the sequences using the VaxiJen tool and the ANTIGENpro tool; predict the allergenicity of the sequences using the Algpred tool and the AllerTop tool; calculate the physicochemical properties of the sequences using the ProtParam server. After removing possible allergens, a new antigen with strong immunogenicity and physicochemical properties meeting the requirements was obtained (see Table 1).

[0041] Table 1 Amino acid and coding sequences of target antigens

[0042]

[0043]

[0044] (2) Design and construction of DNA templates encoding multivalent antigens: Using the PUC57 plasmid as the material, the target sequences were inserted successively following its T7 promoter, including 5’UTR, Kozak sequence, ORF, 3’UTR sequence, polyA tail, and a single restriction site. The ORF includes the coding sequences of multivalent antigens and an adjuvant (granulocyte colony-stimulating factor), which are connected to each other by the coding sequences of P2A peptide and T2A peptide.

[0045] (3) In vitro transcription and modification of mRNA: Add x μl (1 μg) of linear DNA template, 10 μl of NTP Buffer Mix, 2 μl of T7RNA Polymerase Mix, and (8 - x) μl of DEPC water to make a total volume of 20 μl. Add the above components to a nuclease-free centrifuge tube in the above order and mix well. Incubate in a 37°C water bath for 4 h. After that, add 2 μl of DNaseⅠ and continue to incubate in a 37°C water bath for 15 min. Then add x μl (10 μg) of in vitro transcribed RNA, 2 μl of Capping Buffer, 1 μl of GTP, 1 μl of SAM, 1 μl of Vaccinia Capping Enzyme, and (15 - x) μl of DEPC water to make a total volume of 20 μl. Incubate in a 37°C water bath for 30 min. Then take the product and adjust the volume to 50 μL with DEPC water. Add 25 μL of LiCl solution (5 M), mix well, and incubate at -20°C for 30 min. Subsequently, centrifuge at 4°C (13000g) for 15 min. Discard the supernatant, and wash the precipitate with 500 μL of ice-cold 70% ethanol. Centrifuge again under the same conditions, discard the supernatant, and resuspend the precipitate in 50 μL of DEPC water to obtain purified mRNA. Use Nanodrop2000 to detect the purity and concentration of the purified product, and identify it by agarose gel electrophoresis, then store it at -80°C for later use.

[0046] (4) Load the above-obtained IVT mRNA and adjuvant into DCs-targeted lipid nanoparticles to prepare a tumor vaccine. The specific steps are as follows: First, prepare a chitosan acetate solution with a concentration of 2 mg / ml; then, weigh 0.3375 g of D-mannose solution and 0.375 g of triacetoxyborohydride sodium and dissolve them in 5 ml of deionized water to obtain a mixed solution; mix the above solutions in equal volumes and continuously stir magnetically at room temperature for 48 h, then transfer them to a dialysis device with a molecular weight cut-off of 12 - 14 kDa and dialyze for 72 h, replacing the deionized water every 8 h during this period. Freeze-dry to obtain mannosylated chitosan (MC). On this basis, prepare MC-modified Eths, namely Eths MC: Weigh 0.008 g of octadecylamine, 0.02 g of cholesterol, and 0.2 g of egg yolk lecithin, dissolve them in 4 ml of absolute ethanol, stir evenly, transfer to a round-bottom flask, and rotary evaporate on a rotary evaporator to remove ethanol to obtain a thin film. Subsequently, add 20 ml of a 30% ethanol solution containing the above IVT mRNA and CpG, stir until a uniform suspension is obtained, and then perform ultrasonic treatment (power is 100 W, the working time and the interval time are both 3 s, and 5 cycles are carried out) to obtain a drug-loaded Eths solution with uniform particle size; add 20 ml of 1 mg / ml MC (prepared with 1% acetic acid solution), continue to stir for 2 h, and centrifuge at 12,000 rpm for 10 min to remove free MC to obtain an anti-tumor mRNA vaccine that can be transdermally targeted to DCs for delivery. The vaccine patch can be prepared by spraying the vaccine onto a silk fibroin (SF) fiber membrane using the electrospray method.

[0047] Example 2: Study on the immunological activity of the mRNA vaccine

[0048] (1) In vitro induction of DCs maturation by the mRNA vaccine: Inoculate DCs into a 6-well plate (4×10 5 / well), add the vaccine obtained in Example 1 (4), set a positive control group with LPS (1 μg / mL) and a negative control group with PBS. After treatment according to the above method, wash the cells three times with PBS, digest with trypsin and collect the cells, resuspend them with PBS, and add PE-labeled goat anti-mouse monoclonal antibodies (anti-CD80 and anti-CD86) according to the instructions and incubate on ice for 30 minutes. Subsequently, wash with PBS, centrifuge at 1500 rpm / min for 5 min, and repeat 3 times. Finally, resuspend the cells in 300 μl of PBS and detect the proportion of CD80 and CD86 positive cells using a flow cytometer.

[0049] (2) In vitro induction of T cell proliferation by the mRNA vaccine: The CFSE fluorescence labeling method is used to detect T cell proliferation. Incubate the spleen single cell suspension with 5 μM CFSE for 10 minutes, centrifuge and wash, and then co-culture with DC cells at a ratio of 20:1 for 72 hours. Detect the change in CFSE fluorescence intensity by flow cytometry and calculate the T cell proliferation index.

[0050] (3) Study on in vivo induction of DCs maturation and in vivo T cell proliferation by the mRNA vaccine: Select SPF-grade female C57BL / 6 mice at 3 - 5 weeks old, and adopt a three-immunization cycle design: perform transdermal immunization on days 0, 7, and 14 respectively. Key points of the immunization operation: After locally depilating the back of the mouse, moisten the skin with a little pure water, apply the transdermal immunization membrane prepared in Example 1 of the present invention, and fix it appropriately with medical tape. Sacrifice the mice 7 days after the last immunization, prepare single cell suspensions from the spleen and lymph nodes respectively, and detect T cell proliferation and DCs maturation in the lymph nodes by flow cytometry.

[0051] Example 3: Study on the anti-tumor effect of mRNA vaccine

[0052] (1) Experiment on T cell killing of various tumor cells: One week after the third immunization, the mice were sacrificed by cervical dislocation. The spleens were taken to prepare single-cell suspensions, which were mixed and cultured with CFSE-fluorescently labeled tumor cells at a ratio of 50:1 for 24 h. After PI staining, the proportion of CFDA-SE + / PI + positive cells was the T cell tumor cell killing rate.

[0053] (2) Animal tumor-bearing experiment: Mouse melanoma, mouse breast cancer, and mouse pancreatic cancer were used as tumor models. The day when the mice were subcutaneously inoculated with tumors was recorded as day 0. The mice were immunized with the mRNA vaccine of the present invention on days -21, -14, and -7 respectively, and the method was the same as before. On day 0, 1×10 6 tumor cells were subcutaneously inoculated at the root of the right hind leg of each mouse. Once the tumors grew to the size of soybeans, the body weight of the mice was recorded once a day, and the length (L) and width (W) of the tumors were measured. The tumor volume was calculated according to the following formula: V = (L×W 2 ) / 2. On day 15, orbital venous blood was collected (the blood samples were divided into two parts and placed in anticoagulant tubes and clot-promoting tubes respectively. The blood samples in the anticoagulant tubes were stored at 4°C for later use. The blood samples in the clot-promoting tubes were allowed to stand at room temperature for 30 min and then centrifuged at 1000 rpm for 20 min. The upper clear serum was carefully aspirated and stored at -80°C for later use). Subsequently, the mice were sacrificed by cervical dislocation and the tumors were removed. The tumor masses were weighed and photographed. One sample from each group was fixed with 4% paraformaldehyde for 24 h, embedded in paraffin and sectioned. H&E and TUNEL staining were used to analyze the apoptosis of tumor cells, and CD4 / CD8 immunofluorescence staining was used to analyze the infiltration of T lymphocytes in tumor tissues. The remaining tumors were ground to extract T lymphocytes, and T lymphocytes in the spleens and lymph nodes of the mice were extracted. Flow cytometry was used to analyze the immune effect. An Elisa kit was used to detect the secretion levels of cytokines in the serum.

[0054] (3) Evaluation of the biosafety of transcutaneous immunotherapy. The hearts, livers, spleens, lungs, kidneys and other organs of the above experimental mice were collected, fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin and sectioned. H&E staining was used to analyze whether there were pathological changes. A fully automatic blood cell analyzer and a blood biochemical analyzer were used to perform routine blood and blood biochemical analyses on the above obtained whole blood samples and sera respectively to examine whether there were abnormalities in each index.

[0055] Result analysis

[0056] The immunological activity study of the mRNA vaccine showed that the mRNA vaccine loaded with tumor antigens could activate the maturation of DCs, significantly upregulating the expression of the surface maturation markers CD80 and CD86 of DCs. The vaccine could significantly stimulate T cell proliferation. These indicated that the mRNA vaccine could effectively activate immune cells.

[0057] In vitro killing experiments showed that the killing efficiencies of T cells activated by the vaccine against B16F10 (melanoma), Hela (cervical cancer), L1210 cells, Eca-109 cells, and THP-1 cells reached 58%, 44%, 77%, 58%, and 43% respectively. In vivo experiments confirmed that it could promote a significant increase in CD80 / CD86 and CD4 / CD8 positive cells in tumors, spleens, and lymph nodes and inhibit the proportion of Tregs. After vaccination, the expression levels of TNF-α, IFN-γ, IL-12, Gzms-b, and Ki67 in the sera of mice could also be increased. These data indicated that the vaccine could effectively stimulate the body to produce cellular and humoral immune responses and promote tumor cell apoptosis. Blood routine, blood biochemistry, and pathological section analysis of important organs showed that the transcutaneous immunotherapy anti-tumor regimen had good biosafety. The broad-spectrum nature of the vaccine was verified in tumor-bearing experiments in multiple tumor models: the vaccine could inhibit the growth of melanoma, breast cancer, and pancreatic cancer. These data together indicated that the mRNA vaccine provided a new strategy for tumor prevention and treatment by highly activating antigen-specific T cell responses, remodeling the immune microenvironment, and inducing immune memory.

[0058] This specific embodiment is only an interpretation of the present application and is not a limitation to the present application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A broad-spectrum anti-tumor transdermal immune mRNA vaccine, characterized in that, Contain gene sequences of induced pluripotent stem cells sharing antigens with tumors.

2. The broad-spectrum anti-tumor transdermal immune mRNA vaccine according to claim 1, characterized in that, The shared antigens include antigens with amino acid sequences of SEQ NO: 1-14.

3. The broad-spectrum anti-tumor transdermal immune mRNA vaccine according to claim 1, characterized in that, The nucleic acid sequence of the shared antigen has the structure from the N-terminus to the C-terminus: T7 promoter - 5'UTR - Kozak sequence - ORF - stop codon - 3'UTR - polyA - cleavage site; The ORF includes coding sequences for multivalent antigens and adjuvants, which are connected to each other by P2A peptide and T2A peptide coding sequences.

4. The broad-spectrum anti-tumor transdermal immune mRNA vaccine according to claim 1, wherein The pharmaceutical carrier is a dendritic cell-targeting lipid nanoparticle, and the mRNA molecule is encapsulated in the lipid nanoparticle.

5. The broad-spectrum anti-tumor transdermal immune mRNA vaccine according to claim 1, wherein, The tumors are melanoma, pancreatic cancer tumor, breast cancer tumor.

6. The preparation method of the broad-spectrum anti-tumor transdermal immune mRNA vaccine according to any one of claims 1-5, characterized in that: Include the following steps: (1) Screen suitable iPSCs-tumor shared neoantigens through literature research and bioinformatics; (2) Synthesize and modify mRNA encoding multivalent tumor antigens using in vitro transcription technology; (3) Load the above-obtained IVT mRNA and adjuvant into a DCs-targeting lipid nanocarrier to prepare a tumor vaccine, and use the electrospray method to spray the vaccine on a silk fibroin fiber membrane to prepare a vaccine patch.

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