Application of LMP2 antigen mRNA

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of LMP2 antigen mRNA. According to the LMP2 antigen mRNA design, an MHC-I molecular intracellular sequence is adopted, and the efficiency that an expression antigen is presented by MHC-I and MHC-II can be remarkably improved. The method has universality, the designed LMP2 antigen enhanced mRNA has high immunogenicity, and the prepared mRNA vaccine can be used for treating a series of cancers and tumors which are represented by nasopharynx cancer and are caused by virus infection.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to the application of LMP2 antigen mRNA. Background Art

[0002] According to the International Agency for Research on Cancer, in 2018, there were approximately 129,000 new cases of NPC, accounting for 0.7% of all confirmed cases in 2018. However, its global distribution is extremely uneven. The incidence of NPC is higher in men than in women. Characterized by its unique geographic distribution, NPC differs from other head and neck cancers in biology, epidemiology, histology, natural history, and response to treatment, and therefore the treatment of NPC has its own particularities. These findings suggest that a combination of genetic, ethnic, and environmental factors may affect the pathogenesis of NPC.

[0003] Radiotherapy is currently the main treatment for NPC and the basic treatment for non-disseminated NPC. Due to the particularity of the nasopharyngeal anatomy, surgery is not suitable for the initial treatment of NPC. However, radiotherapy causes huge nonspecific damage to the body. Local recurrence and distant metastasis are two reasons for the failure of NPC treatment. Therefore, on the basis of conventional treatment, it is urgent to find new comprehensive treatment methods. In recent decades, with the progress of molecular biology and tumor immunology, the use of molecular biology methods to enhance patients' cellular immunity, prevent the recurrence and metastasis of NPC and serve as a supplement to traditional treatment methods has received more and more attention.

[0004] Viral antigens are the most immunogenic molecules in malignant tumors and the most significant tumor antigens in protective tumor immunity. As a member of the herpesvirus family, Epstein-Barr virus is the first virus discovered to be associated with human tumors. It is now clear that Epstein-Barr virus is one of the main causes of nasopharyngeal carcinoma, lymphoma and lymphoma after immunosuppression. The latent membrane proteins LMP1 and LMP2 expressed by Epstein-Barr virus play multiple tumorigenic roles and can play a role by activating multiple signal transductions and regulating the expression of various tumorigenic genes. In terms of therapeutic vaccines, EBV nuclear antigen EBNA1 and latent membrane antigens LMP1 and LMP2 are the main therapeutic targets.

[0005] LMP2 is one of the viral proteins that is continuously expressed in tissues of Epstein-Barr virus-related tumors such as nasopharyngeal carcinoma, and does not cause B lymphocyte transformation. In vivo and in vitro studies have shown that the Epstein-Barr virus LMP2 gene can well induce specific cellular immune responses. Therefore, LMP2 has become a good target antigen for inducing Epstein-Barr virus-specific cellular immune responses, preventing and treating related tumors. In order to induce a specific cellular immune response against LMP2, it is necessary to first select an LMP2 expression system suitable for therapeutic applications.

[0006] Currently, among the vaccines targeting LMP2, only peptide, DNA vaccines and CTL cell therapy are in the clinical research stage, while there are no reports of mRNA vaccines in the literature. Summary of the invention

[0007] In view of this, one of the purposes of the present invention is to provide a method for enhancing the immunogenicity of EBV mRNA vaccines. The method is universal and can be easily extended to the development of therapeutic mRNA vaccines for other EBV-induced cancers.

[0008] To achieve the above object, the present invention adopts the following scheme:

[0009] A method for enhancing the immunogenicity of EBV mRNA vaccine, wherein the transcription template DNA of mRNA is composed of a promoter, a 5' untranslated region, a secretion signal of an MHC-I type molecule, an antigen coding region, a transmembrane and cytoplasmic domain, a 3' untranslated region, A120 and a terminal sequence after A120 connected in sequence.

[0010] The promoter is T7 or SP6 promoter.

[0011] Furthermore, the nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO:1.

[0012] Furthermore, the nucleotide sequence of the transmembrane and cytoplasmic domains is shown in SEQ ID NO:2.

[0013] Furthermore, the nucleotide sequence of the 3' untranslated region is CTCGAG.

[0014] Furthermore, the nucleotide sequence of A120 is shown in SEQ ID NO:3, and the nucleotide sequence of the terminal sequence of A120 is shown in SEQ ID NO:4.

[0015] The second object of the present invention is to provide an LMP2 immune-enhancing EBV mRNA vaccine, which can be used to prevent and treat a series of cancers caused by the virus infection, represented by nasopharyngeal carcinoma.

[0016] To achieve the above object, the present invention adopts the following scheme:

[0017] An LMP2 immune-enhancing EBV mRNA vaccine, wherein the mRNA transcription template DNA is sequentially connected by a promoter, a 5' untranslated region, a secretion signal of an MHC-I type molecule, an antigen coding region, a transmembrane and cytoplasmic domain, a 3' untranslated region, A120 and a terminal sequence after A120; the nucleotide sequence of the antigen coding region is shown in SEQ ID NO: 5.

[0018] Furthermore, the nucleotide sequence of the mRNA is shown in SEQ ID NO:6.

[0019] Furthermore, the vaccine also includes a targeted nanoformulation carrier that can carry the mRNA.

[0020] Furthermore, the targeted nano-preparation carrier is a liposome carrier.

[0021] Preferably, the liposome carrier is a cationic liposome carrier.

[0022] Cationic liposomes have a positive charge on their surface, and can wrap mRNA molecules inside through electrostatic interaction with the phosphate groups of nucleic acids to form mRNA-lipid complexes. They can also be adsorbed by cell membranes with negative charges on their surfaces, and then through membrane fusion or cellular endocytosis, and occasionally through direct osmosis, mRNA is delivered into cells for further expression.

[0023] Liposomes are composed of lipids (phospholipids) and additives. Lecithin: includes natural phospholipids and synthetic phospholipids. The structural characteristics of phospholipids are a hydrophilic group composed of a phosphate group and a quaternary ammonium salt group, and a lipophilic group composed of two longer hydrocarbon groups; natural phospholipids are mainly lecithin (phosphatidylcholine, PC), which comes from egg yolk and soybean and is neutral; synthetic phospholipids mainly include DPPC (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphatidylethanolamine), DSPC (distearoylphosphatidylcholine), etc., which are all hydrogenated phospholipids, with stable properties, strong antioxidant properties, stable finished products, etc., and are the preferred excipients abroad. Cholesterol and phospholipids are the basic substances that together constitute cell membranes and liposomes. Cholesterol has the function of regulating membrane fluidity, so it can be called the "fluidity buffer" of liposome A.

[0024] Liposomes usually have the following characteristics: 1) targeting and lymphatic orientation; 2) sustained release effect; 3) reduced drug toxicity; 4) improved stability.

[0025] Furthermore, the cationic liposome carrier is prepared by a thin film hydration method, which specifically comprises the following steps:

[0026] 1) Take an appropriate amount of phospholipid material and add it to an organic solvent to dissolve;

[0027] 2) heating in a water bath, and removing the organic solvent by rotary evaporation under reduced pressure to form a film of the phospholipid material;

[0028] 3) adding a hydrating medium to hydrate the phospholipid membrane to obtain a lipid colloidal solution;

[0029] 4) homogenizing the lipid colloid solution obtained in step 3) by probe ultrasound in a water bath or by homogenizing with a homogenizer, microfluidizer or other equipment to obtain a liposome carrier;

[0030] 5) adjusting the concentration of the solution obtained in step 4) to a certain concentration, and then filtering it through a sterile water filter membrane to obtain the cationic liposome carrier.

[0031] Furthermore, the phospholipid material in step 1) is a cationic phospholipid material and a composition thereof with an additive, wherein the cationic phospholipid material is selected from a combination of one or more of DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and its derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, and DOPE, and the additive is selected from a combination of one or more of phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), etc.; and the organic solvent is selected from organic solvents such as ethanol, methanol, chloroform, and ether that can dissolve the phospholipid material.

[0032] Furthermore, in step 2), the water bath heating temperature is 10 to 90° C., and the rotation speed of the reduced pressure rotary evaporation is 10 to 200 rpm.

[0033] Furthermore, in step 3), the time for hydrating the phospholipid membrane is 15 to 240 minutes, the temperature is 10 to 90° C., and the rotation speed is 10 to 200 rpm.

[0034] Furthermore, in step 5), the concentration is 1 to 100 mmol / L.

[0035] The third object of the present invention is to provide a method for preparing the vaccine.

[0036] Specifically, the preparation method of the vaccine is as follows: the prepared liposome carrier solution and the mRNA solution are mixed, and then incubated to obtain the mRNA liposome vaccine.

[0037] Furthermore, the liposome carrier is a cationic liposome.

[0038] Furthermore, the N / P ratio of the phospholipid material and mRNA in the liposome vaccine is 0.5 to 10:1.

[0039] Further, preferably, the N / P ratio of the phospholipid material and mRNA in the liposome vaccine is 1 to 5:1.

[0040] Furthermore, the incubation temperature is 10-40° C., and the incubation time is 10-1000 minutes.

[0041] More preferably, the incubation temperature is 20-30° C., and the incubation time is 30-240 minutes.

[0042] A fourth object of the present invention is to provide the use of the LMP2 mRNA liposome vaccine in anti-tumor treatment, wherein the tumor includes but is not limited to rhinitis tumor.

[0043] The beneficial effects of the present invention are:

[0044] 1) The LMP2 antigen-enhanced mRNA provided by the present invention has strong immunogenicity, and the prepared mRNA vaccine can be used to treat a series of cancers caused by the virus infection, represented by nasopharyngeal carcinoma;

[0045] 2) The method for enhancing the immunogenicity of EBV mRNA vaccine provided by the present invention improves the structure of traditional mRNA, so that the mRNA obtained by this method not only enhances the immunogenicity of the vaccine, but also enables the method to be easily extended to the design of other high-risk EBV mRNAs, and has universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Agarose gel electrophoresis of LMP2 antigen-enhanced mRNA template.

[0047] Figure 2 Denaturing agarose gel electrophoresis of LMP2 antigen-enhanced mRNA.

[0048] Figure 3 This is the gel retardation result of mRNA liposome vaccine.

[0049] Figure 4 This is a graph showing the particle size of LMP2 mRNA. DETAILED DESCRIPTION

[0050] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0051] Example 1 Construction of mRNA transcription vector template

[0052] The mRNA transcription template DNA is connected in sequence by the promoter, the 5' untranslated region, the secretion signal of the MHC-I type molecule, the antigen coding region, the transmembrane and cytoplasmic domain, the 3' untranslated region, A120 and the terminal sequence after A120. The antigen coding region is connected to the corresponding position and sequenced for verification.

[0053] Example 2 In vitro transcription of mRNA

[0054] The template vector of Example 1 was linearized at the tail of the A120 element using Mfe II, and purified by extraction with phenol chloroform and isoamyl alcohol to obtain a linearized template. Electrophoresis was performed to confirm whether the linearization was complete.

[0055] Figure 1 Agarose gel electrophoresis of LMP2 antigen-enhanced mRNA template.

[0056] Perform in vitro transcription reaction according to the table below, and react at 37℃ for 30min. The scale can be directly scaled up according to the demand:

[0057]

[0058] After the reaction is completed, perform DNA template degradation reaction according to the table below, and react at 37℃ for 15min. The scale can be directly scaled up according to the demand:

[0059]

[0060] After the reaction is completed, add 179 μL of nuclease-free water, then add pre-cooled ammonium acetate (5M) of equal volume, precipitate in an ice bath for 15 min, centrifuge at 17000g for 15 min at 4°C. Wash twice with 70% nuclease-free ethanol, evaporate the ethanol, and re-dissolve with nuclease-free water. Measure the concentration with a spectrophotometer.

[0061] Denature RNA according to the table below and incubate at 65°C for 10 min. You can directly scale up according to demand:

[0062]

[0063] Pre-cool in an ice bath for 5 minutes. Perform capping reaction according to the table below, and react at 37℃ for 2 hours. You can directly scale up according to the demand:

[0064]

[0065]

[0066] After the reaction is completed, add 100 μL of nuclease-free water, then add an equal volume of phenol chloroform isoamyl alcohol for extraction, centrifuge at 17000g for 10 minutes, take the supernatant, add an equal volume of pre-cooled ammonium acetate (5M) to precipitate in an ice bath for 15 minutes, and centrifuge at 17000g for 15 minutes at 4°C. Wash twice with 70% nuclease-free ethanol and dissolve to obtain the final mRNA product. Finally, measure the concentration with a spectrophotometer and verify the integrity with electrophoresis.

[0067] Conclusion: If Figure 2 The lanes shown from left to right are GFP mRNA (irrelevant mRNA) and LMP2 mRNA. Denaturing agarose gel electrophoresis showed that LMP2 mRNA was intact and not degraded.

[0068] Example 3 Preparation of cationic liposome carrier

[0069] Table: Preparation formula of cationic liposome carrier

[0070]

[0071] Preparation method: The method for preparing cationic liposome delivery carrier by thin film hydration method is as follows: respectively measure the corresponding prescribed amount of phospholipid material and additive, add them into a 250mL eggplant-shaped bottle, add the corresponding volume of organic solvent, mix and dissolve; in a 37°C water bath, remove the organic solvent by reduced pressure rotary evaporation under light-proof conditions, the speed is 50 rpm, after 2 hours, add the corresponding volume of pure water under light-proof conditions to hydrate the phospholipid film for 45 minutes, the conditions are 60°C, 75rpm / min; the obtained lipid colloid solution is homogenized by an ultrasonic / homogenizer or a high-pressure microfluidizer to obtain a liposome solution; the liposome solution is quantified to a preset volume so that the lipid concentration is 6mM, and filtered through a 0.22μm sterile water filter membrane.

[0072] Example 4 Particle size and potential measurement of cationic liposome carriers

[0073] In this example, the sample prepared by prescription 1 in Example 3 was used as an example to measure the particle size and potential. The measurement methods for other prescriptions are similar. A certain volume of the liposome colloidal solution prepared by prescription 1 (lipid concentration is 6 mM) was measured and diluted with purified water to a lipid concentration of 0.6 mM. The particle size, particle size distribution and potential of the liposome were measured in a laser particle size analyzer (n=3, i.e., each prescription was measured 3 times). The results of the liposome particle size and potential measurement are shown in the following table.

[0074] Particle size Potential 70±3nm 50±3mV

[0075] Conclusion: The particle size of cationic liposomes is about 70nm and the potential is about 50mV.

[0076] Example 5 Preparation of LMP2 mRNA liposome vaccine

[0077] Table: mRNA liposome vaccine preparation recipe and incubation parameters

[0078]

[0079] Note: The cationic liposome carrier solutions in the above table were diluted to a concentration of 6 mmol / L in advance, and the concentration of mRNA in the mRNA solution was 1 mg / mL.

[0080] Preparation method: The cationic liposome carrier prepared in Example 3 was used to prepare the mRNA liposome vaccine. The specific method is as follows: the cationic liposome carrier solution and the mRNA solution were taken in the prescribed amount according to the above table, mixed thoroughly, and incubated at the temperature and time described in the table to obtain the LM2 mRNA liposome vaccine.

[0081] Example 6 Particle size and potential of LMP2 mRNA liposome vaccine

[0082] In this example, the sample prepared in Example 5 was used as an example to measure the particle size and potential. The measurement methods for other prescriptions are similar. A certain volume of the mRNA liposome vaccine colloidal solution of Experimental Example 3 was measured and diluted with purified water to an mRNA concentration of 0.01 mg / mL. The particle size and potential of the mRNA liposome vaccine were measured in a laser particle size analyzer (n=3, i.e., each prescription was measured 3 times). The results of the particle size and potential measurement of the mRNA liposome vaccine are shown in the table below.

[0083] Particle size Potential 140±12nm 41±2mV

[0084] Conclusion: The particle size of the LMP2 mRNA liposome vaccine prepared in Example 5 is about 140 nm, and the potential is about 40 mV. This shows that the particle size increases to a certain extent and the potential decreases to a certain extent after the liposome is loaded with mRNA, which is presumably caused by the binding of mRNA to the surface of the liposome carrier.

[0085] Figure 4 This is a graph showing the particle size of LMP2 mRNA.

[0086] Example 7 Gel retardation experiment

[0087] 1) DNA gel electrophoresis

[0088] Take 0.15g agarose and 15ml water, put them in a conical flask, heat them in a microwave for 2min, cool to about 60℃, add a few drops of Gold View, mix well, pour into the gel plate (control the thickness of the gel to be about 0.5cm), insert the comb, and take out the comb after solidification. Put the gel plate into the electrophoresis tank, use 5μl of linearized plasmid, add 1μl of 6× loading buffer, mix well, and load all 6μl. The DNA marker loading volume is 5μl. 120V, electrophoresis time 25min.

[0089] 2) mRNA gel electrophoresis

[0090] ① Preparation of denatured agarose gel

[0091] Take 36mL of RNase-free water and 0.4g of agarose, place them in a conical flask, heat them in a microwave for 2min, cool to about 60℃, add 4mL of 10*MOPS and mix well, then add 7.5mL of 37% formaldehyde and mix well (volume is about 40mL). Pour into the gel plate (control the thickness of the gel to be about 0.5cm), insert the comb, and remove the comb after solidification. Put the gel plate into the electrophoresis tank, and add the newly configured 4℃ pre-cooled 1*MOPS electrophoresis buffer to the electrophoresis tank to cover the gel.

[0092] ② Electrophoresis sample preparation

[0093] Take 0.3 μL mRNA (0.3 μg) and mix it with 5.7 μL RNase-free water, add 5 μL formaldehyde loading buffer containing ethidium bromide, heat at 70°C for 5 minutes, and then centrifuge it at 4°C.

[0094] ③ Gel electrophoresis

[0095] Add the sample to the gel hole (the sample hole is near the black end of the electrophoresis tank), the added volume of each sample is 10μL, the voltage is set at 200V (current is 300mA, power is 60W), start electrophoresis, and stop electrophoresis when the indicator front reaches 1 / 2 of the gel (about 25min), take out the gel, and observe it in a gel imager. The results are shown in Figure 2 and Figure 3 .

[0096] Conclusion: Lane 1 is mRNA Marker, Lane 2 is E7 / E6, and Lane 3 is LPX. The size of E6 / E7-mRNA is comparable to that of GFP-mRNA, and can be used as a control mRNA for OVA. When N / P=3, no bands appeared in the free mRNA area of ​​each mRNA-loaded preparation, indicating that under this condition, liposomes can fully ensure the integrity of mRNA without degradation, which is in line with experimental expectations.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for enhancing the immunogenicity of EBV mRNA vaccine, wherein the template DNA is composed of a promoter, a 5' untranslated region, a secretion signal of an MHC-I class molecule, an antigen coding region, a transmembrane and cytoplasmic domain, a 3' untranslated region, A120 and a terminal sequence after A120, which are connected in sequence.

2. The method for enhancing the immunogenicity of EBV mRNA vaccine according to claim 1, characterized in that: The promoter is T7 or SP6 promoter; the nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 1; the nucleotide sequence of the transmembrane and cytoplasmic domain is shown in SEQ ID NO: 2; the nucleotide sequence of the 3' untranslated region is CTCGAG.

3. The method for enhancing the immunogenicity of EBV mRNA vaccine according to claim 1, characterized in that: The nucleotide sequence of A120 is shown in SEQ ID NO:3, and the nucleotide sequence of the terminal sequence of A120 is shown in SEQ ID NO:

4.

4. An immunoenhancing mRNA vaccine of LMP2, characterized in that: The mRNA transcription template DNA is composed of a promoter, a 5' untranslated region, a secretion signal of an MHC-I type molecule, an antigen coding region, a transmembrane and cytoplasmic domain, a 3' untranslated region, A120 and a terminal sequence after A120 connected in sequence; the nucleotide sequence of the antigen coding region is shown in SEQ ID NO: 5; the nucleotide sequence of the mRNA transcription template DNA is shown in SEQ ID NO:

6.

5. The mRNA vaccine according to claim 4, characterized in that The vaccine also includes a targeted nanoformulation carrier that can carry the mRNA.

6. The vaccine according to claim 5, characterized in that The targeted nano-preparation carrier is a liposome carrier; the liposome carrier is prepared by conventional liposome preparation methods, including but not limited to thin film hydration method, organic solvent injection method, reverse evaporation method and freeze-thaw method.

7. A method for preparing the vaccine according to any one of claims 4 to 6, characterized in that: The specific preparation method is: mix the prepared liposome carrier solution and mRNA solution, and then incubate to obtain the mRNA liposome vaccine.

8. The preparation method according to claim 7, characterized in that: The N / P ratio of the phospholipid material and mRNA in the liposome vaccine is 1 to 5:

1.

9. Use of the LMP2 mRNA liposome vaccine according to claim 4 in anti-tumor treatment.

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