Personalized mRNA vaccine for treating breast cancer as well as preparation method and application thereof

Abstract

The invention provides a personalized mRNA vaccine for treating breast cancer and a preparation method and application thereof.The personalized mRNA vaccine for treating breast cancer comprises mRNA molecules and lipid nanoparticles, and the amino acid sequence of an expression sequence coded by a gene coding region of the mRNA molecules is shown as any one of SEQ ID NO: 85-SEQ ID NO: 87; the individualized mRNA vaccine is synthesized based on a gene sequencing result of a breast cancer patient, is unique for the breast cancer patient, is an individualized vaccine in a real sense, and has the advantages of being higher in targeting performance, smaller in toxic and side effect, higher in specificity and the like. The immune microenvironment can be improved, the stability is good, the anti-tumor efficiency is high, safety and effectiveness are realized, the quality is controllable, and the research and development period is short; the method is suitable for the technical field of medical preparations.

Description

Technical Field

[0001] The present application relates to the technical field of medical preparations, and specifically, to a personalized mRNA vaccine for treating breast cancer, a preparation method and an application thereof. Background Art

[0002] At present, breast cancer is one of the most serious diseases threatening women's health and has become the number one killer of urban women. Existing technologies, namely conventional treatment methods such as surgery, chemotherapy and radiotherapy, can remove or kill most breast cancer cells in the body, but there are still some residual breast cancer cells or drug-resistant breast cancer cells that become the "culprit" of cancer recurrence and metastasis. The overall treatment effect is limited, and drug resistance or treatment resistance is prone to occur, resulting in poor prognosis for patients, and even unable to obtain a long survival period.

[0003] With the continuous development of tumor treatment technology, immunotherapy has provided an effective auxiliary role in tumor treatment. As a type of immunotherapy, mRNA vaccine has a good tumor immune activation effect. It can also stimulate or mobilize the body's immune system to enhance the anti-tumor immunity of the tumor microenvironment, thereby controlling and killing tumor cells, providing great application prospects for tumor adjuvant treatment.

[0004] Gene mutations are prone to occur during tumor development, resulting in the production of new antigens, which only exist in tumor cells and can become an important target for activating specific tumor immunity. mRNA molecules can produce target proteins or immunogens, activate the body's immune response to fight various pathogens, and mRNA plays an important role in the protein expression process. The mRNA transcribed in the cell nucleus can deliver genetic information to the cytoplasm and further translate into protein. Therefore, personalized mRNA vaccines identify the genetic information in new antigens, transcribe the genetic information into the mRNA sequence by in vitro transcription, and then further modify, process, and package it into a finished vaccine. This vaccine further completes the translation process in the cell, and the generated antigen can activate specific anti-tumor effects in the body. At the same time, since new antigens are only expressed by tumor cells, injection of personalized mRNA vaccines can trigger a true tumor-specific T cell response, thereby preventing "off-target" damage to non-tumor tissues. In addition, new antigens are new epitopes derived from somatic mutations, which may bypass the central tolerance of T cells to their own epitopes, thereby inducing an immune response to tumors. In addition, the neoantigen-specific T cell responses enhanced by personalized mRNA vaccines persist and have the potential to provide post-treatment immune memory, which offers the possibility of long-term prevention of disease recurrence.

[0005] Therefore, designing personalized mRNA vaccines with stronger targeting, less toxic side effects, and the ability to improve the immune microenvironment is expected to achieve the goal of preventing and treating breast cancer specifically, safely, and effectively. Summary of the invention

[0006] In order to solve one of the above-mentioned technical defects, the present application provides a personalized mRNA vaccine for treating breast cancer, a preparation method and its application.

[0007] According to the first aspect of the present application, a personalized mRNA vaccine for treating breast cancer is provided, comprising an mRNA molecule and lipid nanoparticles, wherein the amino acid sequence of the expression sequence encoded by the gene coding region of the mRNA molecule is shown in any one of SEQ ID NO: 85-SEQ ID NO: 87.

[0008] Preferably, the synthesis of the mRNA molecule specifically comprises:

[0009] S10. Collect tumor tissue and healthy blood samples from breast cancer patients for gene sequencing to obtain somatic mutations in tumors;

[0010] S20, using the Immune Epitope Database (IEDB) to evaluate the HLA affinity of the acquired somatic mutations to obtain new antigen peptides;

[0011] S30. Use the tumor antigen database platform CAD (1.0) to perform online analysis of new antigen peptides, preliminarily verify the immunogenicity of new antigen peptides, and screen out new antigen peptides with high affinity to HLA;

[0012] S40, synthesizing the screened new antigen peptides with high affinity to HLA, and then culturing them with the patient's immune cells, and using the enzyme-linked immunosorbent spot (ELISPOT) method to screen out the new antigen peptides with high immunogenicity;

[0013] S50, connecting the screened new antigen polypeptides with high immunogenicity to synthesize mRNA molecules.

[0014] Preferably, in S50, the screened new antigen polypeptides with high immunogenicity are connected to synthesize mRNA molecules, which specifically includes:

[0015] The screened new antigen peptides are arranged in descending order according to the number of immune spots;

[0016] The arranged new antigen peptides are evenly divided into N groups, each group includes m new antigen peptides, and 1≤m≤10;

[0017] The new antigen peptides in each group are connected in descending order of the number of immune spots to synthesize mRNA molecules.

[0018] Preferably, the synthesis of the mRNA molecule further comprises: adding a His tag to the N-terminus of the synthesized mRNA molecule, and simultaneously performing 5'-end capping and 3'-end poly (A) tailing on the synthesized mRNA molecule.

[0019] Preferably, the lipids in the lipid nanoparticles include trimethyl-2,3-dioleyloxypropylammonium bromide (DOTAP), dipalmitoylphosphatidylcholine (DPPC), cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000);

[0020] The mass ratio of the mRNA molecule, DOTAP, DPPC, cholesterol and DSPE-PEG 2000 is 3:10:4:4:1.

[0021] Preferably, the personalized mRNA vaccine comprises the following raw materials: 1.5 mg mRNA molecules, 5 mg DOTAP, 2 mg DPPC, 2 mg cholesterol, 0.5 mg DSPE-PEG 2000, 20 mL citric acid buffer and 1.5 mL anhydrous ethanol.

[0022] According to the second aspect of the present application, a method for preparing a personalized mRNA vaccine for treating breast cancer is provided, comprising the following steps:

[0023] S1. Place DOTAP, DPPC, cholesterol and DSPE-PEG 2000 in a sterile glass bottle, then add anhydrous ethanol to completely dissolve them to obtain a lipid solution;

[0024] S2, purifying the mRNA molecules, dissolving the purified mRNA molecules in a citric acid buffer (pH=4.0), and gently mixing to obtain an mRNA solution;

[0025] S3, injecting the lipid solution at a flow rate of 2 mL / min and the mRNA solution at a flow rate of 0.5 mL / min into the channel of the microfluidic chip, so that the two are mixed in the microfluidic chip to form a mixed solution;

[0026] S4. Transfer the mixed solution to a dialysis bag and dialyze it in 50 mL of PBS (pH=7.4) for 4 hours, replacing PBS every hour to obtain mRNA-loaded lipid nanoparticles, i.e., mRNA vaccines.

[0027] Preferably, the preparation method further comprises: inputting the sequence of the mRNA molecule into the RNAstructure platform, obtaining the secondary structure prediction result of the mRNA molecule, and analyzing the sites where the mRNA molecule is easily bound to lipids; querying the information of the selected lipids on the LipidMaps platform, and obtaining the preparation conditions in S3 according to the structure and properties of the selected lipids and the sites.

[0028] Preferably, in S1, the total lipid concentration of the lipid solution is 10 mM.

[0029] According to the third aspect of the present application, provided is the use of any of the above-described personalized mRNA vaccines for treating breast cancer in the preparation of a drug for treating breast cancer.

[0030] The personalized mRNA vaccine for treating breast cancer provided in this application is synthesized based on the gene sequencing results of breast cancer patients. It is unique to breast cancer patients and is a truly personalized vaccine. In addition, the personalized mRNA vaccine uses the gene sequence of the virus rather than the virus itself, and there is no risk of infection. This personalized mRNA vaccine can not only stimulate CD8 + T cells, promote the expression of IFN-γ and TNF-α, and also stimulate CD8 + T cells have the ability to kill tumor cells. In addition, this personalized mRNA vaccine has stronger targeting, less toxic side effects, and can improve the immune microenvironment. It has the advantages of good stability, high anti-tumor efficiency, safety and effectiveness, controllable quality, and a short R&D cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0032] Figure 1 This is a diagram of the arrangement order of the synthesis of three mRNA molecules;

[0033] Figure 2 This is the gel running verification diagram of three mRNA molecules;

[0034] Figure 3 CD8 corresponding to the three mRNA vaccines + T cell viability bar graph; a is the CD8 of mRNA1 vaccine + T cell viability bar graph; b is the CD8 of mRNA2 vaccine + T cell viability bar graph; c is the CD8 of mRNA3 vaccine + T cell viability bar graph;

[0035] Figure 4 Tumor-specific CD + T cell marker expression bar graph; A is tumor-specific CD8 + The percentage of positive cells expressing TNF-α in T cells is shown in the bar graph; B is the percentage of positive cells expressing TNF-α in T cells; + Histogram of the percentage of positive cells expressing IFN-γ in T cells;

[0036] Figure 5 For CD8 + Histogram of T cell killing efficiency against breast cancer MCF-7B cells;

[0037] Figure 6 This is the survival curve of breast cancer mice treated with three mRNA vaccines. DETAILED DESCRIPTION

[0038] In order to make the technical solutions and advantages in the embodiments of the present application more clearly understood, the exemplary embodiments of the present application are further described in detail below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than an exhaustive list of all the embodiments. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0039] The reagents and materials in the examples, unless otherwise specified, can be obtained from commercial sources; the experimental methods in the following examples, unless otherwise specified, are conventional methods in the art and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0040] RNA sample preservation solution (Thermo Fisher Scientific); breast cancer MCF-7B cells (Shine Biotechnology); C57BL / 6 mice (Scott Biotechnology); sodium heparin anticoagulant (MedChemExpress); AllPrep DNA / RNAMini Kit (QIAGEN); QIAamp DNABlood Mini Kit (QIAGEN); Agilent Sure Select Human All Exon 44Mb (v.2.0, Agilent Technologies); TruSeq RNAAccess Library Prep Kit (Illumina); RNAPurification Kit (TransGen Biotech, Cat: ER701-01); mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific); RNA Purification Kit (TransGen Biotech); Malvern Analyzer (Malvern Panalytical); Quant-iT TM RNA Reagent and Kit (Invitorgen); TBD sample density separation solution (Haoyang Biological Products Technology Co., Ltd.); CD8 + T sorting reagent (BeaverBiosciences); recombinant interferon α2a (Sansheng Pharmaceutical); recombinant interleukin 2 (Sansheng Pharmaceutical); CD3 monoclonal antibody (Tongli Haiyuan Biotechnology); flow cytometry antibody kit (Thermo); Backman flow cytometer (Beckman Coulter); microplate reader (Thermo Fisher Scientific).

[0041] Example 1

[0042] The present application provides a personalized mRNA vaccine for treating breast cancer, comprising mRNA molecules and lipid nanoparticles, wherein the synthesis of the mRNA molecules comprises the following steps:

[0043] S10. Collect tumor tissue and healthy blood samples from breast cancer patients for gene sequencing to obtain somatic mutations in tumors; specifically:

[0044] Collect tumor tissue and healthy blood samples from breast cancer patients, and quickly store the collected tumor tissue in 1 ml RNA sample preservation solution, while the healthy blood samples are stored in blood collection tubes with sodium heparin anticoagulant added;

[0045] DNA and RNA were extracted from tumor tissue using the AllPrep DNA / RNA Mini Kit, and DNA was extracted from healthy blood samples using the QIAamp DNABlood Mini Kit.

[0046] Using high-throughput sequencing technology, Agilent Sure Select Human All Exon44Mb (v.2.0) was used to capture all exons of the extracted DNA to construct a WES sequencing library, and the TruSeq RNA Access Library Prep Kit was used to sequence the extracted RNA to construct an RNA-seq library.

[0047] The WES sequencing library and RNA-seq library were submitted to the Illumina HiSeq×10 platform, and DNA sequencing data and RNA sequencing data with 150 bp paired-end reads were generated using the FASTQ format;

[0048] The DNA sequencing data and RNA sequencing data were aligned with the latest human reference genes. Then, the genome analysis toolkit GATK (v.4.0.10.1) was used to remove duplicate sequences, and the DNA sequencing data in tumor tissues and healthy blood samples were aligned using Mutect2 in GATK to determine somatic mutations in the tumor.

[0049] S20. Use IEDB to evaluate the HLA affinity of the acquired somatic mutations to obtain new antigen peptides; specifically:

[0050] The obtained somatic mutations were analyzed and predicted by IEDB for MHC-I and MHC-II affinity to obtain HLA typing results and immune scores. Then, somatic mutations with strong HLA affinity, IC50 < 50nM or %Rank < 0.5, higher affinity of the mutant epitope to HLA than the corresponding wild type (WT) epitope, and at least 5 reads in the RNA-seq library covering the mutation site were selected as new antigen peptides.

[0051] S30. Use the tumor antigen database platform CAD (1.0) to conduct online analysis of new antigen peptides, preliminarily verify the immunogenicity of new antigen peptides, and screen out new antigen peptides with high affinity to HLA; specifically:

[0052] The new antigen peptide sequences were analyzed online using the tumor antigen database platform CAD (1.0), the different physicochemical characteristics of the peptides were calculated, and the immunogenicity of the new antigen peptides was preliminarily verified in the machine learning model;

[0053] Based on the somatic mutation peptide sequences and HLA typing results, combined with the preliminary verification results of the tumor antigen database platform CAD (1.0), 9-14mer peptide sequences with high affinity to HLA were screened, i.e., new antigen peptides with high affinity to HLA;

[0054] The amino acid sequences of the obtained new antigen polypeptides with high affinity to HLA are shown in Table 1.

[0055] S40. The screened new antigen peptides with high affinity to HLA are synthesized, and then cultured with the patient's immune cells. The ELISPOT detection method is used to detect the level of IFN-γ secretion by the immune cells, and the new antigen peptides with more immune spots than the positive control are screened, that is, the new antigen peptides with high immunogenicity.

[0056] Specifically, 18 new antigen peptides were screened out with more immune spots than the positive control.

[0057] S50, connecting the screened new antigen polypeptides with high immunogenicity to synthesize mRNA molecules; specifically:

[0058] The 18 screened new antigen peptides were arranged in descending order according to the number of immune spots;

[0059] The arranged new antigen peptides were evenly divided into 3 groups, each group including 6 new antigen peptides, as shown in Table 2;

[0060] The new antigen peptides in each group are connected in series in the order of the number of immune spots from high to low, and the mRNA1 molecule, mRNA2 molecule, and mRNA3 molecule are synthesized respectively. The order of the mRNA1 molecule, mRNA2 molecule, and mRNA3 molecule is as follows: Figure 1 As shown;

[0061] A His tag is added to the N-terminus of the synthesized mRNA1 molecule, mRNA2 molecule and mRNA3 molecule, and the amino acid sequence of the expression sequence encoded by the gene coding region of the mRNA1 molecule is shown in SEQ ID NO: 85, the amino acid sequence of the expression sequence encoded by the gene coding region of the mRNA2 molecule is shown in SEQ ID NO: 86, and the amino acid sequence of the expression sequence encoded by the gene coding region of the mRNA3 molecule is shown in SEQ ID NO: 87.

[0062] Specifically, the amino acid sequence of the expression sequence encoded by the coding region of the mRNA1 molecule gene is: QYHSLKLECDKLVSEKSEMQ RHYVMHSQEQ THSQAGSPHG ESESTVHERH 1-50GEAPGDFLQR LLSAGAGMVAGGPLGSESNF GHSSEAPTRT DTMDSMPSQA 51-100MASGTASNSN RSTPACRRRS SRLSTNGTQEILDPDLVVSD L 101-141.

[0063] The amino acid sequence of the expression sequence encoded by the coding region of the mRNA2 molecule gene is: RSSKAHNQKM KLPWGSSISGCDVYAEDSWE ELLIGDVELK RVNGSRSQQW 1-50LLRNITLPEI FTRRSDRFAT TLRKEIQMHRAKLQKSPQPQ KSASAEAAKE 51-100IHYIPSVLIG GPPKVEEAHY ITTQEIQLSE VEQDMD 101-136.

[0064] The amino acid sequence of the expression sequence encoded by the coding region of the mRNA3 molecule gene is: MHLKYGLFST FYMRTSIHDTLGACLRGALT HLPAGLSGLA HLAEDDNLSS 1-50MLHQRTEIPW RACAKYLSRR DHIKDSFHSLWDSVPSLQGE KLYVRKHDKL 51-100KKEAVDRFAE KLTLILQEMD KIVSTFYTVV IPMLNLMIYS LRN101-143.

[0065] Furthermore, the synthesized mRNA1 molecule, mRNA2 molecule and mRNA3 molecule are capped at the 5' end and have a Poly (A) tail structure added at the 3' end to obtain mRNA1 molecule, mRNA2 molecule and mRNA3 molecule with capped and tailed structures.

[0066] Furthermore, the synthesized mRNA1, mRNA2 and mRNA3 molecules were capped at the 5' end using the mMESSAGE mMACHINE T7 kit.

[0067] When implementing it, use The RNA Purification Kit is used to purify in vitro synthesized mRNA molecules, detect the concentration of the purified mRNA molecules, perform electrophoresis in agarose gel, and take photos with a gel imager to verify the size and integrity of the mRNA molecules.

[0068] In the present application, the concentration of the purified mRNA1 molecule is 2.21 μg / μL, the concentration of the purified mRNA2 molecule is 2.28 μg / μL, and the concentration of the purified mRNA3 molecule is 2.36 μg / μL; Figure 2 As shown, the mRNA1 molecule, mRNA2 molecule, and mRNA3 molecule obtained in agarose gel electrophoresis are consistent with the actual nucleotide sizes.

[0069] Table 1 Amino acid sequences of new antigen peptides

[0070] Serial Number Amino acid sequence Serial Number Amino acid sequence SEQ ID NO: 1 ETWIWDLVVVNSSGVAEVGVTVPD SEQ ID NO: 2 RGVTTLPPEAFYLIPWQDWKKYFED SEQ ID NO: 3 EDDNLSSMLHQRTEIPWRACAKYLS SEQ ID NO: 4 MNGLSLSELCCLF SEQ ID NO: 5 PHKHVSGFSSSLWTSSTGDAGGGSR SEQ ID NO: 6 QYHSLKLECDKLVSEKSEMQRHYVM SEQ ID NO: 7 SAFFLEWFSEEAHRVYGDIIHTPAK SEQ ID NO: 8 RLILNEVSLLGSVPGGKVEFKRTLI SEQ ID NO: 9 QQKLSKLKGEPGLVTIHGMAGCGKS SEQ ID NO: 10 SRELYMRHFPFKTLHFYLIRALQLL SEQ ID NO: 11 PQPQKSASAEAAKEIHYIPSVLIGG SEQ ID NO: 12 FIIGCNPTILHVQELYIRAFQKLTD SEQ ID NO: 13 SQSPQHAEMCLHPAGPTFPEEMGET SEQ ID NO: 14 GEAPGDFLQRLLSAGAGMVAGGPLG SEQ ID NO: 15 VRKHDKLKKEAVDRFAEKLTLILQE SEQ ID NO: 16 TLPEPVGTQVSMDEIQNESACGLDV SEQ ID NO: 17 PAEFDAMVELDGNDVRVSSRGKYAE SEQ ID NO: 18 NDGTIVEKYLRENTVQSVKPWLTEI SEQ ID NO: 19 IRVDRNHRVLLNVTDFDLEPQDSCI SEQ ID NO: 20 CPPGFQGSNCEKSVDRCSLQPCRNG SEQ ID NO: 21 PADSLLKYDTPVPVSRNTEKRSPKA SEQ ID NO: 22 SESNFGHSSEAPTRTDTMDSMPSQA SEQ ID NO: 23 WEKDTVSLVSGSIFLITSTGALYIK SEQ ID NO: 24 PSKAGIWRVECLVGEHLHAGMSTLF SEQ ID NO: 25 SLTPQQAQAQGIPLTPQQAQALGVP SEQ ID NO: 26 ETLMAKESTQGHWSYLLTCCIAQKP SEQ ID NO: 27 HSQEQTHSQAGSPHGESESTVHERH SEQ ID NO: 28 AQRERIQECMSQTELLTSLFDL SEQ ID NO: 29 LGGDNERSPRDDDAYLAGLIRQVEE SEQ ID NO: 30 NGSRSQQWLLRNITLPEIF SEQ ID NO: 31 PKKPRSLRIYESPVGISSHEGKVAS SEQ ID NO: 32 KLKNQTAEPLAPQPPAGPSKVEQLQ SEQ ID NO: 33 AVMCLSVVIFGTDIIGNLAVMCIVC SEQ ID NO: 34 MASGTASNSNRSTPAC SEQ ID NO: 35 PAQELLAPQPFPDPPSSVLREGSQEK SEQ ID NO: 36 PESVSCILQEPKGNLAFFQLRKVWG SEQ ID NO: 37 FSKEPAPAEIEQS SEQ ID NO: 38 HTKEELIELKEEDEIPHSELDLETV SEQ ID NO: 39 LTFADELILRKRVLLSRYNPEGLLN SEQ ID NO: 40 FYTALRHLDLSTKEISFLQPGAFQA SEQ ID NO: 41 GKEDAFISSCESTKTVCEMEAVLSA SEQ ID NO: 42 ARDICVIQVPPERRYTDYFVIVSGT SEQ ID NO: 43 RSIEEACLTLQHFNRLGIQAEPLYR SEQ ID NO: 44 PWGTMAWAPRTARVPGRSATAATWK SEQ ID NO: 45 GTTATKIDVYLL SEQ ID NO: 46 ISGCDVYAEDSWEELLIGDVELKRV SEQ ID NO: 47 FSSRVRTEFPLGHYGTAAEVKQAVL SEQ ID NO: 48 RRDHIKDSFHSLWDSVPSLQGEKLY SEQ ID NO: 49 GAESGGSCAWRGF SEQ ID NO: 50 PHTNSGAFVYGSRSFLDKVANGLAV SEQ ID NO: 51 YKLTYSTEVLLDQGKGKLQDSVGYR SEQ ID NO: 52 ASTGDTTPLPVTATSSASTGQDTPL SEQ ID NO: 53 GRVCGLCGNFDDSAINDFATRSRSV SEQ ID NO: 54 RGFHWRQKFLRVTRSAICIQSWWRG SEQ ID NO: 55 FGVGEGPAAPSPA SEQ ID NO: 56 YIHRFTNGGKEKKVAHFGYHWSLME SEQ ID NO: 57 KQEKSLFERSNVVVEIMEKFNTISV SEQ ID NO: 58 IGNWTEISEFILRSFSSLPTEIQSL SEQ ID NO: 59 MDKIVSTFYTVVIPMLNLMIYSLRN SEQ ID NO: 60 QAVVPCRVTVLSTKVTLHREFPAKE SEQ ID NO: 61 RRRSSRLSTNGTQEILDPDLVVSDL SEQ ID No: 62 DTLGACLRGALTHLPAGLSGLAHLA SEQ ID NO: 63 EEIPEEVIQPYQKFSVEVGRLAQET SEQ ID NO: 64 PDPSWPLLFFPLFWALPLLGPV SEQ ID NO: 65 AAKIIRHPKYNRA SEQ ID NO: 66 FRLQAPQASVTAASEQTTEFGIHKP SEQ ID NO: 67 PPKVEEAHYTTTQEIQLSEVEQDMD SEQ ID NO: 68 KDAREILKQIEYHNLFKYVGETQPT SEQ ID NO: 69 NALEKIAMKQYTTNVKRGKEVIEHY SEQ ID No: 70 HYSTKAYSWNISHKYCQNRYTDLVA SEQ ID NO: 71 EFNIMVVGQSGLDKSTMVNTLFKSK SEQ ID NO: 72 TRRSDRFATTLRKEIQMHRAKLQKS SEQ ID NO: 73 LTFTESTHVEIKKFSTEKLLQRITE SEQ ID NO: 74 DGRGYRWWPGRLVISRQLDPAM SEQ ID NO: 75 MHLKYGLFSTFYMRTSIH SEQ ID NO: 76 AYAGRQASQTESVDGPRTRCPVYIY SEQ ID NO: 77 NRNLRKGPSGLAEEINFEDFLTIMS SEQ ID NO: 78 LCKEFTELLSQDGTPHGTSRLAPVL SEQ ID NO: 79 VKLQQLEASYACEEKELLKVMKEYA SEQ ID NO: 80 GDPPKSNQSLGWEDSSKPVSSPDWN SEQ ID NO: 81 RSSKAHNQKMKLPWGSS SEQ ID NO: 82 CWGPFFLHLTLIILCPEHPTCGCIF SEQ ID NO: 83 EDFTVQDPTEAETLTAAIQHISQGS SEQ ID NO: 84 LIHHQRSHSGERHFACRECGKAFRG

[0071] Table 2 ELISPOT spot counting results

[0072] Group 1:

[0073] Sequence ID (SEQ ID) Number of spots SEQ ID NO: 6 182 SEQ ID NO: 27 176 SEQ ID NO: 14 171 SEQ ID NO: 22 169 SEQ ID NO: 34 164 SEQ ID NO: 61 158

[0074] Group 2:

[0075] Sequence ID (SEQ ID) Number of spots SEQ ID NO: 81 147 SEQ ID NO: 46 141 SEQ ID NO: 30 138 SEQ ID NO: 72 136 SEQ ID NO: 11 134 SEQ ID NO: 67 133

[0076] Group 3:

[0077] Sequence number (SEQ ID) Number of spots SEQ ID NO: 75 129 SEQ ID NO: 62 127 SEQ ID NO: 3 123 SEQ ID NO: 48 121 SEQ ID NO: 15 117 SEQ ID NO: 59 115

[0078] Furthermore, the lipids in the lipid nanoparticles include DOTAP, DPPC, cholesterol, and DSPE-PEG 2000. Lipid nanoparticles have the characteristics of good biocompatibility, long-term stability, and high loading capacity, and are currently the most commonly used mRNA encapsulation and delivery materials. Lipid nanoparticles encapsulate mRNA, which can ensure the stability of mRNA and the reliability of transmission. Lipid nanoparticles can also reduce the innate immune effect while delivering mRNA into cells, preventing severe inflammatory reactions.

[0079] Furthermore, the mass ratio of mRNA molecule, DOTAP, DPPC, cholesterol, and DSPE-PEG 2000 is 3:10:4:4:1.

[0080] In specific implementation, the personalized mRNA vaccine includes the following raw materials: 1.5 mg mRNA molecules, 5 mg DOTAP, 2 mg DPPC, 2 mg cholesterol, 0.5 mg DSPE-PEG 2000, 20 mL citric acid buffer and 1.5 mL anhydrous ethanol.

[0081] Example 2

[0082] The present application also provides a method for preparing a personalized mRNA vaccine for treating breast cancer, comprising the following steps:

[0083] S1. Place 5 mg DOTAP, 2 mg DPPC, 2 mg cholesterol and 0.5 mg DSPE-PEG 2000 in a sterile glass bottle, then add 1.5 mL anhydrous ethanol to completely dissolve them to prepare a lipid solution with a total lipid concentration of 10 mM;

[0084] S2. Purify the mRNA molecules, dissolve 1.5 mg of the purified mRNA molecules in 20 mL of citric acid buffer (pH = 4.0), and gently mix to obtain an mRNA solution;

[0085] S3, injecting the lipid solution at a flow rate of 2 mL / min and the mRNA solution at a flow rate of 0.5 mL / min into the channel of the microfluidic chip, so that the two are mixed in the microfluidic chip to form a mixed solution;

[0086] S4. Transfer the mixed solution to a dialysis bag and dialyze it in 50 mL of PBS (pH = 7.4) for 4 hours, replacing PBS every hour to remove anhydrous ethanol and unencapsulated mRNA, thereby obtaining mRNA-loaded lipid nanoparticles, i.e., mRNA vaccines.

[0087] Furthermore, the preparation method also includes: inputting the sequence of the mRNA molecule into the RNAstructure platform, obtaining the secondary structure prediction result of the mRNA molecule, and analyzing the sites where the mRNA molecule is easily bound to lipids; querying the information of the selected lipids on the LipidMaps platform, and optimizing the preparation conditions in S3 according to the structure and properties of the selected lipids and the sites.

[0088] In the specific implementation, the mRNA1 molecules, mRNA2 molecules and mRNA3 molecules purified in Example 1 are all subjected to the above-mentioned method to prepare mRNA1 vaccine, mRNA2 vaccine and mRNA3 vaccine respectively.

[0089] In order to demonstrate the beneficial effects of the mRNA vaccines prepared in this application, the mRNA1 vaccine, mRNA2 vaccine and mRNA3 vaccine prepared in this example were analyzed using the Malvern analyzer and Quant-iT TM RNA Reagentand Kit was used for characterization.

[0090] The particle size and dispersion index (PDI) of the mRNA vaccine were analyzed using a Malvern analyzer. The results showed that the size of the nanoparticles in the mRNA1 vaccine was 78-106nm, and the PDI was 0.134; the size of the nanoparticles in the mRNA2 vaccine was 80-111nm, and the PDI was 0.138; the size of the nanoparticles in the mRNA3 vaccine was 79-110nm, and the PDI was 0.143.

[0091] By Quant-iT TM The RNA Reagent and Kit was used to measure the encapsulation efficiency of mRNA vaccines. The results showed that the encapsulation efficiency of mRNA1 vaccine was 87.3%, the encapsulation efficiency of mRNA2 vaccine was 86.7%, and the encapsulation efficiency of mRNA3 vaccine was 85.9%. It can be seen that mRNA1 vaccine, mRNA2 vaccine and mRNA3 vaccine can all efficiently encapsulate mRNA molecules.

[0092] Example 3

[0093] This example evaluates the cytotoxicity of the mRNA vaccine, and the specific method is as follows:

[0094] 30 mL of peripheral blood donated by volunteers was collected and peripheral blood mononuclear cells (PBMC) were isolated using TBD sample density separation fluid;

[0095] The isolated cells were stained with CD8 + CD8 + T cells were counted at 1 × 10 6 cells / mL, added to the culture medium containing 1000 IU / mL of recombinant interferon α2a;

[0096] The above culture medium was placed in an incubator at 37°C and 5% CO2 for 24 h, and then 1500 IU / mL (final concentration) of recombinant interleukin-2 and 50 ng / mL (final concentration) of CD3 monoclonal antibody were added and cultured for another 24 h to obtain activated CD8 + T cells;

[0097] Activated CD8 + T cells were seeded in 96-well plates (5 × 10 3 After overnight culture, mRNA1 vaccine, mRNA2 vaccine, and mRNA3 vaccine (calculated as mRNA, with a final concentration of 2 μg / mL) were added, and T cell activity was detected after co-incubation for 0 h, 6 h, 12 h, 18 h, and 24 h. The results are as follows Figure 3 shown.

[0098] Depend on Figure 3 It can be seen that mRNA1 vaccine, mRNA2 vaccine and mRNA3 vaccine have no obvious cytotoxicity and have good safety.

[0099] Example 4

[0100] This example detects DC cells loaded with mRNA vaccine to stimulate CD8 + The expression of IFN-γ and TNF-α in T cells is as follows:

[0101] (1) 1×10 6 After culturing the DC cells for 5 days, they were co-cultured with 25 μg of mRNA1 vaccine for 1 day, and then the DC cells were induced to mature for 2 days to obtain DC cells loaded with mRNA1 vaccine;

[0102] Similarly, the above method was used to obtain DC cells loaded with mRNA2 vaccine and DC cells loaded with mRNA3 vaccine, which will not be repeated here.

[0103] (2) Experimental groups 1 to 3 were set up, where experimental group 1 was composed of DC cells loaded with mRNA1 vaccine and CD8 + T cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 3 days, and DC cells loaded with mRNA1 vaccine were added in an amount of 1.53×10 6 ,CD8 + The amount of T cells added to each well was 5.0×10 6 indivual;

[0104] The methods used in experimental groups 2 and 3 were the same as those in experimental group 1, except that in experimental group 2, DC cells and CD8 + T cells were co-cultured, and group 3 was DC cells loaded with mRNA3 vaccine and CD8 + T cells were co-cultured;

[0105] At the same time, a control group was set up, which was CD8 + T cells were cultured alone and CD8 + The amount of T cells added to each well was 5.0×10 6 indivual.

[0106] (3) Each group of cells was fixed using a flow cytometry antibody kit, and then purified TNF-α antibody and purified IFN-γ antibody were added to the control group and each experimental group, respectively. The cells were incubated at room temperature in the dark for 30 min, and the expression rates of TNF-α and IFN-γ in the cells were analyzed using a Backman flow cytometer.

[0107] The experimental results are as follows Figure 4 As shown in Table 3, compared with the control group, the specific CD8 + The expression of T cell markers TNF-α and IFN-γ was upregulated, showing a stronger potential to kill tumor cells. + The expression of TNF-α and IFN-γ in T cells was higher than that in DC cells loaded with mRNA2 or mRNA3 vaccine. + The expression levels of TNF-α and IFN-γ in T cells indicate that the immunogenicity of the new antigen has an impact on the immunogenicity of the mRNA vaccine.

[0108] Table 3 Specific CD8 + The percentage of positive cells for T cell markers TNF-α and IFN-γ

[0109] Control group Experimental Group 1 Experimental Group 2 Experimental Group 3 <![CDATA[CD8 + T cells expressing TNF-α positive cells%]]> 1.5 27.6 23.4 21.8 <![CDATA[CD8 + T cells expressing INF-γ positive cells%]]> 3.6 58.4 53.7 49.8

[0110] Example 5

[0111] This example evaluates the in vitro tumor cell killing ability of mRNA1 vaccine, mRNA2 vaccine and mRNA3 vaccine, specifically including:

[0112] (1) Specific T cell killing experiment in vitro

[0113] DC cells and CD8 + T cells were co-cultured. CD8 + T cells 5×10 6 1.53×10 DC cells loaded with mRNA1 vaccine 6 The cells were cultured in T cell culture medium at 37°C and 5% CO2 for 7 days to obtain CD8 + DC cells loaded with mRNA2 vaccine and DC cells loaded with mRNA3 vaccine were prepared according to the above method to obtain CD8 T cells stimulated by DC cells loaded with mRNA2 vaccine. + T cells, DC cells stimulated by mRNA3 vaccine + T cells.

[0114] Acquisition of specific CD8 + After T cells, breast cancer MCF-7B cells containing luciferase markers were selected as target cells for tumor cell killing evaluation, specifically:

[0115] First, a blank group, control groups 1-3, and experimental groups 4-6 were set up; the blank group was cultured with breast cancer MCF-7B cells alone, and the amount of breast cancer MCF-7B cells added to each well was 1.0×10 5 The control group 1 contained a mixture of breast cancer MCF-7B cells and mRNA1 vaccine, the control group 2 contained a mixture of breast cancer MCF-7B cells and mRNA2 vaccine, and the control group 3 contained a mixture of breast cancer MCF-7B cells and mRNA3 vaccine. The amount of breast cancer MCF-7B cells added to each well in each control group was 1.0×10 5 The final concentration of mRNA vaccine in each well was 10 μg / mL; Experimental group 4 was CD8 stimulated by breast cancer MCF-7B cells and DC cells loaded with mRNA1 vaccine. + T cell co-culture, experimental group 5 is breast cancer MCF-7B cells and DC cells loaded with mRNA2 vaccine stimulated CD8 + T cell co-culture, experimental group 6 is breast cancer MCF-7B cells and DC cells loaded with mRNA3 vaccine stimulated CD8 +T cells were co-cultured, and the amount of breast cancer MCF-7B cells added to each well in each experimental group was 1.0×10 5 CD8 stimulated by DC cells loaded with mRNA vaccine + The amount of T cells added to each well was 1.0×10 5 indivual;

[0116] Then, the blank group, each experimental group and each control group were placed in a cell culture incubator at 37°C and 5% CO2 for 10 h. After that, the fluorescence value was measured by an ELISA instrument and the CD8 + The efficiency of T cells killing tumor cells: the killing efficiency of the experimental group = (1-fluorescein value of the experimental group / fluorescein value of the blank group) × 100%; the killing efficiency of the control group = (1-fluorescein value of the control group / fluorescein value of the blank group) × 100%.

[0117] The experimental results are as follows Figure 5 As shown in the figure, each control group had almost no killing effect on breast cancer MCF-7B cells; DC cells loaded with mRNA vaccine stimulated CD8 + T cells have a significant killing effect on breast cancer MCF-7B cells. By comparison, DC cells loaded with mRNA1 vaccine stimulated CD8 + The killing efficiency of T cells on breast cancer MCF-7B cells (87.3%) was higher than that of CD8 + CD8 T cells and DC cells stimulated by mRNA3 vaccine + The killing efficiency of T cells on breast cancer MCF-7B cells (67.4%, 61.2%) further illustrates that the immunogenicity of the new antigen has an impact on the immunogenicity of the mRNA vaccine.

[0118] (2) Verification of the anti-tumor activity of mRNA vaccines in solid tumor models

[0119] The specific experimental method is as follows: digest breast cancer MCF-7B cells in the logarithmic growth phase, and construct a breast cancer mouse model by subcutaneously injecting breast cancer MCF-7B cells into C57BL / 6 mice. 14 days later, the mice injected with breast cancer MCF-7B cells were randomly divided into 4 groups (n=10), and PBS, mRNA1 vaccine, mRNA2 vaccine, and mRNA3 vaccine were injected into the tail vein once a week. The survival status of the mice was monitored every day for 50 days after the model was established, and the survival curve was drawn, as shown in the figure. Figure 6 shown.

[0120] Depend on Figure 6 It can be seen that the mRNA1 vaccine showed the best improvement in survival, with a survival rate of 50% within 50 days, indicating that this type of mRNA vaccine has a better therapeutic effect.

[0121] The personalized mRNA vaccine for treating breast cancer provided in this application is synthesized based on the gene sequencing results of breast cancer patients. It is unique to breast cancer patients and is a truly personalized vaccine. In addition, the personalized mRNA vaccine uses the gene sequence of the virus rather than the virus itself, and there is no risk of infection. This personalized mRNA vaccine can not only stimulate CD8 + T cells, promote the expression of IFN-γ and TNF-α, and also stimulate CD8 + T cells have the ability to kill tumor cells. In addition, this personalized mRNA vaccine has stronger targeting, less toxic side effects, and can improve the immune microenvironment. It has the advantages of good stability, high anti-tumor efficiency, safety and effectiveness, controllable quality, and a short R&D cycle.

[0122] The present application also provides the use of any of the above-described personalized mRNA vaccines for treating breast cancer in the preparation of drugs for the treatment of breast cancer.

[0123] Although the preferred embodiments of the present application have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0124] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A personalized mRNA vaccine for treating breast cancer, characterized in that: The invention comprises an mRNA molecule and lipid nanoparticles, wherein the amino acid sequence of the expression sequence encoded by the gene coding region of the mRNA molecule is shown in any one of SEQ ID NO: 85 to SEQ ID NO:

87.

2. The personalized mRNA vaccine for treating breast cancer according to claim 1, characterized in that: The synthesis of the mRNA molecule specifically comprises: S10. Collect tumor tissue and healthy blood samples from breast cancer patients for gene sequencing to obtain somatic mutations in tumors; S20, using the immune epitope database to evaluate the HLA affinity of the acquired somatic mutations to obtain new antigen peptides; S30. Use the tumor antigen database platform CAD (1.0) to perform online analysis of new antigen peptides, preliminarily verify the immunogenicity of new antigen peptides, and screen out new antigen peptides with high affinity to HLA; S40, synthesizing the screened new antigen polypeptides with high affinity to HLA, and then culturing them with the patient's immune cells, and screening the new antigen polypeptides with high immunogenicity by ELISA; S50, connecting the screened new antigen polypeptides with high immunogenicity to synthesize mRNA molecules.

3. The personalized mRNA vaccine for treating breast cancer according to claim 2, characterized in that: In the S50, the screened new antigen polypeptides with high immunogenicity are connected to synthesize mRNA molecules, which specifically includes: The screened new antigen peptides are arranged in descending order according to the number of immune spots; The arranged new antigen peptides are evenly divided into N groups, each group includes m new antigen peptides, and 1≤m≤10; The new antigen peptides in each group are connected in descending order of the number of immune spots to synthesize mRNA molecules.

4. The personalized mRNA vaccine for treating breast cancer according to claim 2, characterized in that: The synthesis of the mRNA molecule also includes: A His tag is added to the N-terminus of the synthesized mRNA molecule, and a cap is added to the 5' end and a Poly(A) tail structure is added to the 3' end of the synthesized mRNA molecule.

5. The personalized mRNA vaccine for treating breast cancer according to claim 1, characterized in that: The lipids in the lipid nanoparticles include trimethyl-2,3-dioleyloxypropylammonium bromide, dipalmitoylphosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000; The mass ratio of the mRNA molecule, trimethyl-2,3-dioleyloxypropylammonium bromide, dipalmitoylphosphatidylcholine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 3:10:4:4:

1.

6. The personalized mRNA vaccine for treating breast cancer according to claim 5, characterized in that: Including the following ingredients: 1.5 mg mRNA molecule, 5 mg trimethyl-2,3-dioleyloxypropylammonium bromide, 2 mg dipalmitoylphosphatidylcholine, 2 mg cholesterol, 0.5 mg distearoylphosphatidylethanolamine-polyethylene glycol 2000, 20 mL citric acid buffer and 1.5 mL anhydrous ethanol.

7. The method for preparing a personalized mRNA vaccine for treating breast cancer according to any one of claims 5 or 6, characterized in that: The following steps are involved: S1. Place trimethyl-2,3-dioleyloxypropylammonium bromide, dipalmitoylphosphatidylcholine, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a sterile glass bottle, then add anhydrous ethanol to completely dissolve them to obtain a lipid solution; S2, purifying the mRNA molecules, dissolving the purified mRNA molecules in a citric acid buffer (pH=4.0), and gently mixing to obtain an mRNA solution; S3, injecting the lipid solution at a flow rate of 2 mL / min and the mRNA solution at a flow rate of 0.5 mL / min into the channel of the microfluidic chip, so that the two are mixed in the microfluidic chip to form a mixed solution; S4. Transfer the mixed solution to a dialysis bag and dialyze it in 50 mL of PBS (pH=7.4) for 4 hours, replacing PBS every hour to obtain mRNA-loaded lipid nanoparticles, i.e., mRNA vaccines.

8. The method for preparing a personalized mRNA vaccine for treating breast cancer according to claim 7, characterized in that: Also includes: The sequence of the mRNA molecule is input into the RNAstructure platform to obtain the secondary structure prediction results of the mRNA molecule and analyze the sites where the mRNA molecule is prone to bind to lipids; The information of the selected lipids was queried on the LipidMaps platform, and the preparation conditions in S3 were obtained based on the structure and properties of the selected lipids and the site.

9. The method for preparing a personalized mRNA vaccine for treating breast cancer according to claim 7, characterized in that: In S1, the total lipid concentration of the lipid solution is 10 mM.

10. Use of the personalized mRNA vaccine for treating breast cancer according to any one of claims 1 to 6 in the preparation of a medicament for treating breast cancer.

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