Mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine as well as preparation method and application thereof

Abstract

The invention discloses a mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine and a preparation method and application thereof.The breast cancer therapeutic tumor neoantigen mRNA vaccine comprises an antigen epitope composition, the antigen epitope composition comprises at least one of seven polypeptide fragments combined with MHC class I molecules and three polypeptide fragments combined with MHC class II molecules, and the antigen epitope composition comprises at least one of three polypeptide fragments combined with MHC class II molecules; the amino acid sequences of the seven polypeptide fragments combined with the MHC class I molecules are respectively shown as SEQ ID NO.1-SEQ ID NO.7, and the amino acid sequences of the three polypeptide fragments combined with the MHC class II molecules are respectively shown as SEQ ID NO.8-SEQ ID NO.10. The invention further discloses a preparation method of the polypeptide fragments combined with the MHC class I molecules. The vaccine has an obvious inhibition effect on tumor growth.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine and a preparation method and application thereof. Background Art

[0002] Breast cancer is a malignant tumor that occurs in the mammary glandular epithelial tissue. It is the most common malignant tumor in women worldwide and poses a serious threat to women's health. Studies on its pathogenesis and metastasis mechanisms have shown that it is a tumor with highly heterogeneous molecular typing and clinical characteristics. Treatments for breast cancer include surgery, endocrine therapy, targeted therapy, chemotherapy, and radiotherapy. Immunotherapy has achieved certain results, but there is still a lack of screening criteria for people who are good at immunotherapy and biomarkers that predict efficacy.

[0003] One of the biggest difficulties in conquering tumors is the widespread heterogeneity of tumors. Not only within tumor tissues, but even for the same type of tumor, there are huge differences between different patients. During the rapid growth and proliferation of cancer cells, they often do not have time to repair the errors that occur during DNA replication, so many new mutant proteins will appear, which are called tumor neoantigens. The principle of personalized tumor neoantigen vaccines is to discover specific tumor neoantigens in the patient's body, and then develop vaccines to specifically activate the immune system, so that T cells can kill tumor cells expressing such neoantigens, and achieve truly personalized precision treatment. The combination of immunotherapy and personalized tumor neoantigen mRNA vaccines has made significant clinical progress, bringing new treatment options to patients with tumors such as melanoma and non-small cell lung cancer.

[0004] At present, there is a lack of preclinical evaluation methods for the combination therapy of tumor neoantigen vaccines and other types of tumor therapeutic drugs, which makes it impossible to effectively evaluate the efficacy of combined treatment. In the study of breast cancer, the mouse breast cancer cell line 4T1 is usually used as a tumor model. 4T1 can be easily transplanted into mice, is highly tumorigenic and invasive, and can spontaneously metastasize to distant organs. The tumor growth and metastatic spread of 4T1 cells in BALB / C mice are very similar to those of human breast cancer, and are widely used in allogeneic tumor models of human breast cancer. 4T1 is used as a tumor model to screen and identify tumor neoantigens, and the prepared tumor neoantigen vaccine mainly activates CD4 + T cells, but the tumor suppression effect has not been reported (Kreiter, S. et al. Nature 520, 692–696 (2015).).

[0005] To solve this problem, it is necessary to develop a tumor neoantigen mRNA vaccine for the mouse 4T1 breast cancer model for preclinical evaluation of the efficacy of the tumor neoantigen vaccine in combination with other therapeutic methods. Summary of the invention

[0006] The purpose of the present invention is to provide a tumor neoantigen mRNA vaccine for a mouse 4T1 breast cancer model and a preparation method and application thereof, which has a significant inhibitory effect on tumor growth.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In the first aspect of the present invention, a mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine is provided, and the mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine comprises: an antigen epitope composition, the antigen epitope composition comprises at least one of 7 polypeptide fragments binding to MHC-I class molecules and 3 polypeptide fragments binding to MHC-II class molecules, the amino acid sequences of the 7 polypeptide fragments binding to MHC I class molecules are respectively shown as SEQ ID NO.1-SEQ IDNO.7, and the amino acid sequences of the 3 polypeptide fragments binding to MHC II class molecules are respectively shown as SEQ ID NO.8-SEQID NO.10.

[0009] Furthermore, the antigen epitope composition includes two or more of 7 polypeptide fragments binding to MHC class I molecules and 3 polypeptide fragments binding to MHC class II molecules, which are connected in series via a linker.

[0010] Furthermore, the linker is selected from GSGS.

[0011] Furthermore, the mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine also includes encapsulation materials.

[0012] Furthermore, the encapsulating material includes at least one of ionic cationic lipids, phosphatidylcholine, cholesterol and polyethylene glycol lipids.

[0013] In the second aspect of the present invention, a method for preparing the mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine is provided, the preparation method comprising:

[0014] The 31 amino acid polypeptide fragments containing antigen epitopes as shown in SEQ ID NO.1 to SEQ ID NO.10 are connected in series through a linker, and then codon optimized to obtain a DNA sequence;

[0015] Expanding and culturing the recombinant plasmid constructed by the DNA sequence in Escherichia coli, and collecting the bacterial cells;

[0016] The bacterial cells are subjected to alkaline lysis, neutralization, clarification, concentration, liquid replacement, purification, linearization, enzyme digestion and template purification to obtain a high-quality template;

[0017] The template is subjected to in vitro transcription reaction in vitro to obtain mRNA stock solution, which is then encapsulated into lipid nanoparticles through an ethanol lipid mixture to obtain a therapeutic tumor neoantigen mRNA vaccine for mouse 4T1 breast cancer.

[0018] As an optional embodiment, the linker is GSGS.

[0019] As an optional embodiment, the connection order in the series connection is not limited. Preferably, the series connection is from left to right according to the neoantigen score, and the MHC-I class molecules (nucleic acid molecules encoding 7 polypeptide fragments that bind to the MHC-I class molecules) are preferentially connected, and then the class II molecules (nucleic acid molecules encoding 3 polypeptide fragments that bind to the MHC-II class molecules) are connected.

[0020] In the third aspect of the present invention, the use of the mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine in the preparation of anti-breast cancer drugs is provided.

[0021] The present invention has the following advantages and beneficial effects:

[0022] 1. The tumor neoantigen mRNA vaccine of the present invention uses high-throughput sequencing technology to perform exon sequencing and RNA sequencing on 4T1 tumor cell samples, analyze the mutation sites in tumor cells, perform amino acid and transcript annotation and differential gene expression analysis on the mutation sites, and then use bioinformatics methods to screen out tumor neoantigens based on the comprehensive score of factors affecting the immunogenicity of neoantigens such as the affinity of mutant peptides binding to MHC-I and MHC-Ⅱ molecules, the cleavage ability of proteasomes to mutant proteins, and the intracellular transport level of peptides. The top 10 MHC-I and 10 MHC-II molecule mutant peptides with the top 10 comprehensive scores are extended to 31 amino acids with the mutation site as the center, prepared into mRNA single chains through linker series connection, and further prepared into mRNA vaccines. By verifying the immunogenicity of tumor neoantigens through the IFN-γELISPOT in vitro experimental method, 10 immunogenic neoantigens were screened out.

[0023] 2. We connected these 10 immunogenic neoantigens in series through linkers to prepare single-stranded mRNA and prepared it into an mRNA vaccine. Compared with the existing technology, the length of this vaccine is 31 amino acids, which makes the neoantigen more likely to be sheared in the body, thereby increasing the possibility of being recognized, bound and presented by MHC molecules, thereby effectively activating T cells, especially CD8 +T cells, so an unexpected therapeutic effect was achieved in the 4T1 tumor model, significantly inhibiting the growth of mouse tumors, with a tumor (volume) inhibition rate TGI (The Tumor Growth Inhibition value) of 74.4%. The present invention provides a reliable evaluation model for preclinical personalized tumor neoantigen vaccines and other types of tumor treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0025] Figure 1 This is a flowchart for the prediction and screening of tumor neoantigens and a schematic diagram of the 10 antigen concatenations.

[0026] Figure 2 The IFNγ-ELISPOT method was used to screen neoantigens with immunogenicity.

[0027] Figure 3 The tumor inhibitory effect of tumor neoantigen mRNA vaccine preparation in the mouse 4T1 tumor model.

[0028] Figure 4 The mRNA-LNP-10 4T1 tumor neoantigen vaccine significantly activated CD4 + and CD8 + T cells, where A is CD3 + T cell ratio, B is CD4 + Activation ratio, C is CD8 + Activation ratio. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0030] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0031] The present application will be described in detail below with reference to embodiments and experimental data.

[0032] Example 1. Prediction and screening of neoantigens in tumors

[0033] 1. Whole exome sequencing and mRNA gene expression determination

[0034] Mouse genomic DNA is randomly broken into fragments of approximately 180-280 bp in length by a Covaris ultrasonic crusher, and end repair and single-base A tail are added. Specific adapters are then connected to both ends of these fragments to construct a mouse DNA library. Next, after pooling, the mouse library with different index tags will be liquid-phase hybridized with up to hundreds of thousands of biotin-labeled specific probes provided in the AgilentSureSelect Mouse All Exon Kit. These probes are designed to cover a total of millions of exon regions of approximately 20,000 genes in the mouse genome, ensuring effective capture of target exons. After hybridization, the DNA fragments hybridized with the probes are selectively adsorbed by magnetic beads, thereby achieving enrichment of the exon region. After elution and purification, the captured exons are linearly amplified by PCR, and when the library quality meets the standard, the IlluminaHiseq platform is sequenced according to the effective concentration of the library and the data output requirements. After obtaining the original sequencing sequence (Sequenced Reads), somatic mutations were identified by comparing it with the reference genome (GRCm38), and a total of 1,167 SNVs (single nucleotide variants) were identified.

[0035] We used bioinformatics to conduct a comprehensive analysis of the immunogenicity of the peptide sequences corresponding to these mutations and the wild-type sequences, and then selected the top 10 peptides that could be bound and presented by MHC-I and II molecules.

[0036] 2. Preparation of 4T1 tumor neoantigen mRNA vaccine

[0037] (1) mRNA synthesis

[0038] We took each predicted antigen and its mutated amino acid as the center, extended the peptide length to 31 amino acids, connected 20 31 amino acid new antigen amino acid sequences in series through the linker GSGS, and optimized the amino acid sequence (sequence SEQID NO: 12) through codons to obtain a DNA sequence (sequence SEQ ID NO: 11), inserted the synthesized DNA sequence into the BglI and MluI restriction sites of the pUC57-Kan plasmid vector, transferred it into E.Coli and selected high-expression positive clones. After shaking flask expansion culture, the bacteria were collected. After alkaline lysis, neutralization, clarification, concentration and liquid replacement, purification, linearization and restriction digestion, and template purification, high-quality templates were obtained for in vitro transcription. The templates all include a DNA template containing an open reading frame surrounded by 5' and 3' untranslated region (UTR) sequences, and end with a sequence encoding a poly A tail.

[0039] The template is transcribed in vitro using optimized T7 RNA polymerase and transcription conditions to obtain mRNA.

[0040] (2) Purification of mRNA

[0041] The synthesized mRNA was purified by oligo-dT affinity, the buffer was exchanged to sodium acetate by lateral flow filtration, and sterile filtered. RNA integrity was determined by microfluidic parallel capillary electrophoresis (Fragment Analyzer system5200, Agilent), and the concentration, pH value, residual DNA, protein, and double-stranded RNA impurities of the solution were also determined. The 5' end capping efficiency and 3'-polyadenosine (poly A) tail of mRNA were analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0042] (3) Preparation of mRNA vaccine particles

[0043] SM102: DSPC: cholesterol: DMG-PEG2000 were dissolved and mixed in ethanol at a molar ratio of 48:10:40.5:1.5. The lipid phase flow rate of the microfluidic device was set to 6 ml / min, and the mRNA phase flow rate was set to 18 ml / min. The mRNA mixed solution and the lipid mixed solution were encapsulated at a flow rate ratio of 3:1. After encapsulation, the sample was collected by ultrafiltration and liquid exchange to obtain the mRNA vaccine (mRNA4T1-20).

[0044] The product has been analytically characterized, including determination of particle size and polydispersity, encapsulation efficiency, pH, endotoxin, and mRNA concentration (Table 1), making it suitable for in vitro and in vivo scientific studies at the preclinical stage.

[0045] Table 1. Vaccine characterization

[0046] serial number project result 1 pH 7.3 2 Particle size 112nm 3 Dispersion coefficient 0.29 4 Encapsulation efficiency 85％ 5 mRNA content 160μg / ml 6 Bacterial endotoxins 7EU / ml

[0047] (4) Screening and identification of tumor neoantigens

[0048] BALB / c mice were immunized with mRNA4T1-20 (12ug was injected intramuscularly into the right back of the hind limb), and a booster immunization was performed on the 7th day. 14 days after the immunization, the spleen of the mice was obtained and the spleen single cell suspension was prepared.

[0049] 2×10 5 Freshly isolated splenocytes were inoculated into a 96-well plate coated with polyvinylidene fluoride (PVDF) containing IFNγ capture antibody. Cells were stimulated with peptides synthesized from predicted neoantigens, with a final concentration of 1 mg / mL for each peptide. The negative control wells were culture medium without peptides, and the positive control was 10ul of dissolved positive stimulant (concentration 5ug / mL). After overnight stimulation, the plates were washed, and biotinylated IFNγ detection antibodies were added in sequence. ImmunoSpot Analyzer was used for imaging and ImmunoSpot software was used for quantification. T cell epitopes of mutant antigens were screened out. If the number of spots in the negative control wells was 0-5 spots, the number of spots in the negative control wells was subtracted from the number of spots in the experimental wells to be ≥6; if the number of spots in the negative control wells was ≥6 spots, the number of spots in the experimental wells must be ≥2 times the number of spots in the negative control wells. Using this as the positive standard, a total of 7 of the 20 neoantigen peptides were positive, coming from mutant antigens that could be presented by MHC-I class molecules and 3 mutant antigens that could be presented by class II molecules, indicating that they had potential immunogenicity.

[0050] Table 2 Immunogenic polypeptide sequences of the present invention

[0051]

[0052] Example 2: Detection of the tumor-suppressing effect of 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine in 4T1 mice

[0053] 1. Preparation of therapeutic tumor neoantigen mRNA vaccine for mouse 4T1 breast cancer

[0054] The 31-mer polypeptide fragments shown in SEQ ID NO.1-SEQ ID NO.10 were connected in series through a linker (GSGS) (sequence SEQ ID NO.12) and prepared into a tumor neoantigen mRNA vaccine (mRNA-4T1-10) as described in Example 1.

[0055] 2. Detection of the anti-tumor effect of mRNA vaccine against neoantigens of 4T1 breast cancer in mice

[0056] 1. Establish a 4T1 tumor model in BALB / c female mice. The injection volume of 4T1 cells was 50 μl / mouse (1 million cells / mouse). The mice were divided into a treatment group and a control group, with 10 mice in each group.

[0057] 2. Starting from the fourth day after tumor cell inoculation, mice were treated with the selected mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine (mRNA-4T1-10) prepared above, with a dose of 12 μg / mouse;

[0058] 3. Administer medication on the 4th, 7th, and 11th days after tumor implantation;

[0059] 4. Measure the tumor diameter with a vernier caliper every 2-3 days. The tumor volume is calculated as: V = 0.5a × b 2 , a and b represent the long and short diameters of the tumor, respectively;

[0060] 5. Tumor growth curves were drawn based on tumor size, with each data point represented by mean tumor volume ± standard error (SEM), n = 10;

[0061] The results are as follows Figure 3 As shown, the therapeutic tumor neoantigen mRNA vaccine mRNA-LNP-10 for mouse 4T1 breast cancer has significant anti-tumor activity. Compared with the existing technology, it significantly inhibited tumor growth in the 4T1 tumor model, and the tumor inhibition efficacy reached 74.4% with TGI.

[0062] Example 3. Tumor neoantigen mRNA vaccine significantly activates CD8 + T cells

[0063] At the end of the experiment, mouse spleen cells were obtained and digested into a single cell suspension with a concentration of 1×10 6 cells / 100 μL, and flow cytometry was used to detect the immune cell components in the spleen of mice.

[0064] Mouse spleen was dissected and ground on a disposable sterile mesh. A disposable sterile culture dish was placed under the mesh. The spleen was soaked in a small amount of 1x PBS and gently ground with the rubber stopper of a 5mL syringe. 2ml of PBS was added to rinse the mesh. The collected cell suspension was transferred to a 1.5mL centrifuge tube and centrifuged at 1800rpm for 5min. The supernatant was discarded and resuspended with 1ml of red blood cell lysis buffer. Lysis was performed for 8min. 10ml of PBS was added to terminate the lysis and centrifuged at 1800rpm for 5min. The supernatant was discarded and resuspended with 1ml of PBS and transferred to a 1.5ml centrifuge tube. The cells were counted to a concentration of 1×10 6cells / 100μl, then add CD3, CD4, and CD8 antibodies, and incubate at 4℃ in the dark for 30 minutes. After the antibody incubation is completed, add 1ml 1×PBS, centrifuge at 4℃, 1800rpm for 5 minutes, remove the supernatant, resuspend the cells with 200μl 1xPBS buffer, transfer to a flow tube, and detect on a flow cytometer.

[0065] Analysis of CD3 + T cells, CD3 + CD4 + T cells, CD3 + CD8 + The percentage of T cells. Oneway ANOVA statistical method was used to perform statistical analysis on the data differences between the two groups. Error bars represent the standard error of the mean (SEM). *p<0.05; **p<0.01; ***p<0.001.

[0066] The results are as follows Figure 4 As shown, the CD3 + T cells, CD3 + CD4 + T cells, CD3 + CD8 + The proportion of T cells increased significantly compared with the control group. These data indicate that the 4T1 tumor vaccine preparation produced a very effective T cell response in tumor-bearing mice, producing CD4 + T cells and CD8 + T cells exert anti-tumor immune responses.

[0067] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0068] Although the preferred embodiments of the present invention 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 that fall within the scope of the present invention.

[0069] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine, characterized in that: The mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine includes: an antigen epitope composition, which includes at least one of 7 polypeptides bound to MHC-I class molecules and 3 polypeptide fragments bound to MHC-II class molecules, the amino acid sequences of the 7 polypeptide fragments bound to MHC I class molecules are respectively shown as SEQ ID NO.1-SEQ ID NO.7, and the amino acid sequences of the 3 polypeptide fragments bound to MHC II class molecules are respectively shown as SEQ ID NO.8-SEQ ID NO.

10.

2. A mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine according to claim 1, characterized in that: The antigen epitope composition comprises two or more of 7 polypeptide fragments binding to MHC-I molecules and 3 polypeptide fragments binding to MHC-II molecules.

3. A mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine according to claim 2, characterized in that: The multiple polypeptide fragments are connected in series via a linker.

4. A mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine according to claim 3, characterized in that: The linker is GSGS.

5. The therapeutic tumor neoantigen mRNA vaccine for mouse 4T1 breast cancer according to claim 1, characterized in that: The mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine also contains encapsulated materials.

6. A mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine according to claim 5, characterized in that: The encapsulating material includes at least one of ionic cationic lipids, phosphatidylcholine, cholesterol and polyethylene glycol lipids.

7. A method for preparing a therapeutic tumor neoantigen mRNA vaccine for mouse 4T1 breast cancer according to any one of claims 1 to 6, characterized in that: The preparation method comprises: The 31 amino acid polypeptide fragments containing antigen epitopes as shown in SEQ ID NO.1 to SEQ ID NO.10 are connected in series through a linker, and then codon optimized to obtain a DNA sequence; Expanding and culturing the recombinant plasmid constructed by the DNA sequence in Escherichia coli, and collecting the bacterial cells; The bacterial cells are subjected to alkaline lysis, neutralization, clarification, concentration, liquid replacement, purification, linearization, enzyme digestion and template purification to obtain a high-quality template; The template is subjected to in vitro transcription reaction in vitro to obtain mRNA stock solution, which is then encapsulated into lipid nanoparticles by encapsulation materials to obtain mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine.

8. The method for preparing the mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine according to claim 7, characterized in that: The linker includes a flexible linker.

9. The method for preparing the mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine according to claim 7, characterized in that: The DNA sequence is shown in SEQ ID NO:

11.

10. Use of the mouse 4T1 breast cancer therapeutic tumor neoantigen mRNA vaccine described in any one of claims 1-6 in combination with other anti-tumor treatment methods in the preparation of preclinical efficacy evaluation products.

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