CN112516296A - New antigen RNA and immune adjuvant poly (I: C) composite vaccine and construction method thereof - Google Patents

Abstract

The invention provides a new antigen RNA and immune adjuvant Poly (I: C) composite vaccine, which comprises a PEG-PLL-PLLeu self-assembled nano micelle, a new antigen RNA vaccine loaded on the PEG-PLL-PLLeu self-assembled nano micelle and an immune adjuvant Poly (I: C); wherein, the nucleotide sequence of the neoantigen RNA is shown as SEQ ID NO. 1. The new antigen RNA and immune adjuvant poly (I: C) composite vaccine provided by the invention has the advantages of good stability, high translation efficiency, easy transfer of expression products and good treatment effect.

Description

New antigen RNA and immune adjuvant poly (I: C) composite vaccine and construction method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel antigen RNA and immune adjuvant poly (I: C) composite vaccine and a construction method thereof.

Background

The lung cancer is the malignant tumor with the highest incidence in China, according to the statistical data of the national cancer center, the number of new lung cancer cases and lung cancer lethal cases in China is increased, and the lung cancer is at the top of all malignant tumors no matter the incidence or the mortality, thus seriously threatening the life health of people.

Non-small cell lung cancer (NSCLC) is the major pathological type of lung cancer, accounting for approximately 80%. Of the diagnosed NSCLC, more than half of patients with advanced stage inoperable are only eligible to receive drugs or best supportive treatment, and the treatment of advanced NSCLC has been the focus of research. Since the last century, a platinum-based combination chemotherapy has been the first choice, but it only marginally improved patient survival at the expense of significant toxic side effects, and the 5-year survival rate for advanced NSCLC remains only 2.8%. Since the beginning of this century, with the discovery of NSCLC driver gene mutation, a series of small molecule Tyrosine Kinase Inhibitors (TKIs) drugs for driver gene mutation such as Epidermal Growth Factor Receptor (EGFR) and Anaplastic Lymphoma Kinase (ALK) were applied to clinical application in sequence, so that the 5-year survival rate of the population with positive driver gene sensitive mutation in advanced NSCLC was improved to 14.6%.

However, efficacy against driver mutations is limited to the sensitive mutant population. Meanwhile, malignant tumors are continuously cloned and evolved to generate new drug resistance mutation, so that the generation of small molecule targeted drug resistance in a short time is caused. In the late days, immunotherapy has been a hot spot due to the success of the development of immune checkpoint inhibitor drugs (ICIs). In the research aiming at the NSCLC, the apoptosis molecule-1 (PD-1) and the ligand thereof (PD-L1) and the related drugs of cytotoxic T lymphocyte antigen-4 (CTLA-4) achieve good clinical curative effect and are approved to be used for treating the advanced NSCLC at home and abroad. However, the overall effectiveness of ICIs is only around 20%, probably because the therapeutic effects of ICIs require the body to have an effective anti-tumor immune response and an appropriate immune microenvironment. At present, people with advanced NSCLC without sensitive mutation and with poor ICIs treatment effect still use chemotherapy as a main means, and have poor prognosis, so that the overall 5-year survival rate of the advanced NSCLC is still below 5 percent, and a new treatment means is urgently needed to be searched.

In fact, immunotherapy, in addition to ici drugs, anti-tumor vaccines and adoptive immune cell therapy are also viable approaches. In principle, an anti-tumor immune response must be elicited by a tumor antigen. The current research considers that the antigen expressed by the tumor mutant gene, neoantigen (neoantigen), is specific to tumor cells, can avoid central tolerance of T cells and has the capability of inducing immune response. A series of reports of Nature and Cancer Research prove that the neoantigen-based tumor vaccine treatment has considerable effective rate and good clinical application potential. Depending on the form of vaccine used, the current research directions can be divided into three categories: peptide vaccines, nucleic acid vaccines and neoantigen loaded DC cell vaccines. Research evidence shows that the RNA vaccine form in the nucleic acid vaccine has advantages in cost and safety, and unlike peptide vaccines, the RNA vaccine is designed without considering HLA restriction, and has great clinical potential.

However, the efficacy of tumor neoantigen RNA vaccines is limited by three important factors: design of RNA sequence. Research shows that the sequence of the new antigen RNA vaccine needs to find out the specific mutation of tumor tissue by comparing exon sequencing results of the tumor tissue and normal tissue, and then further perform immunogenicity verification to determine the RNA sequence for preparing the vaccine. 2. Load delivery of neoantigen RNA vaccines. Because RNA is readily degraded by enzymes, neoantigen RNA vaccines need to be delivered from specific vectors, which is desirable to both protect RNA from degradation and enhance targeted delivery. 3. Is suitable for tumor immune microenvironment where immune cells exert efficacy.

In order to search for a new treatment mode of advanced NSCLC, the invention aims to take mouse Lewis lung cancer cell C57BL6 mouse transplantation tumor as a model object, select a new antigen RNA vaccine as an incision point, and expect to successfully prepare a new antigen RNA vaccine and a composite nano preparation of an immune adjuvant.

Disclosure of Invention

The technical problem is as follows: in order to solve the defects of the prior art, the invention aims to provide a novel antigen RNA and immune adjuvant poly (I: C) composite vaccine and a construction method thereof.

The technical scheme is as follows: the invention provides a new antigen RNA and immune adjuvant Poly (I: C) composite vaccine, which comprises a PEG-PLL-PLLeu self-assembled nano micelle, a new antigen RNA vaccine loaded on the PEG-PLL-PLLeu self-assembled nano micelle and an immune adjuvant Poly (I: C); wherein, the nucleotide sequence of the neoantigen RNA is shown as SEQ ID NO. 1.

Preferably, the molar ratio of the PEG-PLL-PLLeu self-assembled nano-micelle to the neoantigen RNA vaccine is between 1:1 and 10: 1; the molar ratio of the neoantigen RNA vaccine to the Poly (I: C) is between 1:1 and 5: 1.

The preparation method of the PEG-PLL-PLLeu self-assembly nano micelle comprises the following steps:

(1) synthesis of PEG-PLL-PLLeu: performing ring-opening polymerization on amino polyethylene glycol (NH2-PEG) and LLZ-NCA to obtain a PEG-pLLZ polymer; further ring-opening polymerization of the PEG-pLLZ polymer and the LLEu-NCA to obtain PEG-pLLZ-pLLEu; performing deprotection reaction to obtain PEG-PLL-PLLeu;

(2) self-assembly of PEG-PLL-PLLeu: dissolving PEG-PLL-PLLeu with concentration of 1mg/ml, using ultrapure water as solvent, magnetically stirring overnight, and performing ultrasonic treatment after complete dissolution to obtain the PEG-PLL-PLLeu self-assembled nano micelle.

The preparation method of the antigen RNA vaccine comprises the following steps:

(1) selecting pCI-neo (pCI-neo mammalian expression vector) plasmid as a vector, inserting the DNA sequence of the RNA vaccine of claim 1 between pCI-neo plasmid XhoI and NotI enzyme cutting site, and amplifying to obtain plasmid for loading RNA vaccine sequence;

(2) cutting the plasmid loaded with the RNA vaccine sequence by using a Sap I endonuclease to linearize the plasmid;

(3) using in vitro transcription kit (mMESSAGE mMACHINE)^TMT7 ULTRA Transcription Kit) in vitro Transcription of the linearized plasmid;

(4) the transcription product is then purified by a kit (MEGAclear)^TMTransfer Clean-Up Kit) to obtain the RNA vaccine.

The invention also provides a construction method of the novel antigen RNA and immune adjuvant poly (I: C) composite vaccine, which comprises the following steps:

(1) preparing the PEG-PLL-PLLeu self-assembled nano micelle:

(1.1) Synthesis of PEG-PLL-PLLeu: performing ring-opening polymerization on amino polyethylene glycol (NH2-PEG) and LLZ-NCA to obtain a PEG-pLLZ polymer; further ring-opening polymerization of the PEG-pLLZ polymer and the LLEu-NCA to obtain PEG-pLLZ-pLLEu; performing deprotection reaction to obtain PEG-PLL-PLLeu;

(1.2) self-assembly of PEG-PLL-PLLeu: dissolving PEG-PLL-PLLeu with concentration of 1mg/ml, using ultrapure water as solvent, magnetically stirring overnight, and performing ultrasonic treatment after complete dissolution to obtain PEG-PLL-PLLeu self-assembled nano micelle;

(2) preparation of antigen RNA vaccine:

(2.1) selecting pCI-neo (pCI-neo mammalian expression vector) plasmid as a vector, inserting the DNA sequence of the RNA vaccine of claim 1 between pCI-neo plasmid XhoI and NotI enzyme cutting site, and amplifying to obtain plasmid loaded with RNA vaccine sequence;

(2.2) cutting the plasmid loaded with the RNA vaccine sequence by using Sap I endonuclease to linearize the plasmid;

(2.3) Using in vitro transcription kit (mMESSAGE mMACHINE)^TMT7 ULTRA Transcription Kit) in vitro Transcription of the linearized plasmid;

(2.4) the transcription product is further purified by a kit (MEGAclear)^TMTranscription Clean-Up Kit) to obtain RNA vaccine;

(3) co-loading of PEG-PLL-PLLeu to neoantigen RNA and the immune adjuvant poly (I: C): and co-incubating the PEG-PLL-PLLeu micelle solution, the RNA vaccine and the aqueous solution of Poly (I: C) at 37 ℃ to obtain the new antigen RNA and immune adjuvant Poly (I: C) composite vaccine.

Preferably, the method for constructing the composite vaccine of the neoantigen RNA and the immune adjuvant poly (I: C) comprises the following steps:

(1) preparing the PEG-PLL-PLLeu self-assembled nano micelle:

(1.1) Synthesis of PEG-PLL-PLLeu: the molar ratio of NH2-PEG to LLZ-NCA is 1:10 to 1:50, the two are dissolved in dry dimethyl sulfoxide (dry DMF, wt 10%), stirred for 24-96h under the protection of nitrogen at 30-80 ℃, and then precipitated by excessive ethyl ether to obtain PEG-PLLZ. Dissolving PEG-PLLZ and LLEu-NCA in dry dimethyl sulfoxide (dried DMF, wt 10%) at 30-80 deg.C under protection of nitrogen, stirring for 24-96 hr, and precipitating with excessive diethyl ether to obtain PEG-PLLZ-PLLeu;

dissolving PEG-PLLZ-PLLeu in trichloroacetic acid (wt 5%) and HBr (wt 33%), stirring at 0-30 deg.C under nitrogen protection for 24-96 hr, and precipitating with excessive diethyl ether to obtain crude product;

then dialyzing the crude product by a dialysis membrane with the molecular weight of 3500Da for 24-96 hours respectively in ammonia water (wt 0.1%) and distilled water to obtain a PEG-PLL-PLLeu final product;

(1.2) self-assembly of PEG-PLL-PLLeu: dissolving PEG-PLL-PLLeu with concentration of 0.5-5mg/ml, using ultrapure water as solvent, magnetically stirring overnight, and performing ultrasonic treatment after complete dissolution to obtain PEG-PLL-PLLeu self-assembled nano micelle;

(2) preparation of antigen RNA vaccine:

(2.1) selecting pCI-neo (pCI-neo mammalian expression vector) plasmid as a vector, inserting the DNA sequence of the RNA vaccine of claim 1 between pCI-neo plasmid XhoI and NotI enzyme cutting site, and amplifying to obtain plasmid loaded with RNA vaccine sequence;

(2.2) cutting the plasmid loaded with the RNA vaccine sequence by using Sap I endonuclease to linearize the plasmid;

(2.3) Using in vitro transcription kit (mMESSAGE mMACHINE)^TMT7 ULTRA Transcription Kit) in vitro Transcription of the linearized plasmid;

(2.4) the transcription product is further purified by a kit (MEGAclear)^TMTranscription Clean-Up Kit) to obtain RNA vaccine;

(3) co-loading of PEG-PLL-PLLeu to neoantigen RNA and the immune adjuvant poly (I: C): and co-incubating the PEG-PLL-PLLeu micelle solution, the RNA vaccine and the aqueous solution of Poly (I: C) at 37 ℃ to obtain the new antigen RNA and immune adjuvant Poly (I: C) composite vaccine.

Has the advantages that: the new antigen RNA and immune adjuvant poly (I: C) composite vaccine provided by the invention has the advantages of good stability, high translation efficiency, easy transfer of expression products and good treatment effect. The RNA vaccine for treating non-small cell lung cancer provided by the invention utilizes codon optimization to design a nucleic acid sequence of a new antigen, utilizes a linker to connect, perfects the insertion of sequences such as SEC (SEC-enhanced-sequence separation), MITD (mitogenic-differentiation-mediated isothermal amplification) and the construction of poly A tail, constructs a plasmid after the sequence design is finished, and obtains a target RNA sequence through linearization and in vitro transcription, namely the new antigen RNA vaccine.

Drawings

FIG. 1 is a screening process of tumor neoantigen;

FIG. 2 is a schematic diagram of the structure of pCI-neo;

FIG. 3 is a drawing of agarose gel electrophoresis of RNA vaccine sequence loaded plasmid after linearization;

FIG. 4 is a gel electrophoresis of the synthesized RNA vaccine.

FIG. 5 is a hydrogen spectrum of PEG-PLL-PLLeu, which indicates that each characteristic peak of the PEG-PLL-PLLeu structure exists correctly.

FIG. 6 is an infrared spectrum of PEG-PLL-PLLeu, which shows two characteristic peaks at 1650 and 1550cm-1, representing amide bonds in polypeptide, and additionally shows a peak at 1105cm-1, which is a characteristic peak of C-O-C in PEG structure, thus proving that the structure of the synthesized sample in the present study is correct.

FIG. 7 shows that the PEG-PLL-PLLeu nanoparticles formed by self-assembly have basically stable particle size and no statistical difference in particle size after being continuously measured for 6 weeks.

FIG. 8 is the electron micrograph of PEG-PLL-PLLeu nano-particle, the average measurement value of its particle size is 86.1 + -8.4 nm.

FIG. 9 is a graph showing the uptake of PEG-PLL-PLLeu by DC cells;

FIG. 10 is a graph showing the results of the cytotoxicity test of the composite preparation against CCK8 cells; the CCK8 is used for detecting the influence of the RNA vaccine on the growth activity of 293T cells, and the results indicate that the cell activity vaccine group and the normal saline group are respectively 78.5 +/-9.8% and 88.5 +/-4.7%, and no significant statistical difference exists.

FIG. 11 shows the levels of IFN α, β, γ, TNF α and IL-6 in serum 3h after administration using the Th1/Th2 cytokine panel 6-plex assay. The results show that no obvious increase of serum cytokine level is seen in the normal saline group after administration, IFN alpha and IL-6 are seen in the PM group, and the levels of the detected cytokines are obviously increased in the R + PM group and the R + P + PM group, but no obvious difference exists between the two groups.

Fig. 12 shows the flow cytometry for detecting the proportion of infiltrating lymphocytes in tumor tissues, including CD4+ T cells, CD8+ T cells, Treg cells and TAM cells, and the results suggest that: the proportion of CD4+ T and CD8+ T cells in the R + PM group and the R + P + PM group is obviously higher than that in the normal saline group and the PM group, and the R + P + PM group is more obviously increased than that in the R + PM group; the proportion of Treg cells of the R + PM group and the R + P + PM group is reduced compared with that of the normal saline group and the PM group, but no obvious difference exists; the proportion of TAM cells was elevated in the R + PM group and the R + P + PM group, but there was no significant difference between the two groups.

Fig. 13 is a growth curve of a model mouse graft, with results suggesting: the growth rates of the R + PM group and the R + P + PM group were slower than those of the saline group and the PM group.

FIG. 14 is a survival curve of a model mouse. And (4) prompting by a result: survival of mice in the R + PM group and the R + P + PM group was significantly better than in the saline group and the PM group.

Detailed Description

The following examples are given to further describe the present invention in detail with reference to specific embodiments. The following examples are intended to illustrate the invention, but not to limit the scope of the invention.

The experimental procedures in the following examples are conventional, except for the specific illustrations. The raw materials and test reagents used in the examples were commercially available products except for those specifically mentioned.

Example 1 neoantigen screening

1. Construction and sequencing of animal models

Non-synonymous mutations characteristic of tumors are detected. The first step in the identification of the tumor neoantigen (neoantigen) is to find non-synonymous mutations characteristic of tumor cells. With the progress of sequencing technology, whole-gene sequencing (WGS) or whole-exon sequencing (WES) performed simultaneously by pairing tumor tissues and normal tissues of the same individual has been realized without difficulty. By comparing the results of the two, non-synonymous mutation sites specific to tumor tissues, including point mutations, indel mutations, and frameshift mutations, can be obtained. Recent research shows that the expression condition of non-synonymous mutation can be further prompted from the transcription level by matching with an RNA sequencing (RNAseq) technology, so that the screening of neoantigen is facilitated.

The invention aims at the research of the advanced NSCLC, and the animal model selects a C57BL6 mouse, and constructs a lung cancer Lewis cell subcutaneous transplantation tumor model of the mouse. Mouse tail tissue and mouse lung cancer Lewis cell strain are taken to carry out Whole Exome Sequencing (WES), and meanwhile transcriptome RNA sequencing (RNAseq) is carried out on the mouse lung cancer Lewis cell. Comparing exon sequencing results of mouse tail tissue cells and lung cancer Lewis cell strains, the unique mutations of the lung cancer Lewis cell strains are 3389, wherein the unique point mutations are 3029, and the unique insertion deletion mutations are 360.

Further comparing the expression conditions of the specific mutations obtained by the comparison in the RNAseq result of the lung cancer Lewis cell strain, defining high expression by taking the RNAseq expression fpkm value of more than 60 as a boundary, and selecting the sites which meet missense mutation and have more than moderate influence on the gene function prediction, thereby screening 59 specific mutations, wherein 56 point mutations and 3 insertion deletion mutations are selected.

The screening procedure is shown in FIG. 1; the 59 mutations specific to mouse lung cancer Lewis cells in C57BL6 are shown in Table 1.

TABLE 1

2. Screening for novel antigens

The number of tumor-specific non-synonymous mutations sought by the WGS/WES combined RNAseq technique is usually numerous, but not all of them have the ability to form neoantigen and induce an anti-tumor immune response. The requirement for neoantigen "quality" is that a stronger HLA binding capacity is essential. Numerous experiments have been conducted to examine the binding ability of various antigenic peptides eluted from HLA molecules to HLA molecules using mass spectrometry-based immunopeptidomics (MS-based immunopeptidomics). These data are studied and summarized into databases by computers, forming various computer tools such as IEBD Analysis Resource, etc., which can predict the binding ability of a particular peptide fragment to HLA class I or class II molecules. At present, the mass spectrometry technology is directly utilized to search for the 'high-quality' neoantigen of individual tumor tissues, but the research has not become a gold standard scheme because of high cost, large tissue demand and lack of sensitivity.

Therefore, In the present study, the non-synonymous mutation sites obtained by sequencing technology are preferably selected from In silico peptide prediction and sequencing to screen the potential neoantigen population.

In this study section, we searched and recorded the peptide sequences corresponding to the unique and highly expressed mutated exon sites of the previously screened Lewis cells by the NCBI resource database. Then according to the screening thought provided by Ugur Sahin et al, the peptide sequences utilize the MHC-II Binding Predictions function of IEBD database to calculate the Binding capacity of each peptide sequence and mouse MHC-II molecules of H2-IAb genotypes. And obtaining the optimal corresponding peptide sequence for preparing the nucleic acid vaccine through computer calculation analysis. The analysis result needs to satisfy: consensus percentile rank results show high MHC class II molecule binding capacity; 2. the high binding capacity peptide fragment needs to contain the detected mutant codon site. Wherein the binding capacity to MHC class II molecules is critical at a consensus percentile rank (consensus percentile rank) of less than 30.

By the above screening conditions we obtained a total of 9 peptide sequences to be subsequently used in tandem synthetic vaccines. The results of the in silico peptide prediction and ranking (Table 2) are shown for the selected col3a1 mutation and the unselected Lonp1 mutation, as examples, and the amino acid sequences of the 9 selected peptides (Table 2).

TABLE 2

TABLE 3

The C57BL6 mouse subcutaneous transplantation tumor model of the mouse lung cancer Lewis cell is constructed, the gene states of the Lewis cell and the C57BL6 mouse are detected by adopting WES, and the unique mutation of the Lewis cell is discovered by comparison. Then missense mutation with high expression and obviously influenced gene expression is screened by means of RANseq, and the mutant peptide fragments are subjected to computer prediction and sequencing by using the MHC-II Binding Predictions function of the IEBD database, so that 9 new antigen combinations for subsequent tandem RNA vaccines are obtained.

Example 2 construction of a neo-antigen RNA vaccine (RNA vaccine for the treatment of non-small cell lung cancer)

In order to construct an RNA vaccine, In Vitro Transcription (IVT) of its entire nucleotide sequence (DNA sequence) needs to be achieved.

The method comprises the steps of selecting a pCI-neo (pCI-neo mammalian expression vector) plasmid as a vector, firstly inserting a DNA sequence of a designed RNA vaccine between a pCI-neo plasmid XhoI and a NotI enzyme cutting site, and amplifying to obtain a plasmid for loading an RNA vaccine sequence. The structure of pCI-neo is schematically shown in FIG. 2.

The plasmid carrying the RNA vaccine sequence is linearized by cutting the plasmid with the Sap I endonuclease, and the linearization is verified by agarose gel electrophoresis, and the result is shown in FIG. 3, which shows that the Sap I endonuclease successfully linearizes the plasmid and can start the in vitro transcription step. Subsequent use of the in vitro transcription kit (mMESSAGE mMACHINE)^TMT7 ULTRA Transcription Kit) for in vitro Transcription of the linearized plasmid, and purifying the Transcription product with a purification Kit (MEGAclear)^TMTranscription Clean-Up Kit) to obtain RNA for subsequent experimentsA vaccine sequence.

The synthesized RNA vaccine was tested for concentration, purity and gel electrophoresis (FIG. 4). The absorbance of the synthesized RNA product A260 is 28.866, the absorbance of A280 is 13.309, the value of A260/A280 is calculated to be 2.17, the purity of the synthesized product is good, and the calculated RNA concentration is 1154.6 ng/ul. Meanwhile, the synthesized RNA is subjected to sequencing detection, and the sequencing result proves the correctness of the RNA sequence obtained by IVT, so that the synthesized RNA can be used as a further RNA vaccine experiment.

The RNA vaccine with a tandem structure of sequences including Cap, UTR, SEC, Linker, Neoantigen, MITD, UTR, poly A and Sap I is designed. The DNA fragment of the above sequence was inserted into pCI-neo plasmid, and linearized by Sap I. And then an in vitro transcription kit and a purification kit are utilized to carry out IVT of linearized plasmids, and finally the designed RNA vaccine fragment is successfully obtained, the concentration purity is good, and the structure is correct.

Example 3 construction of PEG-PLL-PLLeu self-assembled micelle vector

Because of the structural specificity of RNA vaccines, their role is closely related to the mode of administration and delivery. The RNA vaccine provided by the invention is wrapped by the nanoparticles and then administered intravenously, so that the effect is better: on one hand, the nano-particles can protect the loaded RNA from being degraded and damaged; on the other hand, the physicochemical property of the nano particles can realize the effect of slowly and continuously releasing the RNA vaccine. In addition, secondary lymphoid organs such as lymph nodes and spleens are important sites for recognition and activation of T cells and DC cells, and intravenous delivery of nanoparticles is beneficial to distribution of RNA vaccine in the secondary lymphoid organs of the whole body.

1. Synthesis of PEG-PLL-PLLeu

The polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu) can form a cationic micelle through self-assembly, the particle size of the cationic micelle is about 100nm, the stability is good, and the performance requirement of the nanoparticle carrier is met. In addition, because it has a positive charge, it can efficiently load nucleic acid (including DNA, RNA) molecules having a negative charge. The invention adopts PEG-PLL-PLLeu cationic micelle as a carrier to realize the accurate delivery of nucleic acid molecules, has good effect, has the characteristics of good biocompatibility and no obvious cytotoxicity, and is suitable for subsequent transformation and clinical application.

The invention adopts PEG-PLL-PLLeu as a carrier to carry out chemical synthesis. LLZ-NCA and LLEu-NCA were prepared using cyclic anhydride (NCA), N epsilon-benzyloxycarbonyl-L-lysine (LLZ) and L-leucine (LLEu). The PEG-pLLZ polymer is obtained by ring-opening polymerization of aminopolyethylene glycol (NH2-PEG) and LLZ-NCA. And further carrying out ring-opening polymerization by using the PEG-pLLZ polymer and the LLEu-NCA to obtain the PEG-pLLZ-pLLEu. Finally, PEG-PLL-PLLeu is obtained by deprotection reaction. The method is conventional.

Synthesis of PEG-PLL-PLLeu: the molar ratio of NH2-PEG to LLZ-NCA is 1:25(1:10 to 1:50 can also achieve the aim of the invention), the two are dissolved in dry dimethyl sulfoxide (dried DMF, wt 10%), and stirred for 48h (24-96h optionally) under the protection of nitrogen at 60 ℃ (30 ℃ -80 ℃, and then the PEG-PLLZ is obtained by precipitation with excess ether. Dissolving PEG-PLLZ and LLEu-NCA at a molar ratio of 1:1(1:2 to 2:1 are optional) in dried dimethyl sulfoxide (dried DMF, wt 10%), stirring at 60 deg.C (30 deg.C-80 deg.C) under nitrogen protection for 48h (24-96h is optional), and precipitating with excessive diethyl ether to obtain PEG-PLLZ-PLLeu;

dissolving PEG-PLLZ-PLLeu in trichloroacetic acid (wt 5%) and HBr (wt 33%), stirring under nitrogen protection at 15 deg.C (optional at 0 deg.C-30 deg.C) for 48h (optional at 24-96 hr), and precipitating with excessive diethyl ether to obtain crude product;

then dialyzing the crude product by a dialysis membrane with the molecular weight of 3500Da for 48h (optional in 24-96 h) in ammonia water (wt 0.1%) and distilled water respectively to obtain a PEG-PLL-PLLeu final product;

the structural correctness and purity of the obtained product are verified by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance hydrogen spectroscopy (1H-NMR). The results of the relevant measurements are shown in FIGS. 5 and 6, and show that the FT-IR of the synthesized PEG-PLL-PLLeu of the present study has 1650 cm and 1550cm-¹Two characteristic peaks representing amide bonds in the polypeptide and additionally having an amino acid sequence located at 1105cm-¹The peak of (2) is a C-O-C characteristic peak in the PEG structure, and the correct structure of the synthesized sample in the research is proved. 1H-NMR confirmed the good structure and purity of the sample in this study.

2. Self-assembly of PEG-PLL-PLLeu and related characterization

To verify that the former part of the synthesized PEG-PLL-PLLeu can form cationic micelles by self-assembly, and to explore the optimal self-assembly concentration.

The invention can be realized by dissolving PEG-PLL-PLLeu with the concentration of 0.5-5mg/ml, using ultrapure water as a solvent, stirring overnight by magnetic force, and obtaining self-assembled micelles by ultrasonic treatment for 1 hour after complete dissolution.

In the embodiment, PEG-PLL-PLLeu is dissolved at the concentration of 1mg/ml, the solvent is ultrapure water, the mixture is magnetically stirred overnight, and the PEG-PLL-PLLeu self-assembled nano micelle is obtained by ultrasonic after complete dissolution;

the particle size and zeta potential of the self-assembled micelle were measured, and an electron micrograph thereof is shown in FIG. 8. The result shows that the synthesized PEG-PLL-PLLeu in the research can be successfully self-assembled into nano-micelle, the particle size is 86.1 +/-8.4 nm, and the zeta potential is 58.64 +/-2.55 mV. The particle size was measured every week for 6 consecutive weeks, indicating good stability, as shown in fig. 7.

The above provides synthetic neoantigen RNA and a vehicle for immunoadjuvant delivery. The carrier is selected from PEG-PLL-PLLeu reported in the literature, a sample is obtained by utilizing ring opening polymerization, and the structure and the purity of the sample are confirmed by FT-IR and 1H-NMR. And further, the synthesized PEG-PLL-PLLeu is self-assembled into a nano micelle by utilizing a self-assembly reaction, the measured particle size and zeta potential show good performance, and continuous observation indicates that the particle size is stable, so that the method is suitable for subsequent RNA and adjuvant loading and further experiments.

Example 4 preparation of a Complex vaccine of neoantigen RNA and immunoadjuvant poly (I: C)

The microenvironment state of the tumor is critical to the efficacy of anti-tumor immunotherapy. A review was published in Nature journal by Daniel Chen equal to 2017, which classified the microenvironment of malignancies into three types, respectively: 1. type of immunoinflammatory; 2. immune-privileged types; 3. immune desert type. Where only immunoinflammatory tumors are likely to benefit from anti-tumor immunotherapy. Except that neoantigen is used for reconstructing anti-tumor immune response, if the microenvironment of the tumor can be improved simultaneously, the non-immune inflammatory type is converted into the immune inflammatory type, the immune tolerance state of tumor cells can be expected to be reduced, and the killing effect of the anti-tumor immune response on the tumor is enhanced.

The use of immunological adjuvants is an important means of improving the immune microenvironment, which exerts an effect of enhancing immune response through the modulation of antigen and T cell receptor binding signals, costimulatory molecule binding signals, and microenvironment cytokine signals.

Polyinosinic acid-polycytidylic acid (Poly (I: C)) is a high molecular polymer that mimics the structure of viral double-stranded RNA and can act as an immunoadjuvant against both costimulatory molecule signals and microenvironment cytokine signals. Poly (I: C) can stimulate important anti-tumor immune-related cells including DC cells, NK cells and T cells as double-stranded RNA analogues, and can further realize a great deal of positive effects of inducing the release of proinflammatory factors, promoting the maturation and the activation of the DC cells, enhancing the antigen cross-presentation capacity of the DC cells, stimulating the activation of the T cells and the like by combining with Toll-like receptor 3(TLR3), melanoma differentiation-related protein 5(MDA-5) and retinoic acid inducible protein 1(RIG-1) of the cells, so that Poly (I: C) becomes the basis of immune adjuvants and the focus of clinical research.

More importantly, Poly (I: C) is a double-stranded RNA analogue, has negative charge like an RNA vaccine, can be loaded by PEG-PLL-PLLeu nano-micelle according to the same principle, can realize local aggregation and slow release on the tumor after delivery, and effectively improves the tumor immune microenvironment.

1. Co-loading of PEG-PLL-PLLeu to neoantigen RNA and immune adjuvant poly (I: C)

For loading of the neoantigen RNA vaccine and Poly (I: C), the loading method is realized by co-incubating PEG-PLL-PLLeu micelle solution, RNA vaccine and aqueous solution of Poly (I: C) at 37 ℃, and the molar ratio of the PEG-PLL-PLLeu micelle solution to the RNA vaccine to the Poly (I: C) is 10:2: 1. During the incubation, the RNA vaccine and Poly (I: C) will adsorb into the PEG-PLL-PLLeu micelle due to charge interaction.

The particle size and zeta potential in this case, and the stability of the particle size, were measured. The particle diameter of the loaded nano micelle is 68.6 +/-8.5 nm, and the zeta potential is 48.7 +/-5.0 mV.

Preparing a batch of new antigen RNA and immune adjuvant poly (I: C) compound vaccines:

the molar ratio of the PEG-PLL-PLLeu self-assembled nano-micelle to the new antigen RNA vaccine is 1: 1; the molar ratio of the neoantigen RNA vaccine to the Poly (I: C) is 1: 1; the particle diameter of the loaded nano micelle is 64.7 +/-8.2 nm, and the zeta potential is 43.2 +/-4.3 mV.

The molar ratio of the PEG-PLL-PLLeu self-assembled nano-micelle to the new antigen RNA vaccine is 10: 1; the molar ratio of the neoantigen RNA vaccine to the Poly (I: C) is 5: 1; the particle diameter of the loaded nano micelle is 66.8 +/-7.3 nm, and the zeta potential is 43.9 +/-4.8 mV.

The molar ratio of the PEG-PLL-PLLeu self-assembled nano-micelle to the new antigen RNA vaccine is 5: 1; the molar ratio of the neoantigen RNA vaccine to the Poly (I: C) is 3: 1; the particle diameter of the loaded nano micelle is 69.2 +/-7.4 nm, and the zeta potential is 49.2 +/-4.7 mV.

2. Uptake and cytotoxicity detection of combination vaccines

DC cells from C57BL6 mice were isolated, co-cultured with a complex preparation of PEG-PLL-PLLeu micelles, 40ug vaccine RNA and 20ug Poly (I: C) (RNA sequence labeled with Cy 5) for 4h, and then the uptake of DC cells was observed by fluorescence microscopy. The results are shown in fig. 9, suggesting that the fluorescently labeled complex vaccine preparation can smoothly enter DC cells.

Then, the composite preparation containing 200ug PEG-PLL-PLLeu micelle, 40ug vaccine RNA and 20ug Poly (I: C) and 293T cells are co-cultured for 48h, and CCK8 cell activity detection is carried out to judge cytotoxicity, the specific result is shown in figure 6(CCK8 experimental data), and the result shows that the composite preparation of the new antigen RNA vaccine and the immunologic adjuvant Poly (I: C) prepared in the research has no obvious influence on the growth of the 293T cells under the treatment concentration and the toxicity is acceptable.

In the experiment, the synthesized PEG-PLL-PLLeu self-assembled nano-micelle is used as a carrier, the loading of the RNA vaccine and the immunologic adjuvant Poly (I: C) is successfully carried out, the particle size is slightly increased compared with the PEG-PLL-PLLeu self-assembled nano-micelle after the loading, and the zeta potential indicates good stability. The results of co-culture with mouse cell DC cells suggest that the complex formulation can be rapidly taken up by DC cells. Cytotoxicity experiments suggested that the combined preparation at therapeutic doses did not have a significant adverse effect on the growth of 293T cells.

Example 5 animal experiments with neoantigen RNA and immunoadjuvant poly (I: C) Complex vaccine

The local Tumor Microenvironment (TME) contains a variety of different immune cells that have important effects on tumor growth, metastasis and anti-tumor immunotherapy. Tumor metastasis is the major cause of tumor-related death.

Immune cells in the TME of many malignancies are able to create an immunosuppressive environment and promote tumor growth. This type of TME is mostly dominated by tumor-induced myeloid cells, such as tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs). TAMs and TANs produce various cytokines that inhibit anti-tumor immune responses, such as arginase 1, monocyte chemotactic protein 1, IL-6 and IL-8; in addition, they can also up-regulate apoptosis 1(PD-1) and programmed death ligand 1(PD-L1) to inhibit the anti-tumor effect of T cells.

Regulatory T cells (Tregs) are also important factors in TME to promote the immunosuppressive state. For example, studies have reported that the presence of Tregs correlates with high PD-1 expression, and that metastases from prostate cancer patients have high levels of functional Tregs in the bone marrow, leading to an immunosuppressive TME state. Further studies have shown that regulatory B cells (Bregs) are able to transform CD4+ T cells into Tregs and induce TAMs to increase the expression of PD-L1 to further promote the formation of an immunosuppressive microenvironment. In addition, there is evidence that the function of Dendritic Cells (DCs) is also affected in inhibitory TMEs, leading to decreased antigen presentation and impaired anti-tumor immune responses. The relative hypoxic state of TME also affects the migration and chemotactic capacity of Natural Killer (NK) cells, potentially promoting tumor growth, metastasis.

The invention synthesizes a novel composite preparation of PEG-PLL-PLLeu nano micelle loaded with new antigen RNA vaccine and immune adjuvant Poly (I: C). The novel compound preparation is presumed to be capable of initiating anti-tumor immune response, improving tumor immune microenvironment, further inhibiting tumor growth and prolonging survival, in order to verify the presumption, a C57BL6 mouse subcutaneous transplantation tumor model of mouse lung cancer Lewis cells is constructed in the part, and the research is conducted on the influence of a compound vaccine on blood inflammatory factors of a model animal, immune cell typing analysis of tumor tissues of the model animal and the tumor growth and survival condition of the model animal.

1. Effect of the Compound vaccine on blood inflammatory factors of model animals

The groups comprise a normal saline control group (Sal group), a blank nano micelle carrier group (PM group), a pure RNA nano micelle group (R + PM) and a composite nano micelle group (R + P + PM), and are all administered by tail vein injection. Serum IFN alpha, beta, gamma, TNF alpha and IL-6 levels 3h after administration were measured using a Th1/Th2 cytokine panel 6-plex.

The results show that no obvious increase of serum cytokine level is seen in the normal saline group after administration, IFN alpha and IL-6 are seen in the PM group, and the levels of the detected cytokines are obviously increased in the R + PM group and the R + P + PM group, but no obvious difference exists between the two groups, and the results are shown in figure 10.

2. Immunocytotyping analysis of tumor tissue in model animals

The experimental groups are as above, starting from d0 tumors, d3, d7 and d14 are respectively injected into tail vein of each group of preparation, model animals are killed at d15, and tumor tissues are taken for flow cytometry. The cell classification tested included CD4+ T cells, CD8+ T cells, Treg cells, and TAM cells, and further analyzed for typing of TAM cells.

The detection results are shown in fig. 11, and the results indicate that: the proportion of CD4+ T and CD8+ T cells in the R + PM group and the R + P + PM group is obviously higher than that in the Sal group and the PM group, and the R + P + PM group is more obviously increased than that in the R + PM group; the proportion of Treg cells in the R + PM group and the R + P + PM group is reduced compared with that in the Sal group and the PM group, but no obvious statistical difference exists; the proportion of the TAM cells of the R + PM group and the R + P + PM group is increased, and further analysis indicates that the proportion of the TAM II type cells is remarkably reduced.

3. Tumor growth and survival in model animals

Experimental groups were as above, starting from d0 tumors, d3, d7, d14, d21, d28 groups of preparations were injected into the tail vein, and tumor volumes were measured every three days and calculated according to the formula (a2 × b)/2(a, minor axis; b, major axis). And survival of each group of mice was recorded.

The tumor growth curve and survival curve are shown in FIGS. 12 and 13. The results show that the R + PM group and the R + P + PM group can effectively control the growth of the Lewis cells of the mice in the C57BL6 mice, the survival of the mice is prolonged, and the R + P + PM group has more remarkable advantages.

It will be apparent to those skilled in the art that the above description of specific embodiments of the invention is not intended to limit the application of the invention, and that various equivalents and modifications may be made thereto depending on the circumstances. All such substitutions and modifications are intended to be within the scope of the appended claims without departing from the spirit of the invention.

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Claims (6)

1. A new antigen RNA and immune adjuvant poly (I: C) composite vaccine is characterized in that: the composite vaccine comprises a PEG-PLL-PLLeu self-assembled nano micelle, a new antigen RNA vaccine loaded on the PEG-PLL-PLLeu self-assembled nano micelle and an immunologic adjuvant Poly (I: C); wherein, the nucleotide sequence of the neoantigen RNA is shown as SEQ ID NO. 1.

2. The composite vaccine of neoantigen RNA and immunoadjuvant poly (I: C) according to claim 1, wherein: the molar ratio of the PEG-PLL-PLLeu self-assembly nano-micelle to the new antigen RNA vaccine is between 1:1 and 10: 1; the molar ratio of the neoantigen RNA vaccine to the Poly (I: C) is between 1:1 and 5: 1.

3. The composite vaccine of neoantigen RNA and immunoadjuvant poly (I: C) according to claim 1, wherein: the preparation method of the PEG-PLL-PLLeu self-assembly nano micelle comprises the following steps:

(1) synthesis of PEG-PLL-PLLeu: performing ring-opening polymerization on amino polyethylene glycol (NH2-PEG) and LLZ-NCA to obtain a PEG-pLLZ polymer; further ring-opening polymerization of the PEG-pLLZ polymer and the LLEu-NCA to obtain PEG-pLLZ-pLLEu; performing deprotection reaction to obtain PEG-PLL-PLLeu;

(2) self-assembly of PEG-PLL-PLLeu: dissolving PEG-PLL-PLLeu with concentration of 1mg/ml, using ultrapure water as solvent, magnetically stirring overnight, and performing ultrasonic treatment after complete dissolution to obtain the PEG-PLL-PLLeu self-assembled nano micelle.

4. The composite vaccine of neoantigen RNA and immunoadjuvant poly (I: C) according to claim 1, wherein: the preparation method of the antigen RNA vaccine comprises the following steps:

(1) selecting pCI-neo (pCI-neo mammalian expression vector) plasmid as a vector, inserting the DNA sequence of the RNA vaccine of claim 1 between pCI-neo plasmid XhoI and NotI enzyme cutting site, and amplifying to obtain plasmid for loading RNA vaccine sequence;

(2) cutting the plasmid loaded with the RNA vaccine sequence by using a Sap I endonuclease to linearize the plasmid;

(3) in vitro Transcription of the linearized plasmid was performed using an in vitro Transcription Kit (mMESSAGE mMACHINE ™ T7 ULTRA Transcription Kit);

(4) the Transcription product is processed by a purification Kit (MEGAclear ™ Transcription clear-Up Kit) to obtain the RNA vaccine.

5. The method for constructing a composite vaccine of neoantigen RNA and immunoadjuvant poly (I: C) according to claim 1, wherein the method comprises the steps of: the method comprises the following steps:

(1) preparing the PEG-PLL-PLLeu self-assembled nano micelle:

(1.1) Synthesis of PEG-PLL-PLLeu: performing ring-opening polymerization on amino polyethylene glycol (NH2-PEG) and LLZ-NCA to obtain a PEG-pLLZ polymer; further ring-opening polymerization of the PEG-pLLZ polymer and the LLEu-NCA to obtain PEG-pLLZ-pLLEu; performing deprotection reaction to obtain PEG-PLL-PLLeu;

(1.2) self-assembly of PEG-PLL-PLLeu: dissolving PEG-PLL-PLLeu with concentration of 1mg/ml, using ultrapure water as solvent, magnetically stirring overnight, and performing ultrasonic treatment after complete dissolution to obtain PEG-PLL-PLLeu self-assembled nano micelle;

(2) preparation of antigen RNA vaccine:

(2.1) selecting pCI-neo (pCI-neo mammalian expression vector) plasmid as a vector, inserting the DNA sequence of the RNA vaccine of claim 1 between pCI-neo plasmid XhoI and NotI enzyme cutting site, and amplifying to obtain plasmid loaded with RNA vaccine sequence;

(2.2) cutting the plasmid loaded with the RNA vaccine sequence by using Sap I endonuclease to linearize the plasmid;

(2.3) in vitro Transcription of the linearized plasmid using an in vitro Transcription Kit (mMESSAGE mMACHINE ™ T7 ULTRA Transcription Kit);

(2.4) processing the Transcription product by a purification Kit (MEGAclear Transcription clear-Up Kit) to obtain an RNA vaccine;

(3) co-loading of PEG-PLL-PLLeu to neoantigen RNA and the immune adjuvant poly (I: C): and co-incubating the PEG-PLL-PLLeu micelle solution, the RNA vaccine and the aqueous solution of Poly (I: C) at 37 ℃ to obtain the new antigen RNA and immune adjuvant Poly (I: C) composite vaccine.

6. The method for constructing the composite vaccine of neoantigen RNA and immunoadjuvant poly (I: C) according to claim 5, wherein the method comprises the following steps:

(1) preparing the PEG-PLL-PLLeu self-assembled nano micelle:

(1.1) Synthesis of PEG-PLL-PLLeu: the molar ratio of NH2-PEG to LLZ-NCA is 1:10 to 1:50, the two are dissolved in dry dimethyl sulfoxide (10% by weight), stirred for 24-96h at 30-80 ℃ under the protection of nitrogen, and then precipitated by excessive ethyl ether to obtain PEG-PLLZ;

dissolving PEG-PLLZ and LLEu-NCA in dry dimethyl sulfoxide (dried DMF, wt 10%) at 30-80 deg.C under protection of nitrogen, stirring for 24-96 hr, and precipitating with excessive diethyl ether to obtain PEG-PLLZ-PLLeu;

dissolving PEG-PLLZ-PLLeu in trichloroacetic acid (wt 5%) and HBr (wt 33%), stirring at 0-30 deg.C under nitrogen protection for 24-96 hr, and precipitating with excessive diethyl ether to obtain crude product;

then dialyzing the crude product by a dialysis membrane with the molecular weight of 3500Da for 24-96 hours respectively in ammonia water (wt 0.1%) and distilled water to obtain a PEG-PLL-PLLeu final product;

(1.2) self-assembly of PEG-PLL-PLLeu: dissolving PEG-PLL-PLLeu with concentration of 0.5-5mg/ml, using ultrapure water as solvent, magnetically stirring overnight, and performing ultrasonic treatment after complete dissolution to obtain PEG-PLL-PLLeu self-assembled nano micelle;

(2) preparation of antigen RNA vaccine:

(2.1) selecting pCI-neo (pCI-neo mammalian expression vector) plasmid as a vector, inserting the DNA sequence of the RNA vaccine of claim 1 between pCI-neo plasmid XhoI and NotI enzyme cutting site, and amplifying to obtain plasmid loaded with RNA vaccine sequence;

(2.2) cutting the plasmid loaded with the RNA vaccine sequence by using Sap I endonuclease to linearize the plasmid;

(2.3) in vitro Transcription of the linearized plasmid using an in vitro Transcription Kit (mMESSAGE mMACHINE ™ T7 ULTRA Transcription Kit);

(2.4) processing the Transcription product by a purification Kit (MEGAclear Transcription clear-Up Kit) to obtain an RNA vaccine;

(3) co-loading of PEG-PLL-PLLeu to neoantigen RNA and the immune adjuvant poly (I: C): and co-incubating the PEG-PLL-PLLeu micelle solution, the RNA vaccine and the aqueous solution of Poly (I: C) at 37 ℃ to obtain the new antigen RNA and immune adjuvant Poly (I: C) composite vaccine.

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