A recombinant vaccine based on FcRn capable of improving cellular immunity and its application

Abstract

The present invention provides a fusion protein framework, recombinant vaccine, etc. based on FcRn that can improve the level of cellular immunity. The fusion protein comprises at least one target antigen protein, a transmembrane region and a cytoplasmic region of the FcRn protein. The present invention finds that the FcRn framework can direct the antigen to the endosome, which is acidified and then fused with the lysosome, thereby promoting the degradation of the antigen into an antigen peptide. The FcRn-based fusion protein molecular framework and recombinant vaccine of the present invention can effectively enhance the antigen presentation of cells, have strong cellular immunogenicity, and can stimulate or activate CD4+ or CD8+T cells, improve the level of cellular immunity, and provide new technology for the preparation of immune drug design that efficiently induces cellular immunity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to the field of immune medicine technology, and specifically to a recombinant vaccine based on FcRn that can improve cellular immunity levels, a preparation method and an application thereof. Background Art

[0002] Cellular immunity, also known as cell-mediated immunity, is the most effective defense response to eliminate intracellular parasitic microorganisms and is also an effective way to inhibit tumors. T cells are one of the main cell groups that respond to cellular immune responses and can be activated by antigen fragments on the surface of antigen-presenting cells (APCs) and transformed into effector T cells. Since T cells can only recognize fragments of antigens displayed on the cell surface, the major histocompatibility complex (MHC) is a key factor in the antigen presentation process and is indispensable in inducing the host to produce effective adaptive immunity against pathogens. Many current experimental studies have shown that MHC class II molecules are expressed on CD4 + T cell presentation of antigenic peptides is an important process for activating cellular immune responses and plays a key role in inducing immune cells to eliminate tumor cells or pathogens.

[0003] Recombinant vaccines are of particular importance in human and veterinary medicine as agents and drugs for the prevention and treatment of infectious diseases and cancer. Currently, a key factor in evaluating the effectiveness of recombinant vaccines is to detect the degree of activation of specific T cells of immune organisms by the vaccine. However, recombinant vaccines based on conventional structures can only produce weak stimulation to T cells, or even fail to effectively activate T cells, and cannot meet experimental or medical needs.

[0004] Neonatal Fc receptor (FcRn) is encoded by the FCGRT gene and is not only present in the embryonic period, but also widely present in parenchymal cells (epithelium, endothelium, hepatocytes and keratinocytes) and hematopoietic cells in adulthood. Studies have shown that FcRn is mainly present on the membranes of endosomes and lysosomes, and its structure can form non-covalent bonds with β-microglobulin, participating in the circulation between endosomes, lysosomes and cell membranes.

[0005] Regarding the application of FcRn, the prior art US patent document US6086875A (published on July 11, 2000) discloses a method for activating or inhibiting a systemic immune response in a mammal, comprising administering an effective amount of a conjugate of an FcRn binding partner and an antigen selected from the group consisting of antigenic features of pathogens, antigenic features of autoimmune diseases, and antigenic features of allergens to the lumen side of the epithelial barrier of an epithelial tissue expressing the FcRn receptor, wherein the FcRn binding partner is a non-specific IgG or an FcRn binding fragment of IgG. The prior art Chinese patent document CN101316927A (published on December 3, 2008) discloses the use of the FcRn receptor, specifically discloses a method for generating an antibody or a functional part, derivative and / or analog thereof in a cell, comprising providing a cell capable of generating the antibody using the FcRn receptor protein and culturing the cell to achieve the generation of the antibody. Prior art Chinese patent document CN104232667A (published on 2014-12-24) discloses a method for constructing an FcRn-mediated anti-Echinococcus granulosus targeted mucosal vaccine vector, describing the fusion expression of antigens with the Fc domain of mouse IgG2, which can promote antigen recognition by FcRn, but does not involve the construction or application of FcRn-related recombinant vaccines. It can be seen that there is no prior art that discloses that FcRn has specific excellent properties and can be used to construct antigen fusion proteins or recombinant vaccines.

[0006] Therefore, there is still an urgent need in this field for a better fusion antigen architecture and a correspondingly constructed recombinant vaccine to achieve a better ability to promote antigen presentation and improve the level of cellular immunity. Summary of the invention

[0007] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a fusion protein framework, recombinant vaccine, etc. based on FcRn that can improve the level of cellular immunity. The fusion protein of the present invention comprises at least one target antigen protein, a transmembrane region and a cytoplasmic region of the FcRn protein. The present invention finds that the FcRn framework can direct antigens to endosomes, which are acidified and then fuse with lysosomes, thereby promoting the degradation of antigens into antigenic peptides, and can effectively enhance the antigen presentation of cells. The present invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, pharmaceutical uses, application methods, etc.

[0008] One aspect of the present invention provides a fusion protein, characterized in that it comprises:

[0009] (a) at least one target antigen protein; and

[0010] (b) The transmembrane region of the FcRn protein and the cytoplasmic region of the FcRn protein.

[0011] Furthermore, it is characterized in that the FcRn protein is derived from mammals.

[0012] Furthermore, it is characterized in that the amino acid sequences of the transmembrane region and the cytoplasmic region of the FcRn protein are shown in SEQ ID NO:4, the amino acid sequence of the transmembrane region of the FcRn protein is shown in SEQ ID NO:6, and the amino acid sequence of the cytoplasmic region of the FcRn protein is shown in SEQ ID NO:7.

[0013] Furthermore, it is characterized in that the antigen is any one tumor antigen or a combination of tumor antigens.

[0014] Furthermore, it is characterized in that the tumor antigen is selected from one or more of the following groups: BY55 (CD160antigen), SPRY4 (Protein sprouty homolog 4), TCF7 (Transcription factor 7), WNT6 (Protein Wnt-6), Trp1 (Short transient receptor potential channel 1), and Trp2 (Short transient receptor potential channel 2).

[0015] Further, it is characterized in that the BY55 amino acid sequence is shown in SEQ ID NO:13, the SPRY4 amino acid sequence is shown in SEQ ID NO:14, the TCF7 amino acid sequence is shown in SEQ ID NO:15, the WNT6 amino acid sequence is shown in SEQ ID NO:16, the Trp1 amino acid sequence is shown in SEQ ID NO:17, and the Trp2 amino acid sequence is shown in SEQ ID NO:18.

[0016] Furthermore, it is characterized in that the antigen is located at the N-terminus of the transmembrane region of the FcRn protein; the N-terminus of the antigen optionally comprises a signal peptide; and the various parts of the fusion protein can be optionally separated by a linker sequence.

[0017] Furthermore, it is characterized in that the fusion protein comprises, from N-terminus to C-terminus, a signal peptide, an antigen amino acid sequence, an FcRn sequence transmembrane region, and an FcRn sequence cytoplasmic region.

[0018] Furthermore, it is characterized in that the fusion protein comprises any one of the following structures (a)-(d) in sequence from the N-terminus to the C-terminus:

[0019] (a) Signal peptide, target antigen BY55, GS linker, target antigen SPRY4, GS linker, transmembrane region of FcRn protein and cytoplasmic region of FcRn protein;

[0020] (b) Signal peptide, target antigen TCF7, GS linker, target antigen WNT6, GS linker, transmembrane region of FcRn protein and cytoplasmic region of FcRn protein;

[0021] (c) Signal peptide, target antigen Trp1, GS linker, transmembrane region of FcRn protein and cytoplasmic region of FcRn protein;

[0022] (d) Signal peptide, target antigen Trp2, GS linker, transmembrane region of FcRn protein and cytoplasmic region of FcRn protein.

[0023] Further, it is characterized in that the amino acid sequence of the signal peptide is shown in SEQ ID NO:2; the amino acid sequence of the GSlinker is shown in SEQ ID NO:3; the BY55 amino acid sequence is shown in SEQ ID NO:13, the SPRY4 amino acid sequence is shown in SEQ ID NO:14, the TCF7 amino acid sequence is shown in SEQ ID NO:15, the WNT6 amino acid sequence is shown in SEQ ID NO:16, the Trp1 amino acid sequence is shown in SEQ ID NO:17, the Trp2 amino acid sequence is shown in SEQ ID NO:18, the amino acid sequences of the transmembrane region of the FcRn protein and the cytoplasmic region of the FcRn protein are shown in SEQ ID NO:4, the amino acid sequence of the transmembrane region of the FcRn protein is shown in SEQ ID NO:6, and the amino acid sequence of the cytoplasmic region of the FcRn protein is shown in SEQ ID NO:7.

[0024] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it encodes any fusion protein described in the present invention.

[0025] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule of the present invention.

[0026] Furthermore, it is characterized in that the recombinant gene expression cassette comprises, from the 5' end to the 3' end, in sequence: 5'UTR, signal peptide, antigen nucleic acid sequence, GS linker, FcRn sequence transmembrane region and cytoplasmic region, and 3'UTR.

[0027] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule or the recombinant gene expression cassette of the present invention.

[0028] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule, the recombinant gene expression cassette, or the recombinant vector of the present invention.

[0029] Another aspect of the present invention provides a pharmaceutical composition, characterized in that it contains one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention.

[0030] Furthermore, it is characterized in that the pharmaceutical composition also contains a pharmaceutically acceptable carrier.

[0031] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins described in the present invention, and / or one or more recombinant nucleic acid molecules described in the present invention, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention.

[0032] Another aspect of the present invention provides the use of one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more pharmaceutical compositions of the present invention in any of the following:

[0033] (a) preparing a drug for enhancing cellular antigen presentation;

[0034] (b) preparing a medicament for inducing an immune response in an organism;

[0035] (c) preparing a drug for stimulating or activating CD4+ or CD8+ T cells in an organism;

[0036] (d) Preparation of medicines for treatment, vaccination or biological immunization.

[0037] Furthermore, it is characterized in that the drug is a drug for treating cancer.

[0038] Furthermore, it is characterized in that the cancer is leukemia, melanoma, colorectal cancer, gastric cancer, esophageal cancer, head and neck cancer, bladder cancer, cervical cancer, sarcoma, cell tumor, lung cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, pancreatic cancer, liver cancer, breast cancer, myeloma, glioma or lymphoma, etc.

[0039] Another aspect of the present invention provides a method for improving the antigen presentation effect in mammalian APC (antigen presenting cell), characterized in that the method comprises administering one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules, and / or one or more recombinant gene expression cassettes, and / or one or more host cells, and / or one or more recombinant vectors, and / or one or more pharmaceutical compositions to cells and / or other immune organisms. Preferably, the method is for non-therapeutic and diagnostic purposes.

[0040] Another aspect of the present invention provides a method for stimulating or activating CD4+ or CD8+ T cells, characterized in that the method comprises administering one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules, and / or one or more recombinant gene expression cassettes, and / or one or more host cells, and / or one or more recombinant vectors, and / or one or more pharmaceutical compositions to cells and / or other immune organisms. Preferably, the method is for non-therapeutic and diagnostic purposes.

[0041] The FcRn-based fusion molecule architecture and the recombinant vaccine capable of improving cellular immunity of the present invention have the following beneficial technical effects:

[0042] 1. The FcRn-based fusion molecule architecture and recombinant vaccine of the present invention can effectively enhance cellular antigen presentation, have strong cellular immunogenicity, and can stimulate or activate CD4+ or CD8+ T cells to improve cellular immunity, thus providing new technology for the preparation of highly efficient immune drug design that induces cellular immunity.

[0043] 2. Example 4 of the present invention shows that the FcRn framework can direct antigens to endosomes, which are then acidified and fused with lysosomes, thereby promoting the degradation of antigens into antigenic peptides, which are then bound to MHC class I or class II molecules, thereby achieving the purpose of promoting antigen presentation.

[0044] 3. Example 5 of the present invention shows that the recombinant nucleic acid vaccine (vaccine B) based on the present invention can induce a significant antigen-specific cellular immune response, which is far superior to that of vaccines A and C and close to the cellular immunity level caused by positive stimuli.

[0045] 4. Example 6 of the present invention shows that both the recombinant nucleic acid vaccine I and vaccine II based on the present invention can activate the immune system of tumor-bearing mice and provide effective immune protection. After the third injection of the vaccine, the tumor volume was significantly smaller than that of the negative control group, and tumor growth was continuously inhibited.

[0046] 5. The treatment experiment of Example 7 of the present invention shows that the recombinant nucleic acid vaccine III and vaccine IV based on the present invention can both play a therapeutic role and inhibit the growth of tumor volume; the simultaneous administration of vaccine III and vaccine IV has a better effect and can greatly inhibit tumor proliferation, almost achieving complete inhibition.

[0047] 6. The prevention experiment of Example 7 of the present invention shows that the recombinant nucleic acid vaccine III and vaccine IV based on the present invention can play a preventive role, inhibit tumor proliferation, and effectively improve the survival rate of mice. After vaccination with the recombinant nucleic acid vaccine Vaccine III & Vaccine IV based on the present invention, 80% of the mice still survived on the 25th day of tumor bearing.

[0048] 7. Based on the design strategy of the cellular immune antigen presentation architecture of the present invention, the FcRn molecules with high specificity or homology can be selected according to the target animal to construct a cellular immune vaccine. For example, in the mouse immunization experiment, the transmembrane region and cytoplasmic region sequences of human (Homo sapiens) FcRn are highly homologous to those of mice, so they can significantly activate cellular immunity.

[0049] 8. The FcRn-based fusion molecular architecture and recombinant vaccine of the present invention can be used for disease prevention and treatment in humans and animals, and are particularly suitable for the production and development of animal immune drugs. They can significantly improve antigen presentation and cellular immune responses in immune individuals, and are far superior to existing technologies in inducing cellular immune responses, such as existing conventional nucleic acid vaccine molecular architectures. The present invention has broad application prospects.

[0050] 9. The recombinant nucleic acid vaccine based on the present invention can be applied to the development of various tumor vaccines. The cellular immunity induced by the vaccine performs well in prevention, treatment and long-term protection after treatment, and has extremely high research and development and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a schematic diagram of the molecular structure of the fusion protein of the present invention.

[0052] Figure 2 It is a schematic diagram of the gene expression cassette structure containing the fusion protein molecule of the present invention.

[0053] Figure 3 Schematic diagram of a recombinant vector plasmid comprising the gene expression cassette structure of the present invention.

[0054] Figure 4A-4D The quality control peak diagram and purity test results of vaccines A, B, C, and D of the present invention are shown in FIG. Figure 4A This is the quality control peak diagram and purity test results of vaccine A. Figure 4B This is the quality control peak diagram and purity test results of vaccine B. Figure 4C This is the quality control peak diagram and purity test results of vaccine C. Figure 4D This is the quality control peak diagram and purity test results of vaccine D.

[0055] Figure 5 The following are photos showing the co-localization of the expressed proteins in the endoplasmic reticulum, endosomes and lysosomes after DC 2.4 cells were transfected with the vaccine D of the present invention.

[0056] Figure 6 This is a flow chart of the immunization and sampling of vaccines A, B, and C of the present invention in mice.

[0057] Figure 7 It is a comparison chart of the ELISPOT results of the recombinant vaccine B based on the present invention and the vaccines A and C based on conventional molecular frameworks.

[0058] Figure 8 It is the original result graph of ELISPOT based on the recombinant vaccine B of the present invention and the vaccines A and C based on the conventional molecular framework.

[0059] Fig. 9 Schematic diagram of the structure of vaccine I based on the present invention.

[0060] Fig.10 Schematic diagram of the structure of vaccine II based on the present invention.

[0061] Figure 11A-11B ,in Fig.11A This is the mouse immunization process of Example 6 of the present invention; Fig. 11B This is the immunotherapy effect of inhibiting tumor volume of the C1498 acute myeloid leukemia mouse model based on the recombinant vaccine of the present invention in Example 6 of the present invention.

[0062] Fig.12 Schematic diagram of the architecture of vaccine III based on the present invention.

[0063] Fig.13 Schematic diagram of the architecture of vaccine IV based on the present invention.

[0064] Figure 14A-14B ,in Fig.14A This is the mouse immunization process for the treatment experiment in Example 7; Fig. 14B This is the therapeutic effect of the recombinant vaccine of the present invention on the tumor volume of the B16-F10 melanoma mouse model in the treatment experiment of Example 7 of the present invention.

[0065] Figure 15A-Figure 15C ,in Fig.15A This is the mouse immunization process for the prevention experiment in Example 7; Fig. 15B This is the tumor volume prevention effect of the recombinant vaccine based on the present invention on the B16-F10 melanoma mouse model in the prevention experiment of Example 7 of the present invention; Fig. 15CThis is the preventive effect of the recombinant vaccine of the present invention on the survival rate of the B16-F10 melanoma mouse model in the prevention experiment of Example 7 of the present invention. DETAILED DESCRIPTION

[0066] The term "FcRn" refers to the neonatal fragment crystallizable receptor or neonatal Fc receptor, which is a protein encoded by the FCGRT gene in humans. It is an IgG Fc receptor that is structurally related to beta-2-microglobulin. In rodents, FcRn was originally identified as a receptor for the transfer of maternal immunoglobulin G from mothers to their neonatal offspring via breast milk, and is therefore known as the neonatal Fc receptor.

[0067] The term "transmembrane region and cytoplasmic region of FcRn" refers to the protein sequence of the transmembrane domain and cytoplasmic domain of the FcRn protein, generally refers to the C-terminal peptide chain of the FcRn protein, starting from the first amino acid in the transmembrane region and ending with the last amino acid. In some cases, there is a linker sequence between the transmembrane region and the cytoplasmic region peptide chains of FcRn. Exemplarily, the amino acid sequence of the transmembrane region and the cytoplasmic region of the encoded FcRn sequence can be as shown in SEQ ID NO: 4, wherein the amino acid sequence of the transmembrane region of the FcRn sequence can be as shown in SEQ ID NO: 6, and the amino acid sequence of the cytoplasmic region of the FcRn sequence can be as shown in SEQ ID NO: 7, and a sequence having 80% (e.g., 80%, 85%, 90%, 95%, 98%, 99%, 100%) or more homology with its sequence while maintaining the function of the transmembrane region and the cytoplasmic region of FcRn.

[0068] The term "tumor antigen" or "tumor-associated antigen", tumor-associated antigen, TAA, generally includes the meaning known in the art, which includes any molecule expressed on tumor cells (or associated with tumor cell development), known or believed to have an effect on the tumorigenic properties of tumor cells. Many tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined according to techniques and assays familiar to those skilled in the art, such as clonogenic assays, transformation assays, in vitro or in vivo tumor formation assays, gel migration assays, gene knockout analysis, etc. The term "tumor antigen" can refer to a human transmembrane protein, i.e., a cell membrane protein anchored in the lipid bilayer of a cell. Tumor antigen molecules include but are not limited to: EGFR, HER2, HER3, HER4, EpCAM, CEA, TRAIL, TRAIL receptor 1, TRAIL receptor 2, lymphotoxin β receptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast activation protein (FAP), hepsin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), prostate-specific membrane antigen (PSMA), VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2, TNF-α, TNF-like weak inducer of apoptosis (TWEAK), IL-1R. The TAA used in this article is BY55 (CD160antigen), SPRY4 (Protein sprouty homolog 4), TCF7 (Transcription factor 7), WNT6 (Protein Wnt-6), Trp1 (Short transient receptor potential channel 1) or Trp2 (Short transient receptor potential channel 2).

[0069] The term "administration" or "vaccination" refers to administration based on the nucleic acid vaccine or vaccine composition of the present invention preferably via intramuscular or subcutaneous routes, although other routes of administration can also be used, for example, oral, intranasal (e.g., aerosol or other non-injection administration), intralymph node, intradermal, intraperitoneal, rectal or vaginal administration, or by a combination of routes. Administration in the neck muscle of an animal is preferred. Boosting regimens can be used to adjust the dosage regimen to provide optimal immunity.

[0070] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0071] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide", "recombinant protein", "fusion protein", etc.

[0072] The term "recombinant expression vector" refers to a DNA structure used to express a polynucleotide encoding a desired polypeptide, for example. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcription and translation start and stop sequences. The recombinant expression vector is constructed in any suitable manner and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. Both self-replicating and non-self-replicating vectors are included.

[0073] The term "mRNA" refers to messenger RNA, which is translated into Chinese as "messenger ribonucleic acid". It is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template, carries genetic information and can guide protein synthesis.

[0074] The term "5'-UTR", which refers to the "5' untranslated region" or "5'UTR", is a portion of a gene that is transcribed into a primary RNA transcript (pre-mRNA) and is located upstream of the coding sequence. The primary transcript is the initial RNA product, containing introns and exons, produced by transcription from DNA. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes modifying the ends, removing introns, capping and / or shearing off individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of an mRNA is a portion of the mRNA that is not translated into protein and is located upstream of the coding sequence. In a genomic sequence, the 5'UTR is usually defined as the region located between the transcription start site and the start codon. The 5' untranslated region (5'UTR) of vertebrate mRNA can be tens to hundreds of bases in length.

[0075] The term "3'-UTR", meaning "3'-untranslated region" or "3'UTR", refers to a region located at the 3' end of a gene, downstream of the stop codon of the protein coding region, and which is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule. The 3'-untranslated region typically extends from the stop codon of the translation product to a poly(A) sequence that is typically attached after the transcription process. The 3'-untranslated region of mammalian mRNA typically has a homology region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-untranslated region may contain one or more inverted repeats and may fold to produce a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins (e.g., RNA binding proteins) that are known to increase RNA stability.

[0076] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells.

[0077] The terms "individual", "patient" or "subject" include mammals. Mammals include, but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, and horses, etc.), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats, etc.).

[0078] The terms "transformation, transfection, and transduction" have the meanings generally understood by those skilled in the art, namely, the process of introducing exogenous DNA or RNA into a host.

[0079] The term "drug combination" or "pharmaceutical composition" refers to auxiliary materials widely used in the field of drug production. The main purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature and / or has specific functionality, and also to provide a method so that it can be effectively absorbed in the body of a subject. A pharmaceutically acceptable carrier can be an inert filler or an effective component that provides a certain function for the pharmaceutical combination (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, adhesives, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0080] The term "treatment" means that after suffering from a disease, a subject is exposed to (e.g., administered) a recombinant vaccine, composition, etc. according to the present invention, thereby alleviating the symptoms of the disease compared to when the subject is not exposed to the vaccine, composition, etc., and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that the body has symptoms of the disease.

[0081] The term "prevention" means that before a subject develops a disease, by exposing (e.g., administering) the recombinant vaccine, composition, etc. according to the present invention to the subject, the symptoms after the subject develops the disease are alleviated compared to when the subject does not develop the disease. It does not necessarily mean that the disease must be completely suppressed.

[0082] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0083] The present invention discloses a fusion molecule architecture that effectively improves the antigen presentation efficiency of cells, a method for preparing a recombinant vaccine based on the architecture, and an application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0084] In the fusion protein and encoding nucleic acid and elements thereof, preparation methods and applications provided by the present invention, the raw materials and reagents used can all be obtained commercially.

[0085] The present invention is further illustrated below in conjunction with the examples, wherein, as a preferred embodiment, a nucleic acid vaccine framework is selected for preparing a recombinant vaccine.

[0086] Example 1 Construction of the recombinant nucleic acid vaccine of the present invention and the control vaccine

[0087] In order to prepare a recombinant nucleic acid vaccine comprising the fusion molecule of the present invention (the sequence is shown in SEQ ID NO: 1), the molecular structure diagram of the fusion protein of the present invention is as follows: Figure 1 As shown, from the N-terminus to the C-terminus, it includes: a signal peptide, an antigen amino acid sequence, an FcRn sequence transmembrane region, and an FcRn sequence cytoplasmic region. First, a gene expression cassette is constructed. The schematic diagram of the gene expression cassette architecture containing the fusion protein molecule of the present invention is shown in FIG. Figure 2 As shown, from the 5' end to the 3' end, it includes: 5'UTR, signal peptide (the encoded amino acid sequence is shown in SEQ ID NO:2), antigen nucleic acid sequence (i.e., target antigen), GS linker (the encoded amino acid sequence is shown in SEQ ID NO:3), FcRn sequence transmembrane region and cytoplasmic region (wherein, the amino acid sequence of the encoded FcRn sequence transmembrane region and cytoplasmic region is shown in SEQ ID NO:4, wherein the amino acid sequence of the FcRn sequence transmembrane region is shown in SEQ ID NO:6, and the amino acid sequence of the FcRn sequence cytoplasmic region is shown in SEQ ID NO:7), 3'UTR. In this example, the Fluc epitope polypeptide is used as the target antigen, and the sequence encoding the Fluc epitope polypeptide (the encoded amino acid sequence is shown in SEQ ID NO:5) is cloned into the expression cassette as a model antigen. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained by gene synthesis (commissioned to be synthesized by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription to obtain a vector plasmid for preparing a recombinant nucleic acid vaccine. The schematic diagram of the recombinant vector plasmid containing the gene expression cassette architecture of the present invention is shown in FIG. Figure 3 shown.

[0088] According to the above method, vectors (1)-(4) were prepared for the experiment, wherein the recombinant nucleic acid vaccines of the present invention were vaccine B and vaccine D, and the control vaccines were vaccine A and vaccine C.

[0089] (1) Preparation vector of recombinant nucleic acid vaccine (vaccine A) expressing only Fluc epitope peptide

[0090] Step a: synthesizing a Fluc epitope polypeptide gene fragment as an antigen sequence;

[0091] Step b: constructing a nucleic acid vaccine framework vector;

[0092] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine. Preferably, the vector is pUC57, and the vectors in the following vaccines B, C, and D can also be pUC57.

[0093] Step c: Preparation of recombinant plasmid

[0094] The gene synthesized in step a is inserted into the vector structure of step b to obtain a recombinant nucleic acid vaccine preparation vector that only expresses the Fluc epitope polypeptide.

[0095] (2) Preparation of vectors based on the recombinant nucleic acid vaccine (vaccine B) of the present invention

[0096] Step a: As in step a of (1) above, synthesize the gene fragments of the signal peptide-Fluc epitope polypeptide-GS linker-FcRn sequence transmembrane region and cytoplasmic region, wherein the Fluc epitope polypeptide is used as the antigen sequence;

[0097] Step b: As in step b of (1) above, construct a nucleic acid vaccine framework vector;

[0098] Step c: Preparation of recombinant plasmid

[0099] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for expressing the recombinant nucleic acid vaccine preparation based on the present invention.

[0100] (3) Preparation vector of a recombinant nucleic acid vaccine (vaccine C) expressing only the internal reference protein Tubulin

[0101] Step a: As in step a of (1) above, synthesize a mouse Tubulin (mTubulin) gene fragment as an antigen sequence;

[0102] Step b: As in step b of (1) above, construct a nucleic acid vaccine framework vector;

[0103] Step c: Preparation of recombinant plasmid

[0104] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing a recombinant nucleic acid vaccine expressing mTubulin.

[0105] (4) Preparation of vector based on the recombinant nucleic acid (vaccine D) expressing EGFP protein of the present invention

[0106] Step a: as described in step (1) a above, signal peptide-EGFP-GS linker-FcRn sequence transmembrane region and cytoplasmic region gene fragments, wherein EGFP is green fluorescent protein, which is used for cell localization;

[0107] Step b: As in step b of (1) above, construct a nucleic acid vaccine framework vector;

[0108] Step c: Preparation of recombinant plasmid

[0109] The gene synthesized in step a is inserted into the vector structure of step b to obtain a recombinant nucleic acid (vaccine D) preparation vector based on the present invention and expressing EGFP protein.

[0110] Table 1 Amino acid sequences involved in Example 1

[0111] Component name and serial number sequence Fusion protein molecule SEQ ID NO: 1 MTRLTVLALLAGLLASSRAAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPGGSGGGGSGGGNQIIPDTAILSVVPFHHGF GMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLGSGLGGGGSGVLVVGIVIGVLLLTAAAVGGALLWRRMRSGLPAPWISLRGDDTGVLLPTPGEAQDADLKDVNVIPATA Signal peptide SEQ ID NO: 2 MTRLTVLALLAGLLASSRA GS linker SEQ ID NO:3 GGSGGGGSGG FcRn sequence transmembrane region and cytoplasmic region SEQ ID NO: 4 VLVVGIVIGVLLLTAAAVGGALLWRRMRSGLPAPWISLRGDDTGVLLPTPGEAQDADLKDVNVIPATA Fluc epitope polypeptide SEQ ID NO: 5 GNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPL FcRn sequence transmembrane region SEQ ID NO: 6 VLVVGIVIGVLLLTAAAVGGALLW FcRn sequence cytoplasmic region SEQ ID NO: 7 RRMRSGLPAPWISLRGDDTGVLLPTPGEAQDADLKDVNVIPATA mTubulin sequence SEQ ID NO: 19 MRECISIHVGQAGVQIGNACWELYCLEHGIQPDGQMPSDKTIGGGDDSFNTFFSETGAGKHVPRAFVDLEPTVIDEVRTGTYRQLFHPEQLITGKEDAANNYARGHYTIGK EIIDLVLDRIRKLADQCTGLQGFLVFHSFGGGTGSGFTSLLMERLSVDYGKKSKLEFSIYPAPQVSTAVVEPYNSILTTHTTLEHSDCAFMVDNEAIYDICRRNLDIERPTYT NLNRLIGQIVSSITASLRFDGALNVDLTEFQTNLVPYPRIHFPLATYAPVISAEKAYHEQLSVAEITNACFEPANQMVKCDPRHGKYMACCLLYRGDVVPKDVNAAIATIKTK RTIQFVDWCPTGFKVGINYQPPTVVPGGDLAKVQRAVCMLSNTTAIAEAWARLDHKFDLMYAKRAFVHWYVGEGMEEGEFSEAREDMAALEKDYEEVGVDSVEGEGEEEGEEY EGFP sequence SEQ ID NO: 20 MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0112] Example 2 Preparation of the recombinant nucleic acid vaccine of the present invention and the control vaccine

[0113] (1) Preparation of capped mRNA vaccines

[0114] Step a: The vector plasmid used to produce the capped mRNA vaccine in Example 1 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0115] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0116] (2) Preparation of non-capped mRNA vaccines

[0117] Step a: The vector plasmid used to produce the non-capped mRNA vaccine in Example 1 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0118] Step b: The linearized plasmid is subjected to an in vitro non-capped transcription reaction and the template DNA is degraded.

[0119] (3) DNA vaccine preparation

[0120] Step a: Amplify the vector plasmid used to produce the DNA vaccine in Example 1 to obtain a large amount of target plasmid for purification.

[0121] Step b: Use an endotoxin-free plasmid extraction and purification kit to extract and purify the target plasmid.

[0122] Example 3 Quality Control of the Recombinant Nucleic Acid Vaccine of the Present Invention and the Control Vaccine

[0123] Vaccine A, Vaccine B, Vaccine C, and Vaccine D were prepared by the method for preparing capped mRNA vaccines in Example 2. The purity of the produced recombinant nucleic acid was tested, and the purity of the 5 groups of recombinant nucleic acid vaccines used in the experiment was greater than 85%. Figure 4A -D. The specific description is: (1) Figure 4A It was shown that the recombinant nucleic acid (vaccine A) expressing only the Fluc epitope peptide had a purity of 92.6%; (2) Figure 4B It was shown that the purity of the recombinant nucleic acid (vaccine B) based on the present invention was 91.8%; (3) Figure 4C The results showed that the recombinant nucleic acid (vaccine C) expressing only the internal reference protein mTubulin had a purity of 85.9%. (4) Figure 4D The results show that the recombinant nucleic acid (vaccine D) based on the present invention and expressing EGFP protein has a purity of 88.6%. The above purity meets the quality requirements for vaccine production.

[0124] Example 4: Presentation pathway of the vaccine of the present invention in cells

[0125] The vaccine D in Example 3 was transfected into DC 2.4 cells using a cell transfection reagent to detect the co-localization of the proteins expressed by the nucleic acid vaccine based on the framework of the present invention in the endoplasmic reticulum, endosomes, and lysosomes.

[0126] Test results such as Figure 5 As shown, the DAPI coloring area is the cell nucleus, the Calnexin coloring area is the endoplasmic reticulum, the Rab11 coloring area is the endosome, the LAMP-1 coloring area is the lysosome, EGFP is the green fluorescent protein, and the Merge picture is the fusion of the three pictures on the right. Since EGFP is expressed based on the FcRn vaccine architecture of the present invention, it can show the expression, localization and presentation pathway of the vaccine based on the present invention in the cell.

[0127] As can be seen from the result graph, the EGFP expressed based on the FcRn framework of the present invention is mainly localized in the endoplasmic reticulum and endosomes (indicated by the white arrows). Since the EGFP protein is degraded in the lysosomes, the fluorescence signal in the lysosomes is relatively small, but it is still sufficient to show the existence of co-localization. The above results prove the theoretical mechanism of the present invention: the FcRn framework can guide the antigen to the endosome, and the endosome is acidified and then fused with the lysosome, promoting the degradation of the antigen into antigenic peptides, and then binding to MHC class I or class II molecules, thereby achieving the purpose of promoting antigen presentation.

[0128] Example 5 Immune effect of recombinant nucleic acid vaccine in mice

[0129] The high-quality recombinant nucleic acid vaccines A, B, and C obtained in Example 3 were respectively encapsulated with LNPs and used in the mouse immunization experiment of this example.

[0130] The experiment selected 15 female mice of C57BL / 6 strain at 6-8 weeks of age, each weighing about 20 g. Among them, 5 mice were tested in each group, and there were 3 groups in total. The details are shown in Table 2. The dosage in this table and below refers to the amount of active ingredient.

[0131] Table 2 Grouping and treatment of animals in immunization experiments

[0132]

[0133] Note: sc means administration by injection subcutaneously in the groin of mice.

[0134] The mice in the above groups were given primary and secondary immunizations according to the immunization process in Table 2. The mice were killed on Day 28, fresh spleens were obtained, spleen cell suspensions were prepared, and specific T cells in the spleen cell suspensions were detected by ELISPOT. Figure 6 shown.

[0135] The antigen peptide stimuli used in the ELISPOT experiment are shown in Table 3, all of which are stimuli for the exogenous antigen Fluc protein used in vaccines A and B. Among them, the peptide pool is a mixture of Flucpep-1, Flucpep-2 and Flucpep-3. Since the antigen used in vaccine C is the endogenous Tubulin protein of mice (reference group), theoretically no specific cellular immunity against Fluc protein will be generated, and cells treated with PMA and PHA stimulants are used as positive controls; in addition, the reference group cells without any stimulants are used as negative controls.

[0136] Table 3 Peptide stimuli used in ELISPOT experiments

[0137] name Sequence (number) MHC typing Flucpep-1 ITYAEYFEM (SEQ ID NO: 8) H2-Kb Flucpep-2 SVVPFHHGF (SEQ ID NO:9) H2-Kb Flucpep-3 YDLSNLHEI (SEQ ID NO: 10) H2-Db Flucpep-4 LMYRFEEEL (SEQ ID NO: 11) H2-Db Flucpep-5 GALFIGVAVAPANDI(SEQ ID NO:12) H2-IAb

[0138] ELISPOT test results Figure 7 and Figure 8 As shown, Figure 7 This is a comparison chart of the ELISPOT results of the recombinant vaccine B based on the present invention and the conventional molecular framework vaccines A and C. Figure 8 The ELISPOT original result diagrams of the recombinant vaccine B based on the present invention and the conventional molecular framework vaccines A and C. It can be seen from the experimental results that immunizing mice with conventional recombinant nucleic acid (vaccine A) that only expresses Fluc epitope polypeptides can induce mice to produce cellular immunity against exogenous antigens, but the cellular immunity level is very low. Combined with the experimental results of vaccine C (control group), it is proved that the specific cellular immunity induced by vaccine A is not good, which is equivalent to the negative control results. The recombinant nucleic acid vaccine (vaccine B) based on the present invention can induce significant antigen-specific cellular immune response, which is far better than vaccine A and close to the cellular immunity level caused by positive stimuli in the control group. Figure 8 The original ELISPOT graph of the above results is shown as supporting material.

[0139] The reasons for the above results are analyzed. Conventional molecular architectures cannot introduce antigens into the cellular immune presentation pathway in a targeted manner and can only induce low-level antigen-specific cellular immunity. The recombinant nucleic acid vaccine based on the present invention uses the transmembrane region and cytoplasmic region of FcRn as key structural elements to promote antigen presentation, and targets the antigenic polypeptides to aggregate in the MHC II-enriched lysosomes, thereby promoting the efficient presentation of the antigen CD4 epitope. This can be verified from the detection results of Flucpep-5. Subsequently, CD4 + T cell helper CD8 + T cells are activated, thereby obtaining a higher level of antigen-specific cellular immunity. In addition, FcRn has a special recycling property. Proteins that are not degraded in lysosomes will return to the lysosomes through the "recycle" system and be degraded again, which can theoretically achieve a higher protein degradation efficiency.

[0140] Example 6 Immunotherapeutic effect of the recombinant nucleic acid vaccine of the present invention on the C1498 acute myeloid leukemia mouse model

[0141] Based on the results and analysis of Example 5, it can be concluded that the recombinant nucleic acid vaccine based on the present invention can effectively activate antigen-specific cellular immune responses and is significantly superior to the frameworks of other current inventions. In order to verify that the induced cellular immune response can have a therapeutic effect, the C1498 acute myeloid leukemia mouse model was used as an experimental subject to verify that the vaccine based on the present invention can provide a good therapeutic effect.

[0142] The experiment used C57BL / 6 mice to establish a tumor model and verify the efficacy of the vaccine. The specific groups of experimental mice are shown in Table 4, and the drug was administered by subcutaneous injection.

[0143] Table 4 Grouping and treatment of experimental animals in Example 6

[0144] Vaccine Group Number of mice Processing Negative control 6 Not Vaccinated mTubulin 8 Vaccination containing endogenous protein Tubulin Vaccine I 8 Vaccination containing the vaccine according to the present invention Vaccine II 9 Vaccination containing the vaccine II according to the present invention

[0145] Among them, the antigens of vaccine I and vaccine II are tandemly expressed C1498 TAA target peptides, and the antigen targets involved are BY55 (CD160 antigen), SPRY4 (Protein sprouty homolog 4), TCF7 (Transcriptionfactor 7) and WNT6 (Protein Wnt-6). The schematic diagram of the structure of vaccine I is as follows Fig. 9As shown, from 5' to 3' end, it comprises a signal peptide, a target antigen BY55, a GS linker, a target antigen SPRY4, a GS linker, and an FcRn fragment of the present invention (i.e., a transmembrane region and a cytoplasmic region of the FcRn sequence), the amino acid sequence of BY55 is shown in SEQ ID NO: 13, the amino acid sequence of SPRY4 is shown in SEQ ID NO: 14, and the amino acid sequences of other elements are shown in Table 1.

[0146] BY55 amino acid sequence: DGITEKSSQLVFTIEQATPSDSGTYQCCARSQKPEIYIHGHFLSVLVTGNHTEIRQRQRSHPDFSHINGTLSSG (SEQ ID NO: 13).

[0147] SPRY4 amino acid sequence: VGADDRSWVFSYAQRKWHSMLANEKAPIKGIGQPEKVGLLLDYEAKKLSLVDVSRISVIHTLQTDFRGPVAPAFALWDGELLTHSGL (SEQ ID NO: 14).

[0148] The schematic diagram of the structure of Vaccine II is as follows: Fig.10 As shown, from 5' to 3' end, it comprises a signal peptide, a target antigen TCF7, a GS linker, a target antigen WNT6, a GS linker, and an FcRn fragment of the present invention (i.e., a transmembrane region and a cytoplasmic region of the FcRn sequence), the amino acid sequence of TCF7 is shown in SEQ ID NO: 15, the amino acid sequence of WNT6 is shown in SEQ ID NO: 16, and the amino acid sequences of other elements are shown in Table 1.

[0149] TCF7 amino acid sequence: SLTSGSMGQLPHTVSWPSPPLYPLSPSCGYRQHFPAPTAAPGAPYPRFTHPSLMLGSGVPGHPAAIPHPAIVPSSGK (SEQ ID NO: 15).

[0150] WNT6 amino acid sequence: QAPRGRAPPRPSGLLGTPGPPGPTGSPDASAAWEWGGCGDDVDFGDEKSRLFMDAQHKRGRGDIRALVQLHNNEAGRLAVRSH (SEQ ID NO: 16).

[0151] The mice in the above groups were divided into Fig.11A Immunotherapy was carried out according to the immunization schedule shown. The vaccine was injected 4 times in total during the treatment, with each injection dose of 20 μg. The mice were killed on the 18th day, and the tumor volume was measured and statistical analysis was performed.

[0152] Depend on Fig. 11B The statistical results show that both the recombinant nucleic acid vaccine I and vaccine II of the present invention can activate the immune system of tumor-bearing mice and provide effective immune protection. After the third injection of the vaccine, the tumor volume was significantly smaller than that of the negative control group, and tumor growth was continuously inhibited. Vaccine I and vaccine II of the present invention have similar effects and can achieve statistically significant tumor inhibition effects.

[0153] Although the mTubulin group is an endogenous protein and theoretically does not induce tumor-specific immune responses, the recombinant nucleic acid vaccine is delivered by LNP packaging, which may activate nonspecific immune responses and inhibit C1498 tumors, thus showing a weaker tumor inhibition effect in the results.

[0154] Example 7 Immunotherapeutic and preventive effects of the recombinant nucleic acid vaccine of the present invention on a B16-F10 melanoma mouse model

[0155] The B16-F10 melanoma mouse model is a classic model for verifying the efficacy of melanoma vaccines. This example uses this model as an experimental subject to verify that the vaccine based on the present invention can provide good therapeutic and preventive effects.

[0156] The experiment selected C57BL / 6 mice as the animal model for prevention and treatment experiments. The specific groups are shown in Tables 5 and 6. The drugs were administered by subcutaneous injection.

[0157] Table 5 Grouping and treatment of experimental animals in Example 7

[0158] Vaccine Group Number of mice Processing Negative control 9 Not Vaccinated Vaccine III 9 Vaccination containing the vaccine according to the present invention III Vaccine IV 9 Vaccination containing the vaccine IV according to the present invention Vaccine III & Vaccine IV 9 Vaccination containing vaccine III and vaccine IV based on the present invention

[0159] Table 6 Grouping and treatment of animals in the prevention experiment in Example 7

[0160] Vaccine Group Number of mice Processing Negative control 5 Not Vaccinated mTubulin 5 Vaccination containing endogenous protein Tubulin Vaccine III & Vaccine IV 5 Vaccination containing vaccine III and vaccine IV based on the present invention

[0161] Among them, the antigens of Vaccine III and Vaccine IV are the classical antigen epitopes of B16-F10, and the target antigens involved are Trp1 (Short transient receptor potential channel 1) and Trp2 (Short transient receptor potential channel 2), respectively.

[0162] The schematic diagram of the structure of vaccine III is as follows Fig.12As shown, from 5' to 3' end, it includes a signal peptide, a target antigen Trp1, a GS linker, and the FcRn fragment of the present invention (i.e., the transmembrane region and the cytoplasmic region of the FcRn sequence), the amino acid sequence of Trp1 is shown in SEQ ID NO: 17, and the amino acid sequences of other elements are shown in Table 1.

[0163] Trp1 amino acid sequence: HNCGTCRPGWRGAACNQKILTVRRNLLDLSPEEKSHFVRALDMAKRTTHPQFVIATRRLEDILGPDGNTPQFENISVYNYFVWTHYYSVKKTFLGGSGGGGSGGSF RNTVEGYSAPTGKYDPAVRSLHNLAHLFLNGTGGQTHLSPNDPIFVLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNRQYNMVPFWPPVTNTEMFVTAPDNLGYAYEVQW (SEQ ID NO:17).

[0164] The schematic diagram of the structure of vaccine IV is as follows Fig.13 As shown, from 5' to 3' end, it includes a signal peptide, a target antigen Trp2, a GS linker, and the FcRn fragment of the present invention (i.e., the transmembrane region and the cytoplasmic region of the FcRn sequence), the amino acid sequence of Trp2 is shown in SEQ ID NO: 18, and the amino acid sequences of other elements are shown in Table 1.

[0165] Trp2 amino acid sequence: VQTDTRPWSGPYILRNQDDREQWPRKFFNRTCKCTGNFAGYNCGGCKFGWTGPDCNRKKPAILRRNIHSLTAQEREQFLGALDLAKKSIHPDYVITTQHWLGLLGPNGTQPQIANCSVYDFFV WLHYYSVRDTLLGGSGGGGSGGDGTLDSQVMNLHNLAHSFLNGTNALPHSAANDPVFVVLHSFTDAIFDEWLKRNNPSTDAWPQELAPIGHNRMYNMVPFFPPVTNEELFLTAEQLGYNYAVDLSEEEA (SEQ ID NO:18).

[0166] In the treatment experiment, the mice in each group in Table 5 were treated according to Fig.14A The immunotherapy was carried out according to the immune process shown in the figure. 20 μg was injected each time for treatment. The changes in tumor volume were recorded to evaluate the treatment effect. Among them, the vaccine III & vaccine IV group was a combination of vaccine III and vaccine IV, with the active ingredient mass ratio of 1:1 (10 μg: 10 μg).

[0167] Fig. 14B The recombinant vaccine of the present invention has a therapeutic effect on inhibiting tumor volume in a B16-F10 melanoma mouse model in a treatment experiment. Fig. 14B The statistical results show that both the recombinant nucleic acid vaccine III and vaccine IV of the present invention can play a therapeutic role and inhibit the growth of tumor volume; the combined use of vaccine III and vaccine IV (vaccine III & vaccine IV) has a better effect and can greatly inhibit tumor proliferation, almost completely inhibiting it. This result proves that the recombinant nucleic acid vaccine of the present invention can provide a tumor treatment effect.

[0168] Based on the above treatment trial results, Vaccine III and Vaccine IV were the most effective. Therefore, only Vaccine III and Vaccine IV were included in the prevention trial.

[0169] The mice in each group in Table 6 were divided into Fig.15A The immunization was performed according to the immunization schedule shown, with a dose of 20 ug per injection. B16-F10 tumor cells were inoculated on the 22nd day, and then the mice were continuously observed, and the tumor volume and survival rate were recorded and statistically analyzed.

[0170] Fig. 15B The recombinant vaccine of the present invention has a preventive effect on inhibiting tumor volume in a B16-F10 melanoma mouse model in a preventive experiment. Fig. 15C The recombinant vaccine of the present invention has a preventive effect on the survival rate of the B16-F10 melanoma mouse model in the prevention experiment. Fig. 15B and Fig. 15C The statistical results shown show that the recombinant nucleic acid vaccine III and vaccine IV based on the present invention can play a preventive role, inhibit tumor proliferation, and effectively improve the survival rate of mice. Both the negative control group and the mTubulin group died within 25 days after tumor bearing; after vaccination with the recombinant nucleic acid vaccine III & vaccine IV based on the present invention, 80% of the mice still survived on the 25th day of tumor bearing. This result proves that the recombinant nucleic acid vaccine based on the present invention can provide a tumor prevention effect.

[0171] At the same time, the experimental results of the present invention show that the immune protection induced by the recombinant nucleic acid vaccine of the present invention is specific and cannot immunize tumors outside the target. This also indirectly proves that the recombinant nucleic acid vaccine of the present invention activates antigen-specific cellular immune responses by promoting antigen presentation, thereby achieving the effect of prevention and treatment.

[0172] The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that The fusion protein is selected from any one of the following (a)-(d): (a) The fusion protein comprises, from N-terminus to C-terminus: a signal peptide, a target antigen BY55, a GS linker, a target antigen SPRY4, a GS linker, a transmembrane region of the FcRn protein, and a cytoplasmic region of the FcRn protein; (b) The fusion protein comprises, from N-terminus to C-terminus: a signal peptide, a target antigen TCF7, a GS linker, a target antigen WNT6, a GS linker, a transmembrane region of the FcRn protein, and a cytoplasmic region of the FcRn protein; (c) The fusion protein comprises, from N-terminus to C-terminus: a signal peptide, a target antigen Trp1, a GS linker, a transmembrane region of the FcRn protein, and a cytoplasmic region of the FcRn protein; (d) The fusion protein comprises, from N-terminus to C-terminus: a signal peptide, a target antigen Trp2, a GS linker, a transmembrane region of the FcRn protein, and a cytoplasmic region of the FcRn protein; Among them, the amino acid sequence of the signal peptide is shown in SEQ ID NO:2, the amino acid sequence of the GS linker is shown in SEQ ID NO:3, the amino acid sequence of the transmembrane region of the FcRn protein is shown in SEQ ID NO:6, the amino acid sequence of the cytoplasmic region of the FcRn protein is shown in SEQ ID NO:7, the amino acid sequence of the target antigen BY55 is shown in SEQ ID NO:13, the amino acid sequence of the target antigen SPRY4 is shown in SEQ ID NO:14, the amino acid sequence of the target antigen TCF7 is shown in SEQ ID NO:15, the amino acid sequence of the target antigen WNT6 is shown in SEQ ID NO:16, the amino acid sequence of the target antigen Trp1 is shown in SEQ ID NO:17, and the amino acid sequence of the target antigen Trp2 is shown in SEQ ID NO:

18.

2. A recombinant nucleic acid molecule, characterized in that Encoding the fusion protein of claim 1.

3. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule according to claim 2.

4. A recombinant vector, characterized in that: Comprising the recombinant nucleic acid molecule according to claim 2, or the recombinant gene expression cassette according to claim 3.

5. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule according to claim 2, or the recombinant gene expression cassette according to claim 3, or the recombinant vector according to claim 4.

6. A pharmaceutical composition, characterized in that Comprising one or more fusion proteins according to claim 1, and / or one or more recombinant nucleic acid molecules according to claim 2, and / or one or more recombinant gene expression cassettes according to claim 3, and / or one or more recombinant vectors according to claim 4, and / or one or more recombinant host cells according to claim 5.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

8. A recombinant vaccine, characterized in that: Comprising one or more fusion proteins according to claim 1, and / or one or more recombinant nucleic acid molecules according to claim 2, and / or one or more recombinant gene expression cassettes according to claim 3, and / or one or more recombinant vectors according to claim 4, and / or one or more recombinant host cells according to claim 5.

9. Use of one or more fusion proteins (a) and / or fusion proteins (b) according to claim 1, and / or one or more recombinant nucleic acid molecules according to claim 2, and / or one or more recombinant gene expression cassettes according to claim 3, and / or one or more recombinant vectors according to claim 4, and / or one or more recombinant host cells according to claim 5, and / or one or more pharmaceutical compositions according to claim 6 or 7, and / or one or more recombinant vaccines according to claim 8 in the preparation of a medicament for treating acute myeloid leukemia.

10. Use of one or more fusion proteins (c) and / or the fusion protein (d) according to claim 1, and / or one or more recombinant nucleic acid molecules according to claim 2, and / or one or more recombinant gene expression cassettes according to claim 3, and / or one or more recombinant vectors according to claim 4, and / or one or more recombinant host cells according to claim 5, and / or one or more pharmaceutical compositions according to claim 6 or 7, and / or one or more recombinant vaccines according to claim 8 in the preparation of a medicament for treating melanoma.

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