Anti-tumor or anti-virus vaccine with IL-9 as adjuvant

Abstract

The invention relates to the field of biological medicine, in particular to an anti-tumor or anti-virus vaccine with IL-9 as an adjuvant. The invention provides a medicine and / or vaccine for preventing and treating diseases, the medicine and / or vaccine comprises IL-9 and a specific antigen, and experimental results show that compared with other cytokines, IL-9 can significantly enhance the cell and humoral immune intensity induced by antigens such as viruses and tumors, promote immune memory formation and improve the antibody level in serum; compared with vaccines without IL-9, protein vaccines, DNA vaccines or RNA vaccines containing IL-9 can significantly improve the prevention and treatment effect on viruses or tumors, and have wide application prospects in prevention and treatment of diseases caused by tumors, viruses, pathogenic bacteria and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-tumor or anti-viral vaccine using IL-9 as an adjuvant. Background Art

[0002] Immunotherapy includes cell therapy such as CAR-T, TCR-T, TIL, immune checkpoint therapy represented by PD-1 / PD-L1, and cancer vaccine therapy. Cancer vaccine is a therapy that prepares antigens that are highly expressed or specifically expressed by tumors into vaccines to stimulate the body's own anti-tumor immune response. Compared with cell and immune checkpoint therapy, cancer vaccines can be used to prevent cancer recurrence after surgery in addition to treating cancer, and because cancer vaccines are in principle at a different stage of anti-tumor immune response from cell and immune checkpoint therapy, they can be used in combination with cell and immune checkpoint therapy. However, compared with cell and immune checkpoint therapies that have been approved for marketing and have brought clinical benefits to patients, no cancer vaccines with significant effects have been approved for marketing so far.

[0003] The reasons for the unsatisfactory clinical effect of cancer vaccines include: 1. The body's immune tolerance to tumor antigens, so the immune response induced by the vaccine after delivering antigens into the body is not strong enough. 2. The existence of an inhibitory tumor immune microenvironment limits the induced anti-tumor immune response. The inhibitory tumor microenvironment is composed of cells with immunosuppressive functions recruited and induced to differentiate by tumors and molecules from various sources that inhibit the functions of immune cells. There is currently no effective solution to the inhibitory tumor immune microenvironment. However, the body's immune tolerance to antigens delivered by tumor vaccines is usually caused by insufficient affinity of antigen-presenting cells for antigens or the antigen itself can only bind to antigen-presenting cells but cannot activate and induce antigen-presenting cells to mature well. The solution usually requires adding adjuvants that have the effect of recruiting and stimulating antigen-presenting cells to the delivered antigens. At present, adjuvants are mainly divided into two categories based on the delivery method. One is to deliver nucleic acids encoding them and translate adjuvant molecules into the body, which is called molecular adjuvants. The other is natural or synthetic molecules that directly stimulate the activation and maturation of antigen-presenting cells, which is called chemical adjuvants. Amy HaseleyThorne and others have systematically screened and identified soluble protein forms of Flt3L and CD80 as adjuvants to enhance the immune response of vaccines. However, there are still a large number of cytokines that may have adjuvant functions and need further identification and screening. Different cytokines have different mechanisms of acting as adjuvants, and not all cytokines can be used to prepare adjuvants. Therefore, screening cytokines that can be used as adjuvants still has great clinical application significance. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an anti-tumor or anti-viral vaccine using IL-9 as an adjuvant.

[0005] The present invention provides application of interleukin-9 in improving antigen presentation effect.

[0006] The present invention provides a recombinant protein comprising interleukin-9 and an antigen.

[0007] The interleukin 9 has an amino acid sequence as shown in SEQ ID NO: 1;

[0008] The interleukin 9 described in the present invention, also known as IL-9, can be purified from animals such as humans, mice, pigs, rabbits, etc., or obtained by heterologous expression or recombination of engineered bacteria, and the present invention is not limited to this; in the present invention, the amino acid sequence of the interleukin 9 is as shown in SEQ ID NO: 1; further, in the present invention, the amino acid sequence of the interleukin 9 also includes: a fragment having more than 80% homology with the amino acid sequence shown in SEQ ID NO: 1 and having the same or similar functions; or an amino acid fragment of the amino acid sequence shown in SEQ ID NO: 1 that has been truncated, mutated or modified, and the present invention is not limited to this. The results of the present invention show that compared with other cytokines, the IL-9 described in the present invention can significantly enhance the immune response and promote the formation of immune memory.

[0009] In the present invention, the antigen is derived from pathogenic bacteria, viruses or tumors;

[0010] The virus includes at least one of HPV virus, EBV virus, HCV virus, HIV virus, HBV virus, VZV virus, EB virus or coronavirus;

[0011] The tumor includes at least one of glioblastoma, liver cancer, cervical cancer, ovarian cancer, lung cancer, head and neck cancer, prostate cancer, breast cancer, melanoma, blood cancer, colorectal cancer, ovarian cancer, gastric cancer, bladder cancer, neurofibrosarcoma, lymphangiosarcoma or transitional epithelial carcinoma.

[0012] Furthermore,

[0013] The antigen is E2, E5, E6 and / or E7 protein of HPV virus or its mutant epitope, and the HPV virus includes HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56 and / or HPV58;

[0014] The antigen is LMP1, LMP2, EBNA1 or a mutant epitope thereof of EB virus;

[0015] The antigen is the S protein, N protein, E protein, M protein or an epitope thereof of a coronavirus, and the coronavirus is SARS virus, MERS virus and / or SARS-CoV-2;

[0016] The antigen is GPC3 protein and / or AFP protein of liver cancer;

[0017] The antigen is PSA, PSMA, PSCA, PAP and / or STEAP1 of prostate cancer;

[0018] The antigen is a dominant epitope of BCAR3 and / or HER2 of breast cancer;

[0019] The antigen is MAGE-A3, ISR2, NY-ESO-1, Melan A, gp100, Tyrosinase, TRP1 and / or TRP2 of melanoma;

[0020] The antigen is Immunoglobulin idiotype, Immunoglobulin κ-chain and / or Immunoglobulin λ-chain of a blood tumor;

[0021] The antigen is AIM2, HT001, TAF1B, Micoryx and / or TGFβRII of colorectal cancer;

[0022] The antigen is ovarian cancer folate receptor-α;

[0023] The antigens are P53, IDH1 / 2BAGE, GAGE1, GAGE2, CAG3, RAGE, CEA, CDK4, CASP-8, ras, bcr / abl and / or MUC-1 of various oncogenes, tumor suppressor genes and / or tumor-specific antigens.

[0024] Furthermore, the antigen is at least one of E6 and / or E7 of HPV16, GPC3 of liver cancer, or HER2 of breast cancer;

[0025] The HPV E6 has an amino acid sequence as shown in SEQ ID NO: 5;

[0026] The HPV E7 has an amino acid sequence as shown in SEQ ID NO: 6;

[0027] The GPC3 has an amino acid sequence as shown in SEQ ID NO:7;

[0028] The HER2 has an amino acid sequence as shown in SEQ ID NO:8.

[0029] In the present invention, the recombinant protein further comprises an IgE signal peptide and a Flag tag;

[0030] In the present invention, in order to improve the expression effect of IL-9, a signal peptide is added to the N-terminus of IL-9 to promote protein secretion outside the cell. In some embodiments, the signal peptide is an IgE signal peptide. Specifically, its amino acid sequence is shown in SEQ ID NO:4.

[0031] The Flag tag of the present invention is used for protein purification, and the tag is selected from recombinant protein purification tags well known in the art. In some embodiments, the tag is DYKDDDDK, and its amino acid sequence is shown in SEQ ID NO: 11; the Flag tag does not affect the immune effect of the sequence.

[0032] In the present invention, in the recombinant protein, the connection sequence between the antigen and IL-9 can be IL-9-E6E7; it can also be E6E7-IL-9; it can also be IL-9-E7E6; it can also be E7E6-IL-9, and the present invention is not limited to this.

[0033] Furthermore, the antigen and IL-9 described in the present invention may be from the same expression vector or from different expression vectors, and the present invention is not limited thereto.

[0034] The recombinant protein of the present invention comprises an antigen and IL-9; in some specific embodiments of the present invention, HPV16 E6 and E7 proteins, liver cancer GPC3 and breast cancer HER2 proteins are used as antigens and combined with IL-9, and the experimental results show that adding IL-9 as an adjuvant on the basis of the antigen can enhance the immunogenicity of the antigen and promote the formation of immune memory. The recombinant protein can be used for the prevention or treatment of diseases caused by the corresponding antigen.

[0035] The present invention provides a nucleic acid encoding the recombinant protein.

[0036] Furthermore, in the nucleic acid,

[0037] The nucleic acid sequence encoding interleukin-9 is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0038] Furthermore, the nucleic acid also includes: a nucleic acid molecule formed by replacing, deleting or adding one or more nucleotides to the sequence alone or after recombining the sequence, and / or a nucleic acid molecule that is complementary or partially complementary to the sequence alone or after recombining the sequence.

[0039] The nucleic acid encoding the recombinant protein of the present invention may be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The nucleic acid may include nucleotide sequences with different functions, such as coding regions and non-coding regions, such as CMV or CMV / R promoters, transcription terminators, 5'-UTR, 3'-UTR and 3'-terminal PolyA. In the RNA sequence encoding the nucleic acid of the present invention, the 5'-UTR is β-globin-2, as shown in SEQ ID NO:9, the 3'-UTR is 2β-globin, as shown in SEQ ID NO:10, the length of the 3'-terminal PolyA is 120bp, and the structure of the RNA fragment is 5'UTR-E6E7-3'UTR-A(120) and / or 5'UTR-IL-9-3'UTR-A(120). The nucleic acid of the present invention may be linear or circular in topology. The nucleic acid can be, for example, a part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the aid of recombination, enzymatic or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0040] In the present invention, the nucleic acid sequence of the recombinant protein may be optimized or unoptimized, and the present invention is not limited thereto; the optimization includes but is not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and / or restriction sites that may affect cloning, etc.

[0041] The present invention also provides a transcription unit of the recombinant protein, which refers to a DNA sequence starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc.

[0042] The present invention provides a recombinant vector, which comprises a vector backbone and the nucleic acid of the present invention.

[0043] Furthermore, the sources of the vector skeleton described in the present invention include plants, animals, bacteria, fungi, bacteriophages or viruses, which are not limited in the present invention.

[0044] Further, the recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule, which contains the desired coding sequence and the suitable nucleic acid sequence or element that is indispensable for the expression of the operably connected coding gene in a specific host organism. The nucleic acid sequence or element necessary for the expression in the model animal or mammalian cell includes a promoter, a ribosome binding site and possible other sequences. Known eukaryotic cells use promoters, enhancers and terminators. Once transformed into a suitable host, the vector can be replicated and play a role independently of the host genome, or, in some cases, integrate into the genome by itself. In this specification, "plasmid" and "vector" can sometimes be interchanged and general, because plasmid is the most commonly used vector form at present. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and / or only potential genomic insert. In a specific embodiment, the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various eukaryotic expression vectors. For example, its backbone vector can be a pVAX1 series vector, or a pVR series vector (see Chinese Patent ZL202110624820.8).

[0045] The present invention provides a host,

[0046] Its genome is integrated with the nucleic acid described in the present invention; or

[0047] Transfect or transform the recombinant vector of the present invention.

[0048] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0049] The host provided by the present invention may be derived from plants, animals, bacteria, fungi, bacteriophages or viruses, which are not limited in the present invention. The present invention uses a vector constructed by recombinant DNA technology to transform or transfect a host, so that the transformed host has the ability to replicate the protein-encoding vector or express the desired protein.

[0050] Furthermore, the host includes model animals or mammalian cells. The model animals include experimental animals commonly used in the biomedical field such as mice, rats, rabbits, etc., which are not limited in the present invention. The mammalian cells include at least one of CHO cells, BHK cells, Sp2 / 0, HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9, or FD-CHOS.

[0051] The present invention provides a method for preparing the recombinant protein, which comprises culturing the host cell as described in the present invention to obtain a culture containing the recombinant protein.

[0052] The present invention provides the use of at least one of the following I) to V) in the preparation of a drug and / or vaccine for preventing and treating a disease:

[0053] 1), the recombinant protein of the present invention;

[0054] II), the nucleic acid of the present invention;

[0055] III), the recombinant vector of the present invention;

[0056] IV), the host of the present invention;

[0057] V), a culture containing the recombinant protein obtained by the preparation method of the present invention.

[0058] In the present invention, the disease includes at least one of diseases caused by pathogenic bacteria, viruses or tumors.

[0059] The present invention provides a drug and / or vaccine for preventing and treating a disease, wherein the raw materials thereof include at least one of the following i) to v):

[0060] i), the recombinant protein of the present invention;

[0061] ii), the nucleic acid of the present invention;

[0062] iii), the recombinant vector of the present invention;

[0063] iv), the host of the present invention;

[0064] v), a culture medium containing the recombinant protein obtained by the preparation method of the present invention

[0065] The medicine and / or vaccine described in the present invention may be in the form of DNA, RNA or protein, which is not limited in the present invention.

[0066] Furthermore, the medicine and / or vaccine of the present invention further comprises an adjuvant or a buffer; the adjuvant is used to maintain the activity of the vaccine or to help the vaccine to function. The adjuvant includes but is not limited to a carrier, a surfactant, and a reducing agent, etc.; the carrier includes a lentiviral auxiliary vector, an adenoviral auxiliary vector, etc., which assists in assembly or integration; the buffer includes a PBS buffer, a KCl buffer, a NaCl buffer, a Tris buffer, etc.

[0067] Furthermore, the medicine and / or vaccine described in the present invention also includes the transcription mRNA or translation initiation product of the nucleic acid, etc., which is not limited in the present invention.

[0068] Furthermore, the drug and / or vaccine also includes lipid nanoparticles containing the nucleic acid or mRNA. The preparation process of the lipid nanoparticles includes encapsulating the nucleic acid or mRNA using a transfection reagent in an animal body to form a drug or vaccine in the form of lipid nanoparticles containing the nucleic acid or mRNA for the prevention and treatment of viruses or tumors.

[0069] The prevention and treatment described in the present invention include prevention and treatment. Prevention refers to the administration of the product for preventing and treating the disease described in the present invention before the disease occurs, so as to reduce the risk of the disease. The treatment described in the present invention refers to the administration of the product for preventing and treating the disease described in the present invention after the disease occurs, which can improve the symptoms, inhibit the development of the disease, and restore the patient to health. For example, the product described in the present invention is used to prevent and treat tumors, which can increase the antibody level in serum, inhibit tumor growth, reduce tumor volume or slow down the growth rate of tumors. In the embodiment of the present invention, mouse transplanted tumor cells TC-1 were used as experimental subjects to verify the vaccine adjuvant effect of IL-9 and obtain good therapeutic effects.

[0070] The present invention provides a method for preventing and treating a disease, which comprises administering the medicine and / or vaccine of the present invention, wherein the administration method includes oral administration, injection and / or electroporation.

[0071] The diseases include those caused by pathogenic bacteria, viruses or tumors.

[0072] The present invention provides a medicine and / or vaccine for preventing and treating diseases, wherein the medicine and / or vaccine comprises IL-9 and a specific antigen. Experimental results show that, compared with other cytokines, IL-9 can significantly enhance the cellular and humoral immunity induced by antigens such as viruses and tumors, promote the formation of immune memory, and increase the antibody level in serum; compared with vaccines not containing IL-9, protein vaccines, DNA vaccines or RNA vaccines containing IL-9 can significantly improve the prevention and treatment effects of viruses or tumors, and have broad application prospects in the prevention and treatment of diseases caused by tumors, viruses, pathogens, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The diagram shows the effect of various interleukins on enhancing the immune response induced by HPV16 subtype E6E7 protein;

[0074] Figure 2 The expression level of intracellular IL-9 was detected by western blot after the optimized IL-9 was transfected into 293T cells;

[0075] Figure 3 The expression level of IL-9 in the culture medium supernatant was detected by ELISA after the optimized IL-9 was transfected into 293T cells;

[0076] Figure 4 The results show that the nucleic acid-encoded molecular adjuvant IL-9 enhances the immune response induced by HPV16 subtype E6E7 protein;

[0077] Figure 5 The results show that the nucleic acid-encoded molecular adjuvant IL-9 enhances the immune response induced by the liver cancer GPC3 protein;

[0078] Figure 6 The results show that the nucleic acid-encoded molecular adjuvant IL-9 enhances the immune response induced by the breast cancer HER2 protein;

[0079] Figure 7 The results show that the nucleic acid-encoded molecular adjuvant IL-9 enhances the formation of immune memory induced by HPV16 subtype E6E7 protein;

[0080] Figure 8 The timeline of tumor implantation and plasmid therapeutic immunization in mice is shown;

[0081] Fig. 9 The results show that the nucleic acid-encoded molecular adjuvant IL-9 enhances the anti-tumor effect of HPV16 subtype E6E7 protein vaccine. DETAILED DESCRIPTION

[0082] The present invention provides an anti-tumor or anti-viral vaccine with IL-9 as an adjuvant. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted 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 of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0083] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0084] The present invention provides an immunoenhancing technology using IL-9 as an antigen adjuvant. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted 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 of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0085] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0086] The IL-9 described in the present invention is an important cytokine in the human body, which has the effect of promoting lymphocyte proliferation. The present invention's research shows that IL-9 has an adjuvant effect, can significantly enhance the immunogenicity of the antigen used in the vaccine, and stimulate the formation of immune memory.

[0087] In the present invention, the IL-9 can be a human fragment or a fragment of other animal sources, such as mouse, rabbit, monkey, pig, etc. It can be a complete IL-9 or a fragment or mutant having IL-9 activity, which is not limited in this application. In the examples of this application, human IL-9 was used as an experimental subject to verify the adjuvant effect of IL-9 on antigens. The amino acid sequence of the human IL-9 is shown in SEQ ID NO: 1.

[0088] The number of antigens described in the present invention is 1 or more. For example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more. In the research of the present invention, experiments were conducted on the fusion effect of 1 or 2 antigens, and both showed good results.

[0089] The antigens of the present invention are proteins from viruses, bacteria, fungi, parasites and / or tumors. In some embodiments, the antigens are from the capsid protein or non-structural protein of a virus, the membrane protein of a pathogen, flagellin or the surface antigen of a tumor. It can be a complete fragment or an antigenic determinant thereof. It can contain only one antigenic determinant, or it can be composed of multiple antigenic determinants in series, or it can be one antigenic determinant repeated in series twice or more.

[0090] Related studies and multiple experiments have shown that not all interleukin family members can be used with antigen molecules as adjuvants to enhance immunogenicity; only some have the ability to enhance the immunogenicity of antigens, initiate stronger immune responses, and provide options for the preparation of more potent vaccines. For example, related studies have shown that IL-12 can enhance the immunogenicity of antigens to a large extent, but GM-CSF, IL-23, and IL-15 do not have the effect of enhancing immunogenicity. Therefore, which interleukin has an adjuvant effect needs to be strictly verified through experiments.

[0091] The present invention utilizes IL-9 in combination with different antigen proteins to enhance the strength of immune responses induced by various antigen proteins and improve the effects of preventing and treating related diseases.

[0092] The prevention described in the present invention refers to the administration of the drug described in the present invention before the occurrence of a tumor, which can play a role in reducing the risk of tumor occurrence. The treatment described in the present invention refers to the administration of the drug described in the present invention after the occurrence of a tumor, which can inhibit tumor growth, reduce tumor volume or slow down the growth rate of the tumor. In the examples of the present invention, mouse transplanted tumor cells TC-1 were used as experimental subjects to verify the effect of the fusion protein vaccine.

[0093] In the embodiment of the present invention, the method for preparing the fusion protein adopts the method of inducing recombinant host expression, and the culture can be bacteria, cells, culture fluid obtained by culture, or substances obtained by extraction and / or purification of the above culture.

[0094] The present invention provides a product for preventing and treating a disease, wherein the product for preventing and treating a disease comprises a drug and / or a vaccine. The vaccine further comprises a pharmaceutically acceptable carrier, excipient and / or adjuvant. The drug further comprises a pharmaceutically acceptable excipient.

[0095] In the embodiments of the present invention, the amino acid sequences of the involved fragments and the encoded nucleic acid fragments are shown in Table 1:

[0096] Table 1. Amino acid sequences and encoded nucleic acid fragments involved in the present invention

[0097] SEQ ID NO:1 Amino acid sequence of IL-9 protein SEQ ID NO:2 IgE signal peptide replaces the original signal peptide IL-9 DNA sequence SEQ ID NO:3 IgE signal peptide replaces the original signal peptide IL-9 mRNA sequence SEQ ID NO:4 IgE signal peptide SEQ ID NO:5 HPV16 E6 protein amino acid sequence SEQ ID NO:6 HPV16 E7 protein amino acid sequence SEQ ID NO:7 Amino acid sequence of GPC3 protein SEQ ID NO:8 Amino acid sequence of HER2 protein SEQ ID NO:9 5-UTR SEQ ID NO:10 3-UTR SEQ ID NO:11 C-terminal Flag tag SEQ ID NO:12 Nucleotide sequence of HPV16 E6 SEQ ID NO:13 Nucleotide sequence of HPV16 E7 SEQ ID NO:14 Nucleotide sequence of GPC3 protein SEQ ID NO:15 Nucleotide sequence of HER2 protein SEQ ID NO:16 DNA plasmid containing IL-9 sequence

[0098] The amino acid sequence of the natural IL-9 protein is: MLLAM VLTSALLLCSVAGQGCPTLAGILDINFLINKMQEDPASKCHCSANV TSCLCLGIPSDNCTRPCFSERLSQMTNTTMQTRYPLIFSRVKKSVEVLKNNK CPYFSCEQPCNQTTAGNALTFLKSLLEIFQKEKMRGMRGKI (SEQ ID NO: 1);

[0099] The DNA sequence of IL-9 with the original signal peptide replaced by the IgE signal peptide is: atggactggacctggatcctgtttctggtggccgctgccacaagagtgcacagccaaggatgtcctacactggccggaatcctggatatcaacttcctgatcaacaagatgcaagaggaccccgccagcaagtgccactgtagcgccaatgtgaccagctgcctgtgtctgggcatccccagcgacaattgcaccagaccttgcttcagcgagcggctgagccagatgaccaacaccaccatgcagacccggtatcctctgatcttcagccgcgtgaagaaaagcgtggaagtgctgaagaacaacaagtgcccctacttcagctgcgagcagccctgcaatcagaccacagccggaaatgccctgaccttcctgaagtccctgctggaaatcttccagaaagaaaagatgcggggcatgcgcggcaagatctga (SEQ ID NO:2);

[0100] The mRNA sequence of IL-9 with the original signal peptide replaced by the IgE signal peptide is: auggacuggaccuggauccuguuucugguggccgcugccacaagagugcacagccaaggauguccuacacuggccggaauccuggauaucaacuuccugaucaacaagaugcaagaggaccccgccagcaagugccacuguagcgccaaugugaccagcugccugugucugggcauccccagcgacaauugcaccagaccuugcuucagcgagcggcugagccagaugaccaacaccaccaugcagacccgguauccucugaucuucagccgcgugaagaaaagcguggaagugcugaagaacaacaagugccccuacuucagcugcgagcagcccugcaaucagaccacagccggaaaugcccugaccuuccugaagucccugcuggaaaucuuccagaaagaaaagaugcggggcaugcgcggcaagaucuga (SEQ ID NO:3), where u in the sequence list is converted to t;

[0101] The amino acid sequence of the IgE signal peptide is: DWTWILFLVAAATRVHS (SEQ ID NO: 4);

[0102] The amino acid sequence of HPV16 E6 protein is: HQKRTAMFQDPQESGRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNTRGRWTGRCMSCCRSSRTRRETQL (SEQ ID NO: 5);

[0103] The amino acid sequence of HPV16 E7 protein is: HGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAH YNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID NO: 6);

[0104] The amino acid sequence of GPC3 protein is: MQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQAQYASAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFH (SEQ ID NO:7);

[0105] The amino acid sequence of the HER2 protein is: MKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV (SEQ ID NO:8);

[0106] The nucleotide sequence of the 5-UTR is: agagcggccgctttttcagcaagattaagcccagggcagagccatctattgcttacatttgcttctgacacaactgtgttcactagcaacctcaaacagacacc (SEQ ID NO:9);

[0107] The nucleotide sequence of the 3'-UTR is: agctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgc (SEQ ID NO:10);

[0108] The amino acid sequence of the Flag tag is: DYKDDDDK (as shown in SEQ ID NO:11);

[0109] The nucleotide sequence of the HPV16 E6 protein is: caccagaagagaacagccatgttccaggaccctcaggagagcggaaggaagctgccacagctctgtaccgagctgcagaccaccatccacgacatcatcctcgagtgcgtctactgcaagcagcagctgctgcggagagaggtgtacgacttcgccttcagggacctctgcatcgtgtaccgggacggcaatccctacgccgtgggcgacaagtgcctgaagttctacagcaagatcagcgagtaccggcactattgctacagcctgtacggaaccaccctggagcagcagtacaacaagcccctctgcgacctgctcatccgctgcatcaactgccagaagcccctctgtcccgaggaaaagcagaggcacctggacaagaagcagcggttccacaacaccaggggcaggtggaccggccgctgcatgagttgttgtaggagcagcaggaccagaagagagacccagctg (as shown in SEQ ID NO:12);

[0110] The nucleotide sequence of HPV16 E7 protein is: cacggagatacacctacactgcacgagtacatgctggacctgcagccagagaccaccgatctgtacggctacggacagctgaacgacagcagcgaggaggaagacgagattgatggcccagccggacaggcagaaccagatcgggcccactacaacatcgtcaccttctgctgcaagtgcgacagcaccctgagactctgcgtgcagtctacccacgtggacatcaggaccctggaggatctgctgatgggcacactgggcatcgtctgccccatttgtagccagaagcca (as shown in SEQ ID NO:13);

[0111]

[0112]

[0113]

[0114] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0115] Example 1 Screening of interleukin molecules with adjuvant effects.

[0116] According to previous research results, some cytokines have been screened and identified as molecular adjuvants. Therefore, this study screened and identified several unstudied interleukins that may have adjuvant effects. Usually, adjuvants need to have the function of stimulating lymphocyte proliferation or stimulating the activation and maturation of antigen-presenting cells. Based on this, we constructed five interleukins with such functions, IL-7, IL-9, IL-12, IL-21, and IL-33, into the pVR vector, and immunized C57 mice with plasmids of HPV16E6E7 (the amino acid sequence of HPV16 E6 is shown in SEQ ID NO:5; the amino acid sequence of HPV16 E7 is shown in SEQ ID NO:6) proteins on the pVR vector by in vivo electroporation. About fourteen days after immunization, peripheral blood of mice was collected from the inner canthus, peripheral blood mononuclear cells (PBMCs) were separated, and flow cytometry was performed using MHC class I molecule tetramer technology that can specifically recognize E7-specific T cells. The results are as follows Figure 1 As shown, among the five cytokines screened, the addition of IL-9 induced the strongest specific T cell immune response. IL-7, IL-21, and IL-33 also slightly enhanced but did not reach a significant difference. IL-12 played a completely opposite role, significantly inhibiting the specific T cell immune response caused by the E6E7 protein.

[0117] Example 2 Verification of the adjuvant effect of IL-9 on HPV16 E6E7 protein, liver cancer GPC3 protein and breast cancer HER2 protein as immunogens

[0118] After discovering that IL-9 has an adjuvant effect, the present invention first verified the repeatability of the experiment. Given that nucleic acid form of IL-9 is usually injected locally to express IL-9 and then reaches target sites such as tumors through circulation. Therefore, there are high requirements for the expression level and secretion capacity at the injection site. The present invention replaces the original IL-9 signal peptide with the IgE signal peptide to form a new chimeric protein and obtains the DNA and mRNA form IL-9 sequences of SEQ ID NO:2 and SEQ ID NO:3 through codon optimization. And add Intron A in front of the DNA sequence to construct a pVR vector to obtain a DNA form of IL-9 such as SEQ ID NO:16. After transfecting 293T cells, western blot was used to detect the intracellular IL-9 expression level and ELISA was used to detect the IL-9 level in the culture medium supernatant. The results show that the vector and sequence modification of the present invention significantly improved the expression level and secretion level of IL-9, as shown in Figure 2. Figure 2 and Figure 3 As shown. Then, the pVR-E6E7 vector alone or in combination with the pVR-IntronA-IL-9 plasmid was injected into C57 mice again by in vivo electroporation. About fourteen days after immunization, peripheral blood was collected from the inner canthus of the mice, and peripheral blood mononuclear cells (PBMC) were separated and flow cytometry was performed using MHC class I molecule tetramer technology that can specifically recognize E7-specific T cells. The results showed that the immune effect of IL-9 enhancing E6E7 is reproducible, as shown in the following figure. Figure 4 shown.

[0119] In order to prevent the adjuvant effect of IL-9 from being the result of the specific combination of IL-9 and HPV16 E6E7, the present invention continues to construct an expression vector for the antigen GPC3 that is highly expressed in liver cancer cells. For the GPC3 protein, the present invention selects its amino acid sequence as shown in SEQ ID NO:7, as well as an expression vector for the HER2 antigen in breast cancer; for the HER2 antigen, the present invention selects its portion containing the signal peptide, transmembrane domain and intracellular domain as the immunogen, and its amino acid sequence is shown in SEQ ID NO:8. All sequences are constructed into the pVR vector. The pVR-GPC3 and pVR-HER2 vectors are injected into C57 mice again using in vivo electroporation alone or in combination with the pVR-IL-9 plasmid. About fourteen days after immunization, the spleen of the mouse is taken and ground and dispersed into single cells. It is then treated with a red blood cell lysis buffer and counted after the red blood cells are removed. 2 × 10 5 Splenocytes were plated on an ELISPOT plate pre-coated with mouse IFN-r antibody and stimulated with peptide libraries of GPC3 and HER2 corresponding antigen sequences. The stimulation concentration of the peptide library was 10 μg / mL. The stimulation time was 24 hours. After that, the color was developed and the specific spots were counted. The results were as follows: Figure 5 and Figure 6 As shown in the figure, IL-9 enhanced the immune response to GPC3 and HER2 antigens respectively. Therefore, the adjuvant effect of IL-9 is not limited to HPV16 E6E7 protein but has a broad-spectrum adjuvant effect.

[0120] Example 3 Verification of IL-9 Enhancement of Immune Memory Formation Induced by HPV16 E6E7 Antigen

[0121] The immune response induced by vaccine immunization needs to form immune memory after clearing the target to have a long-term effect. Next, the present invention is to examine whether the specific T cells after HPV16 E6E7 immunization contain central memory T cells. Central memory T cells generally highly express CD62L and CD44 molecules. Therefore, the present invention injects the pVR-E6E7 vector alone or in combination with the pVR-IL-9 plasmid into C57 mice again using in vivo electroporation. About fourteen days after immunization, peripheral blood of mice was collected from the inner canthus, and peripheral blood mononuclear cells (PBMCs) were separated and flow cytometry was performed using PE-MHC class I molecule tetramer technology that can specifically recognize E7-specific T cells combined with FITC-CD44 and APC-CD62L flow cytometry antibodies. The results are as follows Figure 7 As shown, IL-9 can significantly increase the proportion of central memory T cells induced by E6E7.

[0122] Example 4 Verification of the therapeutic effect of IL-9 adjuvant vaccine on tumors

[0123] The therapeutic effect of IL-9 adjuvant vaccine on mouse transplanted tumor cell TC-1 homologous transplanted tumor (taking the fusion gene vaccine with HPV16 E6 and E7 proteins as an example, the verification steps for the tumor intervention effect of vaccines containing other antigens are the same):

[0124] In view of the excellent effect of IL-9 combined with E6E7 and other vaccines in cell immunity experiments, we also conducted experimental exploration on the therapeutic effect of TC-1 homologous transplant tumors immunized with IL-9 adjuvant vaccine. In addition to the DNA vaccine form, we also synthesized the mRNA vaccine form of IL-9 and E6E7 and encapsulated them with nanoparticles. The specific process was to connect 5'UTR-E6E7-3'UTR-A(120) and 5'UTR-IL-9-3'UTR-A(120) into the cloning vector pGEM-3Zf(+)(promega) respectively to complete the construction of the in vitro transcription expression system.

[0125] 1. Linearization of recombinant plasmid, single enzyme digestion reaction system as shown in Table 2: reaction conditions are 37°C, 3h

[0126] Table 2. Recombinant plasmid linearization single restriction enzyme digestion reaction system

[0127] composition Volume (μL) XOt 4 Recombinant plasmid 12 μg 10×CutSmart Buffer 5 <![CDATA[dd H2O]]> Supplement Total 50

[0128] 2. In vitro transcription reaction:

[0129] Choose T7-Flash Scribe TM Transcription Kit (Cellscript) was used for in vitro transcription. During the preparation of the in vitro transcription system, UTP was replaced with N1-Methylpseudouridine-5'-Triphosphate (Trilink Biotech).

[0130] The reaction system is shown in the following table. The reaction conditions are: after the first step, 35°C for 30 min; after the second step, 35°C for 15 min.

[0131] Table 3. In vitro transcription reaction

[0132]

[0133]

[0134] 3. mRNA capping: Use the kit ScriptCap TM Cap 1 Capping System (Cell script) was used for operation.

[0135] 4. Cap-mRNA purification: use MEGAclear TM The purification reaction was performed using the Purification Kit (Invitrogen).

[0136] 5. Preparation of liposome nanoparticles LNP-Man:

[0137] DOTAP ((2,3-dioleoyl-propyl) trimethylammonium chloride), DOPE (dioleoylphosphatidylethanolamine), and DSPE-PEG2000 (distearoylphosphatidylethanolamine-polyethylene glycol 2000) were purchased from Shanghai Avite Pharmaceutical Technology Co., Ltd.; DSPE-PEG2000-Man was purchased from Xi'an Haoran Biotechnology Co., Ltd.;

[0138] Nanoparticles were prepared by rotary evaporation. The specific operation process of LNP-Man preparation is as follows:

[0139] 1) Add DOTAP:DOPE:DSPE-PEG2000-Man in a molar ratio of 50:50:1 into a round-bottom flask in sequence, and add 6 mL of chloroform until the solid is fully dissolved;

[0140] 2) Water bath ultrasound for 15 minutes;

[0141] 3) Place the round-bottom flask into the rotary evaporator. The dissolved material in the round-bottom flask must be below the water surface. The speed is 100 rpm and the rotary evaporation is performed for 15 minutes.

[0142] 4) Remove the round-bottom flask, place it in a fume hood, add 8 mL of HEPES buffer, and dissolve the film on the inner wall of the flask. The dissolving reagent is HEPES;

[0143] 5) Water bath ultrasound for 30 min;

[0144] 6) The ultrasonicated solution was filtered through a 0.22 μm filter membrane for three times to obtain the desired liposome nanoparticles LNP-Man.

[0145] 6. Preparation of LNPs / mRNA

[0146] The prepared cationic lipid nanomaterial LNP-Man was mixed with mRNA; equal volumes of 10 mM HEPES buffer were added to LNPs and mRNA before mixing; the mixed LNPs / mRNA was shaken on a vortex shaker for 1 min and allowed to stand at room temperature for 30 min. Figure 8 The mice were inoculated with tumors and immunized with plasmids according to the timeline in the figure. The amount of each immunization was 25 μg. The mice were immunized once every nine days after the tumor inoculation. The tumors were then measured in the same way and the tumor volumes were calculated. The tumor growth curve was plotted as shown in the figure below. Fig. 9 shown.

[0147] The results showed that after combining with IL-9, the pVR-E6E7 plasmid and E6E7-mRNA inhibited tumor growth earlier than the pVR-E6E7 plasmid group and the PVR-IL-9 group. In addition, the transplanted tumors in the pVR-E6E7 plasmid and E6E7-mRNA combined with IL-9 group completely disappeared around the 31st day after tumor implantation, achieving a complete tumor treatment effect.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of interleukin-9 in improving antigen presentation effect.

2. A recombinant protein comprising interleukin 9 and an antigen.

3. The recombinant protein according to claim 2, characterized in that The interleukin 9 has an amino acid sequence as shown in SEQ ID NO: 1; The antigen is derived from pathogenic bacteria, virus or tumor; The virus includes at least one of HPV virus, EBV virus, HCV virus, HIV virus, HBV virus, VZV virus, EB virus or coronavirus; The tumor includes at least one of glioblastoma, liver cancer, cervical cancer, ovarian cancer, lung cancer, head and neck cancer, prostate cancer, breast cancer, melanoma, blood cancer, colorectal cancer, ovarian cancer, gastric cancer, bladder cancer, neurofibrosarcoma, lymphangiosarcoma or transitional epithelial carcinoma.

4. The recombinant protein according to claim 2 or 3, characterized in that The antigen is E2, E5, E6 and / or E7 protein of HPV virus or its mutant epitope, and the HPV virus includes HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56 and / or HPV58; The antigen is LMP1, LMP2, EBNA1 or a mutant epitope thereof of EB virus; The antigen is the S protein, N protein, E protein, M protein or an epitope thereof of a coronavirus, and the coronavirus is SARS virus, MERS virus and / or SARS-CoV-2; The antigen is GPC3 protein and / or AFP protein of liver cancer; The antigen is PSA, PSMA, PSCA, PAP and / or STEAP1 of prostate cancer; The antigen is a dominant epitope of BCAR3 and / or HER2 of breast cancer; The antigen is MAGE-A3, ISR2, NY-ESO-1, Melan A, gp100, Tyrosinase, TRP1 and / or TRP2 of melanoma; The antigen is Immunoglobulin idiotype, Immunoglobulin κ-chain and / or Immunoglobulin λ-chain of a blood tumor; The antigen is AIM2, HT001, TAF1B, Micoryx and / or TGFβRII of colorectal cancer; The antigen is ovarian cancer folate receptor-α; The antigens are P53, IDH1 / 2BAGE, GAGE1, GAGE2, CAG3, RAGE, CEA, CDK4, CASP-8, ras, bcr / abl and / or MUC-1 of various oncogenes, tumor suppressor genes and / or tumor-specific antigens.

5. The recombinant protein according to any one of claims 2 to 4, characterized in that The antigen is at least one of E6 and / or E7 of HPV16, GPC3 of liver cancer, or HER2 of breast cancer; The HPV E6 has an amino acid sequence as shown in SEQ ID NO: 5; The HPV E7 has an amino acid sequence as shown in SEQ ID NO: 6; The GPC3 has an amino acid sequence as shown in SEQ ID NO:7; The HER2 has an amino acid sequence as shown in SEQ ID NO:

8.

6. The recombinant protein according to any one of claims 2 to 5, characterized in that The recombinant protein also includes an IgE signal peptide and a Flag tag; The IgE signal peptide has an amino acid sequence as shown in SEQ ID NO: 4; The amino acid sequence of the Flag tag is shown in SEQ ID NO:

11.

7. A nucleic acid encoding the recombinant protein according to any one of claims 2 to 6.

8. The nucleic acid according to claim 7, characterized in that The interleukin-9 encoding nucleic acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

9. A recombinant vector comprising a vector backbone and the nucleic acid according to claim 7 or 8.

10. The recombinant vector according to claim 9, characterized in that The vector backbone includes pVR.

11. A host, characterized in that Genome integration of the nucleic acid of claim 7 or 8; or Transfect or transform the recombinant vector according to claim 9 or 10.

12. The host according to claim 11, characterized in that The host includes model animals and / or mammalian cells.

13. A method for preparing the recombinant protein according to any one of claims 2 to 6, comprising culturing the host cell according to claim 11 or 12 to obtain a culture containing the recombinant protein.

14. Use of at least one of the following I) to V) in the preparation of drugs and / or vaccines for preventing and treating diseases: I), the recombinant protein according to any one of claims 2 to 6; II), the nucleic acid according to claim 7 or 8; III), the recombinant vector according to claim 9 or 10; IV), the host according to claim 11 or 12; V), a culture containing the recombinant protein obtained by the preparation method according to claim 13.

15. Drugs and / or vaccines for preventing and treating diseases, wherein the raw materials thereof include at least one of the following i) to v): i), the recombinant protein according to any one of claims 2 to 6; ii), the nucleic acid according to claim 7 or 8; iii), the recombinant vector according to claim 9 or 10; iv) the host of claim 11 or 12; v) A culture containing the recombinant protein obtained by the preparation method according to claim 13.

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